strains of yeast modified to produce ethanol from acetic acid and glycerol

专利摘要:
yeast strains modified to produce ethanol from acetic acid and glycerol. the present invention relates to processes for the production of ethanol from lignocellulosic hydrolysates comprising hexoses, pentoses and acetic acid, in which genetically modified yeast cells are used which comprise an exogenous gene encoding an acetaldehyde dehydrogenase and a bacterial gene that encodes an enzyme with nad + linked glycerol dehydrogenase activity. the process is also characterized by the fact that glycerol is present or is fed to the culture medium in which the modified yeast cell ferments hexoses, pentoses, acetic acid and glycerol in ethanol. the invention further relates to yeast cells for use in such processes. yeast cells advantageously comprise genetic modifications that improve the use of glycerol, such as modifications that increase one or more of the activity of dihydroxyacetone kinase and transport of glycerol into the cell. the yeast cell, more preferably, comprises a functional exogenous xylose isomerase gene and / or functional exogenous genes that give the cell the ability to convert l-arabinose into d-xylulose-5-phosphate and which may comprise a genetic modification that increases acetyl coa synthase activity.
公开号:BR112014012963A2
申请号:R112014012963-0
申请日:2012-11-26
公开日:2020-10-20
发明作者:Johannes Adrianus Maria De Bont；Aloysius Wihelmus Rudolphus Hubertus Teunissen；Paul Klaassen；Wouter Willem Antonius Hartman；Van Beusekon
申请人:Dsm Ip Assets B.V；
IPC主号:

专利说明:

