METHOD OF PRODUCTION OF BUTADIENE AND GENETICALLY MODIFIED MICRO-ORGANISM

专利摘要:
modified microorganisms and methods of making butadiene using them. The present invention relates generally to microorganisms comprising one or more polynucleotides that encode enzymes in one or more pathways, which catalyze the conversion of a fermentable carbon source to butadiene. Methods of using the microorganisms in industrial processes are also provided, including for use in the production of butadiene and products derived therefrom.
公开号:BR112014014652B1
申请号:R112014014652-7
申请日:2012-12-17
公开日:2021-07-13
发明作者:Mateus Schreiner Garcez Lopes；Avram Michael Slovic；Iuri Estrada Gouvea；Johana Rincones Perez；Lucas Penderson Parizzi
申请人:Braskem S.A.；
IPC主号:

专利说明:

FOUNDATION
[001] Butadiene (1,3-butadiene, CH2=CH-CH=CH2, CAS 106-99-0) is a linear, conjugated 4-carbon hydrocarbon typically manufactured (together with other 4-carbon molecules) by cracking oil-based hydrocarbon steam. This process involves harsh conditions and high temperatures (at least about 850°C). Other butadiene production methods involve toxic and/or expensive catalysts, highly flammable and/or gaseous carbon sources, and high temperatures. Globally, several million tons of polymers containing butadiene are produced annually. Butadiene can be polymerized to form polybutadiene, or reacted with hydrogen cyanide (prussic acid) in the presence of a nickel catalyst to form adiponitrile, a precursor to nylon. More commonly, however, butadiene is polymerized with other olefins to form copolymers, such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile-butadiene (ABR), or styrene-butadiene (SBR) copolymers. SUMMARY
[002] The present description relates generally to microorganisms (e.g., non-naturally occurring microorganisms, also referred to herein as modified microorganisms) that comprise one or more polynucleotides that encode enzymes in one or more pathways that catalyze the conversion of a carbon source in butadiene, and the Use such microorganisms in industrial processes, including for use in the production of butadiene and products derived therefrom.
[003] The present description provides methods of producing butadiene from a fermentable carbon source, comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene, and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene in the fermentation medium; and expressing one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides that encode the enzymes in a pathway that catalyze the converting one or more intermediates to butadiene in the microorganism to produce butadiene.
[004] In some embodiments that can be combined with any of the above or below mentioned embodiments, enzymes that catalyze the conversion of the fermentable carbon source into one or more intermediates in the pathway for the production of butadiene are disclosed in. any of Tables 1 to 3.
[005] In some embodiments that can be combined with any of the above or below mentioned embodiments, enzymes that catalyze the conversion of one or more intermediates to butadiene are shown in any of Tables 1 to 3.
[006] In some embodiments that can be combined with any of the above or below mentioned embodiments, butadiene is produced via an intermediate of acetyl-CoA and propionyl-CoA; a crotonyl-CoA intermediate; and/or a formic acid intermediate.
[007] In some embodiments that may be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and propionyl-COA to ketovaleryl-CoA code by a ketothiolase including, for example, a ketothiolase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 58-78.
[008] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ketovaleryl-CoA to (R) or (S) code 3- hydroxyalleryl-CoA by an oxidoreductase including, for example, an oxidoreductase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 103-123.
[009] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of (R) or (S) hydroxyalleryl-CoA to the 2-code pentenoyl-CoA by a dehydratase including, for example, a dehydratase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 37-55.
[0010] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of 2-pentenoyl-CoA to 2-pentenoic acid code by a transferase or a hydrolase including, for example, a transferase or a hydrolase encoded by a polynucleotide as set forth in any one of SEQ ID Nos: 1-28 or 29-33, respectively.
[0011] In some embodiments which may be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding the enzymes in . a pathway that catalyzes the conversion of 2-pentenoic acid to the code of butadiene by a decarboxylase, including, for example, a decarboxylase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 79-98.
[0012] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to 4-pentenoic acid code by an isomerase, including, for example, isomerase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 99-102.
[0013] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of 4-pentenoic acid to butadiene code by a decarboxylase, including, by example, a decarboxylase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 79-98.
[0014] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of 2-pentenoyl-CoA to penta-2,4-dienoyl code -CoA by a dehydrogenase including, for example, a dehydrogenase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 124-139.
[0015] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of penta-2,4-dienoyl-CoA to penta-2 code ,4-dienoic acid by a transferase or a hydrolase including, for example, a transferase or a hydrolase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 1-28, or 29-33, respectively.
[0016] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of 2,4- pentenoic acid to butadiene code by a decarboxylase, including , for example, a decarboxylase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 79-98.
[0017] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to the crotonyl alcohol code by an oxidoreductase, including by example, an oxidoreductase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 103-123.
[0018] In some embodiments that may be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonaldehyde code by an oxidoreductase including, for example, an oxidoreductase encoded by a polynucleotide as set forth in any one of SEQ ID NOs; 103-123.
[0019] In some embodiments that may be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of crotonaldehyde to the crotonyl alcohol code by an oxidoreductase or CoA synthetase including, by example, an oxidoreductase synthetase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 103-123 or SEQ ID NOs: 34-36, respectively.
[0020] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of crotonyl alcohol to butadiene code by a dehydratase including, for example, a dehydratase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 37-55.
[0021] In some embodiments that may be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of CO2 to formic acid code by a dehydrogenase including, for example, a dehydrogenase encoded by a polynucleotide as set forth in any one SEQ ID NOs: 124-139.
[0022] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate and CoA to the acetyl-CoA code and formic acid by a encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 58-78.
[0023] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of formic acid to the formyl-CoA code by a transferase or a CoA synthetase including, for example, a transferase or a CoA synthetase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 1-28 or 34-36, respectively.
[0024] In some embodiments which may be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of 2-acetyl-CoA to the acetoacetyl-CoA code by a ketothiolase including , for example, a ketothiolase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 58-78.
[0025] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-CoA and formyl-CoA to the 3,5-ketovaleryl code - CoA by a ketothiolase including, for example, a ketothiolase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 58-78.
[0026] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of 3,5-ketovaleryl-CoA into the code of (R) or ( S)-5-hydroxy-3-ketovaleryl-CoA by an oxidoreductase including, for example, an oxidoreductase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 103-123.
[0027] In some embodiments that may be combined with any of the above or below mentioned embodiments, the one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of (R) or (S)-5-hydroxy-3- ketovaleryl-CoA within the code of (R) or (S)-3,5-dihydroxyalleryl-CoA for an oxidoreductase including, for example, an oxidoreductase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 103-123.
[0028] In some embodiments which may be combined with any of the above or below mentioned embodiments, one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of (R) or (S) - 3,5-dihydroxyalleryl-CoA in the code of (R) or (S)-3-hydroxy-4-pentenoyl-CoA by a dehydratase including, for example, a dehydratase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 37-55.
