OIL YEAST CELL, CULTURE, AS WELL AS METHODS TO CONVERT A CARBOHYDRATE SOURCE TO FATTY ACID OR TRIACY

专利摘要:
oil cell, culture, nucleic acid molecule, expression cassette, as well as method for producing fatty acids and fatty acid derivatives. The present invention relates to methods useful for converting a carbon source to a biofuel or biofuel precursor using engineered microbes. some aspects of this invention pertain to the discovery of a key regulator of lipid metabolism in microbes. some aspects of this invention pertain to microbes designed for the production of biofuel or biofuel precursor.
公开号:BR112012022108B1
申请号:R112012022108-6
申请日:2011-03-02
公开日:2022-01-18
发明作者:Gregory Stephanopoulos；Syed Hussain Imam Abidi
申请人:Massachusetts Institute Technology；
IPC主号:

专利说明:

RELATED ORDER
[001] This application claims priority under 35 U.S.C. § 119(e) to the United States provisional patent application, U.S.S.N. 61/309,782, filed March 2, 2010, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION
[002] The invention, at least in part, relates to the field of converting a carbohydrate source into a biofuel or a biofuel precursor, for example a fatty acid or fatty acid derivative, such as a triacylglycerol, using a engineered cell or microbe. FEDERALLY SPONSORED RESEARCH
[003] Research leading to certain aspects of the invention(s) disclosed herein was supported, at least in part, by Department of Energy grant 69106899. The United States Government has certain rights in the invention. BACKGROUND OF THE INVENTION
[004] Sustainably produced biofuels are an alternative to fossil fuels, and can help alleviate the depletion of readily accessible fossil fuel stocks, while avoiding fuel-associated pollution and greenhouse gas emissions, thereby satisfying a high demand. of affordable energy in a sustainable way. However, the widespread implementation of biofuel production has been thwarted by several problems with current production methods, for example, the competition of biofuel production plants with feed crops for agriculturally valuable acreages, or the use of industrial substrates with only limited supplies as carbon sources. SUMMARY OF THE INVENTION
[005] The growing interest in the sustainability and renewability of fossil fuels has led to the development of a broad spectrum of alternative biofuels from various sources, including lipids synthesized from renewable resources by microbes, such as bacteria or yeast. Lipids useful as a biofuel, or precursors to biofuels, include, for example, fatty acids and their derivatives (eg, triacylglycerols).
[006] The economic viability of microbe-synthesized biofuels, or biofuel precursors, is dependent on employing a suitable microbe of a phenotype, including a combination of multiple beneficial traits, e.g. a metabolism that allows efficient conversion of carbon to biofuel. or biofuel precursor, high rate of biomass formation, high productivity of biofuel, or biofuel precursor, high levels of intracellular accumulation, or secretion of biofuel or biofuel precursor, good tolerance to food stock (carbon source and associated substances ) and synthesized product (eg fatty acid or triacylglycerol), and stability of the biofuel or biofuel precursor, eg at low carbon source concentrations. The conversion yield (gram of oil produced per gram of substrate, eg glucose) is of particular importance. The microbes commonly employed in the production of biofuel or biofuel precursor do not conform to the required phenotype in a sufficient manner to allow for economical industrial scale production of biofuel.
[007] Some aspects of this invention pertain to the design of traits required in a microorganism for the production of biofuel or biofuel precursor. While lipid and fatty acid metabolism has been studied in microbes from the 1930s and 1940s onwards (see, for example, Woodbine, M. 1959, Microbial fat: Microorganisms as potential fat producers. Prog. Ind. Microbiol.1 :181), little progress has been made toward designing desirable phenotypes related to biofuel production in microbes, despite numerous efforts to genetically engineer a microbe, or to optimize the conditions of the production process. Thus, genetic engineering efforts have mainly been directed towards manipulating a target gene upstream of or within the fatty acid synthesis pathway and optimizing fermentation or growth conditions, for example, by supplementing the growth medium. with fatty acids.
[008] A major obstacle to the genetic engineering of microbes is the lack of genomic information and annotation of key metabolic pathway regulators in target microbes, for example in oilseed yeast. As a result, identification and functional annotation of a key regulator that controls carbohydrate to lipid conversion is still lacking in microbes for biofuel production.
[009] Some aspects of this invention relate to the identification of the oleaginous yeast Y. lipolytica as a biofuel production microbe or biofuel precursor. Some aspects of this invention pertain to the discovery of a key regulator of fatty acid metabolism in a microbe. Some aspects of this invention relate to the discovery of stearoyl-CoA desaturase (SCD) as a key carbohydrate regulator for lipid conversion in a microbe. Some aspects of this invention pertain to an isolated nucleic acid that encodes a key regulator of fatty acid metabolism in a microbe. Some aspects of this invention provide an isolated nucleic acid encoding a key regulator of fatty acid metabolism, for example, an SCD gene product, from an oilseed microbe.
[010] Some aspects of this invention relate to the design of a microbe for the production of biofuel by manipulating the activity of a regulator of fatty acid metabolism, for example, by genetic manipulation. Some aspects of this invention pertain to an isolated microbe designed for biofuel production or biofuel precursor. Some aspects of this invention pertain to an isolated microbe optimized for the conversion of a carbohydrate source into a biofuel or biofuel precursor, for example, an oilseed microbe comprising increased activity of an SCD gene product. Some aspects of this invention pertain to a culture of a microbe designed for the production of biofuel or biofuel precursor. Some aspects of this invention pertain to methods of converting a carbohydrate source into a fatty acid or fatty acid derivative using a microbe designed for biofuel production. Some aspects of this invention pertain to a bioreactor for converting carbohydrate to fatty acid, or converting a fatty acid derivative using a microbe designed for biofuel production. Some aspects of this invention provide a method for converting a carbohydrate source, at least partially, into a biofuel or biofuel precursor using an engineered microbe.
[011] Some aspects of this invention pertain to an isolated oilseed cell, comprising a genetic modification that increases expression of one or more genes chosen from the group of Hemoglobin, Cytochrome, GLUT, Malic Enzyme, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, Acyl-CoA synthetase, Pyruvate carboxylase, and AMPK genes, and/or a genetic modification that reduces expression of a gene chosen from the group of JNK2 and delta-12 desaturase. In some embodiments, the isolated oil cell comprises a nucleic acid construct comprising (a) an expression cassette comprising a nucleic acid encoding the gene product under the control of a suitable homologous or heterologous promoter; (b) an expression cassette comprising a nucleic acid encoding an interfering RNA that targets the gene product under the control of a heterologous promoter; and/or (c) a nucleic acid construct inserted into the genome of the cell, the construct comprising a nucleic acid sequence that increases or decreases expression of the gene product. In some embodiments, the heterologous promoter is an inducible promoter or a constitutive promoter. In some embodiments, the nucleic acid construct inhibits or disrupts the natural regulation of a native gene encoding the gene product resulting in overexpression of the native gene. In some embodiments, the nucleic acid construct inhibits or abolishes native gene expression. In some embodiments, the inhibition or disruption of the native gene's natural regulation is mediated by deletion, disruption, mutation and/or substitution of a regulatory region, or a part of a regulatory region that regulates gene expression, or inhibition or abolition of expression. of a native gene is mediated by deletion, disruption, mutation and/or substitution of a coding sequence of the native gene, or of a regulatory region, or a part of a regulatory region that regulates expression of the native gene. In some embodiments, decreased JNK2 and/or delta-12 desaturase gene expression is mediated by constitutive or inducible expression of a nucleic acid that targets a JNK2 and/or delta-12 desaturase gene product and inhibiting gene expression . In some embodiments, nucleic acid targeting the JNK2 and/or delta-12 desaturase transcript will inhibit transcript expression, via an RNAi pathway. In some embodiments, the nucleic acid that targets the JNK2 and/or delta-12 desaturase transcript is a siRNA, a shRNA, or a microRNA. In some embodiments, a decrease in JNK2 or delta-12 desaturase expression is achieved by combining the wild-type gene in the microbe, e.g., by homologous recombination of a nucleic acid construct, e.g., a target vector, with the genomic locus. JNK2 or delta-12 desaturase, thereby disrupting wild-type gene expression. In some embodiments, the nucleic acid construct is inserted into the genome of the cell. In some embodiments, increased or decreased expression of the gene product imparts a phenotype beneficial to the conversion of a carbohydrate source to a fatty acid, fatty acid derivative, and/or TAG to the cell. In some embodiments, the beneficial phenotype is a modified fatty acid profile, a modified triacylglycerol profile, an increased rate of fatty acid and/or triacylglycerol synthesis, an increase in conversion yield, an increased accumulation of triacylglycerol in the cell, and an increased tolerance of osmotic stress, an increased rate of proliferation, an increased cell volume, and/or an increased tolerance of a substance to a lethal concentration and/or inhibition of proliferation of unmodified cells of the same cell type, by cell. In some embodiments, the modified fatty acid profile or modified triacylglycerol profile of the cell exhibits at least a 2-fold increase in the ratio of C18 fatty acids to C16 fatty acids, as compared to unmodified cells of the same cell type. In some embodiments, the modified fatty acid profile or modified triacylglycerol profile of the cell exhibits at least a 2.5-fold increase in the ratio of C18 fatty acids to C16 fatty acids, as compared to unmodified cells of the same type. cell. In some embodiments, the modified fatty acid profile or modified triacylglycerol profile of the cell exhibits at least a 5-fold increase in the ratio of C18 fatty acids to C16 fatty acids, as compared to unmodified cells of the same cell type. In some embodiments, the modified fatty acid profile or modified triacylglycerol profile of the cell exhibits at least a 5-fold increase in the ratio of C18 fatty acids to C16 fatty acids, as compared to unmodified cells of the same cell type. In some embodiments, the cell is viable under conditions of osmotic stress lethal to unmodified cells. In some embodiments, the cell is viable under osmotic stress conditions at a level of 200% of the highest level tolerated by the unmodified cells. In some embodiments, the cell is viable under osmotic stress conditions at a level of 300% of the highest level tolerated by the unmodified cells. In some embodiments, the cell is viable under osmotic stress conditions at a level of 400% of the highest level tolerated by unmodified cells. In some embodiments, the rate of cell proliferation is at least 5-fold, at least 10-fold, at least 20-fold, at least 25-fold, or at least 30-fold increased as compared to unmodified cells of the same cell type. . In some embodiments, the cell volume is increased at least 2-fold as compared to unmodified cells of the same cell type. In some embodiments, the cell tolerates a substance at a concentration lethal to and/or inhibits proliferation of unmodified cells of the same cell type. In some embodiments, the substance is a fermentable sugar and the concentration is at least 80 g/l, at least 100 g/l, at least 150 g/l, at least 200 g/l, at least 300 g/l. In some embodiments, the rate of synthesis of a fatty acid or a triacylglycerol from the cell is at least 5-fold, or at least 10-fold, increased as compared to unmodified cells of the same cell type. In some embodiments, the cell converts a carbohydrate source to a fatty acid or a triacylglycerol at a conversion rate of at least about 20 g/g, at least about 25 g/g, or at least about 30 g/g. g. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a bacterial cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is an oleaginous yeast cell. In some embodiments, the cell is a Y. lipolytica cell.
[012] Some aspects of this invention pertain to a culture, comprising an isolated oilseed cell, comprising a genetic modification that increases the expression of one or more genes chosen from the group of Hemoglobin, Cytochrome, GLUT, Malic Enzyme, ACC, SCD , FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, Acyl-CoA synthetase, Pyruvate carboxylase, and AMPK genes, and/or a genetic modification that reduces expression of a JNK2 and/or delta-12 gene product desaturase, and a source of carbohydrate. In some embodiments, the isolated oil cell is an engineered microbe as provided herein. In some embodiments, the carbohydrate source is a fermentable sugar. In some embodiments, the carbohydrate source is a monomeric sugar. In some embodiments, the carbohydrate source is glucose and glycerol. In some embodiments, the carbohydrate source is not sterilized. In some embodiments, the culture is maintained under non-sterile conditions. In some embodiments, the culture does not comprise an isolated oil cell selective antibiotic and antiproliferative agent. In some embodiments, the carbohydrate source is derived from plant or algae biomass. In some embodiments, the carbohydrate source is derived from cellulose, hemicellulose, starch, glycerol, or a derivative thereof. In some embodiments, the culture additionally comprises a cellulose or hemicellulose hydrolyzing enzyme. In some embodiments, the biomass or cellulose or hemicellulose is pretreated in a fiber expansion procedure in hot water, or dilute acid, or ammonia, with a hydrolyzing enzyme, with a steam pretreatment, and/or a pre-treatment with lime. In some embodiments, the culture comprises a substance at a lethal concentration to unmodified wild-type, unmodified cells of the same cell type as the isolated oil cell. In some embodiments, the substance is a toxic substance generated during pretreatment of the carbohydrate source such as acetic acid, furfural, or aromatic compounds. In some embodiments, the substance is the carbohydrate source. In some embodiments, the substance is a fermentable sugar. In some embodiments, the substance is a monomeric sugar. In some embodiments, the culture comprises the fermentable sugar at a concentration of at least 80 g/l, at least 100 g/l, at least 150 g/l, at least 200 g/l, at least 250 g/l, or at least 300 g/l.
[013] Some aspects of this invention relate to a method, comprising contacting a carbohydrate source with an isolated oilseed cell, the cell comprising a genetic modification that increases the expression of one or more genes chosen from the group of Hemoglobin, Cytochrome, GLUT, Malic Enzyme, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, Acyl-CoA synthetase, Pyruvate carboxylase, and AMPK gene products, and/or a genetic modification that reduces expression of a JNK2 gene and/or a delta-12 desaturase gene, and incubating the contacted carbohydrate source with the cell under conditions suitable for at least partial conversion of the carbohydrate source to a fatty acid or a triacylglycerol by the cell. In some embodiments, the isolated oil cell is an engineered microbe as provided herein. In some embodiments, the carbohydrate source is a sugar, such as glucose, xylose, etc., or starches derived from plant or algae biomass. In some embodiments, the carbohydrate source is derived from cellulose or hemicellulose. In some embodiments, the carbohydrate source is contacted with the cell in the presence of a cellulose hydrolyzing enzyme or hemicellulose. In some embodiments, the carbohydrate source is contacted with the cell in the presence of about 15 IU of cellulose hydrolyzing enzyme or hemicellulose per g of biomass at 55°C for 48 hours. In some embodiments, the biomass or cellulose or hemicellulose is pretreated with a fiber expansion procedure in hot water, or dilute acid, or ammonia, and/or a hydrolyzing enzyme. In some embodiments, the carbohydrate source contacted with the isolated oil cell comprises a substance at a concentration lethal to unmodified cells of the same cell type as the isolated oil cell. In some embodiments, the substance is a toxic substance generated during pretreatment of the carbohydrate source, for example, acetic acid. In some embodiments, the substance is the carbohydrate source. In some embodiments, the carbohydrate source is a fermentable sugar and the concentration of the fermentable sugar is at least 80 g/l, at least 100 g/l, at least 200 g/l, or at least 300 g/l after contact with the oil cell. In some embodiments, the carbohydrate source is contacted with the isolated oil cell under non-sterile conditions. In some embodiments, the carbohydrate source contacted with the isolated oil cell is incubated under non-sterile conditions. In some embodiments, the carbohydrate source contacted with the isolated oil cell is incubated in an open reactor. In some embodiments, the carbohydrate source is contacted with the isolated oil cell and incubated for converting the carbohydrate source to a fatty acid, or a triacylglycerol in a batch-fed process. In some embodiments, the carbohydrate source is contacted with the isolated oil cell and incubated for conversion of the carbohydrate source to a fatty acid, or a triacylglycerol in a continuous process. In some embodiments, the fatty acid or triacylglycerol is extracted from the carbohydrate source contacted with the isolated oil cell by solvent extraction. In some embodiments, the extraction solvent is a hexane extraction solvent. In some embodiments, the fatty acid, or triacylglycerol, is separated from the carbohydrate source contacted with the isolated oil cell and subsequently refined by transesterification.
[014] Some aspects of this invention relate to a method, comprising modification of the fatty acid profile, the triacylglycerol profile, the rate of fatty acid synthesis, the rate of triacylglycerol synthesis, the extent of fatty acid derivative accumulation in the cell, the rate of secretion of fatty acid derivative, the rate of conversion of carbohydrate to fatty acid, or the rate of conversion of fatty acid derivative, the efficient yield of conversion of carbohydrate to fatty acid, or conversion of derivative of fatty acid, osmotic tension tolerance, proliferation rate, cell volume, or tolerance of a toxic substance of a cell for use in converting a carbohydrate source to a fatty acid, or triacylglycerol, by increasing expression of one or more gene product(s) chosen from the group of Hemoglobin, Cytochrome, GLUT, Malic Enzyme, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, and products AMPK gene, and/or decreased expression of JNK2 and/or a delta-12 desaturase gene. In some embodiments, modifying the fatty acid profile, the triacylglycerol profile, the rate of fatty acid synthesis, the rate of triacylglycerol synthesis, the extent of fatty acid derivative accumulation in the cell, or the rate of secretion of fatty acid derivative of the cell, is increasing the amount of a fatty acid, a fatty acid derivative, and/or a triacylglycerol that is synthesized, accumulated, or secreted by the cell. In some embodiments, modifying the carbohydrate to fatty acid conversion efficiency, or fatty acid derivative conversion of the cell is increasing the conversion efficiency by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5 times. In some embodiments, the fatty acid derivative is a triacylglycerol. In some embodiments, modifying the osmotic stress tolerance, or tolerance of a toxic substance of the cell, is conferring tolerance of osmotic stress, or a toxic substance at a lethal level to unmodified cells of the same cell type. In some embodiments, modifying the proliferation rate is increasing the proliferation rate at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold. In some embodiments, the cell volume modification is increasing the cell volume at least 2-fold. In some embodiments, the cell is a yeast cell. In some embodiments, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is Y. lipolytica.
[015] Some aspects of this invention pertain to an isolated nucleic acid molecule comprising a) a nucleotide sequence that encodes SEQ ID NO:1 (Y. lipolytica SCD), or b) a nucleotide sequence that is at least 85% identical to the nucleotide sequence of (a). In some embodiments, the nucleotide sequence encoding SEQ ID NO:1 is SEQ ID NO:2. In some embodiments, the nucleotide sequence is at least 85% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide sequence is at least 95% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide sequence is at least 97.5% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, the nucleotide sequence is at least 99% identical to the nucleotide sequence of SEQ ID NO:2. In some embodiments, a nucleic acid construct is provided that comprises an isolated nucleic acid molecule as described herein, for example, an isolated nucleic acid molecule as described in this paragraph, and an isolated heterologous promoter. In some embodiments, the promoter is a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is a Translation Elongation Factor (TEF) Promoter. In some embodiments, the inducible promoter is a drug inducible promoter. In some embodiments, the isolated nucleic acid molecule includes a modified SDC promoter. In some embodiments, the modification is a complete or partial deletion and/or mutation of a wild-type SDC promoter sequence resulting in a disruption of the feedback inhibition of said SCD promoter in response to high levels of a fatty acid, a fatty acid derivative, and/or a triacylglycerol. In some embodiments, the modification is an insertion of a heterologous sequence into a wild-type SCD promoter region, optionally associated with a deletion, in whole or in part, and/or a mutation of a wild-type SDC promoter sequence, resulting in in a disruption of the feedback inhibition of said SCD promoter in response to high levels of a fatty acid, a fatty acid derivative, and/or a triacylglycerol.
[016] Some aspects of this invention pertain to a vector comprising an expression cassette, for example any of the expression cassettes mentioned herein. Some aspects of this invention pertain to a cell comprising an expression cassette as described herein, or at least a part of a vector as described herein.
[017] The subject matter of this application may involve, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of a single system or article.
[018] Other advantages, features and uses of the invention will become apparent from the following detailed description of non-limiting embodiments of the invention, when considered in conjunction with the accompanying drawings. In cases where this report and a document incorporated by reference include conflicting disclosure, this descriptive report may control. BRIEF DESCRIPTION OF THE DRAWINGS
[019] Figure 1: Fatty acid profile of Yarrowia lipolytica. A) A log phase culture of Y. lipolytica grown in minimal medium was assayed for total free fatty acid (FFA) using gas chromatography mass spectroscopy (GC-MS) in a rocker flask experiment. B) Total FFA was assayed in the same culture under the same conditions during the stationary growth phase. C) Total lipids (FFA and esterified fatty acids) were assayed in the same culture during stationary phase.
[020] Figure 2: Analysis of total lipids in Yarrowia lipolytica. A) Growth of wild type Y. lipolytica strain in minimal medium until 72 hours stationary phase culture and assayed for total lipids using GC-MS in a rocker flask experiment. B) Total lipids were assayed on the growth of a mutant strain to stationary phase (72 hours) and overexpressing SCD, a native Δ9 desaturase under the control of a quasi-constitutive promoter. C) Confocal microscopy on wild-type strain growth to stationary phase was stained with Nile red. D) Growth of mutant strain to stationary phase was stained with Nile red and analyzed with a confocal microscope.
[021] Figure 3: Glucose consumption of mutant Y. lipolytica-1 (over-expressing cytochrome B, hemoglobin, Glut1, and Δ9-desaturase (SCD), (D9, ■)); and wild-type (LS, ♦) in rock-flask pure glucose. Y. lipolytica mutant-1 exhibits faster glucose consumption characteristics, as compared to wild-type Y. lipolytica, and also a complete glucose consumption, as compared to the incomplete consumption observed in wild type.
[022] Figure 4: A) Sugar consumption in Y. lipolytica mutant 1 (over-expressing cytochrome B, hemoglobin, Glut1, and Δ9-desaturase (SCD)), and mutant 2 (over-expressing cytochrome B, hemoglobin, and Glut1) in 72 hours in corn husk hydrolyzate (Hz). B) Oil production in mutant 1 and mutant 2 hours in corn husk Hz.
[023] Figure 5: Comparison of growth characteristics of wild-type and engineered microbes. YL-eng: Y. lipolytica mutant over-expressing Δ9-desaturase (SCD). YL-wild: Y. lipolytica wild type. Cells were grown in minimal medium containing a sugar concentration of 250 g/l. While wild-type cells failed to grow under these conditions, mutant cells were able to tolerate the high level of sugars and grew well, suggesting that higher biofuel or biofuel precursor productivity can be achieved in processes using mutant strains. . Y-axis: OD values. X-axis: time in hours.
[024] Figure 6: Sugar consumption and growth characteristics of a mutant Y. lipolytica over-expressing Δ9-desaturase (SCD). Cells were grown in medium containing 160 g/l of sugar, and the OD and sugar consumption of the culture were monitored. The mutant cells consumed the supplied sugar within 48 hours, and continued to grow after refueling fed a batch of sugars. This figure exemplifies a useful embodiment for production processes of batch fed and semi-continuous biofuel operations.
[025] Figure 7: Lipid production from engineered Y. lipolytica (over-expressing Δ9-desaturase (SCD), Cytochrome B and hemoglobin).
[026] Figure 8: Fatty acid profile of mutant strain (over-expressing Δ9-desaturase (SCD), cytochrome B and hemoglobin; left bar in each set) and wild type Y. lipolytica strain (right bar in each set) after 72 hours of culture.
[027] Figure 9: Growth kinetics of different mutant strains of Y. lipolytica compared to wild-type Y. lipolytica, CB: overexpressing cytochrome B. D9: Over-expressing SCD.
[028] Figure 10: Growth kinetics of different mutant strains of Y. lipolytica compared to wild-type Y. lipolytica at different glucose levels. Wild: Y. lipolytica wild type; C18: Y. lipolytica mutant over-expressing Δ9-desaturase (SCD).
[029] Figure 11: Kinetics of growth and lipid production of (over-expressing Δ9-desaturase (SCD)) and mutant and wild-type Y. lipolytica.
[030] Figure 12: pYLEX1, an expression vector useful for transgene expression in Y. lipolytica (A). The vector, which is well known to those skilled in the art, may include a selection marker, or a defective URA3 marker, which is derived from the URA3 gene of Y. lipolytica, which allows auxotrophy complementation to uracil, such as markers URA3d described by LE DALL et al., Curr. Genet., 26, 38-44 (1994). Expression control sequences are, for example, promoter and terminator sequences that are active in Yarrowia. In some embodiments, the vector comprises an inducible promoter or constitutive promoter. In some embodiments, the genes can be over-expressed in pYLEX1 microbes, for example, by cloning a construct of interest, for example an SCD cDNA under the control of a promoter, into pYLEX1. Exemplary cloning of cytochrome B and hemoglobin cDNAs under the control of a TEF promoter are shown (B, C).
[031] Figure 13: Projected microbe growth in algal biomass. Dried algae were obtained and autoclaved to break down cells and gelatinize starches. Autoclaved cells were enzymatically treated with alpha-amylase to release glucose. The resulting medium was inoculated with our mutant yeast cells containing Δ9-desaturase and Cytochrome, Glut1, and hemoglobin. The graph shows robust growth of Yarrowia mutant in fermentation medium without any additives. Cells obtained OD 43 in 4-5 days. This shows that there is no inhibition of the growth of mutant yeast.
[032] Figure 14: Microscopy of yeast cells grown on seaweed hydrolysates. Cells were grown under the conditions described in Figure 13. Cells were collected and stained with Nile Vernelho to identify oil. The droplets inside the yeast cells represent oil.
[033] Figure 15: Microscopy of yeast cells grown in raw glycerol. Cells were collected and stained with Nile Red to identify oil. The droplets inside the yeast cells represent oil.
[034] Figure 16: Schematic structure of a delta-12 desaturase construct containing delta-12 desaturase gene flanking regions and antibiotic resistance sequence, which is used to generate delta-12 desaturase strains.
[035] Figure 17: Growth of engineered microbes in 3% acetate with addition of 2% glycerol in 84 hours. DETAILED DESCRIPTION INTRODUCTION
[036] In view of diminishing fossil fuel resources, numerous research efforts have been directed towards developing renewable alternatives. A promising approach is to design microbes for the production of biofuels, eg biodiesel or biodiesel precursors such as triacylglycerols, from renewable carbon sources, eg by using microbes that produce fatty acids or fatty acid derivatives. Microalgae as a raw material for biofuel production (Gouveia L, Oliveira AC.J Ind Microbiol Biotechnol. 2009 Feb;36(2):269-74). While some aspects of this invention pertain to the use of photosynthetic microbes, such as algae, for the production of biofuel or biofuel precursor, the use of photosynthetic microbes creates a set of technological challenges (Cadoret JP, Bernard OJ Lipid biofuel production with microalgae: potential and challenges Soc Biol. 2008; 202(3):201-11 ). The focus of research efforts is shifting towards the design of microbes to convert renewable carbon sources, e.g., fermentable sugars derived from biomass (e.g., glucose or sugars from corn or sugar cane), or non-carbohydrate polymers. (e.g. cellulose or hemicellulose) into biofuel or biofuel precursors, in dark fermentation processes.
[037] Economically viable biofuel production requires (i) the identification of a suitable microbe, and (ii) the design of a required and/or desirable phenotype, which may include multiple traits, in the microbe. Examples of such required and/or desirable traits in such a phenotype include, but are not limited to, rapid and efficient biomass production, growth advantage over unwanted microbes, carbohydrate to oil conversion ideally close to theoretical and efficient, and high substrate and end product tolerance. Some of these traits are prerequisites for economically viable microbe-based biofuel production on an industrial scale. Ideally, the engineered microbe should show a combination of beneficial traits conferring a phenotype that allows efficient conversion of an abundant carbon source to a biofuel or biofuel precursor, in a cost-effective manner, at scale. MICROBIAL PRODUCTION OF A BIOFUEL OR BIOFUEL PRECURSOR
[038] Some aspects of this invention pertain to microbe-mediated biofuel or biofuel precursor production. The term "biofuel" refers to a fuel that is derived from a biological source, such as a living cell, microbe, fungus or plant. The term includes, for example, fuel directly obtained from a biological source, for example, by conventional extraction, distillation, or refining methods, and fuel produced by processing a biofuel precursor obtained from a biological source, for example, by modification chemical, such as transesterification procedures. Examples of biofuels that are directly obtained are alcohols, such as ethanol, propanol, and butanol, fat and oil. Examples of biofuels that are obtained by processing a biofuel precursor (e.g. a lipid), are biodiesel (e.g. produced by transesterification of a lipid), and green/modified diesel fuels (e.g. produced by hydrogenation of an oil). Biodiesel, also referred to as fatty acid methyl (or ethyl) ester, is one of the most economically important biofuels today, and can be produced on an industrial scale by transesterification of lipids, in which sodium hydroxide and methanol (or ethanol) react with a lipid, for example a triacylglycerol, to produce biodiesel and glycerol. Feed stocks for industrial-scale biodiesel production include animal fats, vegetable oils, palm oil, hemp, soybean, rapeseed, flax, sunflower, and oilseed algae. In other approaches, biomass is converted by a microbe into a biofuel precursor, eg a lipid, which is subsequently extracted and further processed to produce a biofuel. The term "biomass" refers to material produced by the growth and/or propagation of a living cell or organism, for example, a microbe. The biomass may contain cells, microbes and/or intracellular contents, for example cellular fatty acids and TAGS, as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by a cell, for example, secreted fatty acids or TAGs. Important types of biomass for biofuel production are algae biomass and plant-derived biomass, eg corn husk and wood fiber. In some embodiments, the biomass for biofuel production or biofuel precursor may comprise plant-derived sugars, for example, sugar cane, or sugars derived from corn.
[039] Some aspects of this invention refer to the identification, design and development of a microbial source of lipids for the production of biodiesel on an industrial scale, economically viable, none of which have been reported. The term "lipid" refers to fatty acids and their derivatives. Consequently, examples of lipids include fatty acids (FA, both saturated and unsaturated); glycerides or glycerolipids, also referred to as acylglycerols (such as monoglycerides (monoacylglycerols), diglycerides (diacylglycerols), triglycerides (triacylglycerols, TAGs, or neutral fats); phosphoglycerides (glycerophospholipids); non-glycerides (sphingolipids, sterol lipids, including cholesterol and steroid hormones, prenol lipids, including terpenoids, fatty alcohols, waxes, and polyketides); and complex lipid derivatives (sugar-linked lipids or glycolipids, and protein-linked lipids). from living cells and microbes Some cells and microbes also produce lipids to store energy, for example in the form of triacylglycerols in lipid droplets.
[040] Some aspects of this invention pertain to the identification of a microbe for biofuel production or biofuel precursor based on a suitable lipid metabolism of the microbe. The term "lipid metabolism" refers to the molecular processes that involve the creation or degradation of lipids. Fatty acid synthesis, fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAG storage, and TAG degradation are all examples of processes that are part of a cell's lipid metabolism. Consequently, the term "fatty acid metabolism" refers to all cellular or organismic processes that involve the synthesis, creation, transformation or degradation of fatty acids. Fatty acid synthesis, fatty acid oxidation, TAG synthesis and TAG degradation are examples of processes that are part of a cell's fatty acid metabolism.
[041] The term "triacylglycerol" (TAG, sometimes also referred to as triglyceride) refers to a molecule comprising a single glycerol molecule covalently bonded to three fatty acid molecules, aliphatic monocarboxylic acids, via, ester bonds, one on each of the three hydroxyl (OH) groups of the glycerol molecule. Triacylglycerols are highly concentrated stores of metabolic energy due to their reduced anhydrous nature, and are a suitable food stock for biodiesel production.
[042] Many cells and organisms store metabolic energy in the form of fatty acids and fatty acid derivatives such as TAGs. Fatty acids and their derivatives, such as TAGs, provide an ideal way to store metabolic energy. The energy contained in C-C bonds can be efficiently released by e—oxidation, a reaction formally equivalent to the reverse of fatty acid biosynthesis, but mediated and regulated by different enzymes that constitute a different molecular pathway. Microbes can derive fatty acids from external supply, endogenous turnover, and de novo synthesis. Some aspects of this invention pertain to the identification of a microbe for biofuel production or biofuel precursor based on the microbe's ability to synthesize and store fatty acids or fatty acid derivatives, such as TAGs, efficiently from an externally supplied carbon source. A MICROBE FOR BIOFUEL PRODUCTION
[043] Some aspects of this invention relate to the identification of a microbe suitable for industrial-scale conversion from carbohydrate to lipid for biofuel production or biofuel precursor. No suitable microbes have been identified that would allow economically viable production of biofuel or a biofuel precursor from a carbohydrate source on an industrial scale. Some aspects of this invention pertain to the identification of an oleaginous yeast, Y. lipolytica, as a biofuel production organism or biofuel precursor based on favorable base metabolism of Y. lipolytica.