[001] [001] The present invention relates to metabolic engineering in microorganisms, such as yeasts. In particular, the invention relates to yeast strains that have been modified to produce ethanol from acetic acid and glycerol. In addition to acetic acid and glycerol, the yeast strain can also consume hexoses and pentoses for the production of ethanol. The invention also relates to processes in which the modified strains of the invention produce ethanol from acetic acid and glycerol.
[002] [002] Second generation bioethanol is produced from, for example, lignocellulosic fractions of plant biomass, which are hydrolyzed to free monomeric sugars, such as hexoses and pentoses, for fermentation in ethanol. Lignocellulosic hydrolysates contain large amounts of acetic acid, which is a potent inhibitor of the fermentation capacities of microorganisms used for the production of ethanol, such as yeasts.
[003] [003] Sonderegger et al. (2004, Appl Environ Microbiol 70: 2892-2897) describe the heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in the xylose fermenting Saccharomyces cerevisiae strain. In combination with native phosphoquetolase, Sonderegger et al.
[004] [004] Guadalupe et al. (2009, Appl. Environ.
[005] [005] Yu et al. (2010, Bioresour. Technol.
[006] [006] Lee and Dasilva (2006, Metab Eng. 8 (1): 58-65) disclose the yeast Saccharomyces cerevisiae modified to produce 1,2-propanediol from glycerol by introducing inter alia the expression of the mgs and gldA genes Escherichia coli.
[007] [007] It is an object of the present invention to provide yeasts that are capable of producing ethanol from acetic acid and glycerol (and hexoses and pentoses), as well as the processes in which these strains are used for the production of ethanol and / or other fermentation products.
[008] The sequence identity is defined herein as a relationship between two or more amino acid sequences (polypeptide or protein) or two or more nucleic acid sequences (polynucleotide), as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relationship between the amino acid or nucleic acid sequences, as the case may be, as determined by the correspondence between strands of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes for one polypeptide with the sequence for a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods. The terms "sequence identity" or "sequence similarity" mean that two (poly) peptide or nucleotide sequences, when optimally aligned, preferably along the entire length (of at least that of the shortest sequence in comparison) ) and maximizing the number of matches and minimizing the number of gaps, as in the programs ClustalW (1.83), GAP or
[009] [009] The preferred methods for determining identity are designed to give the greatest correspondence between the tested sequences. Methods for determining identity and similarity are encoded in publicly available computer programs. Preferred computer program methods for determining the identity and similarity between two strings include, for example, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit , BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Mol. Biol.
[010] [010] Preferred parameters for comparing polypeptide sequences include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: BLOSSUM62 by Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10,915-10,919 (1992); Gap penalty: 12; and gap extension penalty: 4. A useful program with these parameters is available to the public as the "Ogap" program from the Genetics Computer Group, located in Madison, WI. The above mentioned parameters are the standard parameters for comparisons of amino acids (along with no penalty for final gaps).
[011] [011] Preferred parameters for the comparison of nucleic acids include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: matches = +10, non-matches = 0; Gap penalty: 50; Gap extension penalty: 3. Available as the Genetics Computer Group Gap program, located in Madison, Wisconsin. Standard parameters for nucleic acid comparisons are shown above.
[012] [012] Optionally, to determine the degree of similarity of amino acids, the person skilled in the art may also take into account so-called "conservative" amino acid substitutions, as will be clear to the qualified person.
[013] [013] The nucleotide sequences of the invention can also be defined by their ability to hybridize to the parts of specific nucleotide sequences described herein, respectively, under moderate hybridization conditions, or preferably under stringent hybridization conditions. Stringent hybridization conditions are defined herein as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and more preferably about 200 or more nucleotides, to hybridize at a temperature of about 65 ° C in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65 ° C in a solution comprising about 0.1 M of salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is carried out overnight, that is, for at least 10 hours and preferably the washing is carried out for at least one hour with at least two changes of the washing solution.
[014] [014] Moderate conditions are defined here as conditions that allow nucleic acid sequences of at least 50 nucleotides, preferably about 200 or more nucleotides, to hybridize at a temperature of about 45 ° C in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is carried out overnight, that is, for at least 10 hours, and preferably the washing is carried out for at least one hour with at least two changes of the washing solution. These conditions will normally allow for specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions, in order to specifically identify sequences ranging in identity between 50% and 90%.
[015] [015] The "nucleic acid construct" or "nucleic acid vector" is here understood to mean a man-made nucleic acid molecule, resulting from the use of recombinant DNA technology. The term "nucleic acid construct", therefore, does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may comprise (or part of it) naturally occurring nucleic acid molecules.
[016] [016] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream from transcription direction of the coding sequence transcription initiation site, and is structurally identified by the presence of a DNA polymerase-dependent RNA binding site, transcription initiation sites and other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to a person skilled in the art to act directly or indirectly in regulating the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated physiologically or by development, for example, by applying a chemical inducer.
[017] [017] The term "selectable marker" is a term familiar to one skilled in the art and is used here to describe any genetic entity that, when expressed, can be used to select a cell or cells that contain the selectable marker. The term "reporter" can be used interchangeably with a marker, although it is used primarily to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers can be dominant or recessive or bidirectional.
[018] [018] As used herein, the term "operably linked" refers to a bonding of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
[019] [019] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules formed by a chain of amino acids, with no reference to a specific mode of action, dimension, 3-dimensional structure or origin.
[020] [020] "Fungi" (fungus in the singular) are here understood as heterotrophic eukaryotic microorganisms that digest food externally, absorbing nutrient molecules into their cells. Fungi are a separate kingdom from eukaryotic organisms and include yeasts, molds and mushrooms. The terms fungi, fungus and fungal, as used herein, thus expressly include yeasts, as well as filamentous fungi.
[021] [021] The term "gene" means a fragment of DNA comprising a region (transcribed region), which is transcribed into an RNA molecule (for example, an mRNA) in a cell, operably linked to suitable regulatory regions (for example, a promoter). A gene will normally be made up of several operationally linked fragments, such as a promoter, a 5 'terminal sequence, a coding region and a 3' untranslated sequence (3 'end), which comprises a polyadenylation site. "Expression of a gene" refers to the process by which a region of DNA that is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, that is, that is capable of being translated into a biologically active protein or peptide.
[022] [022] The term "homologous" when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that, in nature, the nucleic acid molecule or polypeptide is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) signal sequence secretion and / or a terminator sequence than in its natural environment.
[023] [023] The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refer to a nucleic acid or protein that does not occur naturally, as part of the organism, from cells,
[024] [024] The "specific activity" of an enzyme is understood here as the amount of activity of a particular enzyme per amount of total protein in the host cell, usually expressed in units of enzyme activity per mg of protein in the total host cell. In the context of the present invention, the specific activity of a particular enzyme can be increased or decreased, compared to the specific activity of the enzyme in one (otherwise identical) of the wild-type host cell.
[025] [025] "Anaerobic conditions" or an anaerobic fermentation process is defined herein as conditions or a fermentation process carried out in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol / L / h, more preferably, 0 mmol / L / h is consumed (that is, oxygen consumption is not detectable), and in which the organic molecules serve as electron donors and electron acceptors.
[026] [026] The expression of an exogenous acetaldehyde dehydrogenase in yeast allows the yeast to convert acetic acid, which can be present in high quantities in lignocellulosic hydrolysates, in ethanol. The NADH-dependent reduction of acetic acid in ethanol has been proposed as a substitute for the formation of glycerol as a redox sink in S. cerevisiae cultures grown in anaerobic glucose, thus allowing a stoichiometric basis for the elimination of glycerol production ( as a by-product) during industrial ethanol production and, consequently, a higher ethanol yield (Guadalupe et al. supra). However, the stoichiometry of these reactions is such that the reduction of one molecule of acetic acid in ethanol would require two molecules of glycerol not to be produced. The present inventors have found, however, that, in practice, the amount of acetic acid that is normally present in industrial lignocellulosic hydrolysates is such that the amount of NADH needed to be reduced to ethanol exceeds the amount of NADH that becomes available at prevent the production of glycerol in yeasts grown under anaerobic conditions. The present inventors have now surprisingly found that much higher amounts of acetic acid can be reduced to ethanol by simultaneous consumption of glycerol by yeast, rather than preventing its production.
[027] [027] Large amounts of glycerol are generated as a by-product of the production of biodiesel from transesterification reactions using vegetable oils or animal fats and an alcohol. It is therefore expected that the availability of crude glycerol is therefore expected to increase in the coming years, as a result of the growth in biodiesel production worldwide. Consequently, large amounts of glycerol will be available at low cost. The present invention provides means and methods for the valorization of glycerol obtained, for example, as a by-product of the production of biodiesel, converting it into ethanol that can be used as biofuel. At the same time, the present invention solves the problem of large amounts of acetic acid which are present in lignocellulose hydrolysates and which inhibit the fermentation capacity of ethanol-producing yeasts from such hydrolysates. Another advantage of the present invention is that, by leaving the high osmolarity glycerol response pathway intact in the yeast cells of the present invention (as opposed to strains in which (all) the glycerolphosphate dehydrogenase genes are inactivated, as described by Guadalupe et al. supra), more robust strains of yeast are obtained that are better able to deal with the osmotic stress that can occur under industrial fermentation conditions.
[028] [028] In a first embodiment, the invention relates to a fungal host cell that comprises an exogenous gene that codes for an enzyme with the ability to reduce acetyl-CoA to acetaldehyde, a gene that gives the cell the ability to convert the acetic acid in ethanol.
[029] [029] Thus, the enzyme catalyzes the conversion of acetyl-CoA to acetaldehyde (and vice versa) and is also referred to as an acetaldehyde dehydrogenase (NAD-dependent acetylation) or an acetyl-CoA reductase. The enzyme can be a bifunctional enzyme that further catalyzes the conversion of acetaldehyde to ethanol (and vice versa; see below). For convenience, it refers here to an enzyme that has at least the ability to reduce acetyl-CoA in any acetaldehyde or ethanol as an "acetaldehyde dehydrogenase".
[030] [030] The exogenous gene can code for a monofunctional enzyme that has only acetaldehyde dehydrogenase activity (that is, an enzyme that has only the ability to reduce acetyl-CoA in acetaldehyde), such as, for example, the acetaldehyde dehydrogenase encoded by E. coli mhpF gene. A suitable exogenous gene encoding an enzyme with acetaldehyde dehydrogenase activity comprises a nucleotide sequence that encodes an amino acid sequence of at least 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 98 , 99% amino acid sequence identity to SEQ ID NO: 1. Suitable examples of prokaryotes comprising monofunctional enzymes with acetaldehyde dehydrogenase activity are provided in Table 1. The amino acid sequences of these monofunctional enzymes are available in public databases and can be used by the person skilled in the art for the design of codon-optimized nucleotide sequences encoding the corresponding monofunctional enzyme (see for example, SEQ ID NO: 2).
[031] [031] Table 1: Enzymes with acetaldehyde dehydrogenase activity related to E. coli mhpF Organism Amino acid identity (%) Escherichia coli str. K12 substr. 100% MG1655 Shigella sonnei 100% Escherichia coli IAI39 99% Citrobacter youngae ATCC 29220 93% Citrobacter sp. 30_2 92% Klebsiella pneumoniae 342) 87% Klebsiella variicola 87% Pseudomonas putida 81% Ralstonia eutropha JMP134 82%
[032] [032] Preferably, the host cell comprises a gene that codes for a bifunctional exogenous enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activity, a gene that gives the cell the ability to convert acetic acid into ethanol. The advantage of using a bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activity, as opposed to separate enzymes for each of the acetaldehyde dehydrogenase and alcohol dehydrogenase activities, is that it allows the direct channeling of the intermediate between the enzymes that catalyze the reactions consecutive routes offering the possibility of an efficient, exclusive and protected means of metabolite delivery. Substrate channeling therefore decreases the transit time of intermediates, prevents the loss of intermediates by diffusion, protects unstable intermediates from solvents, and prevents entry of intermediates in competitive metabolic pathways. Therefore, the bifunctional enzyme allows a more efficient conversion of acetic acid to ethanol, compared to separate enzymes of acetaldehyde dehydrogenase and alcohol dehydrogenase. Another advantage of using the bifunctional enzyme is that it can also be used in host cells having little or no alcohol dehydrogenase activity under the condition used, such as, for example, anaerobic conditions and / or catabolic repression conditions.
[033] [033] Bifunctional enzymes with acetaldehyde dehydrogenase and alcohol dehydrogenase activity are known in the art in prokaryotes and protozoa, including, for example, bifunctional enzymes encoded by the Escherichia coli adhE and Entamoeba histolytica ADH2 genes (see, for example Bruchaus and Tannich, 1994, J. Biochem. 303: 743-748; Burdette and Zeikus, 1994, J.
[034] [034] Table 2: Bifunctional enzymes with acetaldehyde dehydrogenase and alcohol dehydrogenase activity related to E. coli adhE Organism Amino acid identity (%) Escherichia coli str. K12 substr. MG1655 100% Shigella sonnei 100% Escherichia coli IAI39 99% Citrobacter youngae ATCC 29220 93% Citrobacter sp. 30_2 92% Klebsiella pneumoniae 342) 87% Klebsiella variicola 87% Pseudomonas putida 81% Ralstonia eutropha JMP134 82% Burkholderia sp. H160 81% Azotobacter vinelandii DJ 79% Ralstonia metallidurans CH34 70% Xanthobacter autotrophicus Py2 67%
[035] [035] Table 3: Bifunctional enzymes with acetaldehyde dehydrogenase and alcohol dehydrogenase activity related to Entamoeba histolytica Organism Amino acid identity (%) Entamoeba histolytica HM-1: IMSS 99% Entamoeba dispar SAW760 98% Mollicutes bacterium D7 65% Fusoll mortiferum ATCC 9817 64% Actinobacillus succinogenes 130Z 63% Pasteurella multocida Pm70 62% Mannheimia succiniciproducens MBEL55E 61% Streptococcus sp. 2_1_36FAA] 61%
[036] [036] The encoding of the exogenous gene for the bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activity, for an enzyme that has acetaldehyde dehydrogenase activity, is preferably an expression construct that comprises a nucleotide sequence that encodes the linked enzyme operatively to suitable regulatory expression regions / sequences to ensure expression of the enzyme by transforming the expression construct in the host cell of the invention. Thus, the gene or expression construct will comprise at least one promoter that is functional in the host cell operably linked to the coding sequence. The gene or construct may further comprise a terminal sequence '5 upstream of the coding region and an untranslated 3' sequence (end 3 ') consisting of a polyadenylation site and a transcription termination site downstream of the coding sequence .