[0029] In some embodiments that may be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of (R) or (S)-3-hydroxy-4-pentenoyl -CoA in the 3-hydroxy-4-pentenoic acid code for a transferase or a hydrolase including, for example, a transferase or a hydrolase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 1-28 or 29-33 , respectively.
[0030] In some embodiments that can be combined with any of the above or below mentioned embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of 3-hydroxy-4-pentenoic acid to the butadiene code by a decarboxylase including, for example, a decarboxylase encoded by a polynucleotide as set forth in any one of SEQ ID NOs: 79-98.
[0031] In some modalities that can be combined with any of the above or below mentioned modalities, the microorganism is a bacterium selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
[0032] In some embodiments that can be combined with any of the above or below mentioned modalities, the microorganism is a eukaryote and is a yeast, filamentous fungus, protozoan, or alga.
[0033] In some embodiments that can be combined with any of the above or below mentioned embodiments, the yeast is Saccharomices cerevisiae, Zimomonas mobilis, or Pichia pastoris.
[0034] In some modalities that can be combined with any of the above or below mentioned modalities, the carbon source is sugar cane juice, sugar cane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or mixture thereof.
[0035] In some embodiments that can be combined with any of the above or below mentioned embodiments, the carbon source is a monosaccharide, oligosaccharide, or polysaccharide.
[0036] In some modalities that can be combined with any of the above or below mentioned modalities, butadiene is secreted by the microorganism into the fermentation medium.
[0037] In some embodiments that can be combined with any of the above or below mentioned embodiments, the methods may further comprise recovering the butadiene from the fermentation medium.
[0038] In some modalities that can be combined with any of the above or below mentioned modalities, the microorganism has been genetically modified to express one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates in the pathway for the production of butadiene; and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene.
[0039] In some modalities that can be combined with any of the above or below mentioned modalities, the conversion of the fermentable carbon source to butadiene is ATP positive (e.g. generates an ATP flux per mole of butadiene produced) and can be additionally combined with a pathway that consumes NADH to provide an anaerobic process for the production of butadiene.
[0040] The present disclosure also provides microorganisms comprising one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source into one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene.
[0041] In some modalities that can be combined with any of the above or below mentioned modalities, enzymes that catalyze the conversion of the fermentable carbon source into one or more intermediates in the pathway for the production of butadiene are shown in any of Tables 1 to 3.
[0042] In some embodiments that can be combined with any of the above or below mentioned embodiments, enzymes that catalyze the conversion of one or more intermediates to butadiene are shown in any of Tables 1 to 3.
[0043] In some embodiments that can be combined with any of the above or below mentioned embodiments, butadiene is produced via an intermediate of acetyl-CoA and propionyl-CoA; a crotonyl-CoA intermediate; and/or a formic acid intermediate.
[0044] In some modalities that can be combined with any of the above or below mentioned modalities, the microorganism is a bacterium selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus.
[0045] In some embodiments that can be combined with any of the above or below mentioned embodiments, the microorganism is a eukaryote and is a yeast, filamentous fungus, protozoan, or alga.
[0046] In some embodiments that can be combined with any of the above or below mentioned embodiments, the yeast is Saccharomices cerevisiae, Zimomonas mobilis, or Pichia pastoris.
[0047] In some embodiments that can be combined with any of the above or below mentioned modalities, the microorganism has been genetically modified to express one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates in the pathway for the production of butadiene; and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene.
[0048] These and other embodiments of the present description will be described in more detail hereafter. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The foregoing summary, as well as the following detailed description of the disclosure will be better understood when read in conjunction with the attached figures. For the purpose of illustrating the description, shown in the figures are modalities that are currently preferred. It should be understood, however, that the description is not limited to the precise arrangements, examples and instruments shown.
[0050] Figure 1 illustrates an example route for the production of butadiene from a fermentable carbon source via an intermediate of acetyl-CoA and propionyl-CoA.
[0051] Figure 2 illustrates an example route for the production of butadiene from a fermentable carbon source via a crotonyl-CoA intermediate.
[0052] Figure 3 illustrates an example route for the production of butadiene from a fermentable carbon source via a formic acid intermediate. DETAILED DESCRIPTION
[0053] The present description refers generally to microorganisms (e.g., non-naturally occurring microorganisms; modified microorganisms) that comprise a genetically modified pathway and uses of microorganisms for the conversion of a fermentable carbon source into butadiene (see, Figures 1 to 3). Such microorganisms comprise one or more polynucleotides that encode enzymes that catalyze the conversion of a fermentable carbon source to butadiene via new enzymatic pathways. Optionally, the butadiene produced can subsequently be converted to polybutadiene or any number of other butadiene-containing polymers.
[0054] This description provides, in part, the disclosure of new enzymatic pathways, including, for example, new combinations of enzymatic pathways for the production of butadiene from a carbon source (eg, a fermentable carbon source). The enzymatic pathways described herein allow for the enzymatic production of butadiene via: an acetyl-CoA and propionyl-CoA intermediate; a crotonyl-CoA intermediate; and/or a formic acid intermediate.
[0055] The methods provided here provide end results similar to those of sterilization without the high capital expenditures and higher and ongoing management costs that are typically required to establish and maintain sterility throughout the entire production process. In this regard, most industrial-scale butadiene production processes are operated in the presence of measurable numbers of bacterial contaminants, due to the aerobic nature of their processes. Bacterial contamination of a butadiene production process is believed to cause a reduction in product yield and an inhibition of the growth of the butadiene-producing microorganism. Such disadvantages of the above methods are avoided by the currently disclosed methods as the toxic nature of the butadiene produced reduces contaminants in the production process.
[0056] The enzymatic pathways disclosed herein are advantageous over prior known enzymatic pathways for the production of butadiene in that the enzymatic pathways disclosed herein are ATP positive and when combined with a NADH consumption pathway can provide an anaerobic pathway for butadiene. While it is possible to use aerobic processes to produce butadiene, anaerobic processes are preferred due to the risk incurred when olefins (which are, by nature, explosive) are mixed with oxygen during the fermentation process, especially for butadiene fermentation. In addition, oxygen and nitrogen supplementation in a fermenter requires additional investment for air compressor, fermenters (bubble column or air-lift fermenter), temperature control and nitrogen. The presence of oxygen can also catalyze the polymerization of butadiene and can promote the growth of aerobic contaminants in the fermenter broth. Furthermore, aerobic fermentation processes for the production of butadiene have several disadvantages at the industrial level (where it is technically difficult to maintain aseptic conditions), such as the fact that: (i) greater biomass is obtained by reducing the total carbon yield for the desired products; (ii) the presence of oxygen favors the growth of contaminants (Weusthuis et al., 2011, Trend in Biotechnology, 2011, vol. 29, N°4, 153-158) and (iii) the mixture of oxygen and gaseous compounds, like butadiene, they pose serious explosion hazards, (iv) oxygen can catalyze unwanted olefin polymerization reaction, and finally (v) higher costs of fermentation and purification under aerobic conditions. Furthermore, the butadiene produced by the processes disclosed herein is not diluted by O2 and N2 thus avoiding both costly and time-consuming purification of the butadiene produced.