[044] Y. lipolytica is a non-pathogenic oleaginous yeast that can use a variety of carbon sources, including organic acids, hydrocarbons, and various fats and oils. The term "oilseed" refers to a microbe that can accumulate more than 20% of its dry cell weight as lipid (see C. Ratledge et al., Microbial routes to lipids. Biochem Soc Trans. 1989 Dec;17(6) ):1139-41). In accordance with some aspects of this invention, Y. lipolytica represents a microbe for biofuel production or biofuel precursor, because Y. lipolytica is an obligate aerobe with the ability to assimilate carbohydrates, for example, glucose, or glycerol, as a single source. of carbon, and compared to other yeast strains, Y. lipolytica has a higher flux of glucose to fatty acid and triacylglycerol (TAG) and higher lipid storage capacity. See, for example, Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM, Yarrowia lipolytica as a model for bio-oil production. Prog Lipide Res. 2009 Nov;48(6):375-87. Additionally, Y. lipolytica is one of the most intensively studied 'unconventional' yeast species and genome sequencing, including mitochondrial DNA, from Y. lipolytica was recently completed. Kerscher S, Durstewitz G, Casaregola S, Gaillardin C, Brandt U., The complete mitochondrial genome of Yarrowia lipolytica. Comp Funct Genomics. 2001;2(2):80-90. The availability of genomic sequence data makes genetic manipulation more accessible, even though the functional annotation of genomic sequences is not complete. See, for example, Sokolova L, Wittig I, Barth HD, Schagger H, Brutschy B, Brandt U., LILBID-mass spectrometry of protein complexes from blue-native gels, a sensitive top-down proteomic approach. proteomics. Published online 2010 Feb 1, PMID: 20127694.
[045] In wild-type Y. lipolytica, fatty acid and TAG synthesis from a carbon source is triggered during the stationary growth phase, suggesting a hermetic regulatory mechanism rather than controlling lipid metabolism. This regulatory mechanism controls the amount of lipids that can be synthesized and stored, which significantly limits the production and conversion of feed stock to lipids. Consequently, the metabolic parameters of wild-type Y. lipolytica are not suitable for production of biofuel or biofuel precursor on an economically viable industrial scale. A KEY MICROBIAL REGULATOR OF FATTY ACID METABOLISM
[046] Some aspects of this invention relate to the surprising findings that (i) saturated fatty acids de novo inhibit fatty acid synthesis and TAG storage, via a closed loop of feed stock, and (ii) that over-expression of SCD , a Δ9-desaturase, in a microbe suitable for biofuel production or biofuel precursor, eg Y. lipolytica, is sufficient to exceed this feedback inhibition of fatty acid synthesis and TAG storage, resulting in significantly increased synthesis, storage of fatty acids and/or TAGs.
[047] Some aspects of this invention relate to the surprising discovery that, in addition to effecting increased synthesis and storage of fatty acids and/or TAGs, overexpression of SCD in a microbe additionally confers a beneficial phenotype for biofuel or precursor production. biofuel to a microbe, e.g. Y. lipolytica, including, but not limited to: (i) hyperactivation of the TAG storage pathway, (ii) growth advantage, (iii) continued oil production, (iv) tolerance elevated for carbohydrate source substances (e.g. glucose and other sugars) in the culture medium, and (v) modification of the fatty acid profile, e.g. a change in the proportions of saturated to unsaturated fatty acids favorable for biofuel production or biofuel precursor.
[048] The discovery of SCD as a key regulator of fatty acid metabolism and TAG synthesis in oilseed microbes, according to this invention, has greater implications for processes that aim to convert renewable carbon sources into biofuel or biofuel precursor, with the help of engineered cells. Based on some aspects of this invention, it is now possible to modify the fatty acid and/or TAG profile of a microorganism, for example an oleaginous yeast, such as Y. lipolytica, in a way that confers highly desirable phenotypes for industrial scale conversion. from carbohydrate to biofuel or biofuel precursor, such as notable increase in fatty acid synthesis, synthesis of TAG, fatty acid and TAG, biomass production, and high tolerance of high substrate, product, and/or toxin concentration in the medium culture.
[049] In accordance with some aspects of this invention, the modification of lipid or fatty acid metabolism in a microbe according to methods provided herein, for example, by over-expressing SCD alone or in combination with other genetic or non-genetic modifications , as provided herein, allows for the generation of a microbe optimized for use in the production of biofuel or biofuel precursor processes. Some aspects of this invention pertain to the design of fatty acid metabolism in a microbe, resulting in increased synthesis rate and accumulation of fatty acids and fatty acid derivatives in the microbe.
[050] Natural fatty acid molecules commonly have an unbranched aliphatic chain or tail, from 4 to 28 carbon atoms. Fatty acids are referred to as "saturated" if all carbon atoms in the aliphatic chain are bonded via a CC single bond, or as "unsaturated" if two or more carbon atoms are bonded via a C-C double bond. Unsaturated fatty acids play an important role in regulating membrane fluidity, cellular activity, metabolism, and nuclear events that control gene transcription.
[051] The fatty acid spectrum in yeast consists mostly of C16 and C18 fatty acids, eg palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18) and oleic acid (C18). Palmitic acid is an unbranched saturated fatty acid with an aliphatic chain of 16 carbon atoms (carbon atoms/unsaturated bonds: 16.0). Stearic acid is an unbranched saturated fatty acid with an aliphatic chain of 18 carbon atoms (18.0). Palmitoleic acid is a monounsaturated fatty acid with an aliphatic chain of 16 carbon atoms (16.1). Oleic acid is a monounsaturated fatty acid with an aliphatic chain of 18 carbon atoms (18.1). Minor fatty acid species in yeast include C14 and C26 fatty acids, which play essential roles in protein modification, or as components of sphingolipids and GPI anchors, respectively.
[052] Again fatty acid synthesis utilizes substantial amounts of metabolites, acetyl-CoA, ATP and NADPH, and thus competes with other cellular processes that are dependent on these compounds. NADPH is required for two-step reduction in the fatty acid elongation cycle, linking fatty acid synthesis to the metabolic state of the cell, and results in fatty acid synthesis being restricted to conditions of high energy loading of the cells, indicated by increased increased ATP/AMP ratio, high reduction equivalents, and high acetyl-CoA pool. Almost all subcellular organelles are involved in fatty acid metabolism, indicating that the maintenance of fatty acid homeostasis requires regulation at multiple levels.
[053] Many organisms, including yeast, are able to synthesize de novo fatty acids from a variety of carbon sources. In an initial step, acetyl-CoA is carboxylated by the addition of CO2 to malonyl-CoA by the enzyme acetyl-CoA carboxylase (ACC; encoded by ACC1 and HFA1 in yeast). Biotin is an essential cofactor in this reaction, and is covalently attached to apoprotein ACC by the enzyme biotin:apoprotein ligase (encoded by BPL1/ACC2 in yeast). ACC is a trifunctional enzyme, which houses a biotin carboxyl transporter protein (BCCP) domain, a biotin carboxylase (BC) domain, and a carboxyl transferase (CT) domain. In many bacteria, these domains are expressed as individual polypeptides and assembled into a heteromeric complex. In contrast, eukaryotic ACC, including mitochondrial ACC variants (Hfa1 in yeast) harbor these functions in a single polypeptide. Malonyl-CoA produced by ACC serves as a two-carbon donor in a cyclic series of reactions catalyzed by fatty acid synthase, FAS, and elongases.
[054] In yeast, the individual functions involved in cytosolic fatty acid synthesis are represented as discrete domains in single polypeptide chains or in two different polypeptide chains, respectively. The yeast cytosolic fatty acid synthase (FAS) is a complex composed of two subunits, Fas1 (β subunit) and Fas2 (α subunit) that are organized as a hexameric α6β6 complex. Fas1 harbors acetyl transferase, enoyl reductase, dehydratase, and malonyl-palmitoyl transferase activities; Fas2 contains acyl transporter protein, 3-ketoreductase, 3-ketosynthase and phosphopantetheine transferase activities.
[055] The mitochondrial synthesis of fatty acid in yeast is carried out by a type II FAS system, harboring the individual enzymatic activities in different polypeptides: Acp1, an acyl transporter protein that transports the prosthetic phosphopantetheine group; Cem1, β-ketoacyl-ACP synthase; Oar1, 3-oxoacyl-[acyl carrier protein] reductase; Htd2, 3-hydroxyacyl thioester dehydratase; Etr1, enoyl-ACP reductase. Ppt2 functions like phosphopantetheine:protein transferase, catalyzing the attachment of the phosphopantetheine prosthetic group to apoACP.
[056] The immediate products of de novo fatty acid synthesis are saturated fatty acids. Saturated fatty acids are known to be the precursors of unsaturated fatty acids in eukaryotes, including yeast. Unsaturated fatty acids are generally produced by desaturation of C-C single bonds in saturated fatty acids by specialized enzymes called desaturases. The control mechanisms that control the conversion of saturated fatty acids to unsaturated fatty acids are not well understood. In eukaryotes, unsaturated fatty acids play important roles in regulating membrane fluidity, cellular activity, metabolism, and nuclear events that control gene transcription. Typically, about 80% of yeast fatty acids are monounsaturated, meaning they contain an unsaturated bond in their aliphatic chain.
[057] A critical step in the biosynthesis of monounsaturated fatty acids is the introduction of the first cis-double bond at the Δ9 position (between carbons 9 and 10). This oxidant reaction is catalyzed by stearoyl-CoA desaturase (SCD, also known as delta-9-desaturase, or Δ9-desaturase). Although double bond insertion occurs on several different methylene-interrupted fatty acyl-CoA substrates, preferred SCD substrates are palmitoyl (16.0)- and stearoyl (18.0)-CoA which are converted to palmitoleoyl (16.1)- and oleoyl( 18.1)-CoA, respectively (Ntambi, J. Lipide Res., 1999, 40, 1549-1558).
[058] In S. cerevisiae, a stearoyl-CoA desaturase gene was identified as Ole1 in 1990 ( Stukey JE, et al., J Biol Chem., 1990, 265(33):20144-9 ). The human stearoyl-CoA desaturase gene was partially characterized in 1994, via isolation of 0.76 kb of partial cDNA from human adipose tissue ( Li et al., Int. J. Cancer, 1994, 57, 50 348-352 ). The gene was fully characterized in 1999 and alternative use of polyadenylation sites was found to generate two transcripts of 3.9 and 5.2 kb (Zhang et al., Biochem. J., 1999, 340, 255264). In S. cerevisiae, fatty acid monodesaturation is catalyzed by the endoplasmic reticulum (ER)-resident and essential Δ9-desaturase, Ole1 (Martin CE, Oh CS, Jiang Y, Regulation of long chain unsaturated synthesis of fatty acid in yeast.. Biochim Biophys Acta. 2007 Mar;1771(3):271-85. Epub 2006 Jul 13.
[059] Some aspects of this invention pertain, at least in part, to the identification of the S. cerevisiae Ole1 SCD homolog in Y. lipolytica, as described herein.
[060] Non-limiting examples of representative sequences of Y. lipolytica SCD are given below: >gi|50548053|ref|XP_501496.1| YALI0C05951p [Yarrowia lipolytica] MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKMSQGA YDDKGRHISEQPFTWANWHQHINWLNFILVIALPLSSFAAAPFVSFN WKTAAFAVGYYMCTGLGITAGYHRMWAHRAYKAALPVRIILALFGGG AVEGSIRWWASSHRVHHRWTDSNKDPYDARKGFWFSHFGWMLLV PNPKNKGRTDISDLNNDWVVRLQHKYYVYVLVFMAIVLPTLVCGFGW GDWKGGLVYAGIMRYTFVQQVTFCVNSLAHWIGEQPFDDRRTPRDH ALTALVTFGEGYHNFHHEFPSDYRNALIWYQYDPTKWLIWTLKQVGL AWDLQTFSQNAIEQGLVQQRQKKLDKWRNNLNWGIPIEQLPVIEFEE FQEQAKTRDLVLISGIVHDVSAFVEHHPGGKALIMSAVGKDGTAVFN GGVYRHSNAGHNLLATMRVSVIRGGMEVEVWKTAQNEKKDQNIVSD ESGNRIHRAGLQATRVENPGMSGMAA (SEQ ID NO: 1)> gi | 50548052 | ref | XM_501496.1 | Yarrowia lipolytica YALI0C05951p (YALI0C05951g) mRNA, complete cds ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGT CGACACCATTGCCTCCGGCCGAGATGTCAACTACAAGGTCAAGTA CACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGG GCCGCCACATTTCCGAGCAGCCCTTCACCTGGGCCAACTGGCAC CAGCACATCAACTGGCTCAACTTCATTCTGGTGATTGCGCTGCCT CTGTCGTCCTTTGCTGCCGCTCCCTTCGTCTCCTTCAACTGGAAG ACCGCCGCGTTTGCTGTCGGCTATTACATGTGCACCGGTCTCGGT ATCACCGCCGGCTACCACCGAATGTGGGCCCATCGAGCCTACAA GGCCGCTCTGCCCGTTCGAATCATCCTTGCTCTGTTTGGAGGAGG AGCTGTCGAGGGCTCCATCCGATGGTGGGCCTCGTCTCACCGAG TCCACCACCGATGGACCGACTCCAACAAGGACCCTTACGACGCC CGAAAGGGATTCTGGTTCTCCCACTTTGGCTGGATGCTGCTTGTG CCCAACCCCAAGAACAAGGGCCGAACTGACATTTCTGACCTCAAC AACGACTGGGTTGTCCGACTCCAGCACAAGTACTACGTTTACGTT CTCGTCTTCATGGCCATTGTTCTGCCCACCCTCGTCTGTGGCTTT GGCTGGGGCGACTGGAAGGGAGGTCTTGTCTACGCCGGTATCAT GCGATACACCTTTGTGCAGCAGGTGACTTTCTGTGTCAACTCCCT TGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTC CCCGAGACCACGCTCTTACCGCCCTGGTCACCTTTGGAGAGGGC TACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGCC CTCATCTGGTACCAGTACGACCCCACCA AGTGGCTCATCTGGACC CTCAAGCAGGTTGGTCTCGCCTGGGACCTCCAGACCTTCTCCCAG AACGCCATCGAGCAGGGTCTCGTGCAGCAGCGACAGAAGAAGCT GGACAAGTGGCGAAACAACCTCAACTGGGGTATCCCCATTGAGCA GCTGCCTGTCATTGAGTTTGAGGAGTTCCAAGAGCAGGCCAAGAC CCGAGATCTGGTTCTCATTTCTGGCATTGTCCACGACGTGTCTGC CTTTGTCGAGCACCACCCTGGTGGAAAGGCCCTCATTATGAGCGC CGTCGGCAAGGACGGTACCGCTGTCTTCAACGGAGGTGTCTACC GACACTCCAACGCTGGCCACAACCTGCTTGCCACCATGCGAGTTT CGGTCATTCGAGGCGGCATGGAGGTTGAGGTGTGGAAGACTGCC CAGAACGAAAAGAAGGACCAGAACATTGTCTCCGATGAGAGTGGA AACCGAATCCACCGAGCTGGTCTCCAGGCCACCCGGGTCGAGAA CCCCGGTATGTCTGGCATGGCTGCTTAG (SEQ ID NO: 2)
[061] Stearoyl-CoA desaturase, or SCD, introduces a double bond into the Δ9-C of these CoA-esterified substrate fatty acids. This activity affects the ratio of saturated to unsaturated fatty acids, eg stearic acid to oleic acid. Stearic acid is the primary substrate for SCD; however, other chain-length fatty acids can be processed by SCD as well. In humans, Stearoyl-CoA desaturase has been seen as a lipogenic enzyme not only for its key role in the biosynthesis of monounsaturated fatty acids, but also for its pattern of regulation by diet and insulin (Ntambi, Lipide Res., 1999, 40, 15491558) . The regulation of stearoyl-CoA desaturase is therefore of considerable physiological importance, and its activity is sensitive to dietary changes, hormonal imbalance, developmental processes, temperature changes, metals, alcohol, peroxisomal proliferators, and phenolic compounds (Ntambi, Lipid Res., 1999, 40, 1549-1558).
[062] Animal models have been very useful in investigating the regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids (PUFAs). For example, in adipose tissue from lean and obese Zucker rats, Jones et al. observed a 75% decrease in stearoyl-CoA desaturase mRNA when both groups were fed a diet high in PUFAs relative to a control diet (Jones et al, Am. J. Physiol., 1996, 271, E44-49) . Similar results were obtained with tissue culture systems. In the murine adipocyte cell line 3T3-L1, arachidonic, linolenic, linolenic, and eicosapentanenoic acids decreased stearoyl-CoA desaturase expression in a dose-dependent manner (Sessler et al, J. Biol. Chem., 1996, 271, 29854). -29858).
[063] The molecular mechanisms by PUFAs that regulate stearoyl-CoA desaturase gene expression in different tissues are still poorly understood. Current understanding of the regulatory mechanism involves binding of PUFAs to a putative PUFA binding protein, after which transcriptional repression occurs, via binding of the PUFA binding protein to a cis-acting PUFA response element of the stearoyl gene. -CoA desaturase (SREBP) ( Ntambi, Lipide Res., 1999, 40, 1549-1558 ; Zhang et al, Biochem. J., 2001, 357, 183-193 ).
[064] While the regulation of SCD gene catalytic activity has been investigated in different organisms, the implications of SCD gene expression and regulation on lipid metabolism itself have not been the subject of extensive study. SCD has been cited to affect the ratio of saturated to unsaturated fatty acids, eg from stearic acid to oleic acid.
[065] Some aspects of this invention relate to the surprising discovery that SCD also functions as a key regulator of fatty acid and TAG metabolism in microbes, for example in Y. lipolytica. Some aspects of this invention relate to the surprising discovery that overexpression of an SCD gene product alone not only provides the ratio of saturated to unsaturated fatty acids in the affected cells, but is sufficient to trigger a marked and unexpected increase in the rate of synthesis. of fatty acid and/or TAG, and/or storage. The unexpected finding that manipulation of desaturase expression alone imparts highly desirable phenotypes to microbes, e.g. oilseed yeast cells, for industrial-scale carbohydrate-to-lipid conversion has implications for achieving efficient production of biofuels or biofuel precursors from renewable carbon sources by microbe-mediated fermentation processes. Excessive downregulation of fatty acid synthesis and storage by over-expression of SCD in a microbe not only confers increased rate of fatty acid synthesis and accumulation in the microbe, but also exceeds the restriction of FA/TAG synthesis to phase stationery of a microbe in the culture. Surprisingly, over-expression of SCD in a microbe, e.g. a microbe for biofuel production or biofuel precursor, also confers increased tolerance to high substrate concentrations, e.g. of fermentable sugars, and to substrate-associated toxic substances. , for example, by-products of substrate pretreatment procedures, to the microbe. The phenotypes conferred by the overexpression of SCD, for example the improved tolerance of phenotypes described above, allow obtaining high concentrations of lipids in industrial fermentation processes that convert sugars to lipids. (See Figure 11 for over-regulation of negative FA synthesis by SCD overexpression).
[066] In accordance with some aspects of this invention, manipulation of additional genes may be beneficial for large-scale production of biofuel or biofuel precursor from a carbon source by microbial fermentation. For example, genes that effect the diversion of carbon-containing substrates, eg sugars, for fatty acid synthesis. Accordingly, some aspects of this invention provide methods for manipulating the expression of genes involved in regulating carbon flux into or out of lipid synthesis pathways to achieve an improvement in lipid production parameters.
[067] Some aspects of this invention provide a method for manipulating the expression and/or activity of other gene products that regulate lipid metabolism of microbes for biofuel or biofuel precursor production. Manipulations in accordance with aspects of this invention are aimed at increasing carbohydrate to fatty acid and/or TAG conversion in order to optimize the engineered organism for large-scale lipid production from carbohydrate sources. The manipulations provided in accordance with some aspects of this invention, for example, over-expression, knockout, knock-down, activation and/or inhibition of specific gene products, may be performed alone or in combination, and/or in combination with other manipulations known to those skilled in the art. The term "manipulation" refers to both genetic manipulation, e.g., over-expression, knockout, knock-down, activation and/or inhibition of specific gene products, and non-genetic manipulation, e.g., manipulation of the growth medium. , substrate, substrate pretreatment, pH, temperature, conversion process, etc.
[068] A manipulation of gene expression, also referred to herein as a modulation of gene expression, can be a disruption or inhibition of natural expression regulation, an over-expression, an inhibition of expression, or a complete abolition of expression of a gene. a given gene. An insertion of a heterologous promoter upstream of a native gene sequence, e.g., a native SCD gene sequence, or the deletion of regulatory sequences within a promoter, e.g., regulatory sequences that mediate gene feedback inhibition SCD by saturated fatty acids are examples of disruption or inhibition of natural expression regulation. Strategies for modulating gene expression may include genetic alterations, for example, by recombinant technologies, such as targeting gene or viral transductions, or non-genetic alterations, for example, environmental alterations known to result in upregulation and gene expression, or transient delivery of modulators, e.g. drugs or small RNA molecules to target cells. Methods for genetically and non-genetically altering microbes are well known to those skilled in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002); Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998); and Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), all of which are incorporated herein by reference.
[069] The term "over-expression", as used herein, refers to an increased level of expression of a given gene in a given cell, cell type or cell state, as compared to a reference cell, for example , a wild-type cell of the same cell type, or a cell of the same cell type, but lacking a specific modification, for example, a genetic modification. Forced continuous expression of the SCD gene in Y. lipolytica cells that exhibit concentrations of saturated fatty acids that would inhibit SCD gene expression in wild-type cells is an example of gene overexpression.
[070] The term "knockout", as used herein, refers to the functional disruption of the expression of a gene product, for example, an RNA or protein. This is normally achieved by targeting a respective genomic region with a targeting construct, which recombines with a specific part of said genomic region and either deletes a part of said genomic region and/or inserts a heterologous nucleotide, or nucleotide sequence, resulting in a complete inhibition of expression of a gene product, for example an mRNA or protein, from the recombined gene. In diploids, such homologous recombination events normally only affect one of the two alleles. Homozygosity can be achieved by various strategies, for example, by generating heterozygotes and sorting offspring. In diploid organisms, eg yeast, the term "knockout strain" usually refers to a strain homozygous for a non-functional allele.
[071] The term "knock-down", as used herein, refers to partial inhibition of the expression of a gene product, for example, an mRNA or protein. Various gene knockdown strategies known in the art can be used to inhibit gene expression (e.g., expression of a gene that inhibits or diverts resources away from lipid synthesis pathways, such as ACS2, FAT1, PCS60, and/or AMPK in oleaginous yeast, e.g. in Y. lipolytica). For example, gene knockdown strategies can be used, which make use of RNA interference (RNAi) and/or microRNA (miRNA) pathways including small interfering RNA (siRNA), short hairpin RNA (shRNA), Double stranded RNA (dsRNA), miRNAs, and other small interfering nucleic acid-based molecules known in the art. In one embodiment, vector-based RNAi modalities (e.g., shRNA or shRNA-mir expression constructs) are used to reduce expression of a gene (e.g., of a gene that inhibits or diverts resources outside of synthesis pathways). of lipid, such as ACS2, FAT1, PCS60, and/or AMPK) in a cell (e.g., in an oilseed yeast cell, such as a Y. lipolytica cell). Plasmids isolated in accordance with aspects of this invention may comprise a promoter operably linked to a gene encoding a small interfering nucleic acid, for example, an shRNA. In some embodiments, an isolated plasmid vector can be employed to generate a viral particle, for example, a retrovirus or bacteriophage, capable of infecting a cell, for example, a yeast cell or bacterial cell. Exemplary viruses include adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, phages and others that are known in the art and disclosed herein.
[072] Some aspects of this invention provide a method for manipulating the activity of a stearoyl-CoA-desaturase (SCD) in a microbe for production of biofuel or biofuel precursor. SCD is a Δ9 desaturase that inserts a double bond between C9 and C10 of stearic acid coupled to CoA, a key step in the generation of saturated fatty acids and their derivatives, as described in greater detail here throughout. In some embodiments, the manipulation is overexpression. In some embodiments, manipulation is effected by contacting a microbe for producing biofuel or biofuel precursor with an expression construct comprising a nucleic acid encoding an SCD gene product, e.g., an SCD protein, operably linked. to a heterologous promoter, for example a constitutive promoter or an inducible promoter. In some embodiments, the nucleic acid encoding an SCD gene product comprises the coding sequence of SEQ ID NO: 2. In some embodiments, the SCD is Y. lipolytica SCD, for example, Y. lipolytica SCD comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the microbe is Y. lipolytica. In some embodiments, manipulation of the activity of an SCD in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate-to-lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of carbohydrate-to-lipid conversion, increased lipid storage and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The stearoyl-CoA Desaturase gene and gene product sequences are well known to those skilled in the art. Exemplary and representative gene and gene product sequences can be found under the GeneID entry: 852825 in the NCBI database (www.ncbi.nlm.nih.gov).
[073] Some aspects of this invention provide a method for manipulating the activity of a c-Jun N-terminal kinase 2 (JNK2) gene product in a microbe for biofuel production or biofuel precursor. JNK2 is located in the cytoplasm and catalyzes the breakdown of fatty acids for energy and carbon blockage generation during starvation. JNK2 is required for energy homoeostasis and plays a crucial role in lipase activation in response to low cellular sugar levels. See, Grimard V, Massier J, Richter D, Schwudke D, Kalaidzidis Y, Fava E, Hermetter A, Thiele C., siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. J Lipide Res. 2008 Nov;49(11):2427-40. Epub 2008 Jul 8. In some embodiments, JNK2 activity is abolished or decreased in a microbe for biofuel or biofuel precursor production, for example, by knockout or knockdown, respectively. In some embodiments, JNK2 activity is decreased in a microbe for biofuel or biofuel precursor production in order to increase product stability and/or decrease product catabolism. In some embodiments, a conditional repression system is used and JNK2 activity is repressed during a stage in the production process where the carbohydrate source, for example, a fermentable sugar, is very low. In some embodiments, manipulation of the activity of a JNK2 gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of lipid conversion. carbohydrate to lipid, increased storage of lipid and, increased rate of growth, increased tolerance to high concentrations of a carbon source, or a lipid product. The GNK2 gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 5601 in the NCBI database (www.ncbi.nlm.nih.gov).
[074] Some aspects of this invention provide a method for manipulating the activity of a delta-12 desaturase gene product in a microbe for biofuel or biofuel precursor production. Delta-12 desaturase is involved in the conversion of lipid-containing oleic acid to higher-chain lipids. In some embodiments, it is desirable to avoid or minimize the production of long-chain fatty acids for biofuel production, for example, in view of the cold flow properties of the resulting biofuel. In some embodiments, delta-12 desaturase activity is abolished or decreased in a microbe for biofuel or biofuel precursor production, e.g., by complete or partial gene deletion or knockdown (e.g., knockout), respectively. In some embodiments, delta-12 desaturase activity is decreased in a microbe for biofuel or biofuel precursor production in order to increase product stability, achieve a desirable TAG profile in the microbe, and/or decrease product catabolism. . In some embodiments, a conditional repression system is used to repress delta-12 desaturase activity. In some embodiments, manipulation of the activity of a delta-12 desaturase gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate-to-lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, increased content of C18 fatty acids, increased percentage of C18 fatty acids of the total fatty acid pool in the microbe, improved cold flow properties of produced lipids, oils, or TAGs, growth rate increased, increased tolerance to high concentrations of a carbon source, or a lipid product. The Delta-12 desaturase gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 2909806 in the NCBI database (www.ncbi.nlm.nih.gov).
[075] Some aspects of this invention provide a method for manipulating the activity of a hemoglobin gene product in a microbe for biofuel or biofuel precursor production. For a summary of hemoglobin gene products, including hemoglobin gene products useful in some embodiments of this invention, see, Frey AD, Kallio PT. Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol Rev. 2003 Oct;27(4):525-45. In some embodiments, the activity of a hemoglobin gene product, e.g., a hemoglobin protein, is increased in the microbe, e.g., by over-expression of a hemoglobin protein encoding nucleic acid. In some embodiments, overexpression of hemoglobin in the microbe effects increased oxygen transfer in the microbe. In some embodiments, increased hemoglobin activity results in improved biofuel or biofuel precursor synthesis, due to increased oxygen flux in a highly oxygen-demanding synthesis pathway, for example, the fatty acid synthesis pathway. In some embodiments, manipulation of the activity of a hemoglobin gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of carbohydrate conversion to lipid, increased storage of lipid, and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 7738539 (Deide_12990) in the NCBI database (www.ncbi.nlm.nih.gov).
[076] Some aspects of this invention provide a method for manipulating the activity of a cytochrome gene product in a microbe for producing a biofuel or biofuel precursor, such as a cytochrome B gene product, more specifically a cytochrome gene product. cytochrome B5. In some embodiments, the activity of a cytochrome gene product, e.g., a cytochrome protein, is increased in the microbe, e.g., by over-expression of a nucleic acid-encoding cytochrome protein. In some embodiments, overexpression of cytochrome in the microbe effects increased oxygen transfer in the microbe. In some embodiments, increased cytochrome activity results in improved biofuel or biofuel precursor synthesis, due to increased oxygen flux in a highly oxygen-demanding synthesis pathway, for example, the fatty acid synthesis pathway. In some embodiments, manipulation of the activity of a cytochrome gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate-to-lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of lipid conversion. carbohydrate to lipid, increased storage of lipid and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The cytochrome gene and gene product sequences are well known to those skilled in the art. An exemplary representative gene sequence can be found under the entry for GeneID: 1528 in the NCBI database (www.ncbi.nlm.nih.gov).
[077] Some aspects of this invention provide a method for manipulating the activity of a glucose transporter (GLUT) gene product (GLUT), e.g., a Glut1 gene product, in a microbe for biofuel production or biofuel precursor. . In some embodiments, the activity of a GLUT gene product, for example, a GLUT protein, is increased in the microbe, for example, by over-expression of a GLUT protein encoding nucleic acid. In some embodiments, overexpression of a GLIT protein encoding nucleic acid in the microbe effects increased glucose uptake by the microbe. In some embodiments, increased GLUT activity results in improved biofuel or biofuel precursor synthesis, due to increased glucose uptake. In some embodiments, manipulation of the activity of a GLUT gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of lipid conversion. carbohydrate to lipid, increased storage of lipid and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The GLUT gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 38109 in the NCBI database (www.ncbi.nlm.nih.gov).
[078] Some aspects of this invention provide a method for manipulating the activity of a Pyruvate carboxylase (PC) gene product in a microbe for biofuel production or biofuel precursor. In some embodiments, the activity of a PC gene product, for example, a PC protein, is increased in the microbe, for example, by over-expression of a PC protein encoding nucleic acid. In some embodiments, overexpression of a nucleic acid encoding PC protein in the microbe effects increased glucose uptake by the microbe. In some embodiments, increased PC activity results in improved biofuel or biofuel precursor synthesis, due to increased glucose uptake. In some embodiments, manipulation of the activity of a PC gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of protein conversion. carbohydrate to lipid, increased storage of lipid and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The PC gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID:5091 in the NCBI database (www.ncbi.nlm.nih.gov).
[079] Some aspects of this invention provide a method for manipulating the activity of a Malic Enzyme (ME) gene product in a microbe for biofuel production or biofuel precursor. ME catalyzes the oxidative decarboxylation of (S)-malate to pyruvate, with the concomitant release of carbon dioxide and conversion of NADP+ to NADPH. In some embodiments, the activity of an ME gene product, e.g., an ME protein, is increased in the microbe, e.g., by over-expression of an ME protein encoding nucleic acid. In some embodiments, overexpression of an ME protein encoding nucleic acid in the microbe effects increased levels of NADPH in the microbe, resulting in sufficient levels of reduced metabolites, e.g., NADPH, for increased fatty acid synthesis. In some embodiments, the increased activity of ME in the improved synthesis of biofuel or biofuel precursor, due to increased levels of NADPH. In some embodiments, manipulation of the activity of an ME gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., increased rate of lipid synthesis, increased efficiency of lipid conversion. carbohydrate to lipid, increased storage of lipid and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The gene and gene product sequences of ME are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 17436 in the NCBI database (www.ncbi.nlm.nih.gov).