[037] [037] In one aspect, the invention relates to methods for the preparation or construction of yeast cells of the invention. For this purpose, the conventional genetic and molecular biology techniques that are used are generally known in the art and have been described for example by Sambrook and Russell (2001, "Molecular cloning: a laboratory manual" (3rd edition), Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press) and Ausubel et al. (1987, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York). In addition, the construction of mutated host yeast strains is carried out by means of genetic crosses, sporulation of the resulting diploids, tetra-dissection of the haploid spores containing the desired auxotrophic markers, and purification of colonies of such haploid host yeasts in the selection medium appropriate. All of these methods are standard genetic methods for yeast known to those skilled in the art.
[038] [038] Promoters suitable for expression of the nucleotide sequence encoding the enzyme with acetaldehyde dehydrogenase activity and optionally alcohol dehydrogenase activity (as well as other enzymes of the present invention, see below) include promoters that are preferably insensitive to repression catabolite (glucose), which are active under anaerobic conditions and / or which preferably do not require xylose or arabinose for induction. Promoters having these characteristics are widely available and are known to the person skilled in the art. Suitable examples of such promoters include, for example, promoters of glycolytic genes, such as phosphofrutokinase (PPK), triose phosphate isomerase (TPT), glyceraldehyde-3-phosphate dehydrogenase (GDP, TDH3 or GAPDH), pyruvate kinase (PYK) , phosphoglycerate kinase (PGK), glucose-6-phosphate isomerase promoters (PGI1 promoter) of yeasts. More details on such yeast promoters can be found in (WO 93/03159). Other useful promoters are the promoters of the gene encoding the ribosomal protein (TEF1), the promoter of the lactase gene (LAC4), promoters of alcohol dehydrogenase (ADH1, ADH4, and the like), the enolase promoter (ENO) and the promoter hexose (glucose) carrier (HXT7). Alternatively,
[039] [039] To increase the probability that the enzyme having acetaldehyde dehydrogenase activity and optionally alcohol dehydrogenase is expressed at sufficient levels and in the active form in the transformed host cells of the present invention, the nucleotide sequence that codes for these enzymes, as well as others enzymes of the invention (see below) are preferably adapted to optimize their use of codons from the host cell in question. The adaptability of a nucleotide sequence that encodes an enzyme for the use of codons in the host cell can be expressed as the codon adaptation index (CAI). The codon adaptation index is defined herein as a measure of the adaptability in relation to the use of codons of a gene for the use of codons of highly expressed genes in a particular host cell, or organism.
[040] [040] The nucleotide sequence encodes an enzyme with acetaldehyde dehydrogenase activity and optionally alcohol dehydrogenase, which is preferably expressed in active form in the transformed host cell. Thus, the expression of the nucleotide sequence in the host cell produces an acetaldehyde dehydrogenase with a specific activity of at least 0.005, 0.010, 0.020, 0.050 or 0.10 µmol min-1 (mg of protein) -1, determined as the rate of reduction of acetyl-CoA-dependent NADH in cell extracts of the transformed host cell, at 30 ° C as described in the Examples presented herein.
[041] [041] The host cell to be transformed with a nucleic acid construct comprising a nucleotide sequence that encodes an enzyme with acetaldehyde dehydrogenase and, optionally, alcohol dehydrogenase is preferably a yeast host cell. Preferably, the host cell is a culture cell. The host cell of the invention is preferably a host capable of transporting active or passive pentose (xylose and preferably also arabinose) into the cell. The host cell preferably contains active glycolysis. The host cell may preferably also contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity, so that xylose isomerized from xylose can be metabolized to pyruvate. The host most preferably contains enzymes for converting a pentose (preferably through pyruvate) into a desired fermentation product, such as ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, 1,3-
[042] [042] Yeasts are defined here as eukaryotic microorganisms and include all species in the subdivision Eumycotina (Yeasts: characteristics and identification, J.A.
[043] [043] In another embodiment, the host cell of the present invention further comprises a genetic modification that introduces NAD +-linked glycerol dehydrogenase activity into the cell. Glycerol dehydrogenase encoded by the endogenous yeast gene GCY1 appears to be specific for the cofactor NADP + (CE
[044] [044] Other common names include glycerin dehydrogenase and glycerol: NAD + 2-oxidoreductase.
[045] [045] Preferably, the genetic modification that introduces NAD +-linked glycerol dehydrogenase activity into the host cell is the expression of an NAD +-linked glycerol dehydrogenase that is heterologous to the host cell. More preferably, the nucleotide sequence for the expression of a heterologous glycerol dehydrogenase in the cells of the invention is a sequence that encodes a bacterial glycerol dehydrogenase that use NAD + as a cofactor (CE 1.1.1.6). A suitable example of a bacterial glycerol dehydrogenase linked to NAD + for expression in a host cell of the invention is for example the gldA gene from E. coli described by Truniger and Boos (1994, J Bacteriol. 176 (6): 1796- 1800), whose expression in yeast has already been reported (Lee and Dasilva, 2006, Metab Eng. 8 (l): 58-65). Preferably, the nucleotide sequence encoding a heterologous glycerol dehydrogenase comprises a nucleotide sequence encoding an amino acid sequence of at least 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 98, the amino acid sequence of 99% identity with SEQ ID NO: 7 or a nucleotide sequence encoding an amino acid sequence that has one or more substitutions, insertions and / or deletions, compared to SEQ ID NO: 7.
[046] [046] For the overexpression of the nucleotide sequence encoding glycerol dehydrogenase, the nucleotide sequence (to be overexpressed) is placed in an expression construct in which it is operationally linked to suitable regulatory expression regions / sequences to ensure overexpression of the enzyme glycerol dehydrogenase by transforming the expression construct within the host cell of the invention (see above). Promoters suitable for the (over) expression of the nucleotide sequence encoding the enzyme that has glycerol dehydrogenase activity include promoters that are preferentially insensitive to catabolite (glucose) repression, which are active under anaerobic conditions and / or that, preferably, they do not require xylose or arabinose for induction. Examples of such promoters are given above. The expression of the nucleotide sequence in the host cell produces a specific activity of glycerol dehydrogenase linked to NAD + at least 0.2, 0.5, 1.0, 2.0, or 5.0 U min-1 (mg of protein ) -1, determined in cell extracts from the transformed host cells, at 30 ° C as described in the Examples presented here.
[047] [047] In another embodiment, the host cell of the present invention further comprises a genetic modification that increases the specific activity of dihydroxyacetone kinase in the cell. Transcriptome data showed that endogenous dihydroxyacetone kinase DAK1 is already expressed at high levels in S. cerevisiae. An increase in the activity of dihydroxyacetone kinase in the cells of the invention may therefore not be strictly necessary. However, in a preferred embodiment, for optimal conversion rates, the host cell of the present invention thus comprises a genetic modification that increases the specific activity of dihydroxyacetone kinase in the cell. Dihydroxyacetone kinase is understood here as an enzyme that catalyzes the chemical reaction (EC 2.7.1.29): ATP + glycerone ↔ ADP + glycerone phosphate
[048] [048] Other common names include glycerone kinase, ATP: glycerone phosphotransferase and acetol kinase (for phosphorylation). It is understood that dihydroxyacetone and glycerone are the same molecule. Preferably, the genetic modification causes the overexpression of a dihydroxyacetone kinase, for example, by overexpression of a nucleotide sequence encoding a dihydroxyacetone kinase. The nucleotide sequence encoding the dihydroxyacetone kinase can be endogenous to the cell or it can be a dihydroxyacetone kinase that is heterologous to the cell. The nucleotide sequences that can be used for the overexpression of dihydroxyacetone kinase in the cells of the invention are, for example, the dihydroxyacetone kinase genes of S. cerevisiae (DAK1) and (DAK2) as for example described by Molin et al. (2003, J.
[049] [049] The nucleotide sequences that can be used for the overexpression of a heterologous dihydroxyacetone kinase in the cells of the invention are for example the sequences that code for a bacterial dihydroxyacetone kinase, such as the Dhak gene of Citrobacter freundii for example described by Daniel et al. (1995, J.
[050] [050] For the overexpression of the nucleotide sequence encoding the dihydroxyacetone kinase, the nucleotide sequence (to be overexpressed) is placed in an expression construct in which it is operationally linked to suitable regulatory expression regions / sequences to ensure overexpression of the enzyme dihydroxyacetone kinase by transforming the expression construct within the host cell of the invention (see above). The appropriate promoters for the
[051] [051] In another embodiment, the host cell of the present invention further comprises a genetic modification that increases the transport of glycerin into the cell. Preferably, the genetic modification that increases glycerin transport into the cell is preferably a genetic modification that causes the overexpression of a nucleotide sequence that encodes at least one of a glycerol transport protein and a glycerol channel.
[052] [052] A glycerol transport protein is understood here as a multi-step transmembrane protein that belongs to the superfamily where O-acyltransferases (MBOAT) are attached to the membrane, including for example, the glycerol transport proteins of S.
[053] [053] A glycerol channel is understood here as a member of the family of channel MIP proteins reviewed by
[054] [054] For the overexpression of the nucleotide sequence encoding the glycerol transport protein and / or the glycerol channel protein, the nucleotide sequence (to be overexpressed) is placed in an expression construct in which it is operationally linked to regions / expression regulation sequences suitable to ensure overexpression of the glycerol transport protein and / or the glycerol channel protein by transforming the expression construct in the host cell of the invention (see above). Promoters suitable for (over) expression of the nucleotide sequence encoding the glycerol transport protein and / or the glycerol channel protein include promoters that are preferentially insensitive to catabolite (glucose) repression, which are active under anaerobic conditions and / or that preferably do not require xylose or arabinose for induction. Examples of such promoters are given above. In the cells of the invention, a glycerol transport protein and / or a glycerol channel protein to be overexpressed, preferably overexpressed by at least a factor of 1.1, 1.2, 1.5, 2, 5, 10 or 20, compared to a strain that is genetically identical except for the genetic modification that causes overexpression. Preferably, the glycerol transport protein and / or the glycerol channel protein are overexpressed under anaerobic conditions by at least a factor of 1.1, 1.2, 1.5, 2, 5, 10 or 20, in compared to a strain that is genetically identical except for the genetic modification that causes overexpression. It is to be understood that these overexpression levels can apply to the steady-state level of enzyme activity (specific activity in the cell), to the steady-state level of enzyme protein, as well as to the steady-state level for coding of the transcription for the enzyme in the cell.
[055] [055] In a preferred embodiment of the host cell of the invention, expression of the glycerol channel protein, as defined above, is reduced or inactivated.
[056] [056] In another embodiment, the host cell of the present invention further comprises a genetic modification that increases the specific activity of acetyl-CoA synthase in the cell, preferably under anaerobic conditions, as this activity has limited action under these conditions . Acetyl-CoA synthetase or acetate-CoA ligase (EC 6.2.1.1) is understood here as an enzyme that catalyzes the formation of a new chemical bond between ethyl and coenzyme A (CoA). Preferably, the genetic modification causes the overexpression of an acetyl-CoA synthetase, for example, by overexpression of a nucleotide sequence encoding an acetyl-CoA synthetase. The nucleotide sequence encoding acetyl-CoA synthase can be endogenous to the cell or it can be an acetyl-CoA synthase that is heterologous to the cell. The nucleotide sequences that can be used for the overexpression of acetyl-CoA synthase in the cells of the invention are, for example, the genes for acetyl-CoA synthetase from S. cerevisiae (ACS1 and ACS2) as for example described by De Jong -Gubbels et al. (1998, FEMS Microbiol Lett. 165: 15-20.). Preferably, the nucleotide sequence encoding acetyl-CoA synthetase comprises an amino acid sequence with at least 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% sequence identity of amino acids with at least one of SEQ ID Nos: 13 and 14.
[057] [057] In one embodiment, the nucleotide sequence that is overexpressed encodes an acetyl-CoA synthetase with a high affinity for acetate.
[058] [058] In another embodiment, the nucleotide sequence that is overexpressed encodes an acetyl-CoA synthetase with a high maximum rate (vmax). Use of an acetyl-CoA synthetase with a high maximum rate is preferred for conditions where there is a relatively high concentration of acetic acid in the culture medium, for example, at least 2, 3, 4 or 5 g of acetic acid / L of culture medium. An acetyl-CoA synthetase with a high maximum rate is defined herein as an acetyl-CoA synthetase with a higher maximum rate than the acetyl-CoA synthetase encoded by the ACS1 of S. cerevisiae. Preferably, the acetyl-CoA synthetase with a high maximum rate is acetyl-CoA synthetase, encoded by the ACS2 gene of S. cerevisiae.
[059] [059] For the overexpression of the nucleotide sequence encoding acetyl-CoA synthetase (to be overexpressed) it is placed in an expression construct in which it is operationally linked to suitable regulatory expression regions / sequences to ensure overexpression of the acetyl- CoA synthase with the transformation of the expression construct within the host cell of the invention (see above). Promoters suitable for (over) expression of the nucleotide sequence encoding the enzyme that has acetyl-CoA synthetase activity include promoters that are preferentially insensitive to catabolite (glucose) repression, which are active under anaerobic conditions and / or which preferably do not require xylose or arabinose for induction. Examples of such promoters are given above. In the cells of the invention, an acetyl-CoA synthetase to be overexpressed is overexpressed by at least a factor of 1.1, 1.2, 1.5, 2, 5, 10 or 20, compared to a strain that is genetically identical , except for genetic modification causing overexpression. Preferably, acetyl-CoA synthetase is overexpressed under anaerobic conditions by at least a factor of 2, 5, 10, 20, 50, or 100, compared to a strain that is genetically identical, except for genetic modification causing overexpression . It is to be understood that these levels of overexpression can apply to the steady state level of enzyme activity (specific activity), to the steady state level of the enzyme protein, as well as to the steady state level for encoding transcription. for the enzyme.
[060] [060] In another embodiment, the host cell of the present invention further comprises a genetic modification that reduces the specific NAD + -dependent glycerol-3-phosphate dehydrogenase activity in the cell. Glycerol-3-phosphate dehydrogenase or glycerolphosphate dehydrogenase (EC 1.1.1.8) catalyzes the reduction of dihydroxyacetone phosphate in sn-glycerol-3-phosphate, while oxidizing NADH to NAD +. In the cells of the invention, the specific activity of glycerolphosphate dehydrogenase is preferably reduced by at least a factor of 0.8, 0.5, 0.3, 0.1, 0.05 or 0.01 compared to a strain that is genetically identical, except for genetic modification causing overexpression, preferably under anaerobic conditions.