[0057] It is to be understood that the steps involved in any and all of the methods described herein may be performed in any order and are not to be limited or restricted with the order in which they are particularly described. For example, the present description provides methods of producing butadiene from a fermentable carbon source comprising: providing a fermentable carbon source; contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene, and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene in a fermentation medium; and expressing one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides encoding enzymes in a pathway that catalyzes a converting one or more intermediates to butadiene in the microorganism to produce butadiene. As such, the expression of one or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides encoding enzymes in a pathway which catalyze the conversion of one or more intermediates to butadiene in the microorganism for the production of butadiene can be carried out before or after contact with the fermentable carbon source comprising a microorganism comprising one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene, and one or more polynucleotides encoding enzymes in a pathway that catalyze the conversion of one or more intermediates to butadiene in a fermentation medium.
[0058] It is also to be understood that the microorganisms disclosed herein may comprise the entire pathway described in any of Figures 1 to 3 including, comprising all polynucleotides encoding enzymes that catalyze the conversion of a fermentable carbon source to butadiene. Alternatively, it will also be understood that the microorganisms disclosed herein may comprise one or more of the polynucleotides that encode enzymes that catalyze the conversion of a fermentable carbon source to butadiene in any of Figures 1 to 3 (for example, a microorganism may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polynucleotides encoding enzymes that catalyze the conversion of a fermentable carbon source to butadiene as disclosed in any of Figures 1 to 3.
[0059] In some embodiments, the proportion of grams of butadiene produced in grams of fermentable carbon source. is 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0 .32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 , 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0 .57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 , 0.70x 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0, 82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0, 96, 0.97, 0.98, 0.99 or 1.00,
[0060] In some embodiments, a number of moles of carbon in the butadiene produced comprises 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% , 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%x 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 100% of a number of moles of carbon in the fermentable carbon source.
[0061] As used herein, "butadiene" is intended to mean buta-1,3-diene or 1,3-butadiene (CAS 106 99-0) , with a general formula CH2=CH-CH=CH2, and a mass molecular weight of 54.09 g/mol.
[0062] As used herein, the term "biological activity" or "functional activity", when referring to a protein, polypeptide or peptide, may mean that the protein, polypeptide or peptide exhibits a functionality or property that is useful in relating to some biological processes, pathway or reaction. Biological or functional activity may refer, for example, to an ability to interact with or associated with (for example, bind to) another polypeptide or molecule, or may refer to an ability to catalyze or regulate the interaction of other proteins or molecules (eg enzymatic reactions).
[0063] As used herein, the term "culture" can refer to the growth of a population of cells, eg microbial cells, under conditions suitable for growth, in a liquid or solid medium.
[0064] As used herein, the term "derived from" may encompass the terms originating from, obtained from, obtainable from, isolated from, and created from, and generally indicates that a specified material has its origin in another specific material or has characteristics that can be described with reference to the other specific material.
[0065] As used herein, the term "an expression vector" may refer to a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as an NDA encoding sequence (for example , gene sequence) which is operatively linked to one or more suitable control sequence(s) capable of affecting the expression of the coding sequence in a host. Such control sequences include a promoter to affect transcription, an optional operator sequence to control such transcription, a sequence encoding appropriate mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. The vector can be a plasmid, a phage particle or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host's genome (eg, independent of the vector or plasmid), or it can, in some cases, integrate into the genome itself (eg, integrated vector) . The plasmid is the most commonly used form of expression vector. However, the disclosure is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.
As used herein, the term "expression" can refer to the process by which a polypeptide is produced from a nucleic acid sequence that encodes the polypeptides (for example, a gene). The process includes both transcription and translation.
[0067] As used herein, the term "gene" may refer to a segment of DNA that is involved in the production of a polypeptide or protein (eg, fusion protein) and includes regions preceding and following the coding regions, as well as intervening sequences (introns) between individual coding segments (exons).
[0068] As used herein, the term "heterologous", with reference to a nucleic acid, polynucleotide, protein or peptide, may refer to a nucleic acid, polynucleotide, protein or peptide that does not naturally occur in a specific cell, by example, a host cell. The term is intended to include proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes. In contrast, the term homolog, with reference to a nucleic acid, polynucleotide, protein or peptide, refers to a nucleic acid, polynucleotide, protein or peptide that occurs naturally in the cell.
As used herein, the term a "host cell" may refer to a cell or cell line, including a cell such as a microorganism which is a recombinant expression vector that can be transfected for the expression of a polypeptide or protein (for example, fusion protein). Host cells include progeny from a single host cell, the progeny may not necessarily be completely identical (in morphology or in the full complement of genomic DNA) to the original parent cell due to natural, accidental or deliberate mutation. A host cell can include cells transfected or transformed in vivo with an expression vector.
[0070] As used herein, the term "introduced", in the context of inserting a nucleic acid sequence or a polynucleotide sequence into a cell, can include transfection, transformation or transduction and refers to the incorporation of a sequence of Nucleic acid or polynucleotide sequence in a eukaryotic or prokaryotic cell, wherein the nucleic acid sequence or polynucleotide sequence can be incorporated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into a replicon autonomously, or expressed transiently.
[00711 As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in the naturally occurring strain of said species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids that encode metabolic polypeptides, other nucleic acid additions, nucleic acid deletions, and/or other functional disturbance of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides to the referenced species. Additional modifications include, for example, non-coding regulatory regions where the modifications alter the expression of a gene or operon. Microbial organisms that do not naturally occur in the description may contain stable genetic alterations, which refer to microorganisms that can be cultivated for more than five generations without loss of the alteration. Generally, stable genetic changes include modifications that persist longer than 10 generations, in particular stable modifications will persist longer than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. Those skilled in the art will understand that genetic alterations, including metabolic alterations exemplified herein, are described with reference to a suitable host organism such as E. coli and its corresponding metabolic reactions or a suitable host organism for the desired genetic material, such as genes to a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of capability in genomics, those skilled in the art will easily be able to apply the teachings and guidance provided herein to essentially all other organisms. . For example, the E. coli metabolic alterations exemplified herein can be readily applied to other species by incorporating the same nucleic acid or analog encoding other species other than the mentioned species. Such genetic alterations include, for example, genetic alterations of species homologues, in general, and in particular, orthologous, paralogous or nonorthologous gene shifts.
[0072] As used herein, "butadiene" is intended to mean a linear conjugated diene with the molecular formula C4H6, a general formula CH2=CH-CH=CH2 and a molecular weight of 54.09 g/mol. Butadiene is also known in the art as 1,3-butadiene, but-1,3-diene, biethylene, erythrene, divinyl, and vinylethylene.
[0073] As used herein, the term "operationally linked" can refer to a juxtaposition or arrangement of specific elements that allow them to act in concert to bring about an effect. For example, a promoter can be operably linked to a coding sequence if it controls transcription of the coding sequence.