[080] Some aspects of this invention provide a method for engineering an acetyl-CoA carboxylase (ACC) gene product in a microbe for biofuel or biofuel precursor production, for example, in Y. lipolytica gene products. ACC that mediate the conversion of acetyl-CoA, the major C2-precursor in fatty acid synthesis, to malonyl-CoA, which is considered the first step in fatty acid synthesis, and has also been suggested to be the rate-limiting step in fatty acid synthesis (see Cao Y, Yang J, Xian M, Xu X, Liu W. Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Appl Microbiol Biotechnol. 2010). In some embodiments, the manipulation of ACC activity is the overexpression of ACC. In some embodiments, overexpression of ACC in a microbe increases the rate of fatty acid synthesis and/or confers a beneficial phenotype for large-scale conversion of carbohydrate to biofuel or biofuel precursor, e.g., rate of synthesis of increased lipid, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid and, increased growth rate, increased tolerance to concentrations of a substance, e.g. a carbon source, a biofuel or biofuel precursor, or a substance toxic. The ACC gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 855750 in the NCBI database (www.ncbi.nlm.nih.gov).
[081] Some aspects of this invention provide a method for manipulating the activity of an Acyl-CoA synthetase (ACS) in a microbe for production of biofuel or biofuel precursor. ACSs are a family of enzymes that catalyze the thioesterification of fatty acids with CoA to form activated intermediates (see Lu X, Vora H, Khosla C., Overproduction of free fatty acid in E. coli: implications for biodiesel production Metab Eng. 2008 Nov;10(6):333-9). These intermediates are the precursors to phospholipids, fatty acid cholesterol esters, or fatty acid alcohol esters, such as TAGs. Y. lipolytica contains two known and predicted Acyl-CoA synthetases. In some embodiments of this invention, over-expression of an ACS enzyme in a lipid-producing organism is effected to confer a beneficial phenotype for large-scale carbohydrate-to-lipid conversion, e.g., increased lipid synthesis rate, efficiency increased carbohydrate to lipid conversion, increased lipid storage and/or secretion, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The ACS gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 851245 in the NCBI database (www.ncbi.nlm.nih.gov).
[082] Some aspects of this invention provide a method for manipulating the activity of acetyl-CoA synthetase 2 (ACS2), an enzyme located in the peroxisome and involved in fatty acid degradation, in a microbe for biofuel production or biofuel precursor. In some embodiments, inhibition of ACS2 prevents or inhibits fatty acid degradation by catabolic yeast metabolism, and in some embodiments, such inhibition complements an increase in FAA1 gene product activity for increased secretion of fatty acid into the medium. Y. lipolytica contains ACS2 acetyl-CoA synthetase (see Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM., Yarrowia lipolytica as a model for bio-oil production. Prog Lipide Res. 2009 Nov; 48(6):375-87). In some embodiments, knockout, knock-down, and/or inhibition of ACS2 gene expression product or activity in a microbe is effected to confer a beneficial phenotype for large-scale conversion of carbohydrate to biofuel or biofuel precursor, for example, increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, and, increased growth rate, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or precursor of biofuel, or a toxic substance. ACS2 gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 850846 in the NCBI database (www.ncbi.nlm.nih.gov).
[083] Some aspects of this invention provide a method for manipulating the activity of an FAA1 gene product in a microbe for biofuel or biofuel precursor production. The FAA1 gene product catalyzes the cytoplasmic thioesterification of long-chain fatty acids with CoA to activated intermediates. Y. lipolytica FAA1 is a long-chain fatty acid homolog of S. cerevisiae P30624 FAA1-CoA ligase. This enzyme is involved in generating free fatty acid pooling and fatty acid secretion. In some embodiments, overexpression of an FAA1 gene product in a microbe for biofuel production or biofuel precursor is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, e.g., rate of lipid synthesis increased, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, and, increased growth rate, increased tolerance to high concentrations of a carbon source, or a lipid product. The FAA1 gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 854495 in the NCBI database (www.ncbi.nlm.nih.gov).
[084] Some aspects of this invention provide a method for manipulating the very long chain fatty acid-CoA synthetase (FAT1) activity in a microbe for biofuel or biofuel precursor production. FAT1 is thought to control fatty acid transport and thioesterification of very long-chain fatty acids with CoA. Y. lipolytica contains a very long-chain fatty acid FAT1-CoA synthetase. In some embodiments, inhibition of FAT1 activity, for example by genetic manipulation, prevents synthesis of very long fatty acid derivatives and/or increases free fatty acid assembly. In some embodiments, knockout, knock-down, and/or inhibition of FAT1 gene expression product or activity in a microbe is effected to confer a phenotype beneficial for large-scale conversion of carbohydrate to biofuel or biofuel precursor, for example , increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid and, increased growth rate, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or biofuel precursor , or a toxic substance. The FAT 1 gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 852329 in the NCBI database (www.ncbi.nlm.nih.gov).
[085] Some aspects of this invention provide a method for the manipulation of PCS60, also known as FAT2, AMP binding protein acyl-CoA synthetase, or peroxisomal-CoA synthetase, which is a peroxisomal acyl-CoA synthetase with undefined substrate specificity. . Y. lipolytica contains a homolog of S. cerevisiae PCS60. Inhibition of PCS60 will prevent synthesis of very long fatty acid derivatives and increase in free fatty acid pooling. In some embodiments of this invention, knockout, knock-down, and/or inhibition of PCS60 gene expression product or activity in a microbe is effected to confer a phenotype beneficial for large-scale conversion of carbohydrate to biofuel or biofuel precursor, for example, increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, and, increased growth rate, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or precursor of biofuel, or a toxic substance. The FAT2 gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 852523 in the NCBI database (www.ncbi.nlm.nih.gov).
[086] Some aspects of this invention provide a method for over-expressing ATP citrate lyase (ACLY) in a microbe, e.g., Y. lipolytica, for large-scale production of a biofuel or biofuel precursor. Some microbes suitable for industrial-scale production of biofuel or biofuel precursors, including Y. lipolytica, commonly produce large amounts of citrate. ACLY mediates the conversion of citrate to CoA, a reaction, which, according to some aspects of this invention, can be promoted by over-expression of ACIL (see Holz M, Forster A, Mauersberger S, Barth G., Aconitase over-expression changes the product ratio of citric acid production by Yarrowia lipolytica. Appl Microbiol Biotechnol. 2009 Jan;81(6):1087-96). In some embodiments, overexpression of ACYLA reduces unwanted citrate production and/or provides an additional source of acetyl-CoA for biofuel or biofuel precursor synthesis. In some embodiments, excessive production of citrate is inhibited in a microbe for producing a biofuel or biofuel precursor, including Y. lipolytica. In some embodiments, overexpression of ACYLA in a microbe, e.g., in Y. lipolytica, increases the rate of fatty acid synthesis and/or confers a beneficial phenotype for large-scale conversion of carbohydrate to biofuel or biofuel precursor. , for example, increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, and, increased growth rate, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or biofuel precursor, or a toxic substance. See also Lasserre JP, Nicaud JM, Pagot Y, Joubert-Caron R, Caron M, Hardouin J.Talanta. First complex study of alkane-binding protein complexes in the yeast Yarrowia lipolytica. 2010 Feb 15;80(4):1576-85. The ACYLA gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 108728 in the NCBI database (www.ncbi.nlm.nih.gov).
[087] Some aspects of this invention provide a method for the overexpression of Fatty Acid Synthase (FAS) complex. While ACC is likely to be the rate-limiting enzyme in fatty acid synthesis, other steps have also been suggested to exert control of this pathway, most notably, FAS (see Schweizer E, Kottig H, Regler R, Rottner GJ, Genetic control of Yarrowia lipolytica fatty acid synthetase biosynthesis and function. Basic Microbiol. 1988;28(5):283-92). This complex is a multifunctional polypeptide that elongates the fatty acid chain in the most substrate-intensive process in the total lipid synthesis pathway. In some embodiments, overexpression of ACYLA in a microbe, for example in Y. lipolytica, increases the rate of fatty acid synthesis and/or confers a phenotype beneficial for large-scale conversion of carbohydrate to biofuel or biofuel precursor, for example, increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid, and/or secretion, increased rate of growth, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or biofuel precursor, or a toxic substance. The FAS gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entries for GeneID: 853653 and GeneID: 855845 in the NCBI database (www.ncbi.nlm.nih.gov).
[088] Some aspects of this invention provide a method for inhibiting AMP Activated Protein Kinase (AMPK). AMPK is a regulatory enzyme that regulates the activity of other proteins by phosphorylation in response to the cellular AMP:ADP ratio (see LeeYoung RS, Palmer MJ, Linden KC, LePlastrier K, Canny BJ, Hargreaves M, Wadley GD, Kemp BE, McConell GK Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans Am J Physiol Endocrinol Metab 2006 Sep;291(3):E566-73). In yeast, AMPK has been shown to target ACC as well as INO1, a gene required for an earlier step in lipid biosynthesis. Lack of ACC phosphorylation in AMPK knockout mutants results in hyperactive ACC and fatty acid overproduction. In some embodiments, inhibition of AMPK in a microbe leads to hyperactivation of lipid synthesis. In some embodiments, AMPK activity is completely abolished in a microbe, for example, by knockout of the AMPK gene. In some embodiments, AMPK activity is inhibited in a microbe, for example, by genetic or non-genetic manipulation. Inhibition, as opposed to complete abolition, of AMPK activity can prevent negative effects on other AMPK-regulated cellular processes. In some embodiments, knockout, knock-down, and/or inhibition of AMPK gene expression product or activity in a microbe, e.g., Y. lipolytica, is effected to confer a beneficial phenotype for large-scale conversion of carbohydrate to biofuel or biofuel precursor, for example, increased rate of lipid synthesis, increased efficiency of carbohydrate to lipid conversion, increased storage of lipid and/or secretion, increased rate of growth, increased tolerance to concentrations of a substance, for example, a carbon source, a biofuel or biofuel precursor, or a toxic substance. The AMPK gene and gene product sequences are well known to those skilled in the art. Representative exemplary gene and gene product sequences can be found under the entry for GeneID: 100145903 in the NCBI database (www.ncbi.nlm.nih.gov). ISOLATED NUCLEIC ACIDS
[089] Some aspects of this invention provide nucleic acids encoding a gene product that impart a required and/or desired phenotype for biofuel production or biofuel precursor to a microbe, for example, Y. lipolytica. In some embodiments, the nucleic acid is a nucleic acid derived from Y. lipolytica. In some embodiments, the nucleic acid encodes a desaturase, for example, a Δ9 desaturase. In some embodiments, the nucleic acid encodes Y. lipolytica Δ9 desaturase. In some embodiments, the nucleic acid comprises SEQ ID NO: 1. In some embodiments, the nucleic acid is SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a gene product, e.g., a protein, encoded by SEQ ID NO: 1.
[090] Some aspects of this invention provide a gene product, e.g., a protein, that confers a required and/or desirable phenotype for biofuel production or biofuel precursor to a microbe, e.g., Y. lipolytica. In some embodiments, the protein is the Y. lipolytica protein. In some embodiments, the protein is a desaturase, for example, a Δ9 desaturase. In some embodiments, the protein is a Y. lipolytica Δ9 desaturase. In some embodiments, the amino acid sequence of the protein is the sequence provided in SEQ ID NO: 2.
[091] The term "nucleic acid" refers to a molecule comprising multiple linked nucleotides. "Nucleic acid" and "nucleic acid molecule" are used interchangeably and refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms also include polynucleosides (i.e., a polynucleotide minus a phosphate) and any other organic base containing nucleic acid. Organic bases include adenine, uracil, guanine, thymine, cytosine and inosine. Nucleic acids can be single or double stranded. The nucleic acid may be naturally or non-naturally occurring. Nucleic acids can be obtained from natural sources, or they can be synthesized using a (i.e. synthetic) nucleic acid synthesizer. Nucleic acid isolations are routinely performed in the art and suitable methods can be found in standard molecular biology textbooks. (See, for example, Maniatis' Handbook of Molecular Biology.) The nucleic acid can be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA, cDNA, rRNA, miRNA, PNA, or LNA, or a combination thereof, as hereinafter referred to as described. Non-naturally occurring nucleic acids, such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) may also be used in accordance with some aspects of this invention.
[092] Some aspects of this invention pertain to the use of nucleic acid derivatives. As will be described herein, the use of certain nucleic acid derivatives can increase the stability of the nucleic acids of the invention by preventing their digestion, particularly when they are exposed to biological samples that may contain nucleases. As used herein, a nucleic acid derivative is a non-naturally occurring nucleic acid, or a unit thereof. Nucleic acid derivatives may contain non-naturally occurring elements, such as non-naturally occurring nucleotides and non-naturally occurring support bonds. Nucleic acid derivatives, in accordance with some aspects of this invention, may contain supporting modifications such as, but not limited to, phosphorothioate linkages, phosphodiester modified nucleic acids, phosphodiester and phosphorothioate combinations, nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. The support composition of nucleic acids can be homogeneous or heterogeneous.
[093] Nucleic acid derivatives, in accordance with some aspects of this invention, may contain substitutions or modifications in the sugars and/or bases. For example, some nucleic acid derivatives may include nucleic acids having supporting sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position (e.g. example, a 2'-O-alkylated ribose group). Nucleic acid derivatives may include non-ribose sugars, such as arabinose. Nucleic acid derivatives may contain substituted purines and pyrimidines, such as modified C-5 propyne, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil, and pseudoisocytosine bases. .
[094] In some embodiments, a nucleic acid may comprise a peptide nucleic acid (PNA), a locked nucleic acid (LNA), DNA, RNA, or co-nucleic acids of the above such as DNA-LNA co-nucleic acid .
[095] As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid that is not in its natural environment, for example, a nucleic acid that has been (i) extracted and/or purified from a cell or microbe, for example a bacterium or yeast, by methods known in the art, for example by alkaline lysis of the host cell, and subsequent purification of the nucleic acid, for example by a silica adsorption procedure; (ii) amplified in vitro, for example by polymerase chain reaction (PCR); (iii) produced recombinantly by cloning, for example, a nucleic acid cloned into the expression vector; (iv) shredded and sized separated, for example, by in vitro enzymatic digestion, or by shear, and subsequent gel separation; or (v) synthesized by, for example, chemical synthesis. In some embodiments, an isolated nucleic acid can readily be manipulated by recombinant DNA techniques well known in the art. Consequently, a nucleic acid cloned into a vector, or a nucleic acid delivered to a host cell and integrated into the host genome, is considered isolated, but a nucleic acid in its native state in its natural host, for example, in the host genome, it is not. An isolated nucleic acid can be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure, in that it may comprise only a small percentage of the material in the cell in which it resides. Such nucleic acid is isolated; however, as the term is used here.
[096] Some aspects of this invention pertain to nucleic acids that encode a gene product imparting a required or desirable phenotype to a microbe for biofuel production or biofuel precursor that are linked to a promoter or other transcriptional activation element. In some embodiments, the nucleic acid encoding the gene product and linked to a promoter is comprised of an expression vector or expression construct. As used herein, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, recombinantly or synthetically generated, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host microbe, for example an oilseed yeast. In some embodiments, the expression vector can be part of a plasmid, virus, or nucleic acid fragment. In some embodiments, the expression vector includes encoding nucleic acid to be transcribed operably linked to a promoter. A promoter is a nucleic acid element that facilitates the transcription of a nucleic acid to be transcribed. A promoter is typically located on the same strand and upstream (or 5') of the nucleic acid sequence that its transcription controls. In some embodiments, the expression vector includes the encoding nucleic acid to be transcribed operably linked to a heterologous promoter. A heterologous promoter is a promoter not naturally operably linked to a given nucleic acid sequence. For example, the SCD gene in Y. lipolytica is naturally operably linked to the SCD gene promoter in Y. lipolytica. Any other than the Y. lipolytica wild-type SCD gene promoter operably linked to the SCD gene, or parts thereof, for example, in an expression construct, would therefore be a heterologous promoter.
[097] In some embodiments, the expression vector includes encoding nucleic acid, for example, a nucleic acid encoding an SCD gene product, operably linked to a constitutive promoter. The term "constitutive promoter" refers to a promoter that allows continuous transcription of its associated gene. In some embodiments, the expression vector includes encoding nucleic acid, for example, a nucleic acid encoding an SCD gene product, operably linked to an inducible promoter. The term "inducible promoter", interchangeably used herein with the term "conditional promoter", refers to a promoter that allows transcription of its associated gene only in the presence or absence of biotic or abiotic factors. Drug inducible promoters, e.g. tetracycline/doxycycline inducible promoters, tamoxifen inducible promoters, as well as promoters that depend on a recombination event in order to be active, e.g. cre-mediated recombination of loxP sites, are examples of inducible promoters which are well known in the art.
[098] Methods for delivering expression vectors or expression constructs into microbes, for example into yeast cells, are well known to those skilled in the art. Nucleic acids, including expression vectors, can be delivered to prokaryotic and eukaryotic microbes by various methods well known to those skilled in the relevant biological arts. Methods for delivering nucleic acid to a microbe in accordance with some aspects of this invention include, but are not limited to, chemical, electrochemical and biological approaches, e.g., heat shock transformation, electroporation, transfection, e.g. liposome-mediated transfection, DEAE-Dextran-mediated transfection, or calcium phosphate transfection. In some embodiments, a nucleic acid construct, e.g., an SCD expression construct, is introduced into the host microbe using a vehicle, or vector, for transferring genetic material. Vectors for microbial gene transfer material are well known to those skilled in the art, and include, for example, plasmids, artificial chromosomes, viral vectors. Methods for constructing nucleic acid constructs, including expression constructs comprising constitutive or inducible heterologous promoters, and knockout and knockdown constructs, as well as methods and vectors for delivering a nucleic acid or nucleic acid construct to a microbe, are well known to those skilled in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002); Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998); and Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), all of which are incorporated herein by reference.
[099] In some embodiments, the native promoter of a gene that encodes a gene product conferring a required or desirable phenotype to a microbe, for example the native SCD promoter, is modified in the microbe to alter the regulation of its transcriptional activity. In some embodiments, the modified promoter exhibits increased transcriptional activity as compared to its unmodified counterpart. The term "modified promoter", as used herein, refers to a promoter of the nucleotide sequence from which it has been artificially altered. Nucleotide deletion(s), insertion(s) or mutation(s), alone or in combination, are examples of such artificial changes. Artificial promoter alterations can be effected in a targeted manner, for example, by homologous recombination approaches, such as gene targeting, knockout, knockin, site-directed mutagenesis, or artificial zinc claw nuclease-mediated strategies. Alternatively, such changes may be effected by a random or quasi-random event, such as irradiation nucleotide integration, or non-target of a subsequent selection. Promoter modifications are generally shown to modulate the transcriptional activation properties of the respective promoter. For example, disruption or abrogation of a regulatory element that mediates repression of an SCD promoter in response to elevated extracellular fatty acid levels can lead to continued transcriptional activation of the SCD gene even under conditions of high intracellular fatty acid levels. Similarly, insertion of a constitutively active transcriptional activator element into a conditional promoter region can effect overexpression of the respective gene under normally inhibitory conditions. Methods for target disruption of a native promoter, e.g., a native SCD promoter, in a microbe, for example, for target disruption resulting in an increased rate of transcription, are well known to those skilled in the art.
[0100] In some embodiments, a nucleic acid construct is provided that is useful for knocking out a delta-12 desaturase gene in a microbe for biofuel production or biofuel precursor. In some embodiments, the knockout construct comprises genomic sequences from a microbial delta-12 desaturase gene that flanks a nucleotide sequence that, when inserted into the delta-12 desaturase gene, disrupts expression of a delta-12 desaturase gene product. . In some embodiments, the nucleic acid that disrupts the delta-12 desaturase gene expression product is a marker of antibiotic resistance, for example, a phleomycin resistance gene. In some embodiments, the delta-12 desaturase knockout vector comprises a sequence as provided in SEQ IDNO: 28. Methods of delivering knockout vectors to microbes are well known to those skilled in the art, and methods of effecting homologous recombination in microbes, for example, in yeast, are well known to the person skilled in the art as well. The invention is not limited in this particular. MICROBE DESIGN METHODS
[0101] Some aspects of this invention pertain to the design of a microbe, eg, Y. lipolytica, to exhibit a required and/or desirable phenotype for large-scale production of a biofuel or biofuel precursor. Some aspects of this invention pertain to the metabolic design of the SCD pathway in order to produce a microbe optimized for biofuel production. Some aspects of this invention pertain to the metabolic design of a carbon flux that regulates the gene into or out of a fatty acid synthesis pathway so as to produce a microbe optimized for biofuel production.
[0102] Some aspects of this invention provide methods for greatly increasing the efficiency of Y. lipolytica-mediated carbon source conversion to lipid by modulating native Y. lipolytica lipid metabolism. Some aspects of this invention pertain to the discovery that an overexpression of a gene that increases fatty acid or triacylglycerol accumulation, such as SCD, not only results in an increase in lipid accumulation, but also an increase in the rate of lipid synthesis. lipid, lipid content, and/or growth rate. Markedly and unexpectedly, the modulation of lipid metabolism according to some methods provided by this invention also confers other beneficial characteristics, for example, an increased tolerance for food stock substances, including high concentrations of substrate (e.g. glucose) , and/or toxic substances commonly found to contaminate feed stock, eg pre-treated feed stock. Some non-limiting examples of such contaminating substances are furfural, 5-hydroxymethylfurfural and acetic acid. Some non-limiting examples of feed stock materials that generate toxic contaminants after pretreatment are wood feed stocks, corn husks, and bagasse.
[0103] Some aspects of this invention pertain to the design of required and/or desirable phenotypes in Y. lipolytica, via transcriptional inhibition of a key regulator of lipid metabolism, for example, via transcriptional inhibition of SCD. Manipulation of a key regulator of lipid metabolism, e.g., SCD, in other biofuel producing microbes, e.g., yeast, bacteria, fungi, or algae, is also contemplated.
[0104] In order to design an organism, for example an oilseed yeast, to be useful in industrial scale biofuel productions, a detailed understanding of the molecular mechanisms that control fatty acid and lipid metabolism in the respective organism is essential. Until the present invention, the identification and functional annotation of regulators of fatty acid and lipid metabolism in microorganisms producing oil for biofuel production, eg oleaginous yeast, remain unsolved. Some aspects of this invention provide for the identification and functional annotation of a key regulatory gene, SCD, in the oleaginous yeast Y. lipolytica. Isolated SCD nucleic acid and protein molecules are also provided.
[0105] Some aspects of this invention pertain to the design of a desirable phenotype for biofuel production or biofuel precursor in a microbe by genetic engineering. Some aspects of this invention pertain to the manipulation of a gene involved in biofuel production or a biofuel precursor, for example a fatty acid or a triacylglycerol, in a microbe. Some aspects of this invention pertain to the manipulation of a plurality of genes involved in the production of biofuel or a biofuel precursor in parallel in a microbe.
[0106] In some embodiments, a microbe is engineered to produce biofuel or biofuel precursor by manipulating a single gene, in accordance with methods provided by aspects of this invention, e.g., a Δ9 desaturase (e.g., SCD), GLUT (e.g., Glut1), hemoglobin, cytochrome (e.g., cytochrome B5), Malic Enzyme, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, FAS, AMPK, JNK2, or delta-12 desaturase. In some embodiments, a microbe is engineered to produce a biofuel or biofuel precursor by manipulating a plurality of genes in accordance with methods provided by aspects of this invention, e.g., any combination of two or more than one Δ9 desaturase (e.g., SCD), GLUT (e.g. Glut1), hemoglobin, cytochrome (e.g. cytochrome B5), Malic Enzyme, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, FAS, JNK2, delta-12 desaturase, and/ or AMPK. In some embodiments, a microbe is designed to comprise an increased level of an SCD gene product and further manipulation, e.g., a genetic manipulation, of the expression of an additional gene product, e.g., a GLUT (e.g., Glut1), hemoglobin, cytochrome (e.g., cytochrome B5), Malic Enzyme, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, FAS, JNK2, delta-12 desaturase, or AMPK gene product. In some embodiments, a microbe is designed to comprise an increased level of an SCD gene product from a hemoglobin gene product. In some embodiments, a microbe is designed to comprise an increased level of an SCD gene product and a GLUT gene product, for example, a Glut1 gene product. In some embodiments, a microbe is designed to comprise an increased level of an SCD gene product, a GLUT gene product, e.g., a Glut1 gene product, and a hemoglobin and/or a hemoglobin gene product. cytochrome. In some embodiments, a microbe is designed to comprise an increased level of an SCD and Glut1 gene product, hemoglobin and cytochrome b5, and, optionally, a delta-12 desaturase knockout. In some embodiments, the microbe is Y. lipolytica. MICROBES DESIGNED FOR BIOFUEL PRODUCTION
[0107] Some aspects of this invention pertain to a microbe designed and/or optimized for large-scale biofuel or biofuel precursor production. In some embodiments, an engineered microbe is provided that has been manipulated by a method, or using a nucleic acid or protein provided by some aspects of this invention. In some embodiments, an engineered microbe is provided that overexpresses a gene product that, in accordance with some aspects of this invention, confers a required and/or desirable phenotype for biofuel production or biofuel precursor to the microbe. In some embodiments, a microbe comprising increased SCD gene product activity is provided. In some embodiments, the microbe exhibits an increased rate of fatty acid synthesis, an increased storage of TAG, and/or an additional required or desirable trait.
[0108] In some embodiments, the engineered microbe is an oleaginous yeast, eg Y. lipolytica. In some embodiments, an engineered yeast provided by this invention exhibits one or more highly desirable and unexpected phenotypic characteristics, for example: increased conversion of carbon to oil, for example, at a rate approaching theoretical values, robust growth, continuous production of oil, remarkable production of biomass, and increased tolerance of the carbon source and associated substances.
[0109] In some embodiments, the engineered microbe, e.g., engineered yeast, provided by aspects of this invention, exhibits a carbon to oil conversion rate within the range of about 0.02 g/g (g oil, lipid , or TAG produced/g Glucose consumed) at about 0.3 g/g. In some embodiments, the engineered microbe, e.g., engineered yeast, provided by aspects of this invention, exhibits a carbon to oil conversion of about 0.010 g/g (g TAG produced/g Glucose consumed), about 0.010 g/g. 02 g/g, about 0.025 g/g, about 0.03g/g, about 0.04 g/g, about 0.05 g/g, about 0.06 g/g, about 0 0.07 g/g, about 0.075 g/g, about 0.08 g/g, about 0.09 g/g, about 0.1 g/g, about 0.11 g/g, about from 0.12 g/g, about 0.13 g/g, about 0.14 g/g, about 0.15 g/g, about 0.16 g/g, about 0.17 g /g, about 0.18 g/g, about 0.19 g/g, about 0.2 g/g, about 0.21 g/g, about 0.22 g/g, about 0.23 g/g, about 0.24 g/g, about 0.25 g/g, about 0.26 g/g, about 0.27 g/g, about 0.28 g/g g, about 0.29 g/g, or about 0.3 g/g, or theoretical approach values. In some embodiments, the engineered microbe, e.g., engineered yeast, provided by aspects of this invention, exhibits a carbon to oil conversion rate of at least about 0.010 g/g (g TAG produced/g Glucose consumed) at least about 0.02 g/g, at least about 0.025 g/g, at least about 0.03 g/g, at least about 0.04 g/g, at least about 0.05 g/g, at least about 0.06 g/g, at least about 0.07 g/g, at least about 0.075 g/g, at least about 0.08 g/g, at least about 0.08 g/g 0.09 g/g, at least about 0.1 g/g, at least about 0.11 g/g, at least about 0.12 g/g, at least about 0.13 g/g at least about 0.14 g/g, at least about 0.15 g/g, at least about 0.16 g/g, at least about 0.17 g/g, at least about 0 .18 g/g, at least about 0.19 g/g, at least about 0.2 g/g, at least about 0.21 g/g, at least about 0.22 g/g, at least about 0.23 g/g, at least about 0.24 g/g, at least about 0.25 g/g, at least about 0.26 g/g, at least about 0.27 g/g, at least about 0.28 g/g, at least about 0.29 g/g, or at least about 0.3 g/g, or theoretical approach values.
[0110] In some embodiments, the engineered yeast provided by aspects of this invention exhibits a biomass production that is increased about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold. times, about 15 times, about 20 times, about 25 times, about 30 times, about 32 times, about 35 times, or about 40 times, as compared to wild-type yeast. In some embodiments, the engineered yeast provided by aspects of this invention exhibit tolerance to the carbon source and/or associated substances at concentrations of up to about 150%, up to about 175%, up to about 200%, up to about 225%, up to about 250%, up to about 275%, up to about 300%, up to about 325%, up to about 350%, up to about 375%, up to about 400%, or up to about 500% of that of higher concentrations tolerated by wild-type yeast. Non-limiting examples of carbon source associated substances include toxic substances that contaminate the carbon source, e.g. substances that are generated or used during pretreatment of the carbon source (e.g. acidic substances such as acetic acid, or ammonia).
[0111] The data presented here identify a new step in limiting the rate of lipid accumulation in oilseed yeast, the design of which results in greatly improved characteristics of the engineered microbe with respect to the generation of biofuel from carbohydrate sources (e.g. glucose ). Consequently, the methods and manufacturers provided by the present invention represent a significant advance towards an alternative production of biofuels from renewable carbohydrate sources using microbial fermentation, eg yeast. MICROBIAL CROPS FOR BIOFUEL PRODUCTION
[0112] Some aspects of this invention pertain to a culture of a microbe provided herein or designed in accordance with aspects of this invention, or comprising an isolated nucleic acid or protein provided herein.
[0113] In some embodiments, the culture comprises a microbe provided or designed herein in accordance with aspects of this invention, or comprising an isolated nucleic acid or protein from the list provided herein and a medium, for example, a liquid medium.
[0114] In some embodiments, the culture comprises a microbe provided or engineered herein, in accordance with aspects of this invention, or comprising an isolated nucleic acid or protein provided herein and a carbohydrate source.
[0115] In some embodiments, the culture comprises a microbe provided or engineered herein, in accordance with aspects of this invention, or comprising an isolated nucleic acid or protein provided herein and a salt and/or buffer establishment conditions of salinity, osmolarity, and pH, which are amenable to survival, growth, and/or conversion of carbohydrate to biofuel or biofuel precursor by the microbe.
[0116] In some embodiments, the culture comprises an additional component, for example, an additive. Non-limiting examples of additives are nutrients, enzymes, amino acids, albumin, growth factors, enzyme inhibitors (e.g. protease inhibitors), fatty acids, lipids, hormones (e.g. dexamethasone and gibberellic acid), trace, inorganic compounds (e.g. reducing agents such as manganese), redox regulators (e.g. antioxidants), stabilizing agents (e.g. dimethyl sulfoxide), polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotics ( for example, Brefeldin A), salts (for example, NaCl), chelating agents (for example, EDTA, EGTA), and enzymes (for example, cellulase, dispase, hyaluronidase, or DNase). In some embodiments, the culture may comprise a drug-inducing or inhibiting transcript from an inducible or conditional promoter, for example, doxycycline, tetracycline, tamoxifen, IPTG, hormones, or metal ions.
[0117] While specific culture conditions, e.g. carbon source concentration, will depend on the respective microorganism designed to be cultured, general methods and culture conditions for generating microbial cultures are well known to those skilled in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); and Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002), all of which are incorporated herein by reference. For oil production, the engineered microbe cultures described herein are grown under conditions suitable for oil accumulation, as known in the art.
[0118] In some embodiments, a engineered microbe is provided that exhibits a growth advantage over wild type microbes of the same type and/or over other microbes, e.g. microbes commonly found in microbial cultures contaminated for carbon source conversion to biofuel or biofuel precursor. For example, in some embodiments, a microbe is provided that exhibits an increased proliferation rate as compared to wild-type microbes of the same type or other microbes, and/or an increased tolerance to, or viability under conditions that are toxic or growth-restricted. or proliferation to wild-type microbes of the same type and/or other microbes. In some embodiments, the growth and/or proliferation advantage of an engineered microbe provided by aspects of this invention translates into the possibility of using non-sterile culture and fermentation conditions for biofuel or biofuel precursor production, due to the problem of overgrowth of the culture by contaminating microbes can be avoided or completely abolished. In some embodiments, an engineered microbe provided by aspects of this invention is cultured under non-sterile conditions for production of biofuel or biofuel precursor. For example, in some embodiments, non-sterile feed stock, non-sterile culture medium, non-sterile supplements, or a non-sterile bioreactor (e.g., an open reactor under non-sterile conditions) is used for biofuel or biofuel precursor production. . METHODS FOR PRODUCTION OF BIOFUEL/FOOD STOCK/BIOREATORS
[0119] Some aspects of this invention pertain to methods for producing biofuel or biofuel precursor using modified microbes in accordance with this invention. In some embodiments, methods for producing the biofuel or biofuel precursor on an industrial scale are provided.