[061] [061] Preferably, glycerolphosphate dehydrogenase is reduced in the host cell through one or more genetic modifications that decrease expression or inactivate a gene that encodes a glycerolphosphate dehydrogenase. Preferably, the genetic modifications reduce or inactivate the expression of each endogenous copy of the gene encoding a specific glycerolphosphate dehydrogenase in the cell's genome. A given cell can comprise multiple copies of the gene that encodes a specific glycerolphosphate dehydrogenase with one and the same amino acid sequence as a result of di-, poly- or aneuploidy. In such cases, the expression of each copy of the specific gene encoding glycerolphosphate dehydrogenase is preferably reduced or inactivated. Alternatively, a cell can contain several different (iso) enzymes with glycerolphosphate dehydrogenase activity that differ in the amino acid sequence and which are each encoded by a different gene. In such cases, in some embodiments of the invention, it is preferred that only certain types of isoenzymes are reduced or inactivated, while other types are left unchanged (see below). Preferably, the gene is inactivated by deletion of at least a part of the gene or by disruption of the gene, where, in this context, the term gene also includes any non-coding sequence upstream or downstream of the coding sequence, the deletion ( partial) or inactivation that results in a reduction in the expression of glycerol phosphate dehydrogenase activity in the host cell.
[062] [062] A preferred gene encoding a glycerolphosphate dehydrogenase whose activity is to be reduced or inactivated in the cells of the invention is the S. cerevisiae GPD2 gene as described by Eriksson et al. (1995, Mol. Microbiol 17: 95-107), which encodes the amino acid sequence of SEQ ID NO: 15 and its orthologs in other species. Therefore, a gene encoding a glycerolphosphate dehydrogenase whose activity is to be reduced or inactivated in the cells of the invention, is preferably a gene encoding a glycerolphosphate dehydrogenase having an amino acid sequence of at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 15.
[063] [063] In a preferred embodiment of the invention, the host cell of the present invention comprises a high-osmolarity glycerol response pathway. Preferably, therefore, only the activity of the gene (s) encoding a glycerolphosphate dehydrogenase having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 15 is reduced or inactivated , while at least one endogenous gene encoding a glycerolphosphate dehydrogenase having an amino acid sequence with at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 16 is functional . SEQ ID NO: 16 illustrates the amino acid sequence encoded by the GPD1 gene of S. cerevisiae, as described by Albertyn et al. (1994, Mol. Cell. Biol.
[064] [064] Notwithstanding the foregoing, the inventors have now surprisingly found that the inactivation of GPD1 glycerolphosphate dehydrogenase from S. cerevisiae has a more advantageous effect in reducing glycerol production and increasing glycerol and acetate consumption in comparison with the inactivation of GPD2 glycerolphosphate dehydrogenase of S. cerevisiae. Therefore, in a more preferred embodiment, the host cell of the invention comprises a genetic modification that reduces or inactivates the expression of at least the gene (s) encoding a glycerolphosphate dehydrogenase having an amino acid sequence with at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 16 (GPD1).
[065] [065] In another embodiment, the activity of all genes in the host cell that encode a glycerolphosphate dehydrogenase is reduced or inactivated. In such cells, preferably all copies of endogenous genes encoding a glycerolphosphate dehydrogenase having an amino acid sequence with at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity with SEQ ID NO: 15 or 16 are inactive, or at least reduced in expression.
[066] [066] In another embodiment of the invention, the host cell is not a yeast cell that comprises encoding the exogenous gene for an enzyme with the ability to convert pyruvate and coenzyme-A into format and acetyl-CoA. Preferably, the host cell is not a yeast cell comprising a nucleotide sequence encoding a lyase-shaped pyruvate.
[067] [067] In yet another embodiment of the invention, the host cell is a host cell in which the specific dehydrogenase formate activity is at least 81, 85, 90, 95 or 100% of the specific dehydrogenase formate activity in a strain of the host cell that is genetically identical, except for a genetic alteration selected from the group consisting of: a) (the introduction of) an exogenous gene that codes for an enzyme with acetaldehyde dehydrogenase activity, a gene that gives the cell the ability converting acetic acid to ethanol; b) (the introduction of) a bacterial gene that encodes an enzyme for NAD +-linked glycerol dehydrogenase activity; and c) any of the other genetic modifications described hereinabove. Thus, a preferred host cell of the invention is not a yeast cell comprising a genetic modification that reduces the specific NAD + -dependent dehydrogenase format activity in the cell.
[068] [068] In another preferred embodiment, the host cell of the present invention has at least one of the following: a) the xylose isomerization capacity in xylulose; and b) the ability to convert L-arabinose to D-xylulose 5-phosphate. For a) the cell preferably has a functional exogenous xylose isomerase gene, a gene that gives the cell the ability to isomerize xylose into xylulose. For b) the cell preferably has functional exogenous genes that code for an L-arabinose isomerase, an L-ribulokinase and an L-ribulose-5-phosphate-4-epimerase, genes that together give the cell the ability to isomerize and convert L -arabinosis in D-xylulose 5-phosphate.
[069] [069] Fungal host cells that have the ability to isomerize xylose into xylulose, for example, described in WO 03/0624430 and WO 06/009434. The xylose isomerization capacity in xylulose is preferably conferred to the cell by transformation with a nucleic acid construct comprising a nucleotide sequence that encodes a xylose isomerase. Preferably, the cell thus acquires the ability to isomerize xylose into xylulose directly.
[070] [070] Various xylose isomerases (and their amino acids and nucleotide coding sequences), which can be used successfully to give the cell of the invention the ability to isomerize xylose into xylulose directly have been described in the art. These include the xylose isomerases of Piromyces sp. and other anaerobic fungi that belong to the Neocallimastix, Caecomyces, Piromyces or Ruminomyces (WO 03/0624430), Cyllamyces aberensis (USA 20060234364), Orpinomyces (Madhavan et al., 2008, DOI 10.1007 / s00253-008-1794-6) families , bacterial xylose isomerase of the genus Bacteroides, including, for example B.
[071] [071] Fungal host cells having the ability to convert L-arabinose to D-xylulose 5-phosphate for example, as described in Wisselink et al.
[072] [072] The transformed host cell of the invention preferably further comprises the activity of xylulose kinase so that xylulose isomerized from xylose can be metabolized to pyruvate. Preferably, the cell contains endogenous xylulose kinase activity.
[073] [073] A cell of the invention most preferably comprises a genetic modification that increases the flow of the pentose phosphate pathway, as described in WO 06/009434. In particular, genetic modification causes an increase in the flow of the non-oxidative part of the pentose phosphate pathway. The genetic modification that causes an increase in the flow of the non-oxidative part of the pentose phosphate pathway is understood here to mean a change that increases the flow to at least a factor of 1.1, 1.2, 1.5, 2, 5, 10 or 20, compared to the flow in a strain that is genetically identical, except for the genetic modification that causes the flow to increase. The flow of the non-oxidative part of the pentose phosphate pathway can be measured as described in WO 06/009434.
[074] [074] Genetic modifications that increase the flow of the pentose phosphate pathway can be introduced into the cells of the invention in several ways. These include, for example, achieving higher stationary levels of xylulose kinase and / or one or more of the enzymes in the non-oxidative part of the pentose phosphate pathway and / or a reduced stationary level of nonspecific aldose reductase activity. These changes in stationary activity levels can be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and / or by recombinant DNA technology, for example, by overexpression or inactivation, respectively, of the genes encoding the enzymes or factors that regulate these genes.
[075] [075] In a preferred cell of the invention, the genetic modification comprises the overexpression of at least one enzyme (from the non-oxidative part) of the pentose phosphate pathway. Preferably, the enzyme is selected from the group consisting of enzymes that code for ribulose-
[076] [076] There are several means available in the art for the overexpression of enzymes in the cells of the invention. In particular, an enzyme can be overexpressed by increasing the number of copies of the gene encoding the enzyme in the cell, for example, by integrating additional copies of the gene into the cell's genome, by expressing the gene from a vector of episomal multicopy expression or by introducing an episomal multicopy expression vector comprising multiple copies of the gene. The coding sequence for the overexpression of the enzymes is preferably homologous to the host cell of the invention. However, coding sequences that are heterologous to the host cell of the present invention can also be applied.
[077] [077] Alternatively, overexpression of enzymes in the cells of the invention can be achieved through the use of a promoter that is not native to the coding sequence of the enzyme to be overexpressed, that is, a promoter that is heterologous to the sequence of encoding to which it is operatively linked. Although the promoter is preferably heterologous to the coding sequence to which it is operably linked, it is also preferred that the promoter is homologous, i.e. endogenous to the cell of the invention. Preferably, the heterologous promoter is capable of producing a higher level of steady-state transcription that comprises the coding sequence (i.e., capable of producing more transcription molecules, i.e. mRNA molecules, per unit time) than the promoter that is native to the coding sequence, preferably under conditions where xylose or glucose and xylose are available as carbon sources,
[078] [078] Another preferred cell of the invention comprises a genetic modification that reduces the nonspecific aldose reductase activity in the cell. Preferably, the nonspecific aldose reductase activity is reduced in the host cell through one or more genetic modifications that decrease expression or inactivate a gene encoding a nonspecific aldose reductase. Preferably, genetic modifications reduce or inactivate the expression of each endogenous copy of a gene encoding a nonspecific aldose reductase that is capable of reducing an aldopentose, including xylose, xylulose and arabinose, in the cell's genome. A given cell may comprise multiple copies of genes encoding nonspecific aldose reductase as a result of di-, poly- or aneuploidy, and / or a cell may contain several different (iso) enzymes with aldose reductase activity, which differ in sequence of amino acids and which are each encoded by a different gene. In addition, in these cases, the expression of each gene encoding a non-specific aldose reductase is preferably reduced or inactivated. Preferably, the gene is inactivated by deletion of at least a part of the gene or by disruption of the gene, where, in this context, the term gene also includes any non-coding sequence upstream or downstream of the coding sequence, the deletion ( partial) or inactivation that results in a reduction in the expression of nonspecific aldose reductase activity in the host cell. A nucleotide sequence encoding an aldose reductase, the activity of which is reduced in the cell of the invention and the amino acid sequences of these aldose reductases are described in WO 06/009434 and include for example the (unspecific) aldose reductase genes of the GRE3 gene of S. cerevisiae (Träff et al., 2001, Appl. Environm. Microbiol. 67: 5668-5674) and their orthologists in other species.
[079] [079] An even more preferred transformed host cell according to the invention may further comprise genetic modifications that result in one or more of the characteristics selected from the group consisting of (a) increased transport of xylose and / or arabinose into the cell ; (b) decreased sensitivity to catabolic repression; (c) increased tolerance to ethanol, osmolarity or organic acids; and, (d) reduction in the production of by-products. By-products, it is understood that they are molecules containing carbon that are not those of the desired fermentation product and include, for example, xylitol, arabinitol, glycerol and / or acetic acid. Any genetic modification described here can be introduced by classical mutagenesis and screening and / or selection for the desired mutant, or simply by screening and / or selection of spontaneous mutants with the desired characteristics.
[080] [080] A preferred host cell according to the invention, has the ability to grow on at least one of xylose and arabinose as a carbon / energy source, preferably as the only carbon / energy source, and preferably under conditions anaerobic, that is, the conditions as defined here below for the anaerobic fermentation process. Preferably, when grown in xylose as a carbon / energy source the host cell produces essentially no xylitol, for example, xylitol is produced below the detection limit, or for example, less than 5, 2, 1, 0.5 , or 0.3% of the carbon consumed on a molar basis. Preferably, when grown in arabinose as a carbon / energy source, the cell produces essentially no arabinitol, for example, the arabinitol produced is below the detection limit, or for example, less than 5, 2, 1, 0.5 , or 0.3% of the carbon consumed on a molar basis.
[081] [081] A preferred host cell of the invention has the ability to grow in a combination of: a) at least one of a hexose and a pentose; b) acetic acid; and c) glycerol, at a speed of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h-1, under aerobic conditions, or, more preferably, at a rate of at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h-1, under anaerobic conditions. Therefore, preferably the host cell has the ability to grow on at least one of xylose and arabinose as the sole carbon / energy source at a rate of at least 0.01, 0.02, 0.05, 0.1, 0 , 2, 0.25 or 0.3 h-1 under aerobic conditions, or, more preferably, at a speed of at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h-1, under anaerobic conditions. More preferably, the host cell has the ability to grow in a mixture of one hexose (for example, glucose) and at least one of xylose and arabinose (in a proportion of 1: 1 by weight) as the only carbon / energy source. at a speed of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h-1, under aerobic conditions, or, more preferably, at a speed of at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h-1, under anaerobic conditions.
[082] [082] In one aspect, the invention relates to the use of a yeast cell according to the invention for the preparation of a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy acid -propionic, acrylic acid, 1,3-propane-diol, butanol and products derived from isoprenoids.
[083] [083] In another aspect, the invention relates to a process for the production of a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, 1,3- propane-diol, butanols (1-butanol, 2-butanol, iso-butanol) and products derived from isoprenoids. The process preferably comprises the step of: a) fermenting a medium with a yeast cell, wherein the medium contains or is fed with: a) a source of at least one of a hexose and a pentose; b) a source of acetic acid; and c) a source of glycerol and in which the yeast cell ferments acetic acid, glycerol and at least one of the hexoses and pentoses in ethanol. The yeast cell is preferably a (host) cell as defined above. The process preferably comprises a step in which the fermentation product is recovered. The process can be a batch process, a fed batch process or a continuous process, as is well known in the art. In the process of the invention, the source of glycerol can be any source of carbon that has a lower state than glucose. A carbon source having a lower state than glucose is understood as a carbon source of which the average reduction state per C-mol (of its compounds) is greater than the reduction state per C-mol of glucose . Examples of carbon sources, having a lower state than glucose include, for example alkanols such as propanol and butanol; polyols such as 1,3-propane-diol, butanediol, glycerol, mannitol and xylitol.
[084] [084] In a preferred process, the hexose source comprises or consists of glucose. Preferably, the source of pentose comprises or consists of at least one of xylose and arabinose. Preferably, the medium fermented by the cells of the invention comprises or is fed with (fractions of) hydrolyzed biomass comprising at least one of a hexose and a pentose, such as glucose, xylose and / or arabinose. Hydrolyzed biomass (fractions) comprising hexoses and pentoses in general also comprise acetic acid (or a salt thereof). An example of hydrolyzed biomass to be fermented in the process of the invention is, for example, hydrolyzed lignocellulosic biomass. Lignocellulosic biomass is understood here as plant biomass, which is composed of cellulose, hemicellulose and lignin. Carbohydrate polymers (cellulose and hemicellulose) are strongly linked to lignin. Examples of lignocellulosic biomass to be hydrolyzed for use in the present invention include agricultural waste (including corn straw and cane bagasse), wood waste (including sawmill and paper mill waste), and (municipal) waste paper.