[0074] As used herein, the term "promoter" can refer to a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene. A promoter can be an inducible promoter or a constitutive promoter. An inducible promoter is a promoter that is active under developing environmental or regulatory conditions.
As used herein, the term "a polynucleotide" or "nucleic acid sequence" may refer to a polymeric form of nucleotides of any length and any three-dimensional structure and single-stranded or multi-stranded (e.g. single-stranded, double-stranded, triple-helix, etc.), which contain deoxyribonucleotides, ribonucleotides and/or their analogues or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogues. Such polynucleotides or nucleic acid sequences can encode amino acids (for example, polypeptides or proteins, such as fusion proteins). Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the present description includes those polynucleotides that encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analogue can be used, provided the polynucleotide retains the desired functionality, under conditions of use, including those modifications that increase nuclease resistance (eg, deoxy, 2'-0-Me, phosphorothioates , etc.) Tags may also be incorporated for detection or capture purposes, eg radioactive or non-radioactive tags or anchors, eg biotin. The term polynucleotide includes peptide nucleic acids (PNA). Polynucleotides can be naturally occurring or non-naturally occurring. The terms polynucleotide, nucleic acid and oligonucleotide are used interchangeably herein. Polynucleotides can contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. The nucleotide sequence can be interrupted by non-nucleotide components. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O) R, P(O)OR', COCH2 (formacetal), wherein each R or R' is independently H or substituted or unsubstituted (1-20C) alkyl optionally containing an ether (—0—), aryl, alkenyl bond. , cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be identical. Polynucleotides can be linear or circular, or comprise a combination of linear and circular portions.
As used herein, the term a "protein" or "polypeptide" can refer to a composition comprised of amino acids and recognized as a protein by those skilled in the art. A conventional one-letter or three-letter code for amino acid residues is used herein. Protein and polypeptide are terms used interchangeably herein to refer to amino acid polymers of any length, including those comprising peptides/polypeptides linked (e.g., fused) (e.g., fusion proteins). The polymer can be linear or branched, it can comprise the modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
[0077] As used herein, related proteins, polypeptides or peptides may include variant proteins, polypeptides or peptides. Variant proteins, polypeptides or peptides differ from a parent protein, polypeptide or peptide and/or from each other by a small number of amino acid residues. In some embodiments, the number of different amino acid residues is any of about 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, the variants differ in about 1 to about 10 amino acids. Alternatively or additionally, variants may have a specific degree of sequence identity with a reference protein or nucleic acid, for example, as determined using a sequence alignment tool such as BLAST, ALIGN and CLUSTAL (see below). For example, the variant proteins or nucleic acid can be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity of amino acids with a reference sequence.
As used herein, the term "recovered", "isolated", "purified" and "separated" may refer to a material (e.g., a protein, peptide, nucleic acid, polynucleotide or cell) that is removed from at least one component with which it is naturally associated. For example, these terms can refer to a material that is substantially or essentially free of components that normally accompany it as found in its native state, such as, for example, an intact biological system.
As used herein, the term "recombinant" may refer to sequences of nucleic acids or polynucleotides, polypeptides or proteins, cells and based thereon, which have been manipulated by man such that they are not the same as acids. nucleic, polypeptides, and cells as found in nature. Recombinant may also refer to genetic matter (for example, nucleic acid or polynucleotide sequences, the polypeptides or proteins they encode, and vectors and cells that comprise such nucleic acid or polynucleotide sequences) that have been modified to alter their genetic characteristics. sequence or expression, such as mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another coding sequence or gene, placing a gene under control of a different promoter, expressing a gene from one heterologous organism, expressing a gene at low or high levels, expressing a gene conditionally or constitutively in ways different from its natural expression profile, and the like.
[0080] As used herein, the term "selective marker" or "selectable marker" may refer to a gene capable of expression in a host cell that permits ease of selection from those hosts containing an introduced nucleic acid, polynucleotide or vector sequence . Examples of selectable markers include but are not limited to antimicrobial substances (for example, hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.
As used herein, the term "substantially similar" and "substantially identical" in the context of at least two nucleic acids, polynucleotides, proteins or polypeptides may mean a nucleic acid, polynucleotide, protein or polypeptide comprises a sequence having at least minus about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, compared to a nucleic acid (e.g.,-type wild type), reference polynucleotide, protein or polypeptide. Sequence identity can be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, for example, Altshul et al. (1990) J. Mol Biol 215:403-410, Henikoff et al. (1989) Proc. Natl. Acad. Sci. USA 89:10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90:5873; and Higgins et al. (1988) Gene 73:237). Software for performing BLAST analyzes is publicly available through the National Biotechnology Information Center. In addition, databases can be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci. 85:2444-2448). In some embodiments, substantially identical polypeptides differ only by one or more conservative amino acid substitutions. In some embodiments, substantially identical polypeptides are immunologically cross-reactive. In some embodiments, substantially identical nucleic acid molecules hybridize to one another under stringent conditions (eg, within a range of medium to high stringency).
[0082] As used herein, the term "transfection" or "transformation" can refer to the insertion of an exogenous nucleic acid or polynucleotide into a host cell. The exogenous nucleic acid or polynucleotide can be maintained as an unintegrated vector, for example, a plasmid, or, alternatively, can be integrated into the host cell genome. The term transfecting or transfection is intended to encompass all conventional techniques for introducing nucleic acid or polynucleotide into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran mediated transfection, lipofection, electroporation and microinjection.
[0083] As used herein, the term "transformed", "stably transformed" and "transgenic" can refer to a cell that has a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence integrated into the its genome or as an episomal plasmid that is maintained through several generations.
[0084] As used herein, the term "vector" can refer to a polynucleotide sequence designed to introduce nucleic acids into one or more types of cells. Vectors include cloning vectors, expression vectors, transport vectors, plasmids, phage particles, single and double stranded cassettes, and the like.
As used herein, the term "wild-type", "native" or "naturally occurring" proteins may refer to proteins found in nature. The terms sequence and wild type refer to the sequence of an amino acid or nucleic acid sequence that is found in nature or that occurs naturally. In some embodiments, a wild-type sequence is the starting point of a protein engineering project, eg, production of variant proteins.
[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this description pertains. Singleton et al. Dictionary of Microbiology and Molecular Biology, Second Edition, John Wiley and Sons, New York (1994) and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NI (1991) provide someone with skill with a General dictionary many of the terms used in this description. Furthermore, it will be understood that any of the substrates described in any of the ways herein may alternatively include the anion or cation of the substrate.
[0087] Numeric ranges provided here are inclusive of the numbers defining the range.
Unless otherwise noted, nucleic acid sequences are written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation, respectively.