[0120] A variety of carbon sources can be converted into a biofuel or biofuel precursor using a method provided by some aspects of this invention. Sugars, starches, and fibers are non-limiting examples of suitable carbohydrate sources for conversion methods provided by some aspects of this invention. In accordance with some aspects of this invention, a carbohydrate source may comprise a refined and/or unrefined sugar, starch, and/or fiber, or a combination of any of these. Non-limiting examples of sugars are fermentable sugars, such as glucose, fructose, sucrose, xylose, and lactose. Non-limiting examples of starches are amylase and amylopectin. Non-limiting examples of fibers are plant fibers such as cellulose, hemicellulose and wood fibers. Some aspects of this invention pertain to the use of industrial by-products, intermediates, or waste products, for example, crude plant extracts, molasses, straw, or sawdust as a carbon source. In some embodiments, the carbon source is derived from algae. In some embodiments, the algal biomass is produced specifically for use as a carbon source in microbe-mediated biofuel production or biofuel precursor.
[0121] In some embodiments, methods for producing biofuel or biofuel precursor are provided which include the use of an inexpensive, abundant and readily available carbon source feed stock as the carbon source. In some embodiments, cellulose or hemicellulose is used as the carbon source. In some embodiments, the cellulose or hemicellulose is derived from industrial or waste by-products. In some embodiments, the cellulose or hemicellulose is derived directly from plant or algal biomass. Plant or algae biomass is one of the most abundant food stocks, and comprises a significant amount of non-fermentable sugars and fibers, eg cellulose and hemicellulose. In some embodiments, the biomass feed stock is pretreated to convert the non-fermentable sugar or fiber into a fermentable sugar, thereby making them available for microbe growth and biofuel or biofuel precursor mediated production. microbe. In some embodiments, pretreatment of biomass feed stock includes depolymerizing cellulose and/or hemicellulose components to monomeric sugars using a pretreatment method known to those skilled in the art, for example, a dilute acid or of ammonia fiber expansion (AFEX) (see, for example, Yang B, Wyman CE. Dilute acid and autohydrolysis pretreatment. Methods Mol Biol. 2009;581:103-14; Balan V, Bals B, Chundawat SP, Marshall D , Dale BE, Lignocellulosic biomass pretreatment using AFEX Methods Mol Biol. 2009;581:61-77). Other methods for depolymerizing biomass polymers to monomeric sugars are well known to those skilled in the art, and are contemplated for use in some embodiments of this invention.
[0122] In some embodiments, a biomass feedstock containing non-fermentable sugars is pretreated using a dilute acid method to depolymerize a non-fermentable sugar to a monomeric fermentable sugar. In some embodiments, the biomass is treated with dilute sulfuric acid at moderately mild temperatures for a defined period of time. For example, in some embodiments, the biomass is treated with about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, or about 6% of sulfuric acid. In some embodiments, the biomass is treated at about 30°C, at about 37°C, at about 40°C, at about 50°C, at about 60°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, at about 110°C, at about 120°C, at about 130°C, at about 140°C, at about 150°C, at about 175°C, at about 200°C, or above about 200°C.
[0123] In some embodiments, the resulting hydrolyzate contains insoluble lignin and solubilized cellulosic and hemicellulose polymers. The latter products can be further treated to generate hexose and pentose sugars such as glucose and xylose monomers by methods well known to those skilled in the art, for example, by treatment with cellulase or other hydrolyzing enzymes. In some embodiments, pretreatment of non-fermentable sugars with dilute acid results in the generation of by-products that include toxic compounds that inhibit growth, decrease viability, and/or inhibit production of biofuel or biofuel precursor from non-engineered microbes in accordance with aspects of this invention. In some embodiments, the pre-treated feed stock is washed, supplemented with medium supporting microbial growth and biofuel production or biofuel precursor, and/or super-treated with lime for detoxification.
[0124] In some embodiments, a biomass feed stock containing non-fermentable sugars is pretreated using an AFEX method to depolymerize a non-fermentable sugar to a monomeric fermentable sugar. In some embodiments, the biomass is treated with liquid ammonia at high temperature and pressure for a defined period of time. In some embodiments, the biomass is treated for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes or longer. In some embodiments, the biomass is treated at about 30°C, at about 37°C, at about 40°C, at about 50°C, at about 60°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, at about 110°C, at about 120°C, at about 130°C, at about 140°C, at about 150°C, at about 175°C, at about 200°C, or above about 200°C. In some embodiments, AFEX pretreatment results in the conversion of crystalline cellulose contained in the feed stock to a fermentable amorphous form. In some embodiments, the AFEX pretreated biomass feed stock does not contain significant amounts of toxic by-products that inhibit microbial growth and/or biofuel or biofuel precursor production, and is used without prior detoxification for biofuel or biofuel precursor production. microbial biofuel.
[0125] In some embodiments, the biomass feed stock, with or without pretreatment, is treated with an enzyme that hydrolyzes or depolymerizes sugar polymers, for example, with a cellulase or hemicellulase enzyme. In some embodiments, the feed stock is contacted with the enzyme in a liquid phase and incubated at a temperature that allows the enzyme to catalyze a depolymerization or hydrolyzate reaction for a time sufficient to hydrolyze or depolymerize a significant amount of the non-fermentable sugar. or fiber in the biomass feed stock. In some embodiments, the liquid phase of the enzyme-contacted feed stock, which contains the soluble fermentable sugar fraction, is separated from the solid phase, including non-fermentable sugars and fibers, after incubation for hydrolyzing and depolymerization, for example. , by centrifugation. In some embodiments, the liquid fraction of the feed stock is subsequently contacted with a microbe, for example, a microbe provided by aspects of this invention, for conversion to biofuel or biofuel precursor. In some embodiments, enzymatic conversion of non-fermentable sugars or fiber takes place in a consolidated bioprocess, for example, at the same time and/or in the same reactor as microbial conversion of the produced fermentable sugars to biofuel or biofuel precursor. In some embodiments, the enzymatic conversion is performed first, and the enzyme-contacted feed stock is subsequently contacted with the microbe for production of biofuel or biofuel precursor. In some embodiments, enzymatic and microbial conversions are performed at the same time and in the same reactor.
[0126] In some embodiments, an engineered microbe as provided herein, for example a Yarrowia lipolytica over-expressing an SCD gene and optionally carrying out further modifications as described herein, is grown on acetate as the primary carbon source. For example, in some embodiments, the microbe is grown in a solution of acetic acid at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6% , about 7%, about 8%, about 9%, or about 10%. In some embodiments, the acetate concentration is between about 3%-10%. In some embodiments, cell cultures comprising engineered microbes, as provided herein, that are grown on acetate as the primary carbon source are contacted, or "baptized" with glycerol. In some embodiments, the microbes are intermittently contacted with glycerol. In some embodiments, the microbes are continuously or semi-continuously contacted with glycerol. In some embodiments, the microbes are contacted with glycerol at a concentration of about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%. Contacting the engineered microbes provided here with glycerol provides much needed metabolites for the production of TAGs, as well as reducing the portions needed in the production of fatty acids from carbohydrates. In some embodiments, glycerol contact is performed in the methods of producing biofuel or biofuel precursor using a carbon source other than acetate, for example, any carbon source described herein.
[0127] In some embodiments, large-scale microbe-mediated fermentation processes for carbohydrate and lipid conversion can be carried out in bioreactors. As used herein, the terms "bioreactor" and "fermenter", which are used interchangeably, refer to a shutdown, or partial shutdown, in which a biological and/or chemical reaction occurs, at least part of which involves a new or part of a new organism. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product, e.g. a biofuel or biofuel precursor, e.g. a fatty acid and/or TAG, on a commercial scale. or quasi-commercial. Large-scale bioreactors typically have volumes in the range of liters, hundreds of liters, thousands of liters, or more.
[0128] A bioreactor according to aspects of this invention may comprise a microbe or a microbe culture. In some embodiments, a bioreactor may comprise a spore and/or any type of dormant cell type of any isolated microbe provided by aspects of this invention, for example, in a dry state. In some embodiments, addition of a suitable carbohydrate source to such bioreactors can lead to activation of the dormant cell, for example, to germination of a yeast spore, and subsequent conversion of the carbohydrate source, at least in part, to a biofuel or biofuel precursor.
[0129] Some bioreactors, in accordance with aspects of this invention, may include cell culture systems where microbes are in contact with moving liquids and/or gas bubbles. Microbes or microbe cultures according to aspects of this invention can be grown in suspension or attached to solid phase carriers. Non-limiting examples of carrier systems include microcarriers (e.g. polymer beads, microbeads, and microdisks which may be porous or non-porous), crosslinked beads (e.g. dextran) loaded with specific chemical groups (e.g. , tertiary amine groups), 2D micro transporters including cells trapped in non-porous polymer fibers, 3D transporters (e.g. carrier fibers, hollow fibers, multi-cartridge reactors, and semi-permeable membranes which may comprise porous fibers), micro carriers having reduced ion exchange capacity, encapsulation of cells, capillaries, and aggregates. Carriers can be made from materials such as dextran, gelatin, glass, and cellulose.
[0130] Industrial scale carbohydrate to lipid conversion processes according to aspects of this invention can be operated in continuous, semi-continuous or non-continuous modes. Non-limiting examples of modes of operation according to this invention are batch modes, fed batch modes, extended batch modes, repetitive batch modes, take-out/fill modes, spin-wall modes, centrifuge bottle , and/or perfusion modes of operation.
[0131] In some embodiments, bioreactors can be used that allow continuous or semi-continuous replenishment of substrate stock, e.g., a carbohydrate source and/or continuous or semi-continuous separation of the product, e.g., a secreted lipid , an organic phase comprising a lipid, and/or cells that exhibit a desired lipid content, from the reactor.
[0132] Non-limiting examples of bioreactors, according to this invention, are: stirred tank fermenters, bioreactors stirred by rotary mixing devices, chemical stators, bioreactors stirred by oscillating devices, air lift fermenters, bed reactors conditioned bed reactors, fixed bed reactors, fluidized bed bioreactors, bioreactors employing wave-induced agitation, centrifugal bioreactors, laminator bottles, and hollow fiber bioreactors, laminating apparatus (e.g. bench top, cart mounted, and/or automatic), vertically stacked plates, centrifuge flasks, shaker or rocker flasks, multi-well oscillating plates, MD bottles, T-flasks, Roux bottles, multi-surface tissue culture propagators, modified fermenters, and coated beads (e.g. , beads coated with whey protein, nitrocellulose, or carboxymethyl cellulose to prevent f cell attachment).
[0133] Bioreactors and fermenters, in accordance with aspects of this invention, may optionally comprise a sensor and/or a control system for measuring and/or adjusting reaction parameters. Non-limiting examples of reaction parameters are: biological parameters, eg growth rate, cell size, cell number, cell density, cell type, or cell state, chemical parameters, eg pH, potential redox, reaction substrate and/or product concentration, dissolved gas concentration, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration , concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, concentration of serum, ionic resistance, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals , e.g. buffering agents, adjuvants, or reaction by-products, physical/mechanical parameters, e.g. density, conductivity of, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic parameters such as temperature, intensity /quality of light, etc.
[0134] Sensors capable of measuring the parameters as described herein are well known to those skilled in the relevant mechanical and electronic techniques. Control systems capable of adjusting parameters in a bioreactor based on inputs from a sensor, as described herein, are well known to those skilled in the art of bioreactor design.
[0135] A variety of different microbes provided by aspects of this invention can be grown in a bioreactor suitable for carrying out large-scale conversion of carbohydrate to biofuel or biofuel precursor, in accordance with aspects of the invention, for example, microbes from various sources yeast, such as oilseed yeast, bacteria, algae and fungi.
[0136] Non-limiting examples of yeast cells are Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, S. bayanus, S.K. lactis, Waltomyces lipofer cells. Mortierella alpine, Mortierella isabellina, Hansenula polymorpha., Mucor rouxii, Trichosporon cutaneu, Rhodotorula glutinis Saccharomyces diastasicus, Schwanniomyces occidentalis, S. cerevisiae, Pichia stipitis, and Schizosaccharomyces pombe.
[0137] Non-limiting examples of bacteria are Bacillus subtilis, Salmonella, Escherichia coli, Vibrio cholerae, Streptomyces, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas sp, Rhodococcus sp, Streptomyces sp, and Alcaligenes sp.
[0138] Fungal cells can, for example, be cultured from species such as Aspergillus shirousamii, Aspergillus niger and Trichoderma reesei.
[0139] Non-limiting examples of algal cells are Neochloris oleoabundans, Scenedesmus obliquus, Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, and Spirulina maxima cells.
[0140] The type of carbohydrate source to be employed for conversion to a biofuel or biofuel precursor, in accordance with aspects of this invention, depends on the specific microbe employed. Some microbes provided by aspects of this invention may be able to efficiently convert a specific carbohydrate source, whereas a different carbohydrate source cannot be processed by the same microbe at high efficiency, or at all. In accordance with aspects of this invention, the oilseed yeast Y. lipolytica, for example, can efficiently convert sugars, such as glucose, fructose, sucrose, and/or lactose, and high carbohydrate sources into sugars, for example, molasses, and fiber. of plant in fatty acids and their derivatives.
[0141] In some embodiments, a biofuel or biofuel precursor, for example, a fatty acid or a triacylglycerol, generated from a carbon source feed stock is secreted, at least partially, by a microbe provided by aspects of this invention, for example, an oleaginous yeast, such as a Y. lipolytica cell. In some embodiments, a microbe provided by aspects of this invention is contacted with a carbohydrate source in an aqueous solution in a bioreactor, and secreting biofuel or biofuel precursor forms an organic phase that can be separated from the aqueous phase. The term organic phase, as used herein, refers to a liquid phase comprising a non-polar organic compound, for example, a fatty acid, TAG, and/or other non-polar lipid. The organic phase according to this invention may additionally contain a microbe, a carbohydrate, or other compounds found in other phases found in a respective bioreactor. Useful methods for industrial scale phase separation are well known to those skilled in the art. In some embodiments, the organic phase is continuously or semi-continuously siphoned. In some embodiments, a bioreactor is employed comprising a separator, which continuously or semi-continuously extracts the organic phase.
[0142] In some embodiments, a biofuel or biofuel precursor is accumulated in cells in accordance with aspects of this invention. In some embodiments, cells that have accumulated a desirable amount of biofuel or biofuel precursor are continuously or semi-continuously separated from a bioreactor, for example, by centrifugation, sedimentation, or filtration. Cell sorting may additionally be effected, for example, based on a change in the physical characteristics of the cell, such as cell size or density, by methods known to those skilled in the art. The accumulated biofuel or biofuel precursor may subsequently be extracted from the respective cells using standard extraction methods known to those skilled in the art, for example solvent extraction from hexane. In some embodiments, microbial cells are collected and extracted with 3 times the collected cell volume of hexane. In some embodiments, the biofuel or extracted biofuel precursor is further refined. In some embodiments, a biofuel precursor, for example, a triacylglycerol, is converted to a biofuel, for example, biodiesel, using a method well known to those skilled in the art, for example, a transesterification procedure.
[0143] The function and advantage of these and other embodiments of the present invention will be more fully understood from the examples below. The following examples are intended to illustrate the benefits of the present invention, but not to exemplify the full scope of the invention. EXAMPLES MATERIALS AND METHODS
[0144] Gene constructs: The respective genes, eg GLUT1, hemoglobin, cytochrome, Pyruvate carboxylase, SCD, etc., were cloned into plasmid YLEX (Figure 12) between PmlI and Kpn sites. Restriction sites used were PmlI and KpnI. All cDNA was sequenced and mapped to genomic databases. Exemplary representative sequence database entries that the cloned cDNAs were mapped to include: GLUT1: GeneID: 6513; Hemoglobin: Vitreoscilla stercoraria bacterial hemoglobin gene, ACCESS L77863; Cytochrome: GeneID: 1528, CYB5A cytochrome b5 type A; Pyruvate carboxylase: GeneID: 5091; Stearoyl-CoA desaturase SCD (SCD): GeneID: 710155.