[085] [085] In the process of the invention, the sources of xylose, glucose and arabinose can be xylose, glucose and arabinose, as such (for example, as monomeric sugars) or can be in the form of any oligo- or polymeric carbohydrate containing xylose, glucose and / or arabinose units, such as lignocellulose, arabinans, xylans, cellulose, starch and the like. For the release of xylose, glucose and / or arabinose units from such carbohydrates, appropriate carbohydrases (such as arabinases, xylanases, glucanases, cellulases, amylases, glucanases and the like) can be added to the fermentation medium or can be produced by the modified host cell. In the latter case, the host cell can be modified by genetic engineering to produce and excrete such carbohydrates. An additional advantage of using polymer or oligo- glucose sources is that it allows maintaining a low (or lower) level of free glucose concentration during fermentation, for example, using speed-limiting amounts of carbohydrates, preferably during fermentation. This, in turn, will prevent the repression of the systems necessary for the metabolism and transport of non-glucose sugars, such as xylose and arabinose. In a preferred process, the modified host cell ferment both glucose and at least one of xylose and arabinose, preferably simultaneously, in which case, preferably a modified host cell that is insensitive to glucose repression is used to prevent growth diaáuxico. In addition to a source of at least one of xylose and arabinose (and glucose) as a carbon source, the fermentation medium will also comprise the appropriate ingredient necessary for the growth of the modified host cell. Compositions of fermentation media for growth of eukaryotic microorganisms, such as yeasts, are well known in the art.
[086] [086] In the process of the invention, the medium most preferably comprises and / or is fed with a source of glycerol. Glycerol for use in the process of the present invention can advantageously be the glycerol that is generated as a by-product in the production of biodiesel from transesterification reactions using vegetable oils or animal fats and an alcohol.
[087] [087] The fermentation process can be an aerobic or anaerobic fermentation process. An anaerobic fermentation process is defined herein as a fermentation process carried out in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol / L / h, more preferably 0 mmol / L / h is consumed (that is, oxygen consumption is not detectable), and in which organic molecules serve both as electron donors and electron acceptors. In the absence of oxygen, the NADH produced in glycolysis and in the formation of biomass cannot be oxidized by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor, thus regenerating NAD +. Thus, in a preferred anaerobic fermentation process, pyruvate is used as an electron (and hydrogen) acceptor and is reduced in fermentation products, such as ethanol, as well as in non-ethanol fermentation products, such as lactic acid, 3-hydroxy-propionic acid, acrylic acid, 1,3-
[088] [088] Alternatively, the fermentation process of the present invention can be performed under aerobic conditions limited in oxygen. Preferably, in an aerobic process under limited oxygen conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6, and even more preferably at least 7 mmol / L / h. In a preferred oxygen-limited aerobic fermentation process of the invention, the yeast cell of the invention consumes less than 30, 20, 18, 15, 12, 10, 8 or 5% of the amount of oxygen on a C-molar basis related to the carbon source consumed during the conversion of the carbon source into the fermentation product. The conversion ratio of oxygen consumed in relation to the substrate used on a C-molar base (COS) is understood here as mol of O2 used by C-mol consumed from carbon source. Thus, a process of the invention can be performed under strict anaerobic conditions (i.e., COS = 0.0), or the process of the invention can be performed under aerobic conditions, preferably limited to oxygen, where the COS is preferably lower to 0.3, 0.2, 0.18, 0.15, 0.12, 0.1, 0.08 or 0.05.
[089] [089] The fermentation process is preferably carried out at a temperature that is optimal for the modified cells of the invention. Thus, for most yeast cells, the fermentation process is carried out at a temperature that is less than 42 ° C, preferably less than 38 ° C. For yeast cells, the fermentation process is preferably carried out at a temperature that is less than 35, 33, 30 or 28 ° C and at a temperature that is higher than 20, 22 or 25 ° C.
[090] [090] A preferred fermentation process according to the invention is a process for the production of ethanol, wherein the process comprises the step of fermenting a medium with a yeast cell, in which the medium contains or is fed with: a ) a source of at least one hexose and one pentose; b) a source of acetic acid; and, c) a glycerol source, in which the yeast cell ferments acetic acid, glycerol and at least one of the hexoses and pentoses in ethanol and, optionally, b) recovery of ethanol. The fermentation medium can also be carried out as described above. In the process, the volumetric productivity of ethanol is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 and 10.0 g of ethanol per liter per hour. The ethanol yield of xylose and / or glucose and / or arabinose and / or ethyl and / or glycerol in the process is preferably at least 50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is defined here as a percentage of the maximum theoretical yield, which, for xylose, glucose and arabinose is 0.51 g of ethanol per g. xylose, glucose or arabinose. The maximum theoretical yield for glycerol is 0.50 g. of ethanol per g. of glycerol and for acetic acid, the maximum theoretical yield is 0.77 g. of ethanol per g. of acetic acid.
[091] [091] In this document and in its claims, the verb "to understand" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but that items not specifically mentioned are not excluded. In addition, the reference to an element by the indefinite article "one" or "one" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one, and only one, of the elements . The indefinite article "one" or "one", therefore, usually means "at least one".
[092] [092] All patent and literature references cited in the present description are hereby incorporated by reference in their entirety.
[093] [093] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
[094] [094] Figure 1. The evolution of net glycerol levels (g / l) (ie production minus consumption) over time (hours) is shown for strains RN1041, RN1041 + pRN595, RN1186, RN1187, RN1188 and RN1189 of S.
[095] [095] Figure 2. The evolution of net acetic acid levels (g / l) (ie production minus consumption) over time (hours) is shown for strains RN1041, RN1041 + pRN595, RN1186, RN1187 , RN1188 and RN1189 of S.
[096] [096] Cell extracts for activity assays were prepared from exponentially growing aerobic or anaerobic batch cultures and analyzed for protein content, as described by Abbot et al., (2009, Appl. Environ. Microbiol. 75: 2320-2325).
[097] [097] NAD + dependent acetaldehyde dehydrogenase activity (EC 1.2.1.10) was measured at 30 ° C by monitoring NADH oxidation at 340 nm. The reaction mixture (total volume of 1 ml) contained 50 mM potassium phosphate buffer (pH 7.5), 0.15 mM NADH, and cell extract. The reaction was initiated by the addition of 0.5 mM acetyl-Coenzyme A.
[098] [098] For the determination of glycerol-3-phosphate dehydrogenase activity (EC 1.1.1.8), cell extracts were prepared as described above, except that the phosphate buffer was replaced with triethanolamine buffer (10 mM, pH 5) . Glycerol-3-phosphate dehydrogenase was tested in cell extracts at 30 ° C as previously described (Blomberg and Adler, 1989, J. Bacteriol. 171: 1087-1092,9).
[099] [099] The activity of acetyl-CoA synthase (EC 6.2.1.1) was measured as described by Frenkel and Kitchens (1977, J.
[100] [100] The activity of glycerol dehydrogenase and dihydroxyacetone kinase are measured at 30 ° C in cell extracts, essentially as previously described (Gonzalez et al., 2008, Metab. Eng. 10, 234-245). The enzymatic activities of glycerol dehydrogenase and dihydroxyacetone kinase are reported as µmols of substrate / min / mg of cellular protein.
[101] [101] All modifications begin with the strain RN1008 his- fermenter of xylose and arabinose. His100 RN-, also referred to here as RN1041, is a fermentative strain of arabinose and xylose based on CEN.PK) with the genotype: Mat a, ura3-52, leu2-112, his3 :: loxP, gre3 :: loxP, loxP -Ptpi :: TAL1, loxP-Ptpi :: RKI1, loxP-Ptpi-TKL1, loxP- Ptpi-RPE1, delta :: -LEU2, delta :: Padh1XKS1Tcyc1-URA3-Ptpi- xylA-Tcyc1, delta :: LEU2-AAAara .
[102] [102] The deletion of GPD1 in RN1041 produces the strain RN1197. The GPD2 deletion in RN1041 produces the RN1198 strain.
[103] [103] Primers gpd1uf, gpd1ur, gpd1df and gpd1dr are used for the amplification of fragments of genomic sequences upstream and downstream of the GPD1 gene for their inactivation. Both upstream and downstream GPD1 fragments are cloned into a Blunt TOPO vector (Invitrogen) to obtain pGPD1up and pGPD1down, respectively.
[104] [104] Plasmid pRN593 (SEQ ID NO: 40) is constructed by ligating the Hind III and XbaI-cut fragment of pGPD1up with the hphMX fragment cut with SpeI and BsrGI (collection of plasmids C5YeastCompany) and the fragment cut with BsiWI and NcoI of pGPD1down for the T / A vector (Invitrogen) cut with HindIII and NcoI. Plasmid pRN593 is cut with Kpn1 to obtain the deletion fragment to interrupt the genomic copy (SEQ ID NO: 17). The mixture of linear fragments is used for the transformation of yeast. Transformants are selected for resistance to hygromycin. Correct integration results in the deletion of the GPD1 open reading phase. The integration is verified by PCR with the gpd1cf and gpd1cr primers.
[105] [105] The primers GPD2uf, GPD2ur, GPD2df and GPD2dr are used for the amplification of sequences of gnomic fragments upstream and downstream of the GPD2 gene for their inactivation. A 407bp upstream PCR fragment with an AflII site at the 3 'end (derived from the GPD2 sequence) and a BglII site at the 5' end (for the isolation of the deletion construct) is amplified using GPD2uf, GPD2ur and cloned into pCR2. 1 (top T / A, Invitrogen).
[106] [106] A 417bp PCR fragment downstream with an XhoI site at the 5 'end, and a BglII site at the 3' end is amplified using GPD2df and GPD2dr.
[107] [107] For the final construction the plasmid containing the upstream fragment is cut with AflII and Kpn, the downstream fragment is cut with XhoI and NcoI and the natMX marker (plasmid collection Royal Nedalco) is cut with AflII and XhoI and the fragments are ligated to produce plasmid pRN594 (SEQ ID NO: 36). pRN594 is cut with BglII prior to yeast transformation. Transformants are selected for resistance to nourseotrichin. Correct integration is verified by PCR.
[108] [108] The ACS1 open reading phase is amplified by PCR with the acslf and acslr primers.
[109] [109] This PCR fragment is cut with the restriction enzymes HindIII and BssHII and ligated to the TEF1 promoter fragment cut with Sa1I and HindIII (C5YeastCompany collection) and with the ADH1 terminator fragment cut with BssHII and BsiWI (C5YeastCompany collection). This combined fragment is amplified by PCR with specific promoter and terminator primers and cloned into the Blunt TOPO (Invitrogen) vector to give pACS1.
[110] [110] The ACS2 open reading phase is amplified by PCR with the acs2f and acs2r primers.
[111] [111] This PCR fragment is cut with the restriction enzymes PstI and SalI and ligated to the PGK1 promoter fragment cut with SpeI and PstI (C5YeastCompany collection) and to the PGI1 terminator fragment cut with XhoI and BsiWI (C5YeastCompany collection). This combined fragment is amplified by PCR with specific promoter and terminator primers and cloned into the Blunt TOPO (Invitrogen) vector to give plasmid pACS2.
[112] [112] The ACS1 overexpression construct is cut from pACS1 with the restriction enzymes SalI and BsiWI, the ACS2 overexpression construct is cut from pACS2 with the restriction enzymes SpeI and BsiWI, the KanMX marker is cut with BspEI and XbaI (collection of plasmids C5YeastCompany). These fragments are ligated to the plasmid pRS306 + 2mu ORI (collection of plasmids C5YeastCompany) cut with BspEI and XhoI to give the final plasmid pRN753 (SEQ ID NO: 51). This plasmid is used to transform yeast strains, as indicated in Table 4 and transformants are selected for resistance to G418. Overexpression is verified by qPCR. An alternative plasmid that can be used for the overexpression of ACS1 and ACS2 is pRN500 (SEQ ID NO: 20).
[113] [113] The PGK1 promoter (SpeI-PstI) and the ADH1 termination sequence (AflII-NotI) are added to the codon-optimized synthetic fragments and cloned into pRS303 with 2µ ori cut with SpeI and NotI and the expression construct is cloned in this vector. The expression is verified by qPCR.
[114] [114] For the expression of the E. coli mhpF gene, a yeast PGK1 promoter fragment (SpeI-PstI) and an ADH1 terminator fragment (AflII-NotI) (both from the Nedalco plasmid collection) were ligated into the synthetic fragment optimized for codons encoding E. coli mhpF (SEQ ID NO: 2). pRS 303 with 2µ ori (= pRN347, collection of Real Nedalco plasmids) was cut with SpeI and NotI and the mhpF expression construct was cloned into this vector to produce pRN558 (SEQ ID NO: 29).
[115] [115] For the expression of the E. coli adhE gene, a codon-optimized synthetic fragment encoding E. coli adhE (SEQ ID NO: 4) was cut with XbaI and AflII and ligated with XbaI and AflII cut pRN558 (replacing the E. coli mhpF gene in pRN558) to produce pRN595 (SEQ ID NO: 30).
[116] [116] For Entamoeba histolytica adh2 expression, a codon-optimized synthetic fragment encoding E. histolytica adh2 (SEQ ID NO: 6) is cut with XbaI and AflII and ligated into pRN558 cut with
[117] [117] pRN595 is used for later construction of pRN957 and pRN977 (see below). It is clear that pRN558 and pRN596 can be used in the same way, thereby replacing the expression of E. coli adhE with E. coli mhpF or E. histolytica adh2, respectively.
[118] [118] The yeast expression of E. coli gldA was done by ligating a yeast ACT1 promoter fragment (cut with the restriction enzymes SpeI and PstI), a synthetic ORF (SEQ ID NO: 21) , which codes for E. coli gldA, (cut with PstI and BssHII) and yeast CYC1 terminator fragment (cut with BssHII and BsiWI) together in pCRII blunt (Invitrogen) to produce pRNgldA (SEQ ID NO: 28).
[119] [119] PCR is performed on S. cerevisiae gnomic DNA with primers introducing an XbaI site 5 'from ATG and a SalI site 3' from TAA to produce the fragment of SEQ ID NO: 22. A fragment of DNA comprising the S. cerevisiae TPI1 promoter is ligated upstream of the DAK1 ORF fragment and the DNA fragment comprising the S. cerevisiae PGI1 terminator is ligated downstream of the DAK1 ORF to produce pRNDAK (SEQ ID NO: 38) .
[120] [120] The construction for expression in Citrobacter freundii dhak yeast was done by ligating the yeast TPI1 promoter fragment (cut with the restriction enzymes XhoI and XbaI), a synthetic ORF (SEQ ID NO: 26), which codes for C. freundii dhak, (cut with XbaI and SalI) and a yeast PGI1 terminator fragment (cut with XhoI and BsiWI) together in pCRII blunt (Invitrogen) to give pRNdhaK (SEQ ID NO: 27).
[121] [121] PCR is performed on S. cerevisiae genomic DNA with primers that introduce a HindIII site 5 'from ATG and a BamHI site 3' from TAA to produce the fragment of SEQ ID NO: 23. A DNA fragment comprising the S. cerevisiae TDH3 promoter is ligated upstream of the GUP1 ORF fragment and DNA comprising the S. cerevisiae CYC1 terminator fragment is ligated downstream of the GUP1 ORF.
[122] [122] PCR is performed on S. cerevisiae genomic DNA with primers that introduce an NsiI site 5 'from ATG and a BamHI site 3' from TAA to produce the fragment of SEQ ID NO: 24. A DNA fragment comprising the S. cerevisiae (medium) ADH1 promoter is ligated upstream of the FSP1 ORF fragment and DNA comprising the S. cerevisiae CYC1 terminator fragment is ligated downstream of the FSP1 ORF.
[123] [123] pRN347 is constructed by cloning the 2µ origin of replication (which was amplified by PCR from pYES2) in pRS303 (with the HIS3 gene for complementation).
[124] [124] For the construction of pRN957, the E. coli gldA expression construct is cut from the plasmid pRNgldA with the restriction enzymes SpeI and BsiWI.
[125] [125] For the construction of pRN977, the E. coli gldA expression construct is cut from the plasmid pRNgldA with the restriction enzymes SpeI and BsiWI.
[126] [126] Plasmids pRN957 and pRN977 are used to transform RN1041, RN1197, RN1198 and RN1199 to produce yeast strains, as indicated in Table 4.
[127] [127] Tables 4 A and B: Overview of strains built
[128] [128] The proof of concept of the concomitant reduction in acetic acid and oxidation of glycerol was obtained using a medium containing 1% yeast extract and 1% peptone. The experiments were carried out in culture in chemostat (working volume of 1 liter), at D = 0.05 h-1 and the pH was maintained at 5.5 by automatic addition of either KOH or H2SO4. Glucose (50 g / l) and xylose (50 g / l) were added as a carbon and energy source to the yeast extract peptone medium. For these experiments demonstrating the proof of concept, no arabinose was included. Where relevant, acetic acid was added to the 4 g / L yeast extract peptone medium and 10 g / l glycerol. The temperature was maintained at 32 ° C.