[0089] While the present description is likely to be incorporated in various forms, the following description of various embodiments is made with the understanding that the present description is to be considered as an exemplification of the description, and is not intended to limit the disclosure for the specific embodiments illustrated. Titles are provided for convenience only and are not to be construed as limiting the description in any way. Embodiments illustrated in any title may be combined with embodiments illustrated in any other title.
[0090] The use of numerical values in the specific quantitative values in this application, unless expressly stated otherwise, are shown as approximations as if the minimum and maximum values within the established ranges were both preceded by the word "about". Furthermore, the description of the ranges is intended to be a continuous range including all values between the quoted minimum and maximum values as well as any ranges that may be formed by such values. Also described herein are any and all proportions (and ranges of any such proportions) that may be formed by dividing a depicted numerical value into any other depicted numerical value. Accordingly, the skilled person will appreciate that many of these ratios, ranges, and ratio ranges can be unambiguously derived from the numerical values presented herein and in all cases such ratios, ranges, and ratio ranges represent various embodiments of the present description. Modification of Microorganisms
[0091] A microorganism can be modified (for example, by genetic engineering) by any method known in the art to understand and/or express (for example, including over-expression) one or more polynucleotides (for example, heterologous polynucleotides and/or or non-heterologous polynucleotides) that encode enzymes in one or more pathways that are capable of converting a fermentable carbon source to butadiene. The microorganism can naturally express all enzymes in one or more pathways to convert a fermentable carbon source to butadiene or can be modified to express including, for example, overexpression, one or more enzymes in one or more pathways. In some embodiments, the microorganism may comprise less than all enzymes in such a pathway and polynucleotides encoding the missing enzymes may be genetically introduced into the microorganism. For example, the modified microorganism can be modified to comprise one or more polynucleotides that encode enzymes that catalyze the conversion of a fermentable carbon source (eg, glucose) to one or more intermediates (eg, acetyl-CoA and propionyl- CoA; crotonyl-CoA and/or formic acid) in one pathway for the production of butadiene. Additionally or alternatively, the modified microorganism can be modified to comprise one or more polynucleotides that encode enzymes that catalyze the conversion of one or more intermediates (e.g., acetyl-CoA and propionyl-CoA; crotonyl-CoA and/or formic acid) in butadiene. In some embodiments, a polynucleotide can encode an enzyme that catalyzes a conversion of one or more intermediates in a pathway for the production of butadiene. In some embodiments, polynucleotides can be modified (eg, by genetic engineering) to modulate (eg, increase or decrease) the substrate specificity of the encoded enzyme, or polynucleotides can be modified to alter the substrate specificity of the encoded enzyme. (for example, a polynucleotide encoding an enzyme with a specificity for one substrate can be modified in such a way that the enzyme has specificity for another substrate). Preferred microorganisms can include polynucleotides that encode one or more of the enzymes as shown in any one of Tables 1 to 3 and in Figure 1 to 3.
[0092] A microorganism may comprise one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of acetyl-CoA and propionyl-CoA to butadiene. In some embodiments, one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of acetyl-CoA and propionyl-CoA to butadiene may include, but are not limited to: - one or more polynucleotides that encode the enzymes in a pathway which catalyze the conversion of acetyl-CoA and propionyl-CoA to ketovaleryl-CoA (for example, a thiolase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ketovaleryl-CoA to (R) or (S) 3-hydroxyalleryl-CoA (for example, a hydroxyvaleryl-CoA dehydrogenase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) hydroxyalleryl-CoA to 2-pentenoyl-CoA (for example, a hydroxyvaleryl-CoA dehydratase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoyl-CoA to 2-pentenoic acid (for example, a pentenoyl-CoA hydrolase or transferase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to butadiene (for example, a 2-pentenoic acid decarboxylase d); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to 4-pentenoic acid (eg a transposition of C = C isomerase bonds); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-pentenoic acid to butadiene (for example, a 4-pentenoic acid decarboxylase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoyl-CoA to penta-2,4-dienoyl-CoA (for example a pentenoyl-CoA dehydrogenase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of penta-2,4-dienoyl-CoA to. penta-2,4-dienoic (for example, a penta-2,4-dienoyl-CoA-hydrolase, or transferase); and/or - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2,4-pentenoic acid to butadiene (eg, a pent,2,4-dienoic decarboxylase).
[0093] In some embodiments, the microorganism further comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) into methylmalonyl-CoA and/or acryloyl-CoA.
[0094] In some embodiments, a microorganism that is provided comprises one or more of the above polynucleotides, including all of the above polynucleotides.
[0095] Examples of enzymes that convert acetyl-CoA and propionyl-CoA into butadiene are presented in Table 1 below, as well as the substrates that act on the product and that they produce. The number of enzymes represented in Table 1 correlates with the enzyme numbering used in Figure 1 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene via an acetyl-CoA and propionyl-CoA intermediate. Table 1: Butadiene production via acetyl-CoA and propionyl-CoA intermediates


[0096] A microorganism may comprise one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to butadiene. In some embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to butadiene may include, but are not limited to: - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion crotonyl-CoA in crotonyl alcohol (for example a crotonyl-CoA reductase (bifunctional)); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonaldehyde (for example, a crotonaldehyde dehydrogenase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonaldehyde to crotonyl alcohol (eg a crotonyl alcohol dehydrogenase); and/or - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl alcohol to butadiene (eg, a crotonyl alcohol dehydrogenase).
[0097] In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including, all of the above polynucleotides.
[0098] In preferred embodiments, the microorganism further comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) to crotonyl-CoA.
[0099] In some embodiments, the microorganism may further comprise one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) to 3-hydroxybutyryl-CoA and/or 4 -hydroxybutyryl-CoA. In such embodiments, the microorganism further comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA.
[00100] Examples of enzymes that convert crotonyl-CoA to butadiene are presented in Table 1 below, as well as the substrates that act on and the product they produce. The number of enzymes represented in Table 1 correlates with the enzyme numbering used in Figure 1, which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene via a crotonyl-CoA intermediate. Table 2: Butadiene production via a crotonyl-CoA


[00101] A microorganism may comprise one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of formic acid to butadiene. In some embodiments, one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of formic acid to butadiene may include, but are not limited to: - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of CO2 in formic acid (for example a formate dehydrogenase); - one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of pyruvate and CoA to acetyl-CoA and formic acid (for example, an acetyl-CoA: format C-acetyltransferase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of formic acid to formyl-CoA (for example, a formyl-CoA transferase or synthase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-acetyl-CoA to acetoacetyl-CoA (for example, an acetoacetyl-CoA thiolase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-CoA and formyl-CoA to 3,5-ketovaleryl-CoA (for example a 3,5-ketovaleryl-CoA thiolase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3,5-ketovaleryl-CoA to (R) or (S)-5^hydroxy-3-ketovaleryl-CoA (eg a 3,5 -ketovaleryl-CoA dehydrogenase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S)-5-hydroxy-3-ketovaleryl-CoA to (R) or (S)-3,5-dihydroxyalleryl-CoA (for example, a 5-hydroxy-3-ketovaleryl-CoA) dehydrogenase; - one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of (R) or (S)-3,5-dihydroxyalleryl-CoA to (R) or (S) 3-hydroxy-4-pentenoyl-CoA ( for example a 3,5-hydroxyvaleryl-CoA dehydratase); - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S)-3-hydroxy-i-pentenoyl'-CoA to 3-hydroxy-4-pentenoic acid (eg a 3 -hydroxy-4-pentenoyl-CoA-hydrolase, transferase or synthase); and/or - one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxy-4-pentenoic acid to butadiene (for example, a 3-hydroxy-4-pentenoic acid decarboxylase).