[0145] Representative sequences, e.g., coding sequences, useful for the generation of microbes over-expression are, for example, hemoglobin (bacterial) ATGTTAGACCAACAAACCGTAGACACCAGCAAAGCCACT GTTCCTGTATTGAAAGAGCATGGCGTGACCATTACCACGACGTTTTA CCAAAATTTGTTTGCCAAACATCCTGAAGTACGACCTTTGTTTGACA TGGGTCGCCAAGCATCTTTGGAACAGCCTAAGGCTTTGGCGATGAC GGTTGGGGCGGCGGCACAAAACATTGAAAATTTACCTGCAATTTTG CCTGCAGTACAAAAAATTGCCGTCAAACATTGTCAAGCAGGCGTGG CGGCACGACATTATCCGATTGTGGGTCAAGAATTGTTGGGTGCGAT TAAAGAATTATTGGGTGATGCGGCGACCGATGATATTTTGGATGCG TGGGGCAAGGCTTATGGCGTGATTGCCGATGTTTTTATTCAAGTGG AAGCGGATTTGTACGCTCAAGACGCTGAATAA (SEQ ID NO: 3) CYTOCHROME B (Yarrowia) ATGATCATCAACGGCAAGGTCTACGACATCTCCAGCTT CGTTGACGAGCATCCCGGTGGAGAGGAGGTTCTTCTTGATGCCG GTGGAACTGAGGCCACCAACGCTTTCGACGACGTTGGACACTCT GAGGACGCTTACGGCATCCTTAACGACCTCTATGTCGGTGAGGTT GACCCCAGCGAGGACGTTATCCGAAAGACTCACACTGTCAAGACT TCTTACGAGGACGGCGAGTCTGTTGGTGATGACCACGGATCTTCT TCCATGATCTTCCTCATTGTTGCTGCTGCTGTTGCCGCCGCTGCTT TCTTCTACCTCCAGGGTCAGAAATAA (SEQ ID NO: 4) GLUT (rat) ATGGAGCCCAGCAGCAAGAAGGTGACGGGCCGCCTTAT GTTGGCCGTGGGAGGGGCAGTGCTCGGATCCCTGCAGTTCGGCTA TAACACCGGTGTCATCAACGCCCCCCAGAAGGTAATTGAGGAGTTC TACAATCAAACATGGAACCACCGCTATGGAGAGTCCATCCCATCCA CCACACTCACCACACTCTGGTCTCTCTCCGTGGCCATCTTCTCTGT CGGGGGCATGATTGGTTCCTTCTCTGTGGGCCTCTTTGTTAATCGC TTTGGCAGGCGGAACTCCATGCTGATGATGAACCTGTTGGCCTTTG TGTCTGCCGTGCTTATGGGTTTCTCCAAACTGGGCAAGTCCTTTGA GATGCTGATCCTGGGCCGCTTCATCATTGGAGTGTACTGTGGCCTG ACCACCGGCTTTGTGCCCATGTATGTGGGGGAGGTGTCACCCACA GCTCTTCGTGGAGCCCTGGGCACCCTGCACCAGCTGGGCATCGTC GTTGGGATCCTTATTGCCCAGGTGTTCGGCTTAGACTCCATCATGG GCAATGCAGACTTGTGGCCTCTACTGCTCAGTGTCATCTTCATCCC AGCCCTGCTACAGTGTATCCTGTTGCCCTTCTGCCCTGAGAGCCCC CGCTTCCTGCTCATCAATCGTAACGAGGAGAACCGGGCCAAGAGT GTGCTGAAAAAGCTTCGAGGGACAGCCGATGTGACCCGAGACCTG CAGGAGATGAAAGAAGAGGGTCGGCAGATGATGCGGGAGAAGAAG GTCACCATCTTGGAGCTGTTCCGCTCACCCGCCTACCGCCAGCCCA TCCTCATCGCCGTGGTGCTGCAGCTGTCCCAGCAGCTGTCGGGCA TCAATGCTGTGTTCTACTACTCAACGAGCATCTTCGAGAAGGCAGG The TGTGCAGCAGCCTGTGTATGCCACCATCGGCTCGGGTATCGTCAAC CGGCCTTCACTGTGGTGTCGCTGTTCGTCGTGGAGCGAGCTGGC CGTCGGACCCTGCACCTCATTGGTCTGGCTGGCATGGCGGGCTGT GCTGTGCTCATGACCATCGCCCTGGCCCTGCTGGAGCAGCTGCCC TGGATGTCCTATCTGAGTATCGTGGCCATCTTTGGCTTTGTGGCCTT CTTTGAAGTAGGCCCTGGTCCTATTCCATGGTTCATTGTGGCCGAG CTGTTCAGCCAGGGGCCCCGACCTGCTGCTGTTGCTGTGGCTGGC TTCTCTAACTGGACCTCAAACTTCATCGTGGGCATGTGCTTCCAATA TGTGGAGCAACTGTGTGGCCCCTACGTCTTCATCATCTTCACGGTG CTGCTGGTACTCTTCTTCATCTTCACCTACTTCAAAGTTCCTGAGAC CAAAGGCCGGACCTTCGATGAGATCGCTTCCGGCTTCCGGCAGGG GGGTGCCAGCCAGAGCGACAAGACACCTGAGGAGCTCTTCCACCC TCTGGGGGCTGACTCCCAAGTGTGA (SEQ ID NO: 5) malic enzyme (Yarrowia) ATGTTACGACTACGAACCATGCGACCCACACAGACCAGC GTCAGGGCGGCGCTTGGGCCCACCGCCGCGGCCCGAAACATGTC CTCCTCCAGCCCCTCCAGCTTCGAATACTCGTCCTACGTCAAGGGC ACGCGGGAAATCGGCCACCGAAAGGCGCCCACAACCCGTCTGTCG GTTGAGGGCCCCATCTACGTGGGCTTCGACGGCATTCGTCTTCTCA ACCTGCCGCATCTCAACAAGGGCTCGGGATTCCCCCTCAACGAGC GACGGGAATTCAGACTCAGTGGTCTTCTGCCCTCTGCCGAAGCCAC CCTGGAGGAACAGGTCGACCGAGCATACCAACAATTCAAAAAGTGT GGCACTCCCTTAGCCAAAAACGGGTTCTGCACCTCGCTCAAGTTCC AAAACGAGG TGCTCTACTACGCCCTGCTGCTCAAGCACGTTAAGGA GGTCTTCCCCATCATCTATACACCGACTCAGGGAGAAGCCATTGAA CAGTACTCGCGGCTGTTCCGGCGGCCCGAAGGCTGCTTCCTCGAC ATCACCAGTCCCTACGACGTGGAGGAGCGTCTGGGAGCGTTTGGA GACCATGACGACATTGACTACATTGTCGTGACTGACTCCGAGGGTA TTCTCGGAATTGGAGACCAAGGAGTGGGCGGTATTGGTATTTCCAT CGCCAAGCTGGCTCTCATGACTCTATGTGCTGGAGTCAACCCCTCA CGAGTCATTCCTGTGGTTCTGGATACGGGAACCAACAACCAGGAGC TGCTGCACGACCCCCTGTATCTCGGCCGACGAATGCCCCGAGTGC GAGGAAAGCAGTACGACGACTTCATCGACAACTTTGTGCAGTCTGC CCGAAGGCTGTATCCCAAGGCGGTGATCCATTTCGAGGACTTTGGG CTCGCTAACGCACACAAGATCCTCGACAAGTATCGACCGGAGATCC CCTGCTTCAACGACGACATCCAGGGCACTGGAGCCGTCACTTTGG CCTCCATCACGGCCGCTCTCAAGGTGCTGGGCAAAAATATCACAGA TACTCGAATTCTCGTGTACGGAGCTGGTTCGGCCGGCATGGGTATT GCTGAACAGGTCTATGATAACCTGGTTGCCCAGGGTCTCGACGACA AGACTGCGCGACAAAACATCTTTCTCATGGACCGACCGGGTCTACT GACCACCGCACTTACCGACGAGCAGATGAGCGACGTGCAGAAGCC GTTTGCCAAGGACAAGGCCAATTACGAGGGAGTGGACACCAAGAC TCTGGAGCACGTGGTTGCTGCCGTCAAGCCCCATATTCTCATTGGA TGTTCCACTCAGCCCGGCGCCTTTAACGAGAAGGTCGTCAAGGAGA TGCTCAAACACACCCCTCGACCCATCAT TCTCCCTCTTTCCAACCCC ACACGTCTTCATGAGGCTGTCCCTGCAGATCTGTACAAGTGGACCG ACGGCAAGGCTCTGGTTGCCACCGGCTCGCCCTTTGACCCAGTCA ACGGCAAGGAGACGTCTGAGAACAATAACTGCTTTGTTTTCCCCGG AATCGGGCTGGGAGCCATTCTGTCTCGATCAAAGCTCATCACCAAC ACCATGATTGCTGCTGCCATCGAGTGCCTCGCCGAACAGGCCCCC ATTCTCAAGAACCACGACGAGGGAGTACTTCCCGACGTAGCTCTCA TCCAGATCATTTCGGCCCGGGTGGCCACTGCCGTGGTTCTTCAGG CCAAGGCTGAGGGCCTAGCCACTGTCGAGGAAGAGCTCAAGCCCG GCACCAAGGAACATGTGCAGATTCCCGACAACTTTGACGAGTGTCT CGCCTGGGTCGAGACTCAGATGTGGCGGCCCGTCTACCGGCCTCT CATCCATGTGCGGGATTACGACTAG (SEQ ID NO: 6) Yarrowia Delta (9) -desaturase (Estearoila-CoA desaturase) ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGT CGACACCATTGCCTCCGGCCGAGATGTCAACTACAAGGTCAAGTA CACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGG GCCGCCACATTTCCGAGCAGCCCTTCACCTGGGCCAACTGGCAC CAGCACATCAACTGGCTCAACTTCATTCTGGTGATTGCGCTGCCT CTGTCGTCCTTTGCTGCCGCTCCCTTCGTCTCCTTCAACTGGAAG ACCGCCGCGTTTGCTGTCGGCTATTACATGTGCACCGGTCTCGGT ATCACCGCCGGCTACCACCGAATGTGGGCCCATCGAGCCTACAA GGCCGCTCTGCCCGTTCGAATCATCCTTGCTCTGTTTGGAGGAGG AGCTGTCGAGGGC TCCATCCGATGGTGGGCCTCGTCTCACCGAG TCCACCACCGATGGACCGACTCCAACAAGGACCCTTACGACGCC CGAAAGGGATTCTGGTTCTCCCACTTTGGCTGGATGCTGCTTGTG CCCAACCCCAAGAACAAGGGCCGAACTGACATTTCTGACCTCAAC AACGACTGGGTTGTCCGACTCCAGCACAAGTACTACGTTTACGTT CTCGTCTTCATGGCCATTGTTCTGCCCACCCTCGTCTGTGGCTTT GGCTGGGGCGACTGGAAGGGAGGTCTTGTCTACGCCGGTATCAT GCGATACACCTTTGTGCAGCAGGTGACTTTCTGTGTCAACTCCCT TGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTC CCCGAGACCACGCTCTTACCGCCCTGGTCACCTTTGGAGAGGGC TACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGCC CTCATCTGGTACCAGTACGACCCCACCAAGTGGCTCATCTGGACC CTCAAGCAGGTTGGTCTCGCCTGGGACCTCCAGACCTTCTCCCAG AACGCCATCGAGCAGGGTCTCGTGCAGCAGCGACAGAAGAAGCT GGACAAGTGGCGAAACAACCTCAACTGGGGTATCCCCATTGAGCA GCTGCCTGTCATTGAGTTTGAGGAGTTCCAAGAGCAGGCCAAGAC CCGAGATCTGGTTCTCATTTCTGGCATTGTCCACGACGTGTCTGC CTTTGTCGAGCACCACCCTGGTGGAAAGGCCCTCATTATGAGCGC CGTCGGCAAGGACGGTACCGCTGTCTTCAACGGAGGTGTCTACC GACACTCCAACGCTGGCCACAACCTGCTTGCCACCATGCGAGTTT CGGTCATTCGAGGCGGCATGGAGGTTGAGGTGTGGAAGACTGCC CAGAACGAAAAGAAGGACCAGAACATTGTCTCCGATGAGAGTGGA AACCGAATC CACCGAGCTGGTCTCCAGGCCACCCGGGTCGAGAA CCCCGGTATGTCTGGCATGGCTGCTTAG (SEQ ID NO: 7) pyruvate carboxylase (human) ATGCTGAAGTTCCGAACAGTCCATGGGGGCCTGAGGC TCCTGGGAATCCGCCGAACCTCCACCGCCCCCGCTGCCTCCCCA AATGTCCGGCGCCTGGAGTATAAGCCCATCAAGAAAGTCATGGTG GCCAACAGAGGTGAGATTGCCATCCGTGTGTTCCGGGCCTGCAC GGAGCTGGGCATCCGCACCGTAGCCATCTACTCTGAGCAGGACA CGGGCCAGATGCACCGGCAGAAAGCAGATGAAGCCTATCTCATC GGCCGCGGCCTGGCCCCCGTGCAGGCCTACCTGCACATCCCAGA CATCATCAAGGTGGCCAAGGAGAACAACGTAGATGCAGTGCACC CTGGCTACGGGTTCCTCTCTGAGCGAGCGGACTTCGCCCAGGCC TGCCAGGATGCAGGGGTCCGGTTTATTGGGCCAAGCCCAGAAGT GGTCCGCAAGATGGGAGACAAGGTGGAGGCCCGGGCCATCGCC ATTGCTGCGGGTGTTCCCGTTGTCCCTGGCACAGATGCCCCCATC ACGTCCCTGCATGAGGCCCACGAGTTCTCCAACACCTACGGCTTC CCCATCATCTTCAAGGCGGCCTATGGGGGTGGAGGGCGTGGCAT GAGGGTGGTGCACAGCTACGAGGAGCTGGAGGAGAATTACACCC GGGCCTACTCAGAGGCTCTGGCCGCCTTTGGGAATGGGGCGCTG TTTGTGGAGAAGTTCATCGAGAAGCCACGGCACATCGAGGTGCA GATCTTGGGGGACCAGTATGGGAACATCCTGCACCTGTACGAGC GAGACTGCTCCATCCAGCGGCGGCACCAGAAGGTGGTCGAGATT GCCCCCGCCGCCCACCTGGACCCGCAGCTTCGGACTCGGC TCAC CAGCGACTCTGTGAAACTCGCTAAACAGGTGGGCTACGAGAACG CAGGCACCGTGGAGTTCCTGGTGGACAGGCACGGCAAGCACTAC TTCATCGAGGTCAACTCCCGCCTGCAGGTGGAGCACACGGTCAC AGAGGAGATCACCGACGTAGACCTGGTCCATGCTCAGATCCACGT GGCTGAGGGCAGGAGCCTACCCGACCTGGGCCTGCGGCAGGAG AACATCCGCATCAACGGGTGTGCCATCCAGTGCCGGGTCACCAC CGAGGACCCCGCGCGCAGCTTCCAGCCGGACACCGGCCGCATT GAGGTGTTCCGGAGCGGAGAGGGCATGGGCATCCGCCTGGATAA TGCTTCCGCCTTCCAAGGAGCCGTCATCTCGCCCCACTACGACTC CCTGCTGGTCAAAGTCATTGCCCACGGCAAAGACCACCCCACGG CCGCCACCAAGATGAGCAGGGCCCTTGCGGAGTTCCGCGTCCGA GGTGTGAAGACCAACATCGCCTTCCTGCAGAATGTGCTCAACAAC CAGCAGTTCCTGGCAGGCACTGTGGACACCCAGTTCATCGACGA GAACCCAGAGCTGTTCCAGCTGCGGCCTGCACAGAACCGGGCCC AAAAGCTGTTGCACTACCTCGGCCATGTCATGGTAAACGGTCCAA CCACCCCGATTCCCGTCAAGGCCAGCCCCAGCCCCACGGACCCC GTTGTCCCTGCAGTGCCCATAGGCCCGCCCCCGGCTGGTTTCAG AGACATCCTGCTGCGAGAGGGGCCTGAGGGCTTTGCTCGAGCTG TGCGGAACCACCCGGGGCTGCTGCTGATGGACACGACCTTCAGG GACGCCCACCAGTCACTGCTGGCCACTCGTGTGCGCACCCACGA TCTCAAAAAGATCGCCCCCTATGTTGCCCACAACTTCAGCAAGCT TG CTTCAGCATGGAGAACTGGGGAGGAGCCACGTTTGACGTCGCCA CGCTTCCTGTATGAGTGCCCCTGGCGGCGGCTGCAGGAGCTC CGGGAGCTCATCCCCAACATCCCTTTCCAGATGCTGCTGCGGGG GGCCAATGCTGTGGGCTACACCAACTACCCAGACAACGTGGTCTT CAAGTTCTGTGAAGTGGCCAAAGAGAATGGCATGGATGTCTTCCG TGTGTTTGACTCCCTCAACTACTTGCCCAACATGCTGCTGGGCAT GGAGGCGGCAGGAAGTGCCGGAGGCGTGGTGGAGGCTGCCATC TCATACACGGGCGACGTGGCCGACCCCAGCCGCACCAAGTACTC ACTGCAGTACTACATGGGCTTGGCCGAAGAGCTGGTGCGAGCTG GCACCCACATCCTGTGCATCAAGGACATGGCCGGGCTGCTGAAG CCCACGGCCTGCACCATGCTGGTCAGCTCCCTCCGGGACCGCTT CCCCGACCTCCCACTGCACATCCACACCCACGACACGTCAGGGG CAGGCGTGGCAGCCATGCTGGCCTGTGCCCAGGCTGGAGCTGAT GTGGTGGATGTGGCAGCTGATTCCATGTCTGGGATGACTTCACAG CCCAGCATGGGGGCCCTGGTGGCCTGTACCAGAGGGACTCCCCT GGACACAGAGGTGCCCATGGAGCGCGTGTTTGACTACAGTGAGT ACTGGGAGGGGGCTCGGGGACTGTACGCGGCCTTCGACTGCAC GGCCACCATGAAGTCTGGCAACTCGGACGTGTATGAAAATGAGAT CCCAGGGGGCCAGTACACCAACCTGCACTTCCAGGCCCACAGCA TGGGGCTTGGCTCCAAGTTCAAGGAGGTCAAGAAGGCCTATGTG GAGGCCAACCAGATGCTGGGCGATCTCATCAAGGTGACGCCCTC CTCCAAGATCGTGGGGGACCTGGCCCAGTTTATGGTGCAGAATG GATTGAGCCGGGCAGAGGCCGAAGCTCAGGCGGAAGAGCTGTC CTTTCCCCGC TCCGTGGTGGAGTTCCTGCAGGGCTACATCGGTGT CCCCCATGGGGGGTTCCCCGAACCCTTTCGCTCTAAGGTACTGAA GGACCTGCCAAGGGTGGAGGGGCGGCCTGGAGCCTCCCTCCCT CCCCTGGATCTGCAGGCACTGGAGAAGGAGCTGGTAGACCGGCA TGGGGAGGAGGTGACGCCGGAAGATGTGCTCTCAGCAGCTATGT ACCCCGATGTGTTTGCCCACTTCAAGGACTTCACTGCCACCTTTG GCCCCCTGGATAGCCTGAATACTCGCCTCTTCCTGCAGGGACCCA AGATCGCAGAGGAGTTTGAGGTGGAGCTGGAGCGGGGCAAGAC GCTGCACATCAAAGCCCTGGCCGTGAGCGACCTGAACCGGGCCG GCCAGAGGCAGGTCTTCTTTGAGCTCAATGGGCAGCTGCGGTCC ATCTTGGTCAAGGACACCCAGGCCATGAAGGAGATGCACTTCCAC CCCAAGGCCCTAAAGGACGTGAAGGGCCAGATCGGGGCGCCCAT GCCTGGGAAGGTGATAGACATCAAAGTGGTGGCAGGGGCCAAGG TGGCCAAGGGCCAGCCCCTGTGTGTGCTCAGTGCCATGAAGATG GAGACTGTGGTGACCTCACCCATGGAGGGTACTGTCCGCAAGGT TCATGTGACCAAGGACATGACACTGGAAGGTGACGACCTCATCCT GGAGATCGAGTGA (SEQ ID NO: 8) ACC (Saccharomyces cerevisiae) TTATTTCAAAGTCTTCAACAATTTTTCTTTATCATCGGTA GATAACATCTTGATAACTTCAGATAATCCATCAATAGCATTGTCAT GGTCGCTTCTGATCTTTTTAGCTAAGTCTTGAGCGAATGACTCTAA TTTCAAACCCTTTAGTTTATCGTCCAAAGTTTTGTAGTTTTCTTCAA TCCATGTTGCGACTTGCCTATCATCTTCATGGTCCACTGAAGC AG GGTACCACGATCTAATTCTTGCGATCTTTTCTAATCTTGATGCTTC GCCTACCTGATGGCTCAACCTTTTAATCAAATATTCTTCGTTCAAT CTTCTTCTCAATCTCCAGAAGAAGAAACGACGTGCCTCGGTCCAT TCCAGTTCCTTAGAAATAACACCCTTGGCCACCATACGTGAAGAC CTATCGTGCAAATCAGCAAATTGAAGACTGATTTGTCCGTAAATTG GCAATAGTTCTCTCTCACGATCAGCTAATTGCTTGGATATTTGCTG ATGTACTTCTGGAGCCAAACTCTTGTTGGATAATTGAGATCTCAAT TCTCTGTACTTGTCATCCAATCTGTTCATGGTGTCCAGCAATTTTT CTCTACGGAACTTGATACCAACCATACCTTGTGGTTCCAAAACACC AGCTCTAGCGTTGACGTCGGCATACATTTCCATTTGGTCAGCGTT GATAGTTGGATCGACAACAACCCATGAACCACCTCTTAGTTCACC GGTAGGTGGGATATAGATAATAATTGGTTGTTTGTAATCCACCAAT GCGTCAACAATAAACGAACCATACTTCAAGACTTCGTTGAACATAT CACGTTGACCACCAGAGAAACCTCTCCAGTTGGCCAAAATCATCA TTGGCAATTGTTCACCGTTGTTAAAGTCATTGATAGCTTGAGCAGT CTTGAAGGCGGAGTTTGGATGCCAAACTTGACCAGGTTCTTGAAT TAATGTTTCAGCACTATTTGGATTAGCTGGATCAGCAGGAATCAAG TTCTCGACAGTTCTTGTTTCAACACCAATAACACCCAGTGGAATAC CACCAAGACGGGCTCTACCAACGACAACACCTTTGGCCCATCCTG ACAAAGTTTCAAAGAAAGACCCTTTATCAAACAAACCATATTCAAAT CCACTTTCAGTCTCACGACCTTCAATCATCCATCTTACATCGTAAG TTTCATCATTAGTTGG AGTGAAATCAACTGGTCTATCCCATGTGTC TTTAGTTTCCAAGATAGGAACTGGCATATTACGCTTGGCTGGAACA TAAGACATCCATTCAACAATCTTCTCTACACCAGCTAAATCGTCAA CAGCAGTCAAATGTGAAACACCGTTGTTATACATGATTTGAGTACC ACCCAATTGTAAGTTAGAAGTATAAACTTCTCTACCCAGCATTTTG TTGATTGCAGGAGCACCAGTTAAAATAATTGGCTGGCCTTCGACC TGAATAGCTCTTTGACCCAAACGAACCAAATAAGCACCGATACCG ACGGATCTACAAGTGACTAAGGTGATAGTGAAGATATCGTGGTAA GCCCTTGACGTTGCACCAGCAATTAAACCAGATCCACGTAGACAT TCGACACCTAACCCATCTTCAGAACCAATAATTGTCTTGATGACAA ATCTTTCTTCACCGTTTATAACAGTACGTTCAGTGAGAACAGAATT TTCTTTGTCAAATTTCTTTAAAGTTTCCATACCTTCACTTGTTAAGT ATAAGTATTGGAAGCCCTTGTCCGGATTGGCAGCATCATTCCATG CAACTTGAAATAGTGGAACAATCTCTTCAGCCATACCAATTCTGGC ACCTGAGTTTGCAGCCAAGTAAATTCTTGGGATACCACGCTTTCTA GCATATTCAGTAACCTTATTGAAGAATTCGTCTTCTTGTGGACCAA AGGAACCGATCTTGAATGTGATATCGTTAGCAACAACAACAAATTG ACGGCCTCTTGGATATTCAGGAGTCTTTACAGTAATCTTAAAGGCA ACCATACCAATAGCGTTGGCACCAGGTTCTCTTTCCACCTCAGTTA ATTCGCCGTTTTCATCTTCAATCAACTCGTTGGAAATAAAGAAATC ATCTGTTAACTTAACATCTGCAGAGAAATTTTTCCATTGGGATGAC GATGCTTGGCGGAATAATTCTGGGAAGTCATAG ACATATGTGGTA CCCATCAAGTGTGCCTTATAACGTTTTGGTTGCAACCATTCCTTAA CAGGGTAAGGAGTAGCAATAGGTCTTAAATGCATGGATCCAGGTT TACCCAAAGACTTAAATACCCATTCACCTTTTGCGTTCTTGACTTC GGTGTACATTTCTGTTTTGATAACATAACCAGAAACGTTATTGATC AAGGCACGCAATGGTACTGGGGCACCTGTTTGAGGATCTTTGATG ATGATTCTAATTTCGGCAGAAGAAACACGCAATCTCAACAATCTCT TACCAAATCTTTCTAAGAAACCACCGAAGGCGGCTTCGACATCTTC TGGAGAGATATCAAACACCGCAATGAAGTTGATGAAGATATGATTC AAATCAGAATTTGAAGTGTCGGTGACTTCTAAATTATCCAATATATC ACTCATCAATCTGTTAGCTTCAGAAGTCAGATATTCTTGAATAGAA ATGTCATCACGGATATGACCCGTTCTAATAATACCTCTTGTAAAGA ATCTCTTATCCAATGGAGAAGTCTTACTAACAGCTTCGTAGACATG GATGTTTCTATTATCAGTGAAAATTGGTTTAATGTTGAAGTTGGAC AATCTTCCTAATTCCAGTTGGAAGGCCAAAGCCGGCTCAATGTGA CGAATTGTTTCATTTTCGTTATAATTTGGACCGTTAAAAGTATAATA CTTTGGATAAGACCCATCTTTAAAACCGAACATAAATGTGATACGA CGGATAGAAGCATTGATTAATTCCTGCTTATTCAAATCCAAAATTTC TCTCAACCTTACCAAAATTTCCTCTTCAGATTCGAAACCTTCTGTA GAAGCAACACAAACATTAGCAACATTACTCAACGATGCGGAGCTA CCAGAACGATCAGGAGCAGGTCCGTTAGAAGAAGATTGGTGACG AGGAATAACTTCCAAACTTTGTGACAAAATTTCATCAACATCATCTA AA TGATCCACAGCCATCAAAATACCTTCTCTTAACGGAGATGACTG ACTGTTTGCAACATATGACAAATCTGAAACAGAAACAGCCCTGTTC ATACCCATTTTAGATTTAACAGTTGGAAAGGTGGAGAACGCAGCT GAAGGTAGTTGGAATTTCCATTCAACAATTGGAACTGTGACACCTT CGTGAACTCTAATATCTCCTATGGTGTAAGCACGATAAGCACGAC GAATATAGACTTGAGCAGCTGCAGCAGTCACAACTGGGTCTTGAT GGGTTAGGAATTGAAGTAAAACATCGAACACAACGTAATTAGAATC GATCAAGTCCTTCAAGATATTCAAATCTGGTTCAGAGCGCTTTGGA TTGGATGAGCCATAGGCAACCTTCACAACAGAGGATTTTAAGATAT GTTCAATTTGTTCAGTTCTTTCCTTGACCGAAGGTAAAGCGCCTTG AATCAAAATTTCTCTTGCTTGTAGAGCGACCTTAGCGGTAGCCTTA GATTCTAGTTCAACAATATGTTGTAGAGGAGTAGAGAAAATGGCA GAAACTTTAGAAGATAACTTGCACAATGGTTGATAATGTTTCAAGA TAGCTAGGATCAGGTTATTCTTCGCTGAAACTTTCGAATGAGACAA AACAGTTAGCGCAACTTTATCTAGATCTTTAGGGTTTTCATCACGC AATTTCAGAATGATATTTTCCTCACGAACATTTGGACCATTGAATAA CTTTTCAACTTCGTAATATTCTTCCAAGAAATGGACAAATATAGAAT GTTCATGGGCTTCTAACCCGTTAGAGTACTTATGAGCAATATCCGC CAATGGTTCCACGACGGCGCCCAGCAATTTGTCGGGGTTGTATTC AGGATTCTTCACGGCCATATCAATCAATTTACTTAATTGTCTAGCT GGGAAAACAGCACCACGTCTCAAAGAACGTGCAACTAACTCTTCC ATTTGTTCATCTAGCTTAG CAGGCAATCTTGAATGTAAAGCAGAGA TGTGTAGTTTCCATTCTGAGTAAGGCAGTTTTGGATTTCTCAAAAC CTCTATCAATTGTTGCAAGGAAGCGTTCATAATAACTTGGTTGTCA TAACCCTTCAAAATGTTTTCCAAAGTAGACACTAATGACTTGAATTT ATAGGCAGGTTTGGTTCCTTCGATAACTGGAGAACCAAAATCTGG CAGCATACCTTCAAATGGTAGAGCGTGCTTGACCTTGGATGGATC GTCAAGAGTCATAATAGCCATGATATCACCTGCAACAATGGTAGAA CCAGGTTGCTTTAATAACTGGACGATACCATTTTCTTGAGAAACCA AAGGCATTTGCATTTTCATAACTTCAATTTCTGCATATGGTTGGCC CTTGATAATGTGTTCACCATTTTCCACCAAGAATTTAACCAATTTAC CAGGGGATGGAGTACGCAACTGGGTTGGATCGTTTTCAACTTCCA ACAAAGTAGTCATAGAGTCAACGGATAATCTTGTAGCAGCAACTTC TTCTTTCCAATAGATGGTATGCGATTTACCGCCTATGGCAATCAAA AGACCACCATCAGATAGTTGACGCAGTATGATATCACATTTAGAAC CATTGATAAATAATGTGTAACGGTCATTACCGGATTTAGCTACGGT GAACTTGTATCTTTTACCCTCATGGATAAAATCTACAGGGAACATA GTTTGCAGTAGGTCTTTAGATAGAACTTGTCCCTTTTGTAAGGATT CGATATACTTGTGGCGGGCTTCTTCAGATGCTAAGAAAGCCTTTG TAGCGGCACCGCAAATGACGGCAAGAGTTGGATCAGGCTTTTCAG CGGTCATTTTATGAGTAATCAAATCGTCCAACCAACCGGTGGTAAT AGTGTTATCCTCGAAATCTTCAGTTTCCAAAAGTTTGATCAAGTATT CCACAGTAGTTCTGAAATCACCCCTAATGGACAA TTCCTTCAGGG CAACAACCATGTGTTTCCTGGAAGCTTGTCTATTTTCACCAAAAGC AAAAATATGGCCGAACTGAGAGTCCGAAAAGGAGTGAATATTACC ATTGTTACCCACGGAGAAGTAACCCCAAACATTAGAGGAAGAACG GAAGTTTAGTTCATGCAAAGTACCACCCGATGGCTTGAATCCATC GTTTGGATCTTCTGATGTGATACGACAAGCGGTACAATGACCCTTT GGAATAGGTCTTCTTTGTTTCTTGGTGGCATCTTGAGTTTTGAATT CGAAATCGATTTCTGAGGCAGAATGAGGATTCATACCATATAAAGT TCTAATGTCACTTATTCTATGCATAGGGATACCCATAGCGATTTGT AATTGAGCTGCAGGTAAGTTAACACCGGAGACCATTTCCGTTGTT GGATGCTCGACTTGTAATCTTGGGTTCAATTCTAAAAAGTAGAATT TTCCATCATCATGAGAATATAGATACTCCACGGTACCGGCAGAGA CATAACCGACTAGTTTCCCCAGTCTGACGGCAGCCTTTTCCATCT CGTGAAATGTTTCAGCCTTGGCAATTGTAACTGGTGCTTCTTCGAT AATTTTTTGATGACGTCTCTGAACGGAACAGTCTCTACCGAACAAG GAAATATTTGTACCGTACTGATCTGCTAGCAGTTGAACTTCCAAGT GACGCGCTCTACCGGCCAACTTCATGATGAAAATGGGGGAGCCT GGAATTTCGTTGGCTGCCTGGTGGTATAAAGCGATGAAATCTTCTT CACGTTCAACTTGTCTGATACCTTTACCACCACCACCTTCGGATGC CTTAATCATGACAGGAAAACCAATACGCTTGGCCTTTTGTAAACCA TCTTCAGGAGAGGTACAACAACCCTTTTGATAGATGTCATCGTCGA CAGAGACCAGACCGGTTTTCTCGTCCACGTGAACGGTGTCAACAC CGGTACCAGA CCATGGAATACATGGGACTTTAGCACTTTGAGCGA CAATGGTAGAGGAGATTTTATCACCTAAAGACCTCATGGCGTTAC CTGGAGGCCCAATAAAGATGACTTTCCTCTTAGACTGGGACAATTT TTCAGGCAATAGTGGATTCTCGGAGGCGTGACCCCAGCCAGCCC ATACGGCGTCTACGTCTGCTCTTTCGGCGATGTCTACGATCAAGT CTACGTTAGCGTAGTTGTTATTATTAGTACCACCTGGCACTTCAAT GTATTGATCGGCCATACGGATATATTCTGCGTTGGCCTCCAGATC TTCTGGGGTGGCCATGGCGACGAATTGGACGGTTCTGTCATCGC CGAACGTCTCGTATGCCCATTTTCTGACGGATCTAATTTCTTTCAC GGCGGCAATACCATTATTTGCTATCAGGATCTTGGATATGACCGT GTGACCACCGTGACTCTTAACAAAGTCCCTTAACGGGGACTCCTC TAGTTTATCTACTGTATTGAGGCCAATGAAATGACCTGGAAGTTCT GTATGTCTTTCTGAGTAGTTTGTAATTTCGTACTCCATCTTCTGTG GAGAAGACTCGAATAAGCTTTCTTCGCTCAT (SEQ ID NO: 9) FAA (S. cerevisiae) ATGGTTGCTCAATATACCGTTCCAGTTGGGAAAGCCGC CAATGAGCATGAAACTGCTCCAAGAAGAAATTATCAATGCCGCGA GAAGCCGCTCGTCAGACCGCCTAACACAAAGTGTTCCACTGTTTA TGAGTTTGTTCTAGAGTGCTTTCAGAAGAACAAAAATTCAAATGCT ATGGGTTGGAGGGATGTTAAGGAAATTCATGAAGAATCCAAATCG GTTATGAAAAAAGTTGATGGCAAGGAGACTTCAGTGGAAAAGAAA TGGATGTATTATGAACTATCGCATTATCATTATAATTCATTTGACCA ATTGACCGATATCATGCATGAAATTGGTCGTGGGTTGGTGAAAATA GGATTAAAGCCTAATGATGATGACAAATTACATCTTTACGCAGCCA CTTCTCACAAGTGGATGAAGATGTTCTTAGGAGCGCAGTCTCAAG GTATTCCTGTCGTCACTGCCTACGATACTTTGGGAGAGAAAGGGC TAATTCATTCTTTGGTGCAAACGGGGTCTAAGGCCATTTTTACCGA TAACTCTTTATTACCATCCTTGATCAAACCAGTGCAAGCCGCTCAA GACGTAAAATACATAATTCATTTCGATTCCATCAGTTCTGAGGACA GGAGGCAAAGTGGTAAGATCTATCAATCTGCTCATGATGCCATCA ACAGAATTAAAGAAGTTAGACCTGATATCAAGACCTTTAGCTTTGA CGACATCTTGAAGCTAGGTAAAGAATCCTGTAACGAAATCGATGTT CATCCACCTGGCAAGGATGATCTTTGTTGCATCATGTATACGTCTG GTTCTACAGGTGAGCCAAAGGGTGTTGTCTTGAAACATTCAAATGT TGTCGCAGGTGTTGGTGGTGCAAGTTTGAATGTTTTGAAGTTTGT GGGCAATACCGACCGTGTTATCTGTTTTTTGCCACTAGCTCATATT TTTGAATTGGTTTTCG AACTATTGTCCTTTTATTGGGGGGCCTGCA TTGGTTATGCCACCGTAAAAACTTTAACTAGCAGCTCTGTGAGAAA TTGTCAAGGTGATTTGCAAGAATTCAAGCCCACAATCATGGTTGGT GTCGCCGCTGTTTGGGAAACAGTGAGAAAAGGGATCTTAAACCAA ATTGATAATTTGCCCTTCCTCACCAAGAAAATCTTCTGGACCGCGT ATAATACCAAGTTGAACATGCAACGTCTCCACATCCCTGGTGGCG GCGCCTTAGGAAACTTGGTTTTCAAAAAAATCAGAACTGCCACAG GTGGCCAATTAAGATATTTGTTAAACGGTGGTTCTCCAATCAGTCG GGATGCTCAGGAATTCATCACAAATTTAATCTGCCCTATGCTTATT GGTTACGGTTTAACCGAGACATGCGCTAGTACCACCATCTTGGAT CCTGCTAATTTTGAACTCGGCGTCGCTGGTGACCTAACAGGTTGT GTTACCGTCAAACTAGTTGATGTTGAAGAATTAGGTTATTTTGCTA AAAACAACCAAGGTGAAGTTTGGATCACAGGTGCCAATGTCACGC CTGAATATTATAAGAATGAGGAAGAAACTTCTCAAGCTTTAACAAG CGATGGTTGGTTCAAGACCGGTGACATCGGTGAATGGGAAGCAA ATGGCCATTTGAAAATAATTGACAGGAAGAAAAACTTGGTCAAAAC AATGAACGGTGAATATATCGCACTCGAGAAATTAGAGTCCGTTTAC AGATCTAACGAATATGTTGCTAACATTTGTGTTTATGCCGACCAAT CTAAGACTAAGCCAGTTGGTATTATTGTACCAAATCATGCTCCATT AACGAAGCTTGCTAAAAAGTTGGGAATTATGGAACAAAAAGACAG TTCAATTAATATCGAAAATTATTTGGAGGATGCAAAATTGATTAAAG CTGTTTATTCTGATCTTTTGAAGACAGGTAAAGACCA AGGTTTGGT TGGCATTGAATTACTAGCAGGCATAGTGTTCTTTGACGGCGAATG GACTCCACAAAACGGTTTTGTTACGTCCGCTCAGAAATTGAAAAGA AAAGACATTTTGAATGCTGTCAAAGATAAAGTTGACGCCGTTTATA GTTCGTCTTAA (SEQ ID NO: 10) Acyl-CoA synthetase ATGACTGTTACCCCACAGCACCAAGTCGTCCACGAGGC CAACGGTGTCACCCCAAGACCCACTCCTAAGGAGTTTTTTGACAA ACAGCCCCGTCCTGGCCATATCACCTCCATCGAACAGTACCAGGA ATTATACCAGAAGTCCATCGCCGACCCTGAAGGATTCTTTGGTCC TATGGCCAAGGAGTTGTTGTCGTGGGACAGAGACTTCGACAAGGT CAAGTCCGGTTCTTTGAAGGACGGTGACGTTGCCTGGTTCATTGG CGGCCAGTTGAACGCTTCCTACAACTGTGTAGACAGATGGGCCTA TGCGACTCCAGACAAGACTGCCATCATCTACGAAGCTGACGAAGA AAAGGACTCGTACAAGTTGACCTACGCCGAGTTGTTGAGAGAAGT CTCCAAGGTAGCTGGTGTGTTGAAGAGCTGGGGCATCAAAAAGG GTGATACTGTTGCTATCTACTTGCCAATGACTCCTCAAGCTGTTAT TGCTATGCTCGCTGTAGCCAGATTAGGTGCCATCCACTCGGTTAT CTTTGCAGGTTTCTCTTCTGGTTCCATCAGAGACAGAGTCAACGAT GCTTCTTGTAAGGCTCTTATTACCTGTGACGAAGGTAGAAGAGGT GGTAAGACCGTTAACATCAAGAAATTGTGCGACGAAGCCTTGAAG AGCTGTCCTACTGTAGAAAAGGTGCTTGTTTTCAAGAGAACCGGA AACGAAAATATTGAATTGGAAGAGGGTAGAGATTTCTGGTGGGAT GAAGAAACCGCCAAGTTCTCGGGTTA CTTGCCACCTGTTCCAGTC AATTCTGAAGACCCATTGTTCTTGTTGTATACATCTGGTTCCACTG GTACTCCTAAGGGTGTTGTCCACACCACTGGGGGCTACCTCTTAG GTGCTGCCATGACCACCAAGTACATTTTCGACGTCCACCCAGAAG ACATCTTGTTCACTGCCGGTGATGTCGGTTGGATTACTGGTCACA CCTATGCTTTGTACGGACCTTTGGCTCTCGGTATCCCAACAATCGT