[129] [129] Strains pre-cultures are prepared by inoculating a frozen yeast glycerol stock culture in a YP medium (1% w / v yeast extract and 1% w / v peptone) with the addition of each glucose and xylose sugars (each 1% w / v) at 32 ° C and pH 5.5. After 24 h of incubation, under toxic conditions in shaking flasks, 50 ml of this culture is used to inoculate the cultures in chemostat.
[130] [130] In a steady state of the fermentations (5 volume changes), a sample was taken for analysis of sugar consumption (glucose and xylose), consumption of acetic acid, and metabolite (ethanol and glycerol). The concentrations of ethanol, glycerol and acetic acid are monitored by HPLC analysis. To determine the consumption of sugar, glucose and xylose are determined by analysis of HPAEC (Dionex).
[131] [131] The RN1151 strain is unable to achieve a steady state situation in the medium containing 4 g / L of acetic acid or in the presence or absence of glycerol. If no acetic acid is added to the medium, the organism in a steady state has consumed all glucose and xylose (less than 1 g / L remaining). No glycerol was consumed, but instead, it was produced.
[132] [132] Strains RN1200 and RN1201 are also tested in a medium with acetic acid and with or without added glycerol.
[133] [133] Strains RN1202 to RN1207 are similar to strains RN1200 and RN1201, except that the genes GPD1 and / or GPD2 have been excluded. In the medium containing 4 g / L of acetic acid, the sugars glucose and xylose are consumed almost to completion (less than 1 g / L remaining), if glycerol is added to the medium as is the case for strains RN1200 and RN1201. If no glycerol is added, no steady state is obtained.
[134] [134] Corn fiber hydrolyzate contains: glucose (38 g / l), xylose (28 g / l), arabinose (12 g / l) and acetic acid (4 g / l). It had been prepared by treating corn fibers at 160 ° C and pH 3.0 for 20 minutes, followed by enzymatic hydrolysis by cellulases and hemicellulases. Acetic acid was added to this hydrolyzate resulting in a total concentration of acetic acid, in the hydrolyzate, of 10 g / l. The pH of this hydrolyzate enriched in acetic acid, was restored to pH = 4.5 by adding KOH. Yeast extract was added to this hydrolyzate to reach a final concentration of 5 g / l.
[135] [135] Pre-cultures of the strains are prepared by inoculating a culture of frozen yeast glycerol stock in a YP medium (1% w / v yeast extract and 1% w / v peptone) with the addition of each glucose, xylose and arabinose sugars (each 1% w / v) at 32 ° C and pH 5.5. After 24 h of incubation, under toxic conditions in shaking flasks, 50 ml of this culture is used to inoculate the fermentation cultures. Fermentations are carried out in a fed batch fermentation configuration. Hydrolyzate (with or without 50 g / l glycerol) is pumped into the fermenter. If no glycerol was added, then 40 ml of water was added. During the first 6 hours, the flow rate for the hydrolyzate is adjusted at a speed of 5 ml per hour. For the next 6 hours, the flow rate is fixed at 10 ml per hour. Thereafter, for another 43 hours, the flow rate is fixed at 20 ml per hour. The total volume at the end of the fermentation reaches 1000 ml. These anoxic fed batch fermentations are carried out at about pH = 4.5, with gentle agitation at 100 rpm. The temperature during fermentations is set at 32 ° C. To minimize infection, the hydrolysates are heated for 10 min at 105 ° C before fermentations and the kanamycin antibiotic with a final concentration of 50 µg / ml is added.
[136] [136] At the end of fermentations, after 55 h, a sample was taken for analysis of sugar consumption (glucose, xylose and arabinose), consumption of acetic acid, and metabolite (ethanol and glycerol). The concentrations of acetic acid, ethanol, and glycerol are monitored over time by HPLC analysis. To determine the consumption of sugar, glucose, xylose, arabinose are determined by HPAEC (Dionex) analysis.
[137] [137] The strain RN1151 (= RN1041 supplemented with HIS3) was tested in the hydrolyzate, with or without glycerol. In both cases, the glucose concentration at the end of the fermentation period (55 h) is 35 g / l, while xylose and arabinose remain in their initial concentrations of 28 and 12 g / l, respectively. The quantities of ethanol produced are 2 g / L and acetic acid is present at 9.5 g / L. No consumption of glycerol is detected in the hydrolyzate containing glycerol. The fermentation of sugars is interrupted during the course of the fed batch operation because of rising levels of acetic acid. Initially, no acetic acid is present in the fermenter, but during the pumping of the hydrolyzate that contained toxic levels of acetic acid, the concentration quickly reaches toxic levels.
[138] [138] Strains RN1200 and RN1201 are also tested in hydrolyzate, with or without glycerol. These strains perform distinctly differently from the RN1151 strain. In the hydrolyzate containing glycerol, the sugars glucose, xylose and arabinose are consumed until completion. The levels of acetic acid decrease to 2 g / L and the glycerol concentrations, at the end of the fermentation, are 29.5 g / L, in all three cases. The quantities of ethanol produced by strains RN1200 and RN1201 are 51.7 and 52.2 g / l, respectively. In hydrolyzate that does not contain glycerol, considerably less sugar is consumed. Xylose and arabinose levels are unchanged at 28 and 12 g / l, respectively. Glucose is consumed, but only to a limited extent. At the end of the fermentation, the remaining concentration is 32 g / l, in all three cases, with ethanol reaching a concentration of 3 g / l. The concentration of acetic acid drops to 9.1 g / L at the end of the fermentation, while some glycerol is produced (less than 0.5 g / l). From these results it can be concluded that the expression of the E. coli gldA and adhE genes in combination with the positive regulation of DAK1 or the expression of dhak of C. freundii has a profound effect on the performance of the strains. In the presence of glycerol, they are able to consume glycerol and acetic acid, and produce additional ethanol (compared to the RN1151 strain). In the absence of glycerol, the strains consume some acetic acid. But during fermentation, the acetic acid level rises to toxic levels.
[139] [139] Strains from RN1202 to RN1207 are similar to strains RN1200 and RN1201, except that the GPD1 and / or GPD2 genes have been excluded. In the hydrolyzate containing glycerol, the sugars glucose, xylose and arabinose are consumed until completion, as was the case for the RN1200 strain. The levels of acetic acid similarly decreased to about 2 g / L and the glycerol concentrations at the end of the fermentation are 28 g / L in these three cases. The quantities of ethanol produced by strains RN1202, RN1203, RN1204, RN1205, RN1206 and RN1207 are 51.6, 52.9, 52.1, 52.5, 53.1 and 52.3 g / l, respectively. In hydrolyzate that does not contain glycerol, considerably less sugar is consumed. Xylose and arabinose levels are unchanged at 28 and 12 g / l, respectively. Glucose is consumed, but only to a limited extent. At the end of fermentation, in the hydrolyzate without glycerol, the remaining concentration of glucose is 31 g / l, in all three cases, with ethanol reaching a concentration of 3 g / l. The concentration of acetic acid drops to 9.1 g / L at the end of the fermentation, while some glycerol is produced (less than 0.5 g / l). From these results we can conclude that the exclusion of GPD1 and / or GPD2 genes together with the other modifications in RN1202, RN1203, RN1204, RN1205, RN1206 and RN1207 result in strains that can perform the desired reactions.
[140] [140] Unless otherwise indicated, the methods used are conventional biochemical techniques. Examples of suitable general methodology textbooks include Sambrook et al., Molecular Cloning, a Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
[141] [141] The media used in the experiments were either YEP medium (10 g / L yeast extract, 20 g / L peptone) or solid YNB medium (6.7 g / L yeast nitrogen base, 15 g / L agar), supplemented with sugars as indicated in the examples. For solid YEP medium, 15 g / L of agar was added to the liquid medium, before sterilization.
[142] [142] In the AFM experiments, mineral media was used. The composition of mineral medium was described by Verduyn et al. (Yeast (1992), volume 8, 501-517) and was supplemented with 2.325 g / L of urea and sugars, as indicated in the examples.
[143] [143] Yeast transformation was carried out according to the method described by Schiestl and Gietz (Current Genetics (1989), Volume 16, 339-346).
[144] [144] Genomic DNA was extracted from individual yeast colonies for PCR according to the method described by Looke et al. (BioTechniques (2011), Volume 50, 325-328). AFM procedure
[145] [145] The Alcohol Fermentation Monitor (AFM; Halotec, Veenendaal, Netherlands) is a robust and easy-to-use parallel laboratory bioreactor that allows accurate comparisons of yield and carbon conversion rates for six simultaneous anaerobic fermentations.
[146] [146] The initial culture of the AFM experiment contained 50 mg of yeast (dry weight). To determine this, a calibration curve was made for the RN1041 strain of biomass versus OD700. This calibration curve was used in the experiment to determine the volume of cell culture required for 50 mg of yeast (dry weight).
[147] [147] Prior to the start of the AFM experiment, pre-cultures were grown as indicated in the examples. OD700 was measured for each strain and 50 mg of yeast (dry weight) was inoculated in 400 ml of mineral medium (Verduyn et al. (Yeast (1992), volume 8, 501-517), supplemented with 2.325 g / L of urea and sugars, as indicated in the examples.
[148] [148] The method for determining the assay for glycerol dehydrogenase activity was adopted from Lin and Magasanik (1960) J Biol Chem.235: 1820-1823.
[149] [149] Table 5: Test conditions 1.0 M 800 µl carbonate / bicarbonate buffer pH 10
[150] [150] Cell-free extract was prepared by harvesting cells by centrifugation. The cells were harvested in the exponential phase. The cell pellet was washed once with 1 M carbonate / bicarbonate buffer (pH10) and a cell-free extract was prepared from it by adding glass beads and mixed in a vortex mixer at maximum speed for intervals of 1 minute until the cells were disrupted. This was verified microscopically.
[151] [151] Experiments on anaerobic shake flasks were performed as indicated in the examples.
[152] [152] For each time point, a separate shake flask was inoculated, thus preventing aeration during sampling.
[153] [153] The parent strain used in the experiments described in examples 5 to 8 is RN1041.
[154] [154] RN1041 has been described in WO 2012067510.
[155] [155] The following strains have been constructed:
[156] [156] Table 6: strains built RN1041 Parental strain (see above) RN1067 RN1041 gpd1 :: hphMX RN1068 RN1041 gpd2 :: natMX RN1069 RN1041 gpd1 :: hphMX gpd2 :: natMX RN1186 RN1041 + pRN977 RN1187 RN1067 + RNR1077 RN1069 + pRN977
[157] [157] Deletion of the GPD1 gene (gpd1) and / or the GPD2 gene (gpd2) was performed as described in Example 2.
[158] [158] Strains RN1041, RN1067, RN1068 and RN1069 were transformed with plasmid pRN977. This plasmid contains the following characteristics: the HIS3 gene for selection of transformants, the origin of replication 2µ, the ampicillin resistance marker for selection in E. coli, the E. coli adhE gene under the control of the PGK1 promoter and terminator ADH1, the DAK1 gene from S. cerevisiae under the control of the TPI1 promoter and the PGI1 terminator and the E. coli gldA gene under the control of the ACT1 promoter and the CYC1 terminator. All promoters and terminators are from S. cerevisiae. The sequence of plasmid pRN977 is set out in SEQ ID NO: 39.
[159] [159] After transformation of strains RN1041, RN1067, RN1068 and RN1069, isolated from individual colonies were subjected to colony PCR analysis in order to check for the presence of plasmid pRN977. A representative colony for each transformation was selected for further experimentation. These selected strains are designated RN1186, RN1187, RN1188 and RN1189.
[160] [160] Similarly, transformants were generated with the following specifications:
[161] [161] Table 7: transformers RN1190 RN1041 + pRN957 RN1191 RN1067 + pRN957 RN1192 RN1068 + pRN957 RN1193 RN1069 + pRN957
[162] [162] Plasmid pRN957 is similar to pRN977; however, the DAK1 gene from S. cerevisiae has been replaced by the dhak gene from Citrobacter freundii. The sequence of this plasmid, pRN957, is set out in SEQ ID NO: 37.
[163] [163] As a control strain, strain RN1041 was transformed with plasmid pRN595 (RN1041 + pRN595). This plasmid, pRN595, is similar to pRN977; however, it lacks the gldA and DAK1 genes. The sequence of plasmid pRN595 is set out in SEQ ID NO: 30.
[164] [164] The performance of the constructed strains was tested in an anaerobic experiment with a shake bottle.
[165] [165] An aliquot of the cells was taken from the night cultures for inoculation of the anaerobic cultures. The number of cells was such that the anaerobic culture had an initial optical density at 600 nm of approximately 0.1.
[166] [166] The carbon composition of the mineral medium: 2.5% glucose, 2.5% xylose, 1% glycerol and 2 g / L HAc. The pH was adjusted to a pH of 4.5. The shake flasks were closed with a hydraulic valve, in order to guarantee the anaerobic conditions. For each time point, a separate flask was inoculated.
[167] [167] The results of the net increase or decrease in glycerol, after 94 hours of fermentation, and the consumption of HAc, are shown in the table below.
[168] [168] Table 8: Consumption of glycerol and HAc and values of ethanol production per strain Cepa Increase (+) or Decrease Consumption of HAc Title of (-) liquid glycerol (in grams / ethanol (in grams / liter) liter ) (in grams per liter) RN1041 + 1.47 0.24 23.28 RN1186 - 1.20 0.99 25.32 RN1187 - 1.52 0.99 23.76 RN1188 - 0.80 0.97 25.01 RN1189 - 0.86 0.89 24.85 RN1190 - 0.47 0.71 24.38 RN1191 - 0.80 0.93 24.77 RN1192 + 0.93 0.29 23.60 0.92 24.93
[169] [169] The strain shown in Table 8 as RN1041 was transformed with plasmid pRS323, a standard cloning vector containing the HIS3 gene and a 2µ origin of replication, thus complementing histidine auxotrophy.
[170] [170] The results show: - RN1041 produces glycerol, which makes sense since both the GPD1 and GPD2 genes are active and gldA and DAK1 are not overexpressed. As adhE is not expressed in this strain, the consumption of HAc is low.
[171] [171] Overexpression of a homologous or heterologous dihydroxyacetone kinase, in combination with the overexpression of gldA and adhE, results in a simultaneous consumption of acetate and glycerol under anaerobic conditions.
[172] [172] The experiment described in Example 6 was repeated in a slightly different configuration, that is, the AFM (alcoholic fermentation monitor), which allows the determination of carbon dioxide in real time, during the experiment.
[173] [173] The strains tested were RN1041, RN1041 + pRN595, RN1186, RN1187, RN1188 and RN1189. The RN1041 strain was transformed with plasmid pRS323, a standard cloning vector containing the HIS3 gene, and a 2µ origin of replication, thus complementing histidine auxotrophy.
[174] [174] The cells were harvested and an AFM experiment was started as described above.
[175] [175] Samples were taken at regular intervals and sugars, ethanol, glycerol and HAc were determined by HPLC.
[176] [176] The results are shown in the Table below.
[177] [177] Table 9: consumption of glycerol and HAc and the values of ethanol production per strain at time = 112 hours.
[178] [178] The evolution of glycerol and HAc levels over time are shown in Figures 1 and 2.
[179] [179] Strains RN1041 and RN1041 + pRN595 are showing net glycerol production. Strains RN1186 and RN1188 are initially showing glycerol production; however, after about 24 to 32 hours, the consumption of glycerol started and continued until at the end a net consumption of glycerol was observed.
[180] [180] Strains RN1187 and RN1189 do not exhibit initial glycerol production, as seen with RN1186 and RN1188. After 24 hours, the consumption of glycerol begins. Glycerol consumption is significantly higher in these samples, compared to RN1186 and RN1188. These results indicate that the deletion of the GPD1 gene results in a higher consumption of glycerol than the deletion of the GPD2 gene.
[181] [181] The RN1041 + pRN595 strain is showing higher HAc consumption than the reference RN1041 strain. RN1186 and RN1188 are exhibiting higher HAc consumption than RN1041 + pRN595. This result indicates that the consumption of glycerol reinforces the consumption HAc. This effect is even stronger in strains RN1187 and RN1189.
[182] [182] Cell free extracts (CFE) from strain RN1041 and RN1190 were prepared as described above. The glycerol dehydrogenase activity assay, adopted from the Lin and Magasanik (1960) protocol J Biol. Chem. 235: 1820-1823, was carried out. The results are shown in the Table below.
[183] [183] Table 10: glycerol dehydrogenase activity assay Sample Cofator Increase in A340 / min RN1041 5 µl CFE NAD + 0.00 RN1190 5 µl CFE NAD + 0.02
[184] [184] The strain indicated in table 10 as RN1041 was transformed with plasmid pRS323, a standard cloning vector containing the HIS3 gene and a 2µ origin of replication, thus complementing histidine auxotrophy.
[185] [185] These results indicate that: a) E. gldA
coli, expressed in RN1190, is dependent on NAD +, and b) that the increase in the CFE value resulted in a proportional increase in the conversion rate of NAD +, and therefore, of glycerol to dihydroxyacetone.