[00102] In some embodiments, the microorganism further comprises one or more polynucleotides that encode enzymes in a pathway that source fermentable carbon (eg, glucose) into pyruvate.
[00103] In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including all of the above polynucleotides.
[001041 Examples of enzymes that convert formic acid to butadiene are presented in Table 3 below, as well as the substrates they act on and the product they produce. The number of enzymes represented in Table 3 is correlated with the enzyme numbering used in Figure 3 which schematically represents the enzymatic conversion of a fermentable carbon source to butadiene via a formic acid intermediate. Table 3: Butadiene production via a formic acid intermediate


[00105] A microorganism is also provided that comprises one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of a fermentable carbon source to acetyl-CoA and propionyl-CoA and one or more polynucleotides that encode the enzymes into a pathway that catalyzes the conversion of acetyl-CoA and propionyl-CoA to butadiene including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of a fermentable carbon source (eg, glucose ) in pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to methylmalonyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of methylmalonyl-CoA to propionyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acryloyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acryloyl-CoA to propionyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and propionyl-CoA to ketovaleryl-CoA (eg, a thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ketovaleryl-CoA to (R) or (S) 3-hydroxyvaleryl-CoA (for example, a hydroxyvaleryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S)-3 hydroxyvaleryl-CoA to 2-pentenoyl-CoA (for example, a hydroxyvaleryl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoyl-CoA to 2-pentenoic acid (for example, pentenoyl-CoA hydrolase, a pentenoyl-CoA transferase, or a pentenoyl-CoA synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to 4-pentenoic acid (eg, a transposition of C=C bonds isomerase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-pentenoic acid to butadiene (e.g., 4-pentenoic acid decarboxylase or a 2-petenoic acid decarboxylase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including, all of the above polynucleotides.
[00106] A microorganism is also provided that comprises one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of a fermentable carbon source to ethyl-malonyl-CoA and one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of ethyl malonyl-CoA to butadiene, including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA to acetoacetyl-CoA (for example, acetoacetyl-CoA thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA (for example, a 3-hydroxybutyryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (eg, a crotonase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to ethyl-malonyl-CoA (eg, a crotonyl-CoA carboxylase/reductase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to butyric acid (eg, butyryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of butyric acid to ethyl malonyl-CoA (for example, a butanoyl-CoA:carbon dioxide ligase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ethyl malonyl-CoA to 2-(formol)butanoic acid (for example, an ethyl malonyl-CoA reductase (aldehyde formation); which encode enzymes in a pathway that catalyze the conversion of 2-(formyl)butanoic acid to 2-(hydroxymethyl)butanoic acid (eg, a 2-(formyl)butanoic acid reductase (alcohol formation); one or more polynucleotides that encode enzymes in a pathway that catalyzes the conversion of ethyl malonyl-CoA to 2-(hydroxymethyl)butanoic acid (eg, an ethyl malonyl-CoA reductase (alcohol formation); one or more polynucleotides that encode the enzymes to a pathway that catalyze, the conversion of 2-(hydroxymethyl)butanoic acid to 2-(phosphanyloxymethyl)butanoic acid (eg, a 2-(hydroxymethyl)butanoic acid kinase; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-(phosphanyloxymethyl) butanoic acid co in 2-(diphosphanyloxymethyl)butanoic acid (for example a 2-(phosphanyloxymethyl)butanoic acid kinase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-(diphosphanyloxymethyl) butanoic acid to [(E)-but-2-enoxy]-phosphanyl-phosphane (eg, 2-(diphosphanyloxymethyl)butanoic acid decarboxylase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of [(E)-but-2-enoxy]-phosphanyl-phosphane to butadiene (eg, butadiene synthetase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including, all of the above polynucleotides.
[00107] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source to 4-hydroxybutyryl-CoA and 3-hydroxybutyryl-CoA and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-hydroxybutyryl-CoA and 3-hydroxybutyryl-CoA to butadiene including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a carbon source fermentable (eg glucose) to PEP; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of PEP to oxaloacetate (for example, a PEP carboxykinase or PEP carboxylase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of PEP to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA (for example, a pyruvate dehydrogenase or a ferrodoxine pyruvate oxidoreductase) or oxaloacetate (for example, a PEP carboxykinase or PEP carboxylase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA to acetoacetyl-CoA (for example, an acetoacetyl-CoA thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA (for example, 3-hydroxybutyryl-CoA-dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of oxaloacetate to malate (eg, a malate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of malate to fumarate (eg, a fumarase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of fumarate to succinate (eg, a fumarate reductase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of succinate to succinyl-CoA (for example, a succinyl-CoA transferase or a succinyl-CoA-synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of succinyl-CoA to succinyl-semialdehyde (eg, a succinyl-CoA reductase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of succinyl hemialdehyde to 4-hydroxybutyrate (for example, a 4-hydroxybutyrate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of succinate to 4-hydroxybutyrate (for example, a succinate reductase, phosphopantatheinylase, or 4-hydroxybutyrate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA (for example, a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA-synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (for example, a 4-hydroxybutyryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (eg, a crotonase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonaldehyde (eg, a crotonaldehyde dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonaldehyde to crotonyl alcohol (eg, an alcohol dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonyl alcohol (eg, a crotonyl-CoA reductase (bifunctional), and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl alcohol to butadiene (eg, a crotonyl alcohol dehydratase) In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including, all of the above polynucleotides.
[00108] A microorganism is also provided that comprises one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of a fermentable carbon source to acryloyl-CoA and acetyl-CoA and one or more polynucleotides that encode the enzymes in a pathway that catalyze the conversion of acryloyl-CoA and acetyl-CoA to butadiene, including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose ) in pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to lactate (eg, a lactate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactate to lactoyl-CoA (for example, a lactoyl-CoA transferase or synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactoyl-CoA to acryloyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acryloyl-CoA and acetyl-CoA to 3-keto-4-pentenoyl-CoA (for example, a thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-keto-4-pentenoyl-CoA to (R) or (S) 3-hydroxy-4-pentenoyl-CoA (eg, a 3-keto -4 - pentenoyl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (eg, a 3-hydroxy- 4-pentenoyl-CoA-transferase, a hydrolase, or a synthase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxy-4-pentenoic acid to butadiene (eg, a 3-hydroxy-4-pentenoic acid decarboxylase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including all of the above polynucleotides.