TTTTGAAGGTACTCCAGCCTACCCAGACTTTGGTAGATTCTGGCAA ATTGTCGAAAAGCACAAGGCTACCCACTTCTACGTAGCTCCTACT GCCCTCAGATTGTTGAGAAAGAGTGGCGAGCAAGAGATTCCAAAG TACGACTTGTCTTCTTTGAGAACATTGGGCTCTGTTGGTGAACCTA TCTCCCCTGATATCTGGGAATGGTACAACGAGCACGTTGGACAAG GCAGATGCCACATCTCCGACACCTACTGGCAAACTGAGTCTGGTT CTCACTTCATTGCTCCAATTGCCGGTGTCACTCCAAACAAACCTG GTTCAGCCTCTTTGCCATTCTTTGGTATCGAGACCGCTCTTATTGA TCCAGTTTCCGGCCACGAACTCGAAGGTAACGACATCGAAGGTGT TCTTGCCATCAAGAGCACCTGGCCATCTATGGCTAGATCTGTCTG GAACAACCACACCAAGTACATGGACACATACTTGAACCCATACCC AGGCTACTACTTTACCGGCGACGGTGCTGCCAGAGATCACGACG GCTACTACTGGATTAGAGGTAGAGTCGATGATGTCGTCAATGTGT CTGGTCACAGATTGTCTACTGCTGAAATAGAAGCTGCCCTCATCG AACACAACGGTGTTTCTGAAGCTGCTGTGGTTGGTATTACCGACG ACTTAACTGGTCAAGCCGTAGTTGCCTACGTTGCTCTCAAGAACG AATACGTCGA CAAGATCGCCGGCAAGGAAACCAGCGACGAAGCC TTTGCCTTGAGAAAGGAATTGATCATGACCGTCAGAAAGGAAATC GGACCTTTCGCAGCTCCAAAGAGCGTCATCATTGTCGCCGACTTG CCAAAGACCAGATCTGGTAAGATCATGAGAAGAATCTTGAGAAAG ATCTCTGCCAACGAAGCAGACCAATTGGGTGACATCACCACTTTG TCCAACCCTCAGTCTGTCGTTGGTATAATCGACTCCTTTGCTGCTC AATTTGCTAAGAAATAA (SEQ ID NO: 11) ATGGGGAGACACTTGGCCTTGCTTCTGCTTCTGCTCTTC FAT TTCCTCCAGCATTTTGGAGATGGTGATGGAAGCCAAAGACTTGAAC CGACCCCTTCCCTCCAGTTTACACACGTCCAGTACAATGTCACTGT GCACGAAAACTCGGCCGCAAAGACCTATGTCGGCCACCCTAGAAA AATGGGCATCTACATCTTAGACCCCTCGTGGGAAATAAGGTACAAA ATCATCTCAGGAGACAACGAAAACCTATTCAAAGCGGAAGAGTATG TTCTCGGAGACTTTTGCTTTCTAAGGATAAGAACCAAGGGAGGGAA TACTGCCATCCTGAACCGAGAAGTGAGAGACCATTACACACTGGTA ATCAAAGCAGTTGAAAAAGTCACAGATGCCGAGGCCCGAGCCAAG GTCAGGGTGCAAGTGCTGGATACAAACGACTTACGGCCGTTGTTC TCACCCACGTCCTACAGCGTTTCTCTGCCGGAAAACACAGCCATAA GGACCAGTATCGCAAGAGTCAGTGCCACGGATGCGGACATTGGAA CCAACGGCGAATTTTACTACAGCTTTAAAGACAGAACGGACATGTT TGCCATCCACCCAACCAGTGGTGTGGTTGTTTTGACTGGCAGGCT TGATGTCCTGGAGACCCAGCGCTATGAGCTGGAGATCTTGGCTGT GGAC CGGGGAATGAAGCTGTACGGTAGCAGTGGGGTCAGCAGTC TGGCCAAGCTGACGGTTCACGTGGAGCAGGCTAACGAGTGTGCAC CCGGGATAACCGCCGTGACGTTATCACCATCTGAGCTGGACAAGG ACCCAACGTACGCCATTATCACTGTGGAGGACTGCGATCAGGGTG CCAACGGGGAGATAGCATCTTTGAGCATTGTGGCTGGCGACCTCC TTCAGCAGTTTAAAACGGTGAGGTCTTTCCCAGGGAGTAAAGCATT CAAAGTGAAAGCCGTCGGGGGCGTCGACTGGGACAGCCATCCTTA TGGCTACAACCTGACAGTGCAGGCTAAAGACAAAGGAACTCCTCC GCAGTTTTCCCCTGTGAAAGTCATTCACGTCATTTCTCCTCAGTTCA GAGCTGGCCCGGTCAAGTTTGAAATGGATGTTTACAGAGCTGAGA TCAGTGAGTTTGCCCCTCCACATACACCCGTGGTCCTGGTCAAGG CTATTCCTAGTTATTCCCATTTGAGGTACGTTTTTAAAAGCACTCCT GGAAAACCCAAATTCGGTTTAAATCACAACACGGGTCTCATTTCCA TTTTAGAACCAATTAAAAGGCAGCACACATCCCATTTTGAGCTTGA GGTGACAACAAGTGACAGACGAGCCTCCACCAAAGTCGTGGTCAA AGTTGTAGGTACAAACAGCAACCCCCCGGAGTTTACACAGACCTC GTACAAAGCATCCTTTGATGAGAATGCACCCGTCGGTACCCCGGT CATGAGGGTGAGCGCGGTTGACCCTGACGAGGGGGAGAATGGCT ACGTGACTTACAGTATTGCAAACTTAAATCACGTGCCATTTGTCATC GACCACTTTACGGGTGCTGTGAGTACCTCTGAGAATCTGGACTATG AACTGATGCCTCGAGTCTACACGCTGAGGATTCGTGCTTCCGACT GGGGCTTACCGTACCGCCGGGAAGTTGAAG TCCTTGCCACAATTA CTCTGAATAACCTGAATGACAACACCCCCCTGTTTGAGAAGACAAA CTGTGAAGGGACAATTCCCCGAGACCTGGGTGTAGGGGAGCAGAT AACCACGGTTTCTGCCATTGACGCTGATGAGCTGCAGTTGGTCCG GTACCAGATTGAAGCTGGAAATGAGTTGGATTTGTTTGGCTTAAAC CCCAGCTCTGGTGTGCTGTCATTGAAGCACTCGCTCATGGACGGC TTGGGTGCAAAGGTTTCCTTTCACAGCTTGAGAATCACAGCTACAG ACGGAGAAAATTTTGCCACACCATTATATATCAACCTAACGGTGGC TGCCAGTCGCAAGCCAGTAAACTTGCGGTGTGAGGAGACCGGTGT TGCCAAAATGCTGGCAGAGAAACTCCTGCAGGCGAATAAATTACAC CATCAGGGGGACGCGGAGGATATTTTCTTTGATTCTCACTCCGTCA ACGCCCATGCCCCACAGTTTAGGGGTTCTCTTCCAACAGGAATTGA GGTAAAGGAGGACCTCCCAGTGGGCGCCAGTATACTATTCATGAA TGCTACTGACCTTGACTCTGGCTTCAATGGGAAACTGGTCTATGCT ATCTCTGGAGGGAATGATGACAGTTGCTTTACTGTTGACATGGAAA CAGGAATGCTGAAAGTCCTCTCTCCACTTGACCGAGAAGTAACGG ACAAATACACACTGAACATTACCGTGTATGACCTTGGTATACCCCA GAGGGCTGCCTGGCGCCTTCTGGATGTCACCGTCCTGGATGCCAA TGACAACGCGCCCGAGTTTTTACAGGAGAGCTATTTTGTCGAAGTG AGCGAAGACAAGGAGATAAACAGTGAAATCATCCAGGTAGAGGCC ACCGATAAAGACCTGGGCCCCAGCGGACACGTGACATACGCCATC CTCACGGACACAGAGAAGTTTGCGATCGACAGGGTGACCGGTGTG GTGAAAA TTATCCAGCCTTTGGATCGTGAAGTGCAGCGTGTACATT ACCTGAAGATCGAGGCCAGGGACCAAGCCACAGAGGAACCCTGG CTGTCCTCCACTGTGCTTCTGAAAGTGTCACTCGATGATGTTAATG ACAACCCACCTAGGTTCATTCCACCCAGTTACTCCGTGAAGGTTCG AGAAGACCTACCGGAAGGAACCATCATCATGTGGTTAGAAGCCCA TGACCCTGATGTAGGTCAGTCCAGTCAGGTGAGATACAGCCTCCT GGACCACGGAGAAGGCCACTTCGATGTGGATAAACTCAGCGGGG CAGTGAGAATTGTCCAGCAGCTGGACTTTGAGAAGAAGCAACTGT ATAATCTCACCGTGAGGGCCAAAGACAAAGGGAAGCCGGCGTCTC TGTCTTCCACTGGCTACGTGGAAGTGGAGGTCGTGGACGTGAATG AGAACTTACACGCGCCAGTGTTCTCCAGCTTCGTGGAGAAGGGCA CAGTGAAAGAAGACGTCCCTATGGGCTCATCAGTAATGACCGTGT CAGCTCACGATGAGGACACCGGGAGAGATGGAGAGATCCGGTATT CCATCAGAGATGGCTCTGGTGTTGGTGTTTTCAGGATAGATGAAGA AACAGGTGTCATAGAGACCTCAGATCGACTGGACCGAGAGTCGAC TTCCCACTACTGGCTCACCGTCTACGCCACAGATCAGGGTGTGGT GCCTCTGTCATCCTTCATAGAGGTCTACATAGAGGTTGAGGATGTC AATGACAACGCACCACAGACATCAGAGCCTGTGTATTATCCTGAAA TAATGGAGAATTCACCCAAGGATGTATCTGTGGTCCAGATTGAGGC ATTTGACCCGGATTCCAGCTCCAGTGACAAGCTGACGTACAGAATT ACAAGTGGAAATCCCCAAGGGTTCTTCTCAATACACCCTAAAACAG GTCTCATCACAACCACATCGAGGAAGCTGGACCG AGAGCAGCAGG ATGAACACATTCTGGAAGTTACTGTGACAGACAATGGTGTACCTCC CAGATCCACCATTGCCAGGGTCATTGTGAAAATCCTGGATGAGAAC GACAACAGGCCTCAGTTCCTTCAGAAGTTTTATAAAATCAGGCTCC CGGAGCGAGAAAAAGCTGATGGAGACCGGAGCGCGAAGCGCGAG CCTCTCTACCGAGTCATAGCCGCAGATAAGGATGAAGGGCCCAAT GCCGAGCTCTCCTACAGCATCGAGGAAGGGAACGAGCACGGCCG GTTTTCCATTGAACCCAAGACAGGAGTGGTCTCATCCAAAAAGTTC TCTGCGGCTGGAGAATACGACATTCTTTCTATTAAGGCAATTGACA ATGGGCGCCCCCAGAAGTCATCGACCACCAGACTCCATATTGAAT GGATCTCCAAACCCAAGCCGTCCTTGGAGCCGATTTCGTTTGAGG AATCGGTTTTCTCGTTTACTGTAATGGAGAGTGATCCGGTGGCTCA CATGATCGGCGTGATCTCCGTTGAGCCTCCTGGCATGCCTCTGTG GTTTGACATCATCGGGGGCAACTATGACAGTCACTTTGATGTGGAC AAGGGCACTGGAACCATCATTGTGGCCAAGCCCCTTGACGCAGAG CAGAAGTCCAGCTATAACCTCACAGTGGAGGCGACAGACGGGACC TCCACTATCCTCACCCAGGTACTCATCAAAGTAATAGATACCAATG ACCACCGCCCTCAGTTTTCTACCTCGAAATACGAAGTCTCTGTTCC CGAAGACACAGAGCCAGAAACAGAGATTCTGCAAATCAGCGCCGT AGACAGGGACGAGAAAAACAAACTGATCTACACCCTCCAGAGCAG CATAGATCCAGCAAGTCTCAAGAAATTCCGCCTCGATCCTGCAACA GGCGCTCTCTACACATCTGAGAAGCTCGATCACGAAGCCATTCAC CAGCACGTCCTCAC AGTCATGGTCCGGGATCAGGATGTCCCTGTG AAACGCAACTTTGCCAGAATCATTGTGAATGTCAGTGACATGAATG ACCACTCTCCGTGGTTCACCAGTTCGTCCTATGAAGGGCGGGTTT ATGAGTCGGCAGCCGTGGGCTCGGTCGTGCTACAGGTTACAGCTC TGGACAGAGACAAAGGGAGAAATGCTGAAGTGCTCTACTCCATCG AGTCAGGAAACATTGGAAATTCCTTTACAATCGACCCCATCTTGGG CTCTATAAAAACTGCCAGAGAATTGGATCGAAGTCACCAAGTAGAC TATGATTTAATGGTAAAAGCTACAGACAAAGGGGAGCCACCAATGA GCGAAATGACCTCCGTGCGGATCTCTGTCACCGTCGCCGACAATG CCTCTCCTAAGTTCACATCCAAGGAGTACTCGGCTGAGATTAGTGA AGCCATCAGGATTGGGAGTTTTGTTGGAATGGTCTCTGCTCACAGT CAGTCATCAGTGATGTATGAAGTAAAAGATGGAAATATAGGCGATG CATTTAATATCAATCCACATTCAGGAAGCATCGTCACTCAGAGAGC CTTGGATTTTGAGACACTGCCCATTTATACATTGACAGTACAAGGG ACCAACATGGCCGGCTTGTCCACCAATACAACGGTGGTAGTGCAC ATACAGGATGAGAATGACAACCCTCCAGCTTTCACACGGGCGGAA TATTCAGGATTCATTAGTGAATCAGCCTCAGTCAACAGCGTGGTGC TAACGGATAAGAATGTTCCGCTCGTGATCCGAGCCACCGACGCTG ATCGGGAATCCAATGCTCTGCTCGTCTATCAAATTGTCGAGCCATC TGTGCACAACTATTTTGCCATTGATCCCACCACCGGTGCCATCCAT ACCGTACTGAGTCTGGACTATGAAGAGACACGTGTCTTTCACTTCA CCGTCCAAGTGCATGACATGGGGACGCCTCGTCTG TTTGCTGAGT ATGCAGCAAATGTGACCGTGCATGTGATTGACATCAATGACTGCCC CCCTGTCTTCTCTAAGTCACTGTACGAAGCATCCCTCCTATTGCCG ACGTACAAAGGCGTGAACGTCATCACAGTGAATGCCACAGATGCC GACTCCAGGGCGTTCTCCCAGTTAATATACTCCATCACCAAAGGCA ACATTGGGGAGAAGTTCTCCATGGACCACAAGACTGGCACCATAG CAATTCAGAACACAACCCAGTTACGGAGCCGCTATGAGCTGACCG TCCGCGCCTCCGATGGCCGGTTTACAAGCGTGGCCTCCGTGAGAA TCAACGTGAAGGAAAGCAGAGAGAGTCCTCTCAAGTTTACCCAAG ATGCCTACTCTGCGGTGGTGAAGGAGAACTCCACCGAAGCCAAAA CCTTAGCTGTCATTACCGCGATAGGGAACCCGATTAACGAGCCTTT GTTTTACCGTATCCTCAACCCAGACCGCAGATTTAAAATCAGCCAC ACCTCAGGCGTGTTGTCAACCACTGGGATACCATTTGATCGGGAG CAACAGGAGACGTTTGTTGTGGTGGTAGAGGTGACTAAAGAACGG GAGCCGTCGGCCGTGGCCCACGTTGTGGTGAAGGTCACCGTGGA AGACCAGAATGATAATGCACCCGTGTTTGTCAACCTTCCCTACTAT GCTGTGGTGAAGGTGGATGCTGAGGTGGGCCATGTCATCCGCCA CGTCACTGCCATTGACAGAGACAGTGGCAGAAACGGTGACGTTCA CTACTACCTTAAGGAGCATCATGACCACTTTGAGATTGGACCCTCT GGTGACATTTCTCTGAAAAAGCAATTTGAGCACGACACCTTGAATA AAGAATACCTTGTCACAGTGGTTGCGAAGGACGGGGGGAACCCAG CTTTCTCCGCAGAAGTTCTAGTTCCCATCACCGTCATGAACAAAGC CATGCCCGTGTTTGAA AAGGCTTTCTACAGTGCAGAGATTCCCGAG AACGTCCAGACGCACAGCCCAGTGGTCCACGTCCAAGCCAACAGC CCAGAAGGGTTGAAAGTGTTCTACAGTATCACAGACGGGGACCCT TTTAGTCAGTTTACTATCAACTTCAACACTGGGGTGATAAACGTCAT CGCACCGCTGGACTTTGAGTCCCACCCAGCCTATAAGCTAAGCAT ACGGGCCACTGACTCCCTGACTGGCGCCCACGCTGAAGTGTTTGT TGACATCGTAGTAGAAGACATCAATGACAACCCTCCCGTGTTTGTG CAACAGTCTTACTCGACAACCCTGTCTGAAGCATCTGTCATCGGAG CGCCTATCCTTCAAGTTAGAGCCACCGACTCTGACTCGGAACCAAA TAGAGGGATTTCCTACCAGCTGATTGGAAATCACAGCAAAAGCCAC GATCACTTTCACATAGATAGTCACACTGGGCTGATTTCACTGGTGA GGGCTTTGGATTACGAACAGTTCCAGCAGCACAAGCTGCTCGTAA GGGCTGTTGATGGAGGAATGCCGCCACTGAGCAGCGATGTGGTC GTCACTGTGGATGTCACCGACCTCAACGATAACCCGCCTCTGTTTG AACAACAGGTTTACGAAGCTAGGATCAGTGAGCACGCTGCCCACG GGCATTTTGTGATGTGCGTAAAGGCCTGTGATGCAGATCGCTCAG ACCTAGACAGGCTGGAGTACTCCATTCTGTCCGGCAATGATCACAA GAGCTTTGTCATTGACGGGGAGACAGGAATCATCACGCTCTCCAA CCCGCGCCGCCACACCTTGAAGCCGTTCTATAGTCTCAACGTTTCT GTGTCTGATGGGGTTTTCCGAAGCTCGGCTCGGGTGAATGTCACC GTGATGGGAGGGAATTTGCACAGCCCTGTCTTTCACCAGAATGAG TATGAGGTAGAGCTGGCTGAAAACGCCCCCTTGCACACCC Tggtg GTCCAAGTGAAGGCTACTGACAGAGATTCCGGTATCTACGGCCAC CTGACTTACCACCTTGTAAATGACTTTGCCAAAGACAGGTTTTACG TGAACGACGGAGGGCAGGTCTTCACTCTGGAGAGACTTGATCGAG AGGCTCCAGCAGAGAAAGTGATCTCAGTCCGTTTAATGGCTAAGG ATGCTGGGGGGAAGGTCGCCTTCTGCACTGTCAACGTCATCCTCA CGGACGACAATGACAACGCACCACAGTTTCGCTCAACCAAGTACG AGGTGAACGTGGGGTCCAGCGCCGCCAAAGGGACGTCGGTCGTC AAGGTCTTCGCGAGTGATGCCGATGAGGGGTCGAATGCTGACGTC ACCTACGCCATCGAGGCAGATTCGGAAAGTGTCGAGGAGAACTTG GAAATCAACCAACTGACCGGCCTCATTACTACAAAGGAAAGCTTAA TAGGTTTAGAGAATGAATTCTTCACTTTCTTCGTTAGAGCTGTGGAT AACGGGTCTCCGCCCAAAGAGTCTGTTGTTCCTGTCTATGTTAAAA TACTTCCCCCGGAAGTGCAGCTTCCTAGGTTCTCAGAGCCCTTTTA TACCTATTCCATTTCAGAAGACATGCCTATTGGCACAGAGATTGAC CTCATCCGGGTAGAGCATAGCGGGACTGTTCTCTACACCCTGGTC AAAGGCAATACTCCCGAGAGTAACAGGGACGAGTTCTTTGTGATTG ACCGGCAGAGTGGGAGACTGAAGCTGGAGAAGAGCCTTGACCAC GAGACCACTAAGTGGTATCAGTTTTCCATCCTGGCCAGGTGTACTC TGGATGACTACGAGGTGGTGGCTTCTATAGATGTCAGTATCCAGGT GAAAGACGCTAATGATAACAGCCCAGTTTTGGAGTCCAATCCATAC GAGGCATTTATTGTCGAAAACCTGCCAGCAGGGAGTAGGGTCATC CAGGTCAGAGCATCTGACC TAGACTCAGGAGTCAACGGCCAAGTC ATGTACAGTCTAGATCAGTCCCAAGATGCAGACATCATCGAGTCTT TTGCCATTAACATGGAAACAGGCTGGATTACAACCCTCAAGGAGCT TGACCATGAAGAGAGAGCCAGTTACCAGATTAAAGTGGTTGCCTCA GACCATGGTGAAAAGGTGCAGCTGTCTTCCACCGCCATTGTGGAT GTCACCGTCACTGACGTCAACGACAGCCCGCCTCGATTCACAGCT GAGATTTATAAAGGGACAGTGAGTGAGGATGACCCCCCAGGGGGT GTGATCGCCATCTTGAGCACCACTGACGCCGACTCTGAAGAGATT AACCGACAAGTGTCGTACTTCATAACAGGAGGGGATGCATTGGGA CAGTTTGCTGTGGAAAATATGCAGAATGACTGGAGGGTGTACGTG AAGAAACCTCTCGACAGGGAACAAAAGGACAGTTACCTTCTGACC GTCACTGCAACAGATGGGACCTTCTCTTCCAAAGCTAGAGTTGAAG TCAAGGTTCTCGATGCCAATGATAACAGTCCAGTGTGTGAGAGGA CCGCATATTCTGATGCCATTCCCGAAGACGCTCTTCCGGGGAAGC TGGTCATGCAGGTCTCTGCCACAGATGCAGATATCCGGTCCAACG CGGAGATCACTTACACTTTATTTGGCTCAGGTGCAGAAAAGTTTAA ACTGAATCCAGACACAGGTGAACTGAGAACATTAGCCCTCCTTGAT CGTGAGGAGCAAGCAGTTTATCATCTTCTGGTCAAGGCCACAGAC GGAGGGGGCAGATCCTGTCAGGCAACTATTGTGCTCACGTTAGAA GATGTAAATGACAACACCCCCGAGTTCACCGCGGATCCATACGCC ATCACGGTATTTGAAAACACAGAGCCTGGGACACCGTTGACCAGA GTGCAGGCCACCGATGCAGACGCAGGGTTGAATCGGAAGATTTCC T ACTCACTGCTTGACTCTGCTGACGGGCAGTTCTCCATTAACGAGC AGTCCGGAATTCTTCAGTTGGAAAAGCATTTGGACAGGGAACTACA GGCAGTCTATACTCTCACTTTGAAAGCAGCGGACCAAGGATTGCC AAGGAAATTGACAGCCACTGGCACGGTGGTTGTGTCTGTTTTGGAT ATAAATGACAACCCACCTGTGTTTGAGTACCGTGAATATGGTGCCA CCGTGTCAGAGGACATTGTCATCGGGACCGAAGTTCTCCAGGTGT ACGCAGCCAGTCGGGATATCGAGGCGAATGCAGAAATCACATACG CAATCATAAGTGGGAACGAACACGGAAAATTCAGCATCGATTCTAA GACAGGGGCCATATTTATCATTGAGAACCTGGATTATGAAAGCTCC CATGGCTATTACCTGACTGTGGAAGCCACTGATGGAGGCACGCCC TCGTTGAGTGACGTGGCGACCGTGAACATCAACATCACAGATATTA ACGATAACAGCCCAGTGTTCAGCCAGGACAGCTACACCACAGTGG TCAGCGAAGACGCGGCCCTGGAGCAGCCCGTCATTACAATTATGG CTGATGATGCTGATGGCCCTTCAAACAGCCACATCCTCTACTCCAT TATAGAGGGTAACCAAGGAAGTCCATTCACAATCGACCCTGTCAGA GGAGAAATCAAAGTAACGAAGCCCCTAGACCGCGAAACGATCTCA GGTTATACGCTCACGGTGCAGGCTGCCGACAACGGCAATCCACCC AGAGTCAACACCACCACAGTGAACATCGATGTCTCCGATGTCAAC GACAATGCTCCCCTCTTCTCCAGAGACAACTACAGTGTCATCATCC AGGAAAACAAGCCCGTGGGTTTCAGCGTCCTGAAGCTAGTAGTGA CAGACAAGGACTCGTCCCACAACGGCCCCCCTTTCTCCTTTGCTAT TGTGAGTGGAAATGATGACAACAT GTTTGAGGTGAACCAGCACGG GGTCCTCCTGACAGCGGCAACAGTCAAGAGGAAAGTGAAGGACCA TTACCTTCTGCACGTTAAGGTGGCTGACAATGGAAAGCCTCAGCTG TCTTCGTTGACACACATTGACATCAGGGTTATTGAGGAGAGCATCC ACCCTCCTGCCATTTTGCCACTGGAGATTTTCATCACTGCTTCTGG AGAGGAATACTCAGGCGGGGTCATAGGAAAGATCCATGCCACAGA CCAGGATGTGTATGACACCTTGACGTACAGTCTGGATCCCCACAT GGATGGCCTGTTCTCTGTTTCCAGCACGGGGGGTAAACTGATTGC ACACAGAAAGCTGGATATAGGCCAGTACCTTCTTAATGTCAGCGTG ACAGACGGGAAGTTTACAACGGTGGCTGACATCACCGTGCACATC CAGCAAGTGACCCAGGAGATGCTGAACCACACCATCGCTATCCGA TTTGCAAATCTCACCCCGGAAGAGTTTGTCGGCGACTACTGGCGC AACTTCCAGCGAGCTTTACGCAACATCCTGGGCATCCGGAAGAAC GACATACAGATTGTCAGCTTGCAGCCCTCCGAACCCCACTCCCAC CTTGACGTCTTACTCTTTGTAGAGAAATCAGGGGGCACCCAGATCT CAACGAAACAACTTCTGCACAAGATCAATTCTTCCGTCACGGACAT CGAGGAAATCATTGGCGTGAGGATACTGGATGTGTTCCAGAAACT CTGTGCAGGGCTGGATTGCCCGTGGAAATTCTGTGATGAGAAGGT TTCTGTGGATGAAAACATTATGTCAACTCATAGCACAGCCAGACTG AGTTTTGTGACTCCCCGGCACCATAGAACAGCCGTGTGTCTCTGC AAAGATGGGACATGCCCGCCTGTCCACCAAGGGTGCGAAGATAAC CCCTGTCCTGCAGGATCCGAATGTGTCGCTGATCCCCGAGAAGAG AAGTA CAGCTGTGTGTGTCCTGGTGGCGGGTTCGCCAAATGTCCA GGGAGTTCATCCATAACTTTTACCGGCAGCAGCTTTGTGAAATATC GTCTGATGGAAAATGAAAACCGACTGGAGATGAAGTTGACCATGC GCCTGAGAACCTACTCTTCCCACGCGGTTGTGATGTACGCTCGAG GAACTGACTACAGTATCCTGGAGATTCATACTGGGAGACTGCAGTA CAAATTTGACTGTGGAAGTGGCCCTGGGATCGTCTCTGTTCAGAG CATTCAAGTCAACGATGGGCAGTGGCATGCAGTGTCCCTGGAAGT GGAGGGGAATTATGCAAAATTGGTTCTAGATGAAGTCCACACTGCC TCGGGCACAGCCCCAGGAGCTCTGAAAACCCTCAACCTGGATAAC TACGTAATTTTTGGTGGCCACCTCCGCCAGCAAGGGACAAAACAT GGACGAAACACCCAGGTGGCCAATGGTTTCAGGGGCTGCATGGA CTCTATTTATTTGAATGGGCAGGAGCTACCTTTGAACAACAAACCA AGAGCCTATGCACACATCGAAGAATGGGTGGACCTAGCTCATGGG TGCTTGTTAACTGCCACCGAAGACTGTTCCAGCAACCCTTGTCAGA ATGGAGGCGTCTGCAATCCCTCGCCCACTGGAGGTTATTACTGCA AGTGCAGTGCATTGCACGCAGGGACGTACTGTGAGGTGAGCGTCA ACCCGTGCTCCTCCAACCCCTGCCTCTACGGAGGAACGTGCATGG TAGACAACGGAGGTTTTGTTTGCCAGTGCAGGGGGCTGTACACTG GCCAGAGATGTCAGCTTAGTCCGTACTGCAAAGATGAACCCTGTAA AAATGGTGGAACGTGTTTTGACAGTTTGGATGGTGCTGTCTGTCAG TGTGACTCAGGCTTTAGGGGAGAAAGATGTCAGAGTGACATTGAC GAGTGTGCTGGGAACCCCTGTCGGAACGGGGCC CTTTGCGAGAA CACGCATGGCTCCTATCACTGTAACTGCAGCCAGGAGTACAGAGG GAAGCACTGTGAGGATGCCACTCCCAACCACTACGTGTCCACCCC GTGGAACATCGGACTGGCCGAAGGAATCGGAATTATTGTGTTTATA GCCGGGATATTCTTACTGGTGGTGGTGTTTGTCCTCTGCCGAAAG ATGATCAGTCGGAAGAAGAAACACCAGGCGGAACCTGAAGACAAG CGTTTGGGGCCAACCACGGCTTTCTTACAGAGACCTTACTTTGATT CCAAGCCGAGCAAGAACATTTACTCTGACATCCCGCCCCAGGTGC CCGTGCGTCCCATTTCCTACACTCCGAGCATTCCCAGTGACTCTAG AAACAATCTGGACCGGAACTCGTTTGAAGGCTCGGCAATCCCAGA GCACCCAGAATTCAGCACTTTTAACCCCGAGTCTATGCACGGACAT CGGAAAGCCGTGGCTGTGTGCAGCGTGGCTCCAAACTTGCCTCCC CCACCCCCTTCCAACTCTCCCTCAGACAGCGACTCCATTCAGAAG CCCAGCTGGGACTTCGACTACGACGCTAAAGTGGTGGATCTTGAC CCTTGTCTTTCCAAGAAGCCCCTGGAGGAAAAACCCTCTCAGCCAT ACAGTGCCCGGGAGAGCCTGTCCGAGGTGCAGTCCCTTAGCTCCT TCCAGTCAGAGTCCTGTGATGACAATGGGTACCACTGGGATACAT CAGACTGGATGCCCAGTGTTCCTCTGCCAGACATACAAGAGTTCC CCAATTACGAGGTTATCGATGAGCACACGCCCCTCTACTCAGCTGA TCCAAATGCCATCGACACTGACTATTACCCTGGGGGTTATGACATT GAAAGTGACTTTCCACCCCCACCAGAGGACTTCCCTGCACCCGAT GAACTGCCACCATTGCCTCCAGAATTCAGCGACCAGTTCGAGTCC ATACACCCACCCAGA GACATGCCCGCAGCAGGTAGCTTGGGGTCT TCCTCCAGGAATCGTCAGAGGTTCAACCTGAATCAGTACCTGCCCA ATTTCTACCCCGTCGATATGTCTGAACCTCAGAAACAAGGCGCTGG TGAGAACAGTACCTGTAGAGAACCCTACACTCCCTACCCTCCAGG GTATCAAAGAAACTTCGAGGCGCCCACCATAGAAAACATGCCCAT GTCTGTGTACACCTCTACGGCTTCCTGCTCCGATGTGTCAGCGTG CTGCGAAGTGGAGTCTGAGGTCATGATGAGTGACTACGAGAGCGG GGACGACGGCCACTTTGAAGAGGTGACCATTCCCCCGCTAGATTC CCAGCAGCATACGGAAGTGTGA (SEQ ID NO: 12) ATGAAGATTAAAAAATATGTAACTCCTGTAAAAAGAAAA FAT GCTTTCACCATACTCCAATGGATTTCACTACTGTGTAGTCTATGGT TGATCCCCACTGTACAAAGCAAGGCCGATGAGAAGCACACGGCG ACCCTGGAGTATAGACTAGAGAACCAACTGCAAGATCTATATAGG TTTAGCCATAGTGTATATAATGTTACCATACCAGAAAATAGTCTGG GCAAGACTTACGCCAAGGGAGTATTGCATGAAAGACTGGCCGGC CTGAGAGTTGGCTTGAACGCAGAGGTTAAGTATAGGATAATTAGT GGCGATAAGGAGAAGCTATTTAAGGCCGAGGAGAAACTGGTCGG AGATTTTGCCTTCTTAGCGATTCGAACGCGGACAAATAACGTTGTG CTAAACAGAGAAAAAACTGAGGAATACGTTATAAGAGTGAAGGCA CATGTACATTTGCACGACCGAAATGTATCAAGCTATGAAACGGAG GCGAATATCCACATCAAAGTACTGGATCGCAATGACCTGAGTCCG CTGTTTTATCCGACCCAGTACACCGTTGTTATTCCGGAGGACACG CCCAAATATC AAAGTATTTTAAAGGTCACAGCTGACGATGCTGACC TCGGCATCAATGGGGAAATCTACTACAGCCTCCTGATGGATAGTG AATACTTTGCTATCCATCCAACAACTGGCGAAATTACTCTCCTGCA GCAGCTTCAGTATGCGGAGAACTCGCACTTCGAGCTCACGGTGG TGGCCTACGATCGGGGATCATGGGTGAACCATCAGAACCACCAG GCCAGCAAGACGAAGGTTAGTATTTCGGTGAAACAGGTTAACTTT TACGCTCCAGAGATTTTCACGAAAACCTTCTCGAGCGTGACGCCA ACATCAAACCCTTTGATTTATGGAATTGTACGAGTAAACGACAAAG ACACTGGGATAAATGGCAACATAGGGCGATTGGAAATCGTCGATG GAAATCCGGATGGCACGTTTCTTCTGAAGGCGGCGGAGACCAAA GACGAGTACTACATCGAATTGAATCAGTTTGCCCATCTTAACCAGC AACATTTCATTTACAACTTAACCCTACTGGCGGAGGACCTCGGAA CTCCCCGTCGATTCGCCTACAAATCCGTTCCGATTCAAATCAAGC CCGAGAGCAAAAATATACCCATATTCACACAGGAGATTTACGAAGT ATCCATTCCAGAAACGGCACCCATTAACATGCCTGTGATAAGGCT CAAAGTAAGCGATCCAGATTTGGGCAAAAATGCATTGGTCTACTT GGAAATCGTGGGTGGAAATGAGGGCGACGAGTTCCGAATTAATC CCGATTCGGGAATGTTGTACACAGCAAAGCAACTGGATGCCGAAA AGAAGTCAAGTTATACCTTAACAGTCTCCGCCATTGATCAGGCAAA TGTTGGGTCGCGGAAACAATCTTCAGCCAAGGTGAAAATCAGCGT ACAGGATATGAACGACAATGATCCCATTTTTGAGAATGTCAATAAG GTCATTAGTATCAATGAGAACAACTTGGCTGGCTCGTTTGT TGTGA AGCTTACTGCCAAGGACAGGGATTCTGGTGAAAATTCATACATATC GTATAGTATTGCCAATCTAAATGCGGTTCCATTTGAAATCGATCAC TTTAGCGGTATAGTTAAGACCACATCACTGCTTGACTTTGAAACAA TGAAGCGTAACTATGAGCTGATAATCCGTGCATCCGATTGGGGAT TGCCGTACAGAAGACAGACGGAAATCAAACTGTCCATCGTCGTCA AGGATATCAACGATAATCGGCCGCAGTTTGAACGTGTGAACTGCT ATGGCAAAGTGACCAAATCGGCGCCGATGGGCACCGAGGTATTC GTTACCTCAGCCATTGACTTTGATGCAGGCGATATAATATCCTATA GGTTGAGCGACGGCAACGAGGATGGCTGCTTTAACTTGGACCCC ACATCGGGTTCCCTGTCTATTTCCTGCGACCTGAAGAAAACAACC TTAACAAACCGTATTCTCAAAGTTTCCGCCACGGACGGCACCCAC TTTTCCGATGACTTGATCATCAATGTACACCTAATGCCCGAAGATT TGGGTGGAGATTCCAGTATTCTACATGGTTTTGGATCCTTTGAGTG CCGGGAAACCGGCGTGGCCAGGAGATTGGCGGAAACATTATCGT TGGCCGAAAAAAACAATGTAAAGAGTGCATCGCCATCCGTTTTCA GTGACTTGTCTCTAACACCCAGTCGATATGGCCAAAATGTGCATA GACCAGAGTTCGTGAACTTCCCTCAGGAGCTGTCCATTAACGAAA GTGTCCAATTGGGCGAAACAGTTGCTTGGATAGAGGCCAAAGATC GCGATTTGGGCTACAATGGAAAGCTGGTATTTGCAATTTCAGACG GGGACTACGATTCGGTTTTTCGTATTGATCCAGACCGCGGTGAAC TGCAGATTATTGGATATTTGGATAGAGAGCGTCAAAATGAATATGT TCTCAACATCACCGTCTACGATCT GGGTAACCCGACCAAATCGAC GTCAAAAATGTTGCCAATAACGATCCTCGACGTGAACGATAATCG CCCGGTTATTCAGAAGACGTTGGCCACCTTCCGGCTGACTGAGAG CGCCAGGATAGGAACTGTGGTACACTGCCTTCATGCCACGGATG CGGATTCTGGAATCAATGCTCAGGTGACATATGCCCTGTCGGTTG AGTGCAGCGATTTCACAGTAAATGCTACTACGGGATGTCTTCGTC TGAACAAACCACTGGATCGCGAGAAGCAGGATAACTACGCTCTTC ACATAACTGCCAAGGATGGTGGCAGTCCCGTGCTATCCTCGGAG GCATTGGTTTACGTCCTGGTCGACGATGTCAACGACAACGCGCCC GTTTTCGGAGTGCAAGAGTACATATTTAAGGTGCGCGAAGATCTG CCCCGTGGAACAGTGTTGGCCGTAATCGAGGCGGTGGACGAAGA TATTGGACCCAATGCCGAGATCCAATTCTCTTTGAAAGAGGAGAC CCAGGATGAGGAACTATTCAGAATCGATAAGCACACGGGTGCAAT TAGGACTCAAGGATATCTGGACTATGAGAACAAACAAGTGCACAA CCTTATTGTCAGTGCCATCGATGGCGGAGATCCCTCTCTAACTTC GGACATGTCCATCGTAATAATGATCATCGACGTCAACGAGAACCG ATTTGCGCCCGAATTCGACGACTTTGTGTACGAGGGAAAGGTAAA GGAGAACAAGCCGAAGGGAACGTTCGTAATGAATGTCACAGCAC GGGATATGGACACGGTGGACCTGAACTCCAAGATCACGTACTCAA TAACAGGTGGCGATGGACTGGGAATTTTTGCGGTTAACGACCAAG GTTCAATAACTTCCTTGTCGCAACTCGATGCGGAGACGAAAAACTT TTACTGGCTGACGCTCTGTGCACAGGATTGCGCAATAGTTCCCCT CAGCAATTGTGTGGA AGTTTACATACAAGTCGAAAACGAAAACGAT AACATTCCTCTTACGGACAAACCAGTGTACTACGTTAATGTCACGG AAGCCAGTGTGGAAAATGTGGAGATCATTACCCTAAAGGCTTTCG ATCCCGATATAGATCCCACTCAGACTATAACATATAACATAGTTTC CGGAAATCTTGTCGGGTACTTTGAAATTGATTCGAAAACAGGAGT GATTAAGACGACAGAACGCAAATTGGATAGAGAAAATCAAGCGGA ACATATTTTGGAGGTGGCTATATCAGATAACGGATCTCCAGTACTA TCTTCTACATCGCGAATCGTTGTGTCAGTACTGGATATTAACGATA ACAGCCCCGAGTTTGACCAAAGGGTCTACAAGGTGCAAGTTCCGT CTTCAGCCACAGTCAATCAATCTATTTTTCAGGTTCACGCTATCGA CAGCGACAGTGGCGAAAATGGTCGAATTACCTACTCAATTAAGTC CGGAAAGGGTAAGAATAAATTTCGCATCGATAGCCAAAGGGGCCA TATACATATAGCAAAACCATTGGACTCCGACAATGAGTTTGAGATT CACATCAAGGCTGAGGACAACGGAATTCCTAAAAAGAGTCAAACT GCTAGAGTTAATATTGTTGTAGTTCCTGTAAATCCTAATTCCCAAAA TGCACCGTTGATAGTCAGAAAGACATCCGAAAATGTCGTTGATCTT ACGGAAAATGACAAGCCTGGATTTTTGGTCACTCAAATTTTAGCTG