权利要求:
Claims (19)
[1]
1. Process for the production of ethanol characterized by the fact that it comprises the step of fermenting a medium with a yeast cell, in which the medium contains or is fed with: a) a source of at least one among hexose and pentose; b) a source of acetic acid; and, c) a source of glycerol, in which the yeast cell ferments acetic acid, glycerol and at least one between hexoses and pentoses in ethanol and, optionally, the recovery of ethanol, in which the yeast cell comprises an exogenous gene which encodes an enzyme with acetaldehyde dehydrogenase activity, such a gene gives the cell the ability to convert acetic acid into ethanol, and in which the yeast cell comprises a bacterial gene that encodes an enzyme with NAD + linked glycerol dehydrogenase activity.
[2]
Process according to claim 1, characterized in that the yeast cell is not a yeast cell comprising a genetic modification that reduces the specific NAD + -dependent dehydrogenase activity in the cell.
[3]
3. Process according to claim 1 or 2, characterized by the fact that the exogenous gene that encodes the enzyme with acetaldehyde dehydrogenase activity comprises a nucleotide sequence that encodes an amino acid sequence with at least one of: i) at least at least 64% amino acid sequence identity with SEQ ID NO: 1, ii) at least 76% amino acid sequence identity with SEQ ID NO: 3 and, iii) at least 61% amino acid sequence identity with SEQ ID NO: 5; and wherein the bacterial gene encoding an enzyme with NAD +-linked glycerol dehydrogenase activity comprises a nucleotide sequence that encodes an amino acid sequence with at least 45% amino acid sequence identity to SEQ ID NO: 7.
[4]
Process according to any one of claims 1 to 3, characterized in that the yeast cell comprises a genetic modification that reduces the specific NAD + -dependent glycerol-3-phosphate dehydrogenase activity in the cell.
[5]
5. Process according to claim 4, characterized by the fact that the genetic modification that reduces the specific activity of glycerol-3-phosphate dehydrogenase dependent on NAD + in the cell is a genetic modification that reduces or inactivates the expression of an endogenous gene which encodes a glycerolphosphate dehydrogenase having an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 16.
[6]
Process according to any one of claims 2 to 5, characterized in that the yeast cell comprises a genetic modification that increases the specific activity of dihydroxyacetone kinase, where the genetic modification is the overexpression of a sequence nucleotide encoding a dihydroxyacetone kinase, and preferably, the nucleotide sequence encoding dihydroxyacetone kinase comprises a nucleotide sequence encoding an amino acid sequence with at least 50% amino acid sequence identity with at least , one of SEQ ID NO: 8, 9 and 25.
[7]
Process according to any one of claims 2 to 6, characterized by the fact that the cell further comprises a genetic modification that increases at least one of: i) the specific activity of acetyl-CoA synthase and in which, preferably, the genetic modification is the overexpression of a nucleotide sequence that encodes an acetyl-CoA synthetase; and, ii) transporting glycerol into the cell.
[8]
8. Process according to claim 7, characterized in that the nucleotide sequence that encodes an acetyl-CoA synthetase encodes an acetyl-
CoA synthase with a maximum rate greater than the acetyl-CoA synthase encoded by the ACS1 gene of S. cerevisiae, or where the nucleotide sequence encoding an acetyl-CoA synthase encodes an acetyl-CoA synthase with a greater affinity for acetate than that the acetyl-CoA synthase encoded by the ACS2 gene of S. cerevisiae, and that the genetic modification that increases glycerol transport into the cell is the overexpression of a nucleotide sequence that encodes at least one of a protein from glycerol uptake and a glycerol channel, wherein the nucleotide sequence encoding the glycerol uptake protein preferably comprises a nucleotide sequence encoding an amino acid sequence with at least 50% amino acid sequence identity with at least one of SEQ ID NO: 10 and 11, and wherein, preferably, the nucleotide sequence encoding the glycerol channel comprises a nucleotide sequence encoding to an amino acid sequence with at least 30% amino acid sequence identity to the amino acid sequence between amino acids 250 and 530 of SEQ ID NO: 12.
[9]
Process according to claim 8, characterized in that the nucleotide sequence encoding the Acetyl-CoA synthase is the Acetyl-CoA synthase encoded by the ACS1 or ACS2 gene of S. cerevisiae.
[10]
10. Process according to claim 8 or 9, characterized by the fact that the genetic modification increases the specific activity of acetyl-CoA synthetase under anaerobic conditions.
[11]
Process according to any one of claims 4 to 10, characterized in that the yeast cell comprises at least: i) an exogenous functional xylose isomerase gene, the gene of which gives the cell the ability to isomerize xylose in xylulose; and, ii) exogenous functional genes encoding an L-arabinose isomerase, an L-ribulokinase and an L-ribulose-5-phosphate-4-epimerase, these genes together give the cell the ability to convert L-arabinose to D - xylulose-5-phosphate, and in which, preferably, the yeast cell comprises at least one other genetic modification that results in a characteristic selected from the group consisting of: a) increased specific xylulose kinase activity; b) increased flow of the pentose-phosphate pathway c) reduced non-specific activity of aldose reductase d) increased transport of at least one of xylose and arabinose into the host cell; e) decreased sensitivity to catabolic repression; f) increased tolerance to ethanol, osmolarity or organic acids; and, g) reduction in the production of by-products.
[12]
12. Process according to any of claims 1 to 11, characterized by the fact that the yeast cell is of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera , Schwanniomyces and Yarrowia.
[13]
13. Process according to claim 12, characterized by the fact that the yeast cell belongs to a species selected from the group consisting of S. cerevisiae, S. exiguus, S. bayanus, K.
lactis, K. marxianus and Schizosaccharomyces pombe.
[14]
Process according to any one of claims 1 to 13, characterized in that the medium contains or is fed with a lignocellulosic hydrolyzate.
[15]
15. Process according to any one of claims 1 to 14, characterized by the fact that the yeast cell is fermented under anaerobic conditions.
[16]
16. Process according to any one of claims 1 to 15, characterized in that the terms "increase" and "decrease" are defined as compared to the specific activity of the enzyme in an identical wild-type yeast cell.
[17]
17. Yeast cell characterized by the fact that it comprises: a) an exogenous gene that encodes an enzyme with acetaldehyde dehydrogenase activity, which gives the cell the ability to convert acetic acid into ethanol; and, b) a bacterial gene that encodes an enzyme with NAD +-linked glycerol dehydrogenase activity.
wherein the yeast cell is not a yeast cell comprising a genetic modification that reduces the specific NAD + dependent dehydrogenase formate activity in the cell.
[18]
18. Yeast cell according to claim 17, characterized by the fact that the yeast cell is as defined in any one of claims 2 to 16.
[19]
19. Use of a yeast cell as defined in claim 17 or 18, characterized by the fact that it is in the preparation of ethanol.