[00109] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) to acetyl-CoA and 3-hydroxy-propionyl-CoA and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and 3-hydroxypropionyl-CoA to butadiene, including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion from a source of fermentable carbon (eg glucose) to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to lactate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactate to lactoyl-CoA (for example, lactoyl-CoA-transferase or synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactoyl-CoA to acryloyl-CoA (for example, lactoyl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acryloyl-CoA to 3-hydroxy-propionyl-CoA (for example, acryloyl-CoA hydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to 3-hydroxypropionate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxypropionate to 3-hydroxy-propionyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and 3-hydroxy-propionyl-CoA to 5-hydroxy-3-ketovaleryl-CoA (for example, a thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 5-hydroxy-3-ketovaleryl-CoA to (R) or (S) 3,5-dihydroxy-valeryl-CoA (eg, a 5 -hydroxy-3-ketovaleryl-CoA dehydrogenase),* one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) 3,5-dihydroxy-valeryl-CoA to (R) or (S) of 3-hydroxy-4-pentenoyl-COA (for example, a 3,5-hydroxyvaleryl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (eg, a 3-hydroxy- 4-pentenoyl-CoA hydrolase, transferase, or synthase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxy-4-pentenoic acid to butadiene (eg, a 3-hydroxy-4-pentenoic acid decarboxylase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides, including all of the above polynucleotides.
[00110] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg glucose) to acetoacetyl-CoA and formyl-CoA and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-CoA and formyl-CoA to butadiene, including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a carbon source fermentable (eg glucose) to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA and formate (for example, a pyruvate formate lyase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA to acetoacetyl-CoA (eg, thiolase); one or more polynucleotides that encode the enzymes in. a pathway that catalyzes the conversion of CO2 to formate (eg, formate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the format to formyl-CoA (for example, a formyl-CoA transferase, or formyl-CoA synthase); a. or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of formyl-CoA and acetoacetyl-CoA to 3,5-ketovaleryl-CoA (eg, a thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3,5-ketovaleryl-CoA to 5-hydroxy-3-ketovaleryl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 5-hydroxy-3-ketovaleryl-CoA to (R) or (S) 3,5-divaleryl-CoA (eg, a 5-hydroxy -3-Ketovaleryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S)-3,5-dihydroxy-valeryl-CoA to (R) or (S) 3-hydroxy-4-pentenoyl -CoA (for example a 3,5-hydroxyvaleryl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) 3-hydroxy-4-pentenoyl-CoA to 3-hydroxy-4-pentenoic acid (eg, a 3-hydroxy- 4-pentenoyl-CoA-hydrolase, transferase, or synthase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxy-4-pentenoic acid to butadiene (eg, a 3-hydroxy-4-pentenoic acid decarboxylase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides, including all of the above polynucleotides.
[00111] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg glucose) to acetyl-CoA and 3-hydroxy-propionyl-CoA and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and 3-hydroxypropionyl-CoA to butadiene, including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion from a source of fermentable carbon (eg glucose) to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acryloyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acryloyl-CoA to 3-hydroxy-propionyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to 3-hydroxypropionate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxypropionate to 3-hydroxypropionyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and 3-hydroxy-propionyl-CoA to 5-hydroxy-3-ketovaleryl-CoA (for example, a thiolase); one or more of the polynucleotides that encode enzymes in a pathway that catalyze the conversion of 5-hydroxy-3-ketovaleryl-CoA to (R) or (S) 3,5-dihydroxy-valeryl-CoA (eg, a 5-hydroxy-3-ketovaleryl-CoA-dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of (R) or (S) 3,5-dihydroxy-valeryl-CoA to 3,5-hydroxypentanoic acid (eg, a 3,5 acid - hydroxypentanoic kinase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3,5-hydroxypentanoic acid to 3,5-hydroxypentanoic phosphate (for example, a 3,5-hydroxypentanoic acid kinase); one or more polynucleotides that encode enzymes that catalyze via a conversion of 3,5-hydroxypentanoic acid phosphate to hydroxypentanoic acid 3,5-diphosphate (for example, a 3,5-hydroxypentanoic acid phosphate kinase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3,5-hydroxypentanoic acid diphosphate to 1-butenyl-4-diphosphate (for example, a hydroxypentanoic acid diphosphate decarboxylase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 1-butenyl-4-diphosphate to butadiene (eg, a butadiene synthase). In some embodiments, a microorganism is provided that comprises one or more of the above polynucleotides including all of the above polynucleotides.
[00112] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg glucose) to ethyl malonyl-CoA and one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of ethyl malonyl-CoA to butadiene including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA to acetoacetyl-CoA (for example, an acetoacetyl-CoA thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetoacetyl-COA to 3-hydroxybutyryl-CoA (for example, a 3-hydroxybutyryl-CoA dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA (eg, a crotonase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA-se to ethyl-malonyl-CoA (e.g., a crotonyl-CoA carboxylase/reductase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to butyric acid (eg, butyryl-CoA-dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of butyric acid to ethyl-malonyl-CoA (eg, a butanoyl-CoA:-carbon dioxide ligase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ethyl malonyl-CoA to 2-hydroxy-butanoic acid (for example, an ethyl malonyl-CoA reductase, an alcohol dehydrogenase, or an aldehyde dehydrogenase); one or more polynucleotides which they encode. enzymes in a pathway that catalyze the conversion of 2-hydroxymethyl-butanoic acid to 2-butenyl 4-diphosphate (eg, a 2-hydroxy-methyl-butanoate kinase, a hydroxymethyl butanoate-phosphate kinase, or a 2-hydroxy- methyl butanoate diphosphate decarboxylase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-hydroxy-methyl-butanoic acid to 2-butenyl 4-phosphate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-butenyl 4-phosphate to butadiene, and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-butenyl 4-diphosphate in butadiene (for example butadiene synthetase). In some embodiments, a microorganism that is provided comprises one or more of the above polynucleotides including all of the above polynucleotides.