TCGATGATGACAACGACCAGCTGTGGTACAACATTTCCAATGGCA ATGACGACAATACCTTTTACATTGGCCAAGACAACGGAAACATACT GCTTTCAAAATATTTGGACTACGAGACCCAACAGTCCTATAATCTG ACTATCAGCGTCACTGATGGCACATTCACAGCGTTTACTAATCTTT TGGTTCAAGTGATCGATATTAATGACAACCCCCCT CAGTTCGCTAA AGATGTGTATCATGTCAATATATCCGAAAATATTGAAGAGGAATCA GTTATAATGCAACTCCACGCCACTGACAGAGATGAGGACAAGAAG CTATTCTATCACCTGCACGCAACTCAGGATCCGTCGTCGCTGGCA TTGTTCCGAATCGATTCCATAAGTGGAAATGTCATTGTCACTCAGA GATTGGATTTTGAAAAGACTGCGCAGCATATACTCATCGTTTTTGT TAAGGATCAAGGAGCGCCTGGAAAAAGAAACTATGCCAAGATAAT TGTAAACGTGCATGACCACAACGACCATCATCCAGAATTTACTGCT AAAATAATTCAAAGTAAGGTTCCCGAAAGCGCAGCTATTGGCTCTA AGTTAGCCGAAGTGAGGGCCATAGATAGAGATAGTGGTCACAATG CCGAGATCCAGTACTCGATTATCACGGGTAACGTGGGTAGTGTGT TTGAGATTGATCCGACTTTCGGTATAATCACATTGGCTGGCAACTT GAATATCAACAAGATCCAGGAGTACATGCTTCAAGTGAAGGCCGT AGATCTGGGAAATCCACCGCTGTCATCGCAGATTCCGGTACACAT CATTGTCACCATGTCCGAGAACGATCCTCCGAAGTTCCCAACCAA CAACATTGCCATTGAAATATTCGAAAACCTGCCCATCGGAACATTT GTTACTCAAGTCACCGCTCGGTCGTCGTCATCCATATTCTTCAATA TTATTTCCGGCAACATCAACGAAAGCTTCCGCATTAACCCATCTAC TGGAGTTATTGTTATCAATGGAAATATCGACTATGAATCCATCAAA GTATTCAACCTTACGGTTAAAGGAACCAATATGGCAGCCGAGTCA TCCTGCCAAAATATAATTATACATATCCTAGATGCTAACGATAATAT TCCGTATTTCGTTCAAAATGAATATGTTGGAGCATTACCCGAATCC GCCGCTATT GGATCTTACGTACTGAAAGTACACGACTCATCAAAA GATCATTTAACATTACAAGTTAAGGATGCGGATGTCGGAGTAAAC GGAATGGTTGAATACCACATAGTTGACGATCTGGCAAAAAACTTTT TTAAAATAGATTCGACAACTGGCGCTATTGAACTGTTACGACAATT GGACTATGAAACAAACGCTGGTTATACCTTTGACGTTACGGTTAGT GATATGGGAAAGCCCAAACTACATTCCACTACAACTGCACATGTG ACGATTCGTGTCATAAATGTTAACGATTGTCCTCCAGTATTTAATG AGCGTGAACTCAATGTAACTTTGTTCCTTCCAACTTTTGAGAATGT GTTTGTAAGACAAGTTAGCGCAAAGGATGCTGATAACGATACCTTA AGGTTTGATATTGTGGATGGAAACACCAACGAATGTTTCCAGATC GAAAAATACACCGGAATAATTACAACACGAAATTTTGAAATACTAA ATAACGAAAATGATCGGGACTATGCCTTGCACGTCCGTGCCTCCG ACGGAATTTTCTCTGCAATTTTAATAGTTAAAATTAAGGTTTTGTCC GCCATCGATTCGAATTTCGCATTCCAACGTGAATCGTACAGATTTT CTGCATTTGAAAATAACACAAAGGTAGCTACCATTGGATTGGTGAA CGTAATAGGAAACACACTGGACGAAAACGTTGAGTATCGCATCCT GAACCCAACACAATTGTTTGATATTGGAATCAGTTCGGGAGCCCT AAAAACCACTGGAGTTATTTTCGATCGCGAAGTAAAGGATTTGTAC AGACTCTTCGTGGAAGCAAAGTCAATGCTATACGACGGCATGAAT TCAAATGTTCGCAGAGCAGTAACGTCCATAGATATATCCGTCTTGG ATGTGAACGACAATTGCCCCTTGTTTGTCAATATGCCCTATTATGC CACAGTCTCTATTGACGATCCAAAAGGAA CGATTATTATGCAGGTC AAGGCCATTGACTTGGACAGTGCAGAAAACGGCGAAGTTCGGTAC GAACTTAAGAAGGGCAATGGGGAGTTGTTCAAACTGGACCGCAAA TCTGGGGAGTTATCCATAAAGCAGCATGTCGAAGGTCATAACCGA AACTATGAATTGACAGTGGCTGCCTATGATGGCGCCATAACACCA TGCTCCTCGGAAGCTCCTCTGCAGGTTAAGGTTATAGATCGTTCG ATGCCCGTTTTTGAAAAGCAGTTTTATACTGTTAGCGTCAAGGAAG ACGTGGAAATGTACTCAGCCCTTTCCGTATCCATTGAAGCAGAAA GTCCCCTGGGAAGGAGTTTAATTTACACAATATCTTCCGAGAGTCA ATCGTTTGAAATTGATTACAACACGGGATCAATTTTTGTCGTAAAT GAATTGGATTACGAGAAAATAAGCTCACACGATGTTTCCATTCGAG CGACTGACAGTCTTTCTGGTGTTTATGCTGAAGTCGTTTTATCTGT TTCCATTATGGATGTCAATGACTGCTATCCAGAAATTGAGAGTGAT ATATACAACCTAACCATTCCGGAAAATGCATCGTTTGGAACACAAA TTCTGAAGATTAATGCAACTGATAACGACTCGGGAGCAAATGCAA AACTTTCCTATTACATTGAGTCCATTAATGGGCAAAATAATTCAGAA CTGTTTTACATTGACGTCACAGACGGAAATCTGTATTTAAAGACTC CATTGGACTATGAACAAATCAAGTATCATCATATAGTCGTTAACGT AAAGGACCATGGATCGCCATCATTAAGTTCCCGATCAAACGTATTT ATAACAGGTAGAATTCTATGTCGCTTTATCTCTTACAAACTAATTTA TGATTCTATTATTCCAGTTAAAGACTTAAACGACAACGCTCCATGT TTCGTTGAGCCGTCGTACTTCACCAAAGTGTCAGTGGCAGCTGTT C GTGGACAATTTGTTGCTTTACCTAAAGCATACGATAAGGATATTT CCGATACCGATTCTCTGGAATACAAAATTGTTTACGGAAATGAATT GCAAACCTATAGTATTGATAAGCTAACAGGAGTGATTTCCCTTCAA AATATGTTAAATTTCACTGATAAAAGTAGCACAGTCTTGAATATTTC CGTCTCCGATGGAGTTCATACGGCATATGCCCGGCTCAAAATATC CTTATTGCCAGAAAACGTTTACAGTCCACTGTTTGATCAAAGTACT TATGAGGCTCAAGTACCTGAAAACTTGCTACACGGTCATAATATAA TCACGGTAAAAGCATCGGATGGAGACTTTGGCACCTACGCCAATC TTTACTACGAAATAGTTTCGGAGGAAATGAAAAAAATCTTTCTCAT CGACCAAACGACGGGTGTAATAACCTCAAAAGTAACTTTCGACCG TGAAAAAAAGGATGAGTACGTGGTGCTACTGAAGGTGTCCGACGG TGGCGGAAAATTCGGATTTGCCTCTCTCAAGGTCATAGTCGTCGA CGTGAACGATAACGTTCCTTACTTCCTATTGAAGGAATACAAAATG GTTGTTAGCACAACAGTGGAAGCAAACCAAACTATCCTGACGGTC AAAGCCAAAGACGACGATATTGTTGATAATGGATCGGTGCATTTC CAAATTGTTCAAAAATCCAACGATAAGGCAGTAAAGGATGTAATCG AAATCAACGAGAAAACTGGGGATATTGTGTTTAAAAGCAAGGCGG AATCTTACGGAGTGAACTCATATCAGTTTTTCGTTCGCGCTTCCGA TCGCGGTGAACCTCAATTTCATTCGGAAGTTCCAGTGTCAATCGA AATAATCGAGACTGATGCCAATATTCCCACTTTTGAGAAATCGTCA GTTCTACTAAAGATCATAGAGTCAACGCCACCAGGAACCGTGCTA ACGAAGCTACATATGATTGGAAA CTATACGTTCAAATTCTCAATAG CAGCGGATCAGGATCACTTCATGATATCCGATAGTGGTGAACTGA TCCTTCAGCAGACATTGGACAGGGAGCAGCAAGAGTCGCACAATT TGATTGTAGTGGCGGAAACTTCCACGGTTCCCGTTTTTTTCGCCTA CGCTGATGTTTTGATTGACGTTAGGGACGAAAATGATAACTATCCC AAGTTTGACAACACATTCTACAGTGCCAGTGTTGCGGAAAACAGT GAAAAGGTGATATCCTTGGTGAAAGTATCGGCCACAGATGCGGAC ACTGGGCCAAATGGCGACATTCGCTACTACTTGGAAAGTGATACT GAAAACATTCAAAATATTTTTGACATTGACATTTACTCTGGCTGGAT CACCTTGCTAACCTCCTTGGACAGAGAAGTTCAGTCCGAGTACAA TTTCAAAGTAATTGCTGCCGATAATGGCCACCCAAAGCATGATGC AAAAGTACCTGTAACTATCAAAATCGTAGACTATAATGATAACGCA CCAGTATTTAAGTTGCCTATCGAAGGGCTTTCTGTTTTCGAAAACG CGCTGCCTGGCACGGTTTTAATCAACTTACTCCTAATTGATCCCGA TATCGAGAAACAGGAAATGGATTTCTTTATCGTTTCTGGGGACAAG CAAGCCCAGTTTCAGATCGGTAAGAGCGGAGAGTTATTTATTGCC AAACCATTAGATCGCGAACAACTCATGTTCTACAACTTAAGCATAA TAGCCACTGATGGAAAATTCACTGCCAAAGCCAATGTGGAAATAG ATGTAAAAGACATAAACGACAATACGCCTTACTGCCTAAAACCCCG CTATCATATCTCCACTAATGAATCAATCTCGATTGGAACTACACTC GTTGAGGTCAAGGCGATTGACTTTGATTTTCAAAGCAAACTGCGC TTCTATCTTTCGGGCAAAGGTGCGGACGACTTCAGTATAGGAAAG GAAAGTGGCATCCTGAAGGTGGCAAGCGCACTGGATCGGGAGAC AACCCCCAAGTACAAATTGGTCGCACATGTACAGGATGGCAAGGA CTTTACGCAAGAGTGTTTCTCGGAAATAATCATCACGGTCAATGAC ATAAATGACAATATGCCCATTTTCTCAATGGCTCAATATAGAGTGA GTGTACCCGAGGATGCACAACTGAACACATTGATCACGAAAGTGC ACGCGATGGATAAGGATTTCGGGGTAAATAGACAAATCAAATACT CGCTAATGGGTGAAAACCATGATTATTTCAAAATATCAAAATCGAC TGGTATCATAAGGCTGCACAAAAGTCTCGATCGTGAAACAATTTCA TTGTTTAATCTCACTGTGAAGGCGGAGGACTGTGGCGTTCCAAAA CTACACTCCATTGCAACAGTTGCTGTGAACATATTGGACATTAATG ACAATCCACCCGAGTTCAGTATGCGTCAGTATTCGTGCAAAATTCT GGAAAACGCCACACACGGCACAGAAGTGTGCAAAGTTTATGCCAC TTCGATAGATATTGGGGTAAATGCGGATATTCACTACTTCATAATG AGTGGCAACGAGCAGGGGAAGTTCAAAATGGATTCCACGACGGG CGACTTGGTGCTAAATGCAACCTTGGACTATGAAATGTCCAAGTTT TACTTCTTGACCATTCAAGCAATCGATGGCGGCACTCCACCGCTT AGCAACAATGCATATGTGAACATCTCTATTCTGGACATTAATGACA ACAGTCCCACGTTTCTGCAAAACCTGTACCGCATTAATGTCAATGA AGATATTTTCGTGGGCTCCAAGATTCTGGACGTCAAAGCCACGGA CGAAGATTCAGATGTAAATGGTCTTGTAACTTACAACATTGAAAGA GGCGACAATATAGGCCAGTTTTCAATAGATCCGAAAAACGGAACA ATTAGCGTTTCGAGGCCATTAGAT CGTGAGACTATTTCGCACTACA CTCTTGAAATTCAAGCCTGTGATCAGGGAGATCCTCAGAGATGCA ACAGTGTTCCAATCAATATAAACATTTTGGACACTAACGATAATGC ACCCATATTTTCCAGCTCTAACTACAGTGTAGTACTTCAAGAAAAC CGACTTCTGGGCTATGTATTCCTTACCTTCAAGATATCAGACGCAG ACGAAACACCCAATACCACGCCATACACCTTCGATATTAGGTCTG GAAATGAGGGTGGGCTTTTCCGGCTGGAGCAAGATGGTTCCTTGA GAACGGCCTCGCGATTTAATCACAATCTGCAGGACGAATTCGTGA TTCAAGTTCGAGTTTTCGACAACGGCACACCTCCATTATATTCCGA TGCCTGGGTGGTTGTGAAAATAATTGAAGAAAGCCAATACCCGCC CATTGTCACACCCCTAGAAGTAACCATAAATTCATTCGAGGACGAT TTTTCGGGCGCATTCATTGGCAAAGTTCATGCCTCGGATCAGGAC AAGTATGATGAATTGAACTTTAGTTTGGTGTCCGGTCCCGATGACA TGTATCAGAGCTCGAAGCTGTTCAACATTTCCAACAACACGGGAA AGATCTATGCCATATCCAACCTGGATATTGGTCTGTACAAGCTAAA TGTGTCCGTTTCGGATGGTAAATTTCATGTGTTCTCCATTGTCAAA ATCAACGTGGAACTGGTAACCAATGATATGCTAAAAGAGTCGGTT GTCATTCGATTCAGAAGGATTTCAGCATCTGAGTTTCTGCTGAGTC ACAGGAAAACCTTTATGCGCTCCATTCGCAATATAATGCGATGTCG CCAAAAGGATGTAATTCTCATCACCCTTCAATCGGATTATCAAAAA GCATCACAACATGCTGTGGGTAATCGACGAGCCAGGTCCATTGAC TCCGATTTGAACGTGGTGTTTGCAGTGCGAAAGCAGCAAATAATA CCCGATTCCGATGAATTCTTCACAAGTGATGAAATTCGGCAGACA CTGATAGACAAGAAGAACGAGATTGAAAACGAAACCAACCTGGTG GTGGAGGATGTACTACCATCCACCTGTCAAAGCAACAAAAACGAC TGCGTTCACGGGGAATGCAAACAGATATTACAGATCCTGAAGAAC AACGTTACCACCACCTTTACGGATGTGATTAGTTTTGCTGCTCCAT CTTACATTCCGGTGAATACGTGTGTCTGTCGACCAGGATTCGATG GAAAGCACTGCAAAGAGACTGTGAATGCCTGCTCCACGGATCCAT GTTCCCCGCAGAGGATCTGCATGCCGTCTGGCTCGGCTTTGGGT TACCAATGTGTGTGTCCCAAGGGATTTTCAGGAACCTACTGCGAG CGGAAGTCTTCGAAGTGCAGCAATGAGTCCTGTGACATGGGTCTA TTCACTGCGGTGTCCTTTGGCGGAAAGAGCTATGCCCACTACAAG ATCAACAAGGTGAAGGCGAAGTTCACGCTGGAAAACGGGTTTTCC TACTCCCTGCAGATAAGAACTGTGCAACAAACTGGGACTCTGCTG TATGCCAGCGGCAAGGTGGACTACAACATCCTGGAGATCATAAAC GGAGCTGTTCAGTACAGATTCGATTTGGGCTCGGGCGAGGGAGT CATCAGTGTGTCCAGCATTAACATCTCTGACGGCGAGTGGCATCA AATCAGCCTAGAGCGGTCCCTCAATAGTGCCAAAGTGATGGTGGA CAACAAGCACGTCTCCCATGGCAGTGCTCCGGGTGTGAATGGCA TCCTGAACATCCAGTCGAACGATATCTTTGTAGGCGCCGAGGTTC GTCCGCATCCATCGATAATTGGCTACGAGGATATTCAGCGTGGCT TCATCGGTTGCATGGCAAACATCAAAATAGCCAAAGAGTCGCTGC CATTGTACATTTCCGGTGGGAGTACCATTGCTGCCT TGAAACGTTT TACGAATGTCGAGTTCAAGTGCGATCCGTCGAATGTTCTGGTGCG CCTGGGCATTTGCGGATCTCAGCCGTGTGCCAATAGTGGAATCTG CAAGGAACTCGATACGGACGTGTTTGAATGCGCCTGTCAGCCCC GATATTCCGGCAAGCATTGCGAGATTGATTTGGACCCTTGCTCAT CGGGACCCTGCTTGTTTGGCGGCAGGTGCGACTACCACGGACCG AACAACTACAGCTGCACGTGTCCCATCCACTTATCCGGAAAGAGG TGTGAGTACGGCAAGTTCTGCACGCCGAACCCGTGCAAAAACGG TGGCATTTGCGAGGAAGGCGATGGAATATCGCACTGCATGTGCC GCGGCTACACGGGACCCACTTGTGAGATCGATGTGGATGAGTGC GAGAACCAGCCGTGCGGCAATGGAGCGACCTGCATCAATGAACC CGGAAGTTTCCGTTGCATTTGTCCATCTTATCTCACAGGAGCCAG CTGCGGCGATCCCCTGTATTCGAACTCTATTTCTACAAAGCTGAA GAACTTTTCTATAGAGCACATTAGCGGGATCATTTCCGGCGTGGC CGTGGTACTGGTCATCATCAGTTGTGTCCTGTGTTGCGTGGTGTT GAAGAGGAGTTCCTCTTCAAAGCGAAGGAACCGACTAGAAAAGGA CAAGAACAAGTCGTCGTACAAGGAGGCGAACTTGAACTCACTGGT GGACAAGGACAATTACTGCAAACCAAACGTAAAGTTGAGTAACTT GGAGGTTAACCAGCGTCCAATTAGCTACACAGCAGTTCCAAATGA CAACCTAGTCCTGAGCAATAGGAATTTTGTAAATAACTTAGACATC TTGCGTAGCTACGGTTCGGCCGGCGATGAACTGGAAAATGTGCC ATTCGAGTACCAGAAGGTTAATCGAAACAAACAGCATGTGAACATA AACTCCTGCCATTCAACCGATGCAGATA ATGCCTACAAACAAGAAT GGTGCGAGCAAATGCATTTAAGAACCTTCAGTGAAAATAAACTGAA CAATGAACTTAAACGGGATTTCGGACCATCTGTGAGTCGCTTTTCA ACTGGGAAACTAATCCAAGTTGAAATGCCCAACGTGTGCCACTCT TCCAGTGCGAATTTCGTTGATTATTCAGCTCTTGCCAATGGTCAGT ATCATTGGGACTGTTCCGACTGGGTTCGCAAAAGCCATAATCCCT TGCCAGATATAACCGAAGTTCCTGGAGCAGAAATAGCTGATTCGT CGAGCTTACACAGCAACGATAGCAACGAGTCCAAGTCGAAGAAAG CCTTTTTCGTGCACAGGGAAGACGGAGATGTTGATCCGACGAGG GATATAGCCGCGTTGAATGAGGATATCGGATCGGAGTATTTGGAC TCGGAGGCAGAGAGCTGCTTGGAGCCGTTTATGTTGCCAAGATCA AGTAATCAGCCACTTTCAAGACTGAGTTCTTTTAATAATATCGAGA ATGAAGACTATAAATCAAATACAGGCAAAGTATATTTAAGACATCC TGATTCGTATTTACCGACGATGCATTTTCCAAGTGAGACCGATGG GGAAAGCTCTATGACCGAGGGGCCGATTTCTAGGATGGAAATAAA AACCAGGAGGACGATAAGTGAAAATTCAGAGGAGGCATACCTATT TCCATGCACTGTCGGAGAAATTGGATCCAACAGCAACATTTCGGT TCGACTGTGTGAAATTGAAGATTCTGAGTTGGAGGAGTTTTTACCA CAACAACAAACAAACAATTAA (SEQ ID NO: 13) PCS (Saccharomyces cerevisiae) CTACAACTTACTCTTATTTCTGCTGCTCTTAGCAAAAGTT TCTGCGATAACTCTTCTCTGGATTTTACCTGTAGCGGTTTTTGGTAG CTTATCAACAAAGTACACCTTGGTTGGAATTTTGAA AGAGGCTAGG TGCTTCTTTAAGAAGTTCACCAGTTCTTCGTAGGTCATTTTTTCTCC CTTCTTCAAAACAATGGCGGCTTGAACTACTTGGCCGTACATATCG TCGGGAACACCAAATGCAACGGCTTCATCGATCTTTGGATGCGATA GCATAATGCCGTCGAGCTCAATGGGTGAAATCTTTTCACCACCCCT GTTGATAAGCTCTTTGATTCTGCCTGTAAGGACCAAAAACCCCTCA GGGTCGAAATAACCTTGGTCACCGGTTCTGAAATAGTTCTCTCTCT TGGTGAAGTTCTCCTTGTTAGCTTTTGGATTATTAGCATACCCCAAA GTGACGTTTTCGCCTCTGATGGAAACTTCGCCGACTTTGCCCGGG GGCAAGACATTGTCATTGTCATCTAGAATGACGACGGTGACTCCTT GTGGCTGGCCCACAGTACCAGGCTTTCTCTTTCCTGGAGGCAGAT TGTTTGAGGTCATTTGATGTGATGCTTCGGTCATCGCATAGGCCTC CAAGACAGGTGCATTGAATTCCTTCTCCAGCTTATGGAACGTTGCT GGAGCCAAAGCAGAAGAACACGATCTGATGAATCTAATGTGTGGG AAAGGGTTTGGTTTGGGCATGTTCAGCATAATCATGCTTATTGTGG GAACGCAACTGAACCAATTACAGTTGTACTTAACAAATTGGTCCCA GAATAACTTTGGATGGAATCCATCGGGAACCACAACAGAACCCTGA GTTCTAAAAGTGGAAAGTAAAACACCAATTAACCCATGGACGTGGA AAAGAGGCATCACGACATAAGATCTGTCCAAGGGCGTTAGCTTGTA AGTGTTAGCAATGTTCAACGTGCTTCTCACAATGTTCAAATGTAACA AAGGCACCGTTTTTGGAGTGGAGGTGGTACCACTGGTATGCAAAA TCAGGGCAACGTCACTGGAACGGGCAAACCCAGGGAATTTAACGG GATT TGTGTTGACAAATTTGGCGTTGTTCAAAGACCGGTAAATAAC CCTTTTGTAGTTGTCCTCTGGAGAGTATATATCATACTCTACCCTAA ACCTGGTCGCATCGAAGGCCAGCTCTACGATAAAACATCCAAACG TGGAGGCAGATTTTAGAATTTCAGAACTCTGTAACTTTGTGGTACC CTTTGGGACGCAAATCGCCTTAGATTTCAGGTCATTCAAATAAAAAT TGAACTCCTTTTCCTTATAATTGGGATTCAAGGGCGCGCCAATTTTA GCGTCCATAGTAGCACCGAGGAAAGCGACGATAAATTCCAGCCCA TTACGCATGGATATCGCCACTGTATCTTGTCTGAAAACAGCTCCGT ACAATGGAGAATTAGGATTTGTGAACATGGTCTGGAAGTGACCCAC CATGTGGGATAGATCCCTGTAGGTCACCTGAGTGTCCGTTTCAGG AACAATAACGGCGACATTATCGGATACGCTAAAAGTATCGTTGAAC GAAGCAGTAACAGTAGCGGCACTTGTCAT (SEQ ID NO: 14) ACLY (Homo sapiens): GCGAGCCGATGGGGGCGGGGAAAAGTCCGGCTGGGC CGGGACAAAAGCCGGATCCCGGGAAGCTACCGGCTGCTGGGGT GCTCCGGATTTTGCGGGGTTCGTCGGGCCTGTGGAAGAAGCTGC CGCGCACGGACTTCGGCAGAGGTAGAGCAGGTCTCTCTGCAGCC ATGTCGGCCAAGGCAATTTCAGAGCAGACGGGCAAAGAACTCCTT TACAAGTTCATCTGTACCACCTCAGCCATCCAGAATCGGTTCAAGT ATGCTCGGGTCACTCCTGACACAGACTGGGCCCGCTTGCTGCAG GACCACCCCTGGCTGCTCAGCCAGAACTTGGTAGTCAAGCCAGA CCAGCTGATCAAACGTCGTGGAAAACTTGGTCTCGTTGGGGTCAA CCTCACTCTGGATGGGGTCA AGTCCTGGCTGAAGCCACGGCTGG GACAGGAAGCCACAGTTGGCAAGGCCACAGGCTTCCTCAAGAAC TTTCTGATCGAGCCCTTCGTCCCCCACAGTCAGGCTGAGGAGTTC TATGTCTGCATCTATGCCACCCGAGAAGGGGACTACGTCCTGTTC CACCACGAGGGGGGTGTGGACGTGGGTGATGTGGACGCCAAGG CCCAGAAGCTGCTTGTTGGCGTGGATGAGAAACTGAATCCTGAGG ACATCAAAAAACACCTGTTGGTCCACGCCCCTGAAGACAAGAAAG AAATTCTGGCCAGTTTTATCTCCGGCCTCTTCAATTTCTACGAGGA CTTGTACTTCACCTACCTCGAGATCAATCCCCTTGTAGTGACCAAA GATGGAGTCTATGTCCTTGACTTGGCGGCCAAGGTGGACGCCAC TGCCGACTACATCTGCAAAGTGAAGTGGGGTGACATCGAGTTCCC TCCCCCCTTCGGGCGGGAGGCATATCCAGAGGAAGCCTACATTG CAGACCTCGATGCCAAAAGTGGGGCAAGCCTGAAGCTGACCTTG CTGAACCCCAAAGGGAGGATCTGGACCATGGTGGCCGGGGGTG GCGCCTCTGTCGTGTACAGCGATACCATCTGTGATCTAGGGGGTG TCAACGAGCTGGCAAACTATGGGGAGTACTCAGGCGCCCCCAGC GAGCAGCAGACCTATGACTATGCCAAGACTATCCTCTCCCTCATG ACCCGAGAGAAGCACCCAGATGGCAAGATCCTCATCATTGGAGG CAGCATCGCAAACTTCACCAACGTGGCTGCCACGTTCAAGGGCAT CGTGAGAGCAATTCGAGATTACCAGGGCCCCCTGAAGGAGCACG AAGTCACAATCTTTGTCCGAAGAGGTGGCCCCAACTATCAGGAGG GCTTACGGGTGATGGGAGAAGTCGGGAAGACCACTGGGATCCCC ATCCATGTCTTTGGCACAG AGACTCACATGACGGCCATTGTGGGC ATGGCCCTGGGCCACCGGCCCATCCCCAACCAGCCACCCACAGC GGCCCACACTGCAAACTTCCTCCTCAACGCCAGCGGGAGCACAT CGACGCCAGCCCCCAGCAGGACAGCATCTTTTTCTGAGTCCAGG GCCGATGAGGTGGCGCCTGCAAAGAAGGCCAAGCCTGCCATGCC ACAAGATTCAGTCCCAAGTCCAAGATCCCTGCAAGGAAAGAGCAC CACCCTCTTCAGCCGCCACACCAAGGCCATTGTGTGGGGCATGC AGACCCGGGCCGTGCAAGGCATGCTGGACTTTGACTATGTCTGCT CCCGAGACGAGCCCTCAGTGGCTGCCATGGTCTACCCTTTCACTG GGGACCACAAGCAGAAGTTTTACTGGGGGCACAAAGAGATCCTG ATCCCTGTCTTCAAGAACATGGCTGATGCCATGAGGAAGCACCCG GAGGTAGATGTGCTCATCAACTTTGCCTCTCTCCGCTCTGCCTAT GACAGCACCATGGAGACCATGAACTATGCCCAGATCCGGACCATC GCCATCATAGCTGAAGGCATCCCTGAGGCCCTCACGAGAAAGCT GATCAAGAAGGCGGACCAGAAGGGAGTGACCATCATCGGACCTG CCACTGTTGGAGGCATCAAGCCTGGGTGCTTTAAGATTGGCAACA CAGGTGGGATGCTGGACAACATCCTGGCCTCCAAACTGTACCGC CCAGGCAGCGTGGCCTATGTCTCACGTTCCGGAGGCATGTCCAA CGAGCTCAACAATATCATCTCTCGGACCACGGATGGCGTCTATGA GGGCGTGGCCATTGGTGGGGACAGGTACCCGGGCTCCACATTCA TGGATCATGTGTTACGCTATCAGGACACTCCAGGAGTCAAAATGA TTGTGGTTCTTGGAGAGATTGGGGGCACTGAGGAATATAAGATTT GCCGGGGCATCAAGGAGG GCCGCCTCACTAAGCCCATCGTCTGC TGGTGCATCGGGACGTGTGCCACCATGTTCTCCTCTGAGGTCCAG TTTGGCCATGCTGGAGCTTGTGCCAACCAGGCTTCTGAAACTGCA GTAGCCAAGAACCAGGCTTTGAAGGAAGCAGGAGTGTTTGTGCC CCGGAGCTTTGATGAGCTTGGAGAGATCATCCAGTCTGTATACGA AGATCTCGTGGCCAATGGAGTCATTGTACCTGCCCAGGAGGTGC CGCCCCCAACCGTGCCCATGGACTACTCCTGGGCCAGGGAGCTT GGTTTGATCCGCAAACCTGCCTCGTTCATGACCAGCATCTGCGAT GAGCGAGGACAGGAGCTCATCTACGCGGGCATGCCCATCACTGA GGTCTTCAAGGAAGAGATGGGCATTGGCGGGGTCCTCGGCCTCC TCTGGTTCCAGAAAAGGTTGCCTAAGTACTCTTGCCAGTTCATTGA GATGTGTCTGATGGTGACAGCTGATCACGGGCCAGCCGTCTCTG GAGCCCACAACACCATCATTTGTGCGCGAGCTGGGAAAGACCTG GTCTCCAGCCTCACCTCGGGGCTGCTCACCATCGGGGATCGGTT TGGGGGTGCCTTGGATGCAGCAGCCAAGATGTTCAGTAAAGCCTT TGACAGTGGCATTATCCCCATGGAGTTTGTGAACAAGATGAAGAA GGAAGGGAAGCTGATCATGGGCATTGGTCACCGAGTGAAGTCGA TAAACAACCCAGACATGCGAGTGCAGATCCTCAAAGATTACGTCA GGCAGCACTTCCCTGCCACTCCTCTGCTCGATTATGCACTGGAAG TAGAGAAGATTACCACCTCGAAGAAGCCAAATCTTATCCTGAATGT AGATGGTCTCATCGGAGTCGCATTTGTAGACATGCTTAGAAACTG TGGGTCCTTTACTCGGGAGGAAGCTGATGAATATATTGACATTGG AGCCCTCAATGGCA TCTTTGTGCTGGGAAGGAGTATGGGGTTCAT TGGACACTATCTTGATCAGAAGAGGCTGAAGCAGGGGCTGTATCG TCATCCGTGGGATGATATTTCATATGTTCTTCCGGAACACATGAGC ATGTAA (SEQ ID NO: 15) FAS (Mycobacterium subsp.bovis bovid): ATGAGTCAGACGGTGCGCGGTGTGATCGCACGACAAAA GGGCGAACCCGTTGAGCTGGTGAACATTGTCGTCCCGGATCCCG GACCCGGCGAGGCCGTGGTCGACGTCACCGCCTGCGGGGTATGC CATACCGACCTGACCTACCGCGAGGGCGGCATCAACGACGAATAC CCTTTTCTGCTCGGACACGAGGCCGCGGGCATCATCGAGGCCGTC GGGCCGGGTGTAACCGCAGTCGAGCCCGGCGACTTCGTGATCCT GAACTGGCGTGCCGTGTGCGGCCAGTGCCGGGCCTGCAAACGCG GACGGCCCCGCTACTGCTTCGACACCTTTAACGCCGAACAGAAGA TGACGCTGACCGACGGCACCGAGCTCACTGCGGCGTTGGGCATC GGGGCCTTTGCCGATAAGACGCTGGTGCACTCTGGCCAGTGCAC GAAGGTCGATCCGGCTGCCGATCCCGCGGTGGCCGGCCTGCTGG GTTGCGGGGTCATGGCCGGCCTGGGCGCCGCGATCAACACCGGC GGGGTAACCCGCGACGACACCGTCGCGGTGATCGGCTGCGGCGG CGTTGGCGATGCCGCGATCGCCGGTGCCGCGCTGGTCGGCGCCA AACGGATCATCGCGGTCGACACCGATGACACGAAGCTTGACTGGG CCCGCACCTTCGGCGCCACCCACACCGTCAACGCCCGCGAAGTC GACGTCGTCCAGGCCATCGGCGGCCTCACGGATGGATTCGGCGC GGACGTGGTGATCGACGCCGTCGGCCGACCGGAAACCTACCAGC AGGCC TTCTACGCCCGCGATCTCGCCGGAACCGTTGTGCTGGTGG GTGTTCCGACGCCCGACATGCGCCTGGACATGCCGCTGGTCGACT TCTTCTCTCACGGCGGTGCGCTGAAGTCGTCGTGGTACGGCGATT GCCTGCCCGAAAGCGACTTCCCCACGCTGATCGACCTTTACCTGC AGGGCCGGCTGCCGCTGCAGCGGTTCGTTTCCGAACGCATCGGG CTCGAAGACGTCGAGGAGGCGTTCCACAAGATGCATGGCGGCAA GGTATTGCGTTCGGTGGTGATGTTGTGA (SEQ ID NO: 16) AMPK (Homo sapiens): AGTTCCTGGAGAAAGATGGCGACAGCCGAGAAGCAGAA ACACGACGGGCGGGTGAAGATCGGCCACTACATTCTGGGTGACAC GCTGGGGGTCGGCACCTTCGGCAAAGTGAAGGTTGGCAAACATGA ATTGACTGGGCATAAAGTAGCTGTGAAGATACTCAATCGACAGAAG ATTCGGAGCCTTGATGTGGTAGGAAAAATCCGCAGAGAAATTCAGA ACCTCAAGCTTTTCAGGCATCCTCATATAATTAAACTGCACCAGGT CATCAGTACACCATCTGATATTTTCATGGTGATGGAATATGTCTCAG GAGGAGAGCTATTTGATTATATCTGTAAGAATGGAAGGAAATCTGA TGTACCTGGAGTAGTAAAAACAGGCTCCACGAAGGAGCTGGATGA AAAAGAAAGTCGGCGTCTGTTCCAACAGATCCTTTCTGGTGTGGAT TATTGTCACAGGCATATGGTGGTCCATAGAGATTTGAAACCTGAAA ATGTCCTGCTTGATGCACACATGAATGCAAAGATAGCTGATTTTGG TCTTTCAAACATGATGTCAGATGGTGAATTTTTAAGAACAAGTTGTG GCTCACCCAACTATGCTGCACCAGAAGTAATTTCAGGAAGATTGTA TGCAGGCCCAGAGGTA GATATATGGAGCAGTGGGGTTATTCTCTA TGCTTTATTATGTGGAACCCTTCCATTTGATGATGACCATGTGCCAA CTCTTTTTAAGAAGATATGTGATGGGATCTTCTATACCCCTCAATAT TTAAATCCTTCTGTGATTAGCCTTTTGAAACATATGCTGCAGGTGGA TCCCATGAAGAGGGCCACAATCAAAGATATCAGGGAACATGAATG GTTTAAACAGGACCTTCCAAAATATCTCTTTCCTGAGGATCCATCAT ATAGTTCAACCATGATTGATGATGAAGCCTTAAAAGAAGTATGTGA AAAGTTTGAGTGCTCAGAAGAGGAAGTTCTCAGCTGTCTTTACAAC AGAAATCACCAGGATCCTTTGGCAGTTGCCTACCATCTCATAATAG ATAACAGGAGAATAATGAATGAAGCCAAAGATTTCTATTTGGCGAC AAGCCCACCTGATTCTTTTCTTGATGATCATCACCTGACTCGGCCC CATCCTGAAAGAGTACCATTCTTGGTTGCTGAAACACCAAGGGCAC GCCATACCCTTGATGAATTAAATCCACAGAAATCCAAACACCAAGG TGTAAGGAAAGCAAAATGGCATTTAGGAATTAGAAGTCAAAGTCGA CCAAATGATATTATGGCAGAAGTATGTAGAGCAATCAAACAATTGG ATTATGAATGGAAGGTTGTAAACCCATATTATTTGCGTGTACGAAG GAAGAATCCTGTGACAAGCACTTACTCCAAAATGAGTCTACAGTTA TACCAAGTGGATAGTAGAACTTATCTACTGGATTTCCGTAGTATTGA TGATGAAATTACAGAAGCCAAATCAGGGACTGCTACTCCACAGAGA TCGGGATCAGTTAGCAACTATCGATCTTGCCAAAGGAGTGATTCAG ATGCTGAGGCTCAAGGAAAATCCTCAGAAGTTTCTCTTACCTCATC TGTGACCTCACTTGACTCTTCTCCTG TTGACCTAACTCCAAGACCT GGAAGTCACACAATAGAATTTTTTGAGATGTGTGCAAATCTAATTAA AATTCTTGCACAATAA (SEQ ID NO: 17)
[0146] The vector was transformed into Po1g Yarrowia lipolytica strain and selected on leucine-deficient agar plates. Colonies were sorted for the correct insert into the genome using PCR. Δ9-FW AATGGTGAAAAACGTGGACCAAGTGGA (SEQ ID NO: 18) Δ9-REV ATGGATCCCTAAGCAGCCATGCCAGACATAC (SEQ ID NO: 19) GLUT1-FW AATGGAGCCCAGCAGCAAGAAGGTGA (SEQ ID NO: 20) GLUT1-REV AATGGGTACCTCACACTTGGGAGTCAGCC (SEQ ID NO: 21) Hemoglobin FW AGAGACCGGGTTGGCGGCGCA (SEQ ID NO: 22) Hemoglobin REV CAGCGTCTTGAGCGTACAAA (SEQ ID NO: 23) Cytochrome FW A ATGATCATCAACGGCAAGGTCT (SEQ ID NO: 24) Cytochrome REV TTATTTCTGACCCTGGAGGTAGAAG (SEQ ID NO: 25) Pyruvate carboxylase FW AATGCTTAAGTTCCGAACAGT (SEQ ID NO: 26) PyruvatoCGAT carboxylate - CCAGGATG (SEQ ID NO: 27)
[0147] The resulting colony was grown in YPD medium (total medium: yeast extract, peptone, dextrose) and YNB medium (minimum medium, containing all nutrients but no amino acids, and no nitrogen or carbon source). When grown on YNB medium was used, nitrogen was provided as ammonium sulfate and carbon was provided as glucose at a Carbon to Nitrogen ratio of 150. This C/N ratio is necessary to trigger oil buildup. After depletion of excess nitrogen, the sugar is channeled to oil accumulation in the yeast.