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US10100320B2|2018-10-16|Pentose sugar fermenting cell

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WO2014033019A1|2014-03-06|Yeast strains engineered to produce ethanol from acetate

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BR112014002709A2|2021-01-26|pentose sugar fermentation cell

同族专利:

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公开号 | 公开日

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US20180251798A1|2018-09-06|

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EP3321368A2|2018-05-16|

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US9988649B2|2018-06-05|

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EP3321368A3|2018-05-23|

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US10941421B2|2021-03-09|

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ES2648865T3|2018-01-08|

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JP2015504309A|2015-02-12|

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DK2785849T3|2018-01-08|

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PL2785849T3|2018-02-28|

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CN104126011A|2014-10-29|

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AU2012346662A1|2014-06-05|

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WO2013081456A3|2013-08-15|

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CA2857247C|2019-07-23|

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EP2785849A2|2014-10-08|

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WO2013081456A2|2013-06-06|

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AU2012346662B2|2016-07-28|

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CN104126011B|2017-07-11|

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EP2785849B1|2017-09-27|

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CN107189954A|2017-09-22|

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US20150176032A1|2015-06-25|

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EA201491056A8|2015-02-27|

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MX2014006446A|2014-09-01|

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EA201491056A1|2014-09-30|

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CA2857247A1|2013-06-06|

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KR20140099251A|2014-08-11|

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法律状态:
2020-11-03| 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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2021-10-05| B06A| Patent application procedure suspended [chapter 6.1 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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2022-01-25| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-03-03| 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 26/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161564932P| true| 2011-11-30|2011-11-30|

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EP11191333.1|2011-11-30|

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US61/564,932|2011-11-30|

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EP11191333|2011-11-30|

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PCT/NL2012/050841|WO2013081456A2|2011-11-30|2012-11-26|Yeast strains engineered to produce ethanol from acetic acid and glycerol|

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