[0148] Oil Collection: Cells were grown in nitrogen-restricted growth medium. After 72 hours, cells are collected and dried at 60°C for 2 days. Cells were directly treated with 1% sulfuric acid and methanol for 24 hours at 90°C. The oil was converted to FAME (fatty acid methyl esters) and extracted with hexane. The hexane extraction is repeated twice to recover 95% FAME. The hexane fraction is evaporated and resuspended in 5 ml of hexane. 10 μl of the fraction is injected into GC-MS to quantify FAME.
[0149] Cell cultures were collected and prepared for fatty acid analysis as described previously (Voelker and Davies, 1994). The fatty acid content of each sample was quantified by GC-MS using a simple quadruple MS with an electron impact ionization source. The GC column was a 30 m long HP-5 MS (5% phenyl)-methylpolysiloxane with an ID of 0.25 mm and a film thickness of 25 µm. GC elution conditions were as follows: 100°C as the starting temperature (5 min), 15 min ramp to 250°C, held at 250°C for 10 min. EXAMPLE 1
[0150] A qualitative profile of total free fatty acid (FFA) pool was probed in lipolytica culture grown in log and stationary growth phases using GC-MS (Figure 1 A-C). The largest group of FFA is comprised of saturated palmitic and stearic acids and unsaturated oleic acid. A comparison of FFA profiles in the two growth phases revealed absence of oleic acid in the stationary phase, while similar peak intensities of stearic acid and oleic acid were observed in the log phase (Fig.1 A, B). Analysis of total lipids (FFA + lipids) during the stationary phase recovered a partial amount of oleic acid, suggesting that oleic acid is being directed towards the formation of TAG (Fig. 1C). The remaining oleic acid group is used for downstream polyunsaturated fatty acids and therefore cannot be redeemed. Therefore, oleic acid is channeled to TAG formation in a temporal mode during stationary growth phase that coincides with the activation regulation of intracellular TAG storage pathway. This suggests that a checkpoint mechanism may exist to monitor oleic acid levels to regulate oil buildup. EXAMPLE 2
[0151] Since in mouse SCD it is essential for lipogenesis (see, for example, Regulation of stearoyl-CoA desaturases and role in metabolism. Prog Lipide Res. 2004 Mar;43(2):91-104) and is reported to be important for the synthesis of unsaturated fatty acids in many organisms, the role of Y. lipolytica SCD as a rate limiting step in TAG accumulation was tested. Protein sequence analysis of the Saccharomyces cerevisiae OLE1 gene encoding SCD against Y. lipolytica protein sequences revealed a protein with 51% identity. Y. lipolytica desaturase contains three histidine boxes and a cytochrome b5 domain typical for other stearoyl-CoA desaturases. Since desaturase enzymes are highly regulated at the level of gene transcription (see, for example, Regulation of stearoyl-CoA desaturase by poly-insaturated fatty acids and cholesterol. James M. Ntambi. Journal of Lipide Research, Vol. 40, 1549 -1558, September 1999) and during the log phase and stationary phase of cell growth (see Mol Cell Biol Res Commun. 1999 Apr;1(1):36-43), native gene expression of Y. lipolytica desaturase with a quasi-constitutive promoter. A single copy of the modified gene was stably integrated into the genome. The GC-MS profile between the mutant and wild type strains showed a significant increase in the ratio of unsaturated fatty acids to saturated fatty acids (Fig. 2 A, B). Confocal microscopy of intracellular lipids stained with Nile red showed a correlation between elevated unsaturated fatty acids and excess TAG accumulation (Fig. 2C: wild type, Fig. 2D: overexpressing SCD). In many cases, the entire cell volume of the SCD overexpanding cells is completely filled with TAG (Fig. 2D). These findings provide evidence for a key regulatory gene that surprisingly is sufficient to induce intracellular TAG superaccumulation by altering the ratio of unsaturated to saturated fatty acids.
[0152] Confocal imaging of the growing and stationary cells revealed a colliding difference in the pattern of oil accumulation. the intracellular TAG mobility of stationary phase oil-rich mutant cells after re-entry into mitotic cell cycle was tested. Stationary phase cells were batch fed with minimal medium containing the highest concentration of sugars (300 g/l). Cells efficiently re-entered log phase and followed rapid growth and biomass production consuming all sugars within 96 hours. Interestingly, image analysis showed extracellular oil with excess mutant strain accumulation even during the log phase, which is atypical for oleaginous yeast. Although wild-type cells were unable to grow at high sugar concentrations, continued production of oil and yeast-like bodies were absent from the log phrase even at growth-friendly sugar concentrations.
[0153] Taken together, these results establish a continuous batch fed process using high concentrations of sugar, and suggest that the engineered yeast strain is capable of accumulating oil continuously during the log phase and stationary growth phase. EXAMPLE 3
[0154] Two types of mutant yeast were generated, which overexpress the following genes: Mutant 1: SCD, Hemoglobin, Glut1, Cytochrome; Mutant 2: Hemoglobin, Glut1, Cytochrome. The respective genes were cloned into plasmid YLEX between PmlI and Kpn sites. The vector was transformed into Po1g Yarrowia lipolytica strain and selected on Leucine-deficient agar plates. Colonies were sorted for the correct insert into the genome using PCR. The resulting colony was grown on YPD medium and YNB medium with a carbon to nitrogen (C/N) ratio of 150. This C/N ratio is necessary to trigger oil buildup. After depletion of excess nitrogen, the sugar is channeled to oil accumulation in the yeast.
[0155] In order to measure maximum oil accumulation, cells were grown in nitrogen restricted growth medium. After 72 hours, cells were collected and dried at 60°C for 2 days. Cells were directly treated with 1% sulfuric acid and methanol for 24 hours at 90°C. The oil was converted to FAME (fatty acid methyl esters) and extracted with hexane. The hexane extraction was repeated twice to recover 95% FAME. The hexane fraction was evaporated and resuspended in 5 ml of hexane. 10 μl of the fraction was injected into GC-MS to quantify FAME. The maximum oil accumulation in the mutant strains was 80 grams/l.
[0156] The glucose entry kinetics of mutant 1 ("D9") and wild-type yeast ("LS") were compared. Figure 3 shows that mutant 1 consumed all of the provided sugar after 72 hours, so wild-type yeast only consumed about 70% of the provided sugar. It has been observed that wild-type strains do not consume all the sugars even over an extended period of time.
[0157] It was then determined whether the mutant strains can use biomass hydrolysates as a carbon source. A 2 L bioreactor was fitted containing corn husk hydrolysates (Hz) in the presence of 1% yeast extract. Hz contains 20 grams of glucose. Added (batch fed) 180 g of glucose to conc. end of 200 g/l. It was determined that wild type cannot grow in the Hz toxic biomass. Mutant 1 and mutant 2 cells were grown in rocker flask at a final OD of 3 in 50 ml. The overnight culture was added to the respective bioreactor and fermentation was carried out for 72 hours at 30°C. The two reactors, one with mutant 1 and the other with mutant 2, were operated under identical conditions. Agitation was 800 rpm and the pH was adjusted to 5.5.
[0158] Both strains consumed around 50% of the glucose supplied in 72 hours due to limitation of some nutrient factors in the medium (Figure 4A, showing mutant strain 1). The reason for 50% sugar consumption in both strains is due to the presence of Glut1 which is known to transport glucose into the cell. Mutant 1 consumed 123 grams of glucose, so mutant 2 consumed 105 grams of sugar. This result shows that the mutant cells can consume almost 50% of the sugars, and resist Hz toxicity very well compared to the wild type, which does not grow well and consumes less than 10 grams of sugars in previous experiments. The mutant strains showed robust growth and good consumption of sugars. The remaining sugars were not consumed due to some deprivation of nutrient factors (see, Figure 5 and 6).
[0159] Mutant 1 (with overexpressed gene combination) showed increased oil synthesis as compared to mutant 2. Mutant 1 produced 26 grams of oil per liter (Figure 4B), and mutant 2 produced 14 grams of oil per litre. This suggests that overexpression of a combination of genes not only results in increased consumption of supplied sugars, but also increased production of more oil, a useful biofuel precursor. EXAMPLE 4
[0160] The growth advantage, total lipid production, carbohydrate to lipid substrate conversion efficiency, and substrate tolerance between the engineered strain and the wild-type strain in a 2-liter fermenter vessel were then measured.
[0161] The total amount of lipid was measured using GC-MS (Figure 7). A 10-fold higher lipid production (80 g/l) was observed in the engineered strain as compared to the wild-type strain, representing a 20-fold increase over the Yarrowia lipolytica strain described by others (S. Papanikolaou I. Chevalot, M. Komaitis, I. Marc G. Aggelis, Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures Appl Microbiol Biotechnol. 2002 Mar;58(3):308-12.). The dominant species of monounsaturated fatty acid was oleic acid which increased 8.5-fold (g/l) as compared to the control strain (Figure 8). The ratio of total unsaturated to saturated fatty acids was significantly increased, total unsaturated fatty acids are not increased over saturated fatty acids; however, few of them are in the case of c18.1 (see Figures 7 and 8). The conversion efficiency of sugar into oil of the mutant strain was determined to be 0.28 g/g, approaching theoretical values when considering the sugar used for biomass production.
[0162] A remarkable and unexpected 32-fold growth advantage was observed between the engineered strain and the wild-type strain (Fig. 9). The growth characteristic of the mutant strain remains the same at sugar concentrations that were osmotic-lethal to the wild-type strain (Fig. 10). Higher sugar tolerance is particularly important for gravity top fermentation commonly employed in industrial biofuel production. Previously, an inverse correlation was observed between higher biomass production and lipid accumulation in Yarrowia lipolytica cultures (Papanikolaou S, Chevalot I, Komaitis M, Marc I, Aggelis G. Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl Microbiol Biotechnol. 2002 Mar;58(3):308-12.). Therefore, the link between higher biomass production and excess lipid accumulation in our engineered strain was unexpected. Since fat storage is mainly used for membrane synthesis and budding activities (FEBS J. 2008 Nov;275(22):5552-63), a possibility that cells in the log phase re-direct the flow of excess lipid towards membrane synthesis, via activation of cell division pathways and/or lipid secretion to extracellular medium. This would compensate for excess lipid production earlier in the intracellular accumulation of lipid following entry into the stationary phase of the cell cycle. In fact, higher biomass production was coupled with lipid secretion during the early growth phase.
[0163] Figure 11 shows the growth and lipid production kinetics of mutant and wild-type Y. lipolytica. Not only does the mutant strain exhibit a strong growth advantage, but it also produces a significantly higher amount of fatty acids as compared to the wild-type (control) strain.
[0164] Taken together, these results demonstrate efficient metabolic design of oilseed yeast to exhibit highly desirable multiple phenotypes in glucose as a single carbon source. EXAMPLE 5
[0165] The regulatory mechanism of SCD that enhances the diverse phenotypic traits of the mutant strain has been probed. Given the low sequence identity of the Yarrowia lipolytica desaturase gene to similar functional genes in the nematode Caenorhabditis elegans and the mouse, the cross-cloned SCD species for fatty acid specificity in Yarrowia lipolytica was tested. The SCD in C. elegans and mouse has similar specificity towards stearic acid, showed higher biomass production, similar to mutants that overexpress native Yarrowia gene. Confocal imaging confirmed excess oil accumulation during the stationary growth phase. These results suggest that desaturase activity towards oleic acid synthesis is linked to TAG super-accumulation. Since SCD in yeast is known to be regulated at the transcriptional and post-transcriptional level (see Tabor DE, Kim JB, Spiegelman BM, Edwards PA, Identification of conserved cis-elements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase J Biol Chem. 1999 Jul 16;274(29):20603-10; Shimano H, Sterol regulatory element-binding protein family as global regulators of lipid synthetic genes in energy metabolism. Vitam Horm. 2002;65:167-94) , the inhibition of oleic acid feedback in the desaturase gene was investigated as a possible regulatory niche. A single copy of a native desaturase gene with a 1kb upstream sequence including the promoter region was stably integrated. The mutant strain accumulates excessive oil and has a growth and sugar tolerance advantage as with the previous mutant. This shows that, unlike baker's yeast, oil accumulation is not modulated with promoter sequences that trigger desaturase expression. This means that the downregulation of the desaturase gene in Yarrowia is transcriptionally independent and possibly occurs at the metabolite level. This data provides the first mechanistic insight into oil regulation, via the excess inhibitory effects of oleic acid on oleaginous yeast. EXAMPLE 6
[0166] Engineered microbes provided here can be grown on various substrates. Figure 13 shows robust growth of a mutant strain of Y. lipolytica on algal biomass as the carbohydrate source. Figure 14 shows oil accumulation in engineered microbial cells grown in algal biomass. Figure 15 shows oil accumulation in engineered cells grown on crude glycerol. EXAMPLE 7
[0167] Delta-12 desaturase is responsible for converting lipid-containing oleic acid to higher chain lipids. For the proposed biofuel production, C18 chain fatty acids such as stearic acid and acid are preferred in view of the cold flow properties of diesel fuel. It is therefore desirable, in some embodiments, to block or inhibit the conversion of C18 fatty acids to longer chain fatty acids.
[0168] This can be achieved by inhibiting or blocking the expression of the wild-type delta-12 desaturase gene in the host microbe, for example, a microbe over-expressing a Δ9 desaturase (SCD). For this purpose, a nucleic acid construct was generated to knock out wild-type delta-12 desaturase in Yarrowia lipolytica. A schematic structure of the knockout construct is shown in Figure 16. The vector comprises genomic sequences from the delta-12 desaturase gene that flank a fleomycin resistance gene (eg, ZeocinTM). The construct sequence is shown below.
[0169] vector sequence delta-12 desaturase knockout: CCAACAGACCGACCATAGAAATGGATTCGACCACGCAG ACCAACACCGGCACCGGCAAGGTGGCCGTGCAGCCCCCCACGG CCTTCATTAAGCCCATTGAGAAGGTGTCCGAGCCCGTCTACGACA CCTTTGGCAACGAGTTCACTCCTCCAGACTACTCTATCAAGGATAT TCTGGATGCCATTCCCCAGGAGTGCTACAAGCGGTCCTACGTTAA GTCCTACTCGTACGTGGCCCGAGACTGCTTCTTTATCGCCGTTTTT GCCTACATGGCCTACGCGTACCTGCCTCTTATTCCCTCGGCTTCC GGCCGAGCTGTGGCCTGGGCCATGTACTCCATTGTCCAGGGTCT GTTTGGCACCGGTCTGTGGGTTCTTGCCCACGAGTGTGGCCACT CTGCTTTCTCCGACTCTAACACCGAGAGACCGGGTTGGCGGCGC ATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAA TTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACC CCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGC GTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACT AGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAA TAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAA GGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTC TCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTT CTGAGTATAAGAATCATTCAAAATGGCCAAGTTGACCAGTGCCGTT CCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCT GGACCGACCGGCTCGGGTTCTCCCGGGACTT CGTGGAGGACGAC TTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGC GGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGT GGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGT CGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCG AGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGA CCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACT GATCCATGGCCTGTCCCCACGTTGCCGGTCTTGCCTCCTACTACC TGTCCATCAATGACGAGGTTCTCACCCCTGCCCAGGTCGAGGCTC TTATTACTGAGTCCAACACCGGTGTTCTTCCCACCACCAACCTCAA GGGCTCTCCCAACGCTGTTGCCTACAACGGTGTTGGCATTTAGGC AATTAACAGATAGTTTGCCGGTGATAATTCTCTTAACCTCCCACAC TCCTTTGACATAACGATTTATGTAACGAAACTGAAATTTGACCAGA TATTGTTGTAAATAGAAAATCTGGCTTGTAGGTGGCAAAATGCGGC GTCTTTGTTCATCAATTCCCTCTGTGACTACTCGTCATCCCTTTAT GTTCGACTGTCGTATTTCTTATTTTCCATACATATGCAAGTGAGAT GCCCGTGTCCTGGCCATCACCTACCTGCAGCACACCGACCCCAC TCTGCCCCACTACCACGCCGACCAGTGGAACTTCACCCGAGGAG CCGCCGCCACCATCGACCGAGAGTTTGGCTTCATCGGCTCCTTCT GCTTCCATGACATCATCGAGACCCACGTTCTGCACCACTACGTGT CTCGAATTCCCTTCTACAACGCCCGAATCGCCACTGAGAAGATCA AGAAGGTCATGGGCAAGCACTACCGACACGACGACACCAACTTCA TCAAGTCTCTTTACACTGTCGCCC GAACCTGCCAGTTTGTTGAAG GTAAGGAAGGCATTCAGATGTTTAGAAACGTCAATGGAGTCGGAG TTGCTCCTGACGGCCTGCCTTCTAAAAAGTAGAGCTAGAAATGTTA TTTGATTGTGTTTTAACTGAACAGCA (SEQ ID NO: 28)
[0170] A number of genes including Δ9 desaturase, Glut1, hemoglobin and cytochrome b5, were over-expressed in Yarrowia lipolytica delta-12 desaturase knockout cells to additionally increase sugar flux into the cell and increase oil content. A marked increase in cell size was observed with up to 95% by volume of cells filled with oil. EXAMPLE 8
[0171] Yarrowia lipolytica over-expressing SCD was grown in 3% acetic acid solution for 148 hours (Figure 17). Cell cultures were contacted with 2% glycerol at about 84 hours to provide glycerol to trigger fatty acid production. The last one is the bottleneck in oil production using acetate as a feed stock. A marked increase in oil production was observed by confocal laser microscopy using a contact of glycerol in acetate medium showing a new process to efficiently produce oils with better economic factors.
[0172] While various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages herein described, and each such variation and/or modification is intended to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are significant to be exemplary and that actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or will be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is therefore to be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and equivalents thereof, the invention may be practiced otherwise than as specifically described and claimed. . The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, are included within within the scope of the present invention.
[0173] All definitions, as defined and used herein, shall be understood to control dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of defined terms.
[0174] The indefinite articles "a" and "an", as used herein, in the specification and claims, unless clearly stated, shall be understood to mean "at least one."
[0175] The phrase "and/or," as used herein in the specification and claims, is to be understood to mean "either or both" of the elements so joined together, i.e. elements that are present together in some cases, and separately present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those specifically identified elements, unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B", when in conjunction with open-ended language, such as "comprising", may refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0176] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be interpreted as being inclusive, that is, including at least one, but also including more than one, of a number of, or list of elements, and optionally additional unlisted items. Only terms clearly stated to the contrary, such as "only one of", or "exactly one of", or, when used in the claims, "consisting of", shall refer to the inclusion of exactly one element of a number, or list of elements. . In general, the term "or", as used herein, should only be interpreted as indicating exclusive alternatives (i.e., "either or the other, but not both") when preceded by exclusivity terms, such as "either", " one of", "only one of", or "exactly one of". "Consisting essentially of", when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0177] As used herein in the specification and claims, the phrase "at least one", in reference to a list of one or more elements, shall be understood to mean at least one element selected from any one or more of the elements in the element list, but not necessarily including at least one of each and every element specifically listed within the element list, and not excluding any combinations of elements in the element list. This definition also allows elements to optionally be present other than the specifically identified elements within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and/or B") may refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[0178] It should also be understood that, unless clearly stated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited. . REFERENCES 1. J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001), 978-0879695774 2. David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005), 9780879697280 3. John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink , Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11, 2004), 978-0121827786 4. Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002), 978-0123106711 5. Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002), 978-0123106728 6. Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998), 978-0126662603 7. Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), 978-1439802960
[0179] All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference, in their entirety as if each individual publication or patent were specifically and individually indicated. to be incorporated by reference. In the event of conflict, this application, including any definitions herein, will control.

权利要求:
Claims (18)
[0001]
1. Isolated oilseed yeast cell, characterized in that it comprises: a genetic modification that increases the expression of stearoyl CoA desaturase (SCD) relative to an unmodified cell of the same type, wherein the genetic modification comprises: (i) a constitutive or inducible promoter operably linked to a sequence encoding SCD; or (b) a modified SCD promoter, wherein the modification is a complete or partial deletion and/or mutation of a wild-type SCD promoter sequence, resulting in a disruption of the feedback inhibition of the wild-type SCD promoter; and/or wherein the genetic modification is at least one additional copy of an SCD gene; and a genetic modification that reduces delta-12 desaturase expression referring to an unmodified cell of the same type, wherein the genetic modification is the deletion, disruption, mutation, and/or replacement of a coding sequence of a native delta-12 desaturase gene, or a regulatory region, or a part of a regulatory region that regulates expression of a native delta-12 desaturase gene.
[0002]
2. Isolated oleaginous yeast cell according to claim 1, characterized in that the increased or decreased expression of the gene product confers a phenotype conducive to increased conversion of a carbohydrate source to a fatty acid, fatty acid derivative and /or triacylglycerol (TAG) to the cell, referring to an unmodified cell of the same type.
[0003]
3. Isolated oleaginous yeast cell according to claim 2, characterized in that the phenotype is a modified fatty acid profile, a modified TAG profile, an increased rate of fatty acid and/or triacylglycerol synthesis, an increase in conversion yield, an increased accumulation of triacylglycerol in the cell, and an increased tolerance of osmotic stress, an increased proliferation rate, an increased cell volume, and/or an increased tolerance of a substance at a lethal concentration to and/or inhibition of proliferation of unmodified cells of the same cell type by the cell.
[0004]
4. Isolated oleaginous yeast cell according to claim 3, characterized in that the cell is viable under conditions of lethal osmotic tension to unmodified cells.
[0005]
5. Isolated oleaginous yeast cell according to claim 3, characterized in that the cell proliferation rate is at least 5-fold increased, as compared to unmodified cells of the same cell type.
[0006]
6. Isolated oleaginous yeast cell according to any one of claims 3 to 5, characterized in that the cell tolerates a substance at a lethal concentration for and/or inhibition of proliferation of unmodified cells of the same cell type.
[0007]
7. Isolated oleaginous yeast cell according to any one of claims 3 to 6, characterized in that the rate of synthesis of a fatty acid or a TAG of the cell is at least 5-fold increased, as compared to unmodified cells of the same cell type.
[0008]
8. Isolated oilseed yeast cell according to claim 1, characterized in that the oilseed cell is Yarrowia lipolytica.
[0009]
9. Culture, characterized in that it comprises the oleaginous yeast cell, as defined in claim 1.
[0010]
10. Culture according to claim 9, characterized in that it also comprises a carbohydrate source.
[0011]
11. Method for converting a carbohydrate source into fatty acid or triacylglycerol, characterized in that it comprises: contacting a carbohydrate source with an isolated oleaginous yeast cell, as defined in claim 1, and incubating the contacted carbohydrate source with the cell under conditions suitable for at least partial conversion of the carbohydrate source to a fatty acid, or a triacylglycerol, by the cell.
[0012]
12. Method according to claim 11, characterized in that the carbohydrate source contacted with the isolated oleaginous yeast cell comprises a substance at a lethal concentration for non-modified cells of the same cell type, according to the yeast cell isolated oilseed.
[0013]
13. Method according to claim 12, characterized in that the substance is the source of carbohydrate.
[0014]
14. Method according to claim 13, characterized in that the carbohydrate source is a fermentable sugar, and the concentration of the fermentable sugar is at least 80 g/l after contact with the oil cell.
[0015]
15. Method according to claim 11, characterized in that the oleaginous yeast cell is Yarrowia lipolytica.
[0016]
16. Method for modifying in a yeast cell the fatty acid profile, the triacylglycerol profile, the fatty acid synthesis rate, the triacylglycerol synthesis rate, the extent of fatty acid derivative accumulation, the secretion rate of fatty acid derivative, the carbohydrate to fatty acid conversion rate or fatty acid derivative conversion rate, the efficient yield of carbohydrate to fatty acid conversion, or fatty acid derivative conversion, the osmotic stress tolerance, the proliferation rate, cell volume, or tolerance of a toxic substance of a cell for use in converting a carbohydrate source into a fatty acid or triacylglycerol, characterized in that it comprises increasing in the yeast cell the expression of stearoyl CoA desaturase (SCD) referring to an unmodified cell of the same type by introducing a nucleic acid construct comprising an expression cassette comprising a nucleic acid that encodes the SCD under the control of a suitable homologous or heterologous promoter; and/or at least one additional copy of an SCD gene, and decreasing in the yeast cell the expression of a delta-12 desaturase gene referring to an unmodified cell of the same type by introducing deletion, disruption, mutation and/or replacement of a coding sequence of a native delta-12 desaturase gene, or of a regulatory region, or a part of a regulatory region that regulates expression of the native delta-12 desaturase gene.
[0017]
17. Method according to claim 16, characterized in that the modification of the fatty acid profile, the triacylglycerol profile, the fatty acid synthesis rate, the triacylglycerol synthesis rate, the extent of the accumulation of derivative of fatty acid in the cell, or the rate of fatty acid derivative secretion from the yeast cell is increasing the amount of a fatty acid, a fatty acid derivative, and/or a triacylglycerol that is synthesized, accumulated, or secreted by the cell.
[0018]
18. Method according to claim 16, characterized in that the yeast cell is Yarrowia lipolytica.

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法律状态:
2020-10-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-12-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-04-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-07-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-01-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/03/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US30978210P| true| 2010-03-02|2010-03-02|

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US61/309,782|2010-03-02|

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PCT/US2011/026903|WO2011109548A2|2010-03-02|2011-03-02|Microbial engineering for the production of fatty acids and fatty acid derivatives|

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