[00113] A microorganism is also provided that comprises one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a fermentable carbon source (eg, glucose) into lactate and acetyl-CoA and oxalacetate and one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactate and acetyl-CoA and oxalacetate to butadiene including, but not limited to: one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of a carbon source fermentable (eg glucose) to PEP; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of PEP to pyruvate; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of pyruvate to acetyl-CoA; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactate to lactoyl-CoA (for example, a lactate CoA-transferase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of lactoyl-CoA to acryloyl-CoA (for example, a lactoyl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acryloyl-CoA to propionyl-CoA (for example, an acryloyl-CoA oxidoreductase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of propionyl-CoA to ketovaleryl-CoA (eg, a thiolase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of ketovaleryl-CoA to 2-pentenoyl-CoA (for example, a ketovaleryl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoyl-CoA to 2-pentenoic acid (for example, a pentenoyl-CoA hydrolase, transferase, or synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to butadiene (eg, a 4-pentenoic acid decarboxylase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 2-pentenoic acid to 4-pentenoic acid (eg, a polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-pentenoic acid to butadiene ( for example, a 4-pentenoic acid decarboxylase/; one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of an oxalacetate to malate (eg, a malate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of malate to fumarate (eg, a fumarase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of fumarate to succinate (eg, a fumarate reductase); encode the enzymes in a pathway that catalyze the conversion of succinate to succinyl-CoA (eg, a succinyl-CoA transferase synthase); one or more polynucleotides that encode the enzymes into a the pathway that catalyzes the conversion of succinyl-CoA to semialdehyde succinate (eg, a succinyl-CoA reeducatase); one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of hemialdehyde succinate to 4-hydroxybutyrate (eg, a hydroxybutyrate dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze a conversion of 4-hydroxybutyrate to 4-hydroxybutyryl-CoA (for example, a 4-hydroxybutyryl-CoA transferase, or a 4-hydroxybutyryl-CoA synthase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA (for example, a 4-hydroxybutyryl-CoA dehydratase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonaldehyde (eg, a crotonaldehyde dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to crotonyl alcohol (for example, a crotonyl-CoA reductase or a bifunctional alcohol dehydrogenase); one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonaldehyde to crotonyl alcohol (for example, an alcohol dehydrogenase); and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl alcohol to butadiene (eg, a crotonyl alcohol dehydratase).
[00114] Any of the microorganisms provided herein may optionally comprise one or more polynucleotides encoding enzymes that allow balanced redox conversion of a fermentable carbon source to butadiene.
[00115] The microorganism can be an archaea, bacteria, or eukaryote. In some embodiments, the bacteria is Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus including, for example, Pelobacter propionicus, Clostridium propionicum, Clostridium acetobutylicum, Lactobacillus, Propionibacterium acidipropionici, or Propionibacterium freudenre. In some embodiments, the eukaryote is a yeast, filamentous fungus, protozoan, or alga. In some embodiments, the yeast is Saccharoinices cerevisiae, Zimomonas mobilís, or Pichia pastoris.
[00116] In some embodiments, the description contemplates modification (eg manipulation) of one or more of the enzymes provided herein. Such modification can be performed to redesign the substrate specificity of the enzyme and/or to modify (e.g., reduce) its activity against other substrates in order to increase its selectivity for a particular substrate. Additionally or alternatively, one or more enzymes as provided herein can be manipulated to alter (e.g., increase, including, for example, increase its catalytic activity or its substrate specificity) one or more of its properties.
[00117] Any of the enzymes (eg the polynucleotide encoding the enzyme) can be modified (eg mutated or diversified) to expand or change its substrate specificity (eg change the substrate specificity of an enzyme from one substrate to another substrate) by any method known in the art. Such methods include, but are not limited to EpPCR Pritchard et al., J.Theor. Biol. 234:497-509 (2005)); Rolling Circle Amplification (epRCA) Fuji! et al., Nucleic Acids Res. 32:el45 (2004); and Fuji! et al., Nat. Protoc. 1:2493-2497 (2006)); DNA or Family Shuffling Stemmér, Proc. Natl. Academic Sci. U.S.A. 91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP) Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); and/or Random Priming Recombination (RPR) Shao et al., Nucleic Acids Res. 26:681 683 (1998)).
[00118] Examples of additional methods for mutagenesis of a polynucleotide include heteroduplex recombination ( Volkov et al., Nucleic Acids Res. 27:el8 (1999), and Volkov et al., Methods Enzymol, 328:456-463 (2000) ); Random Chimeragenesis on Transient Templates (RACHITT) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated Templates (RETT) (Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffiling (DOGS) (Bergquist and Gibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol.Eng. 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY) (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Tio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY) (Lutz et al., Nucleic Acids Res 29.-E16 (2001)); SCRATCHY (Lutz et al., Proc. Natl. Acad. Sci. U.S.A 98:11248-11253 (2001)); Random Drift Mutagenesis(RNDM) (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagensis (SeSaM) (Wong et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004) and Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT (Muller et al., Nucleic Acids Res. 33:ell7 (2005)). Examples of additional methods include Sequence Homolgy-Independent Protein Recombination (SHIPREC) (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation Mutagensis™ (GSSM™) (Kretz et al., Methods Enymol. 388:3-11 (2004)); Combinatorial Mutagenesis Cassette (CCM) (Reidhaar-Olson et al., Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al., Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM) (Reetz et al., Angew. Chem. Int. Ed. Engl. 40:3589-3591 (2001)); and the Mutator Strains technique (Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001); Low et al., J. Mol. Biol. 260:359-3680 (1996)). Other examples of methods include Look-Through Mutagesis (LTM) (Rajpal et al., Proc. Natl. Acad. Sci. U.S. 102:8466 8471 (2005)); Gene Reassembly (Tunable GeneReassembly™ (TGR™) Techonolgy supplied by Verenium Corporaiton), in Silico Protein Design Automation (PDA) (Hayes et al., Proc. Natl. Acad. Sci. U.S.A 99:15926-15931 (2002)); and Interactive Saturation Mutagenesis (ISM) (Reetz et al., Nat. Protoc. 2:891-903 (2007), and Reetz et al., Angew. Chem. Int. Ed. Engl. 45:7745-7751 (2006) ).
[00119] In some embodiments, sequence alignment and comparative protein modeling can be used to alter one or more of the enzymes described herein. Homology modeling or comparative modeling refers to the construction of an atomic-resolution model of the desired protein from its primary amino acid sequence and three-dimensional structure of a similar experimental protein. Such a model can allow the substrate binding site of the enzyme to be defined, and the identification of specific amino acid positions that can be substituted with another natural amino acid in order to reorient its substrate specificity.
[00120] Variants or sequences having substantial identity or homology to the polynucleotides encoding enzymes as described herein may be used in the practice of the description. Such sequences may be referred to as variants and modified sequences. That is, a polynucleotide sequence can be modified and still retain the ability to encode a polypeptide that exhibits the desired activity. Such variants or modified sequences are therefore equivalent. Generally, the variant or modified sequence may comprise at least about 40% to 60%, preferably about 60% to 80%, more preferably about 80% to 90%, and most preferably about 90% to 95% sequence identity to native sequence.
[00121] In some embodiments, a microorganism can be modified to express, including, for example, overexpressing, one or more enzymes as provided herein. The microorganism may be modified by genetic engineering techniques (e.g. recombinant technology), classical microbiological techniques, or a combination of such techniques and may also include naturally occurring genetic variants to produce a genetically modified microorganism. Some such techniques are generally described, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press.
[00122] A microorganism may include a microorganism into which a polynucleotide has been inserted, deleted or modified (i.e. mutated, for example, by the insertion, deletion, substitution and/or inversion of nucleotides), such that such modifications provide the desired effect of expression (e.g., overexpression) of one or more enzymes as provided herein within the microorganism. Genetic modifications that result in an increase in gene expression or function may be referred to as amplification, overproduction, overexpression, activation, augmentation, addition, or upregulation of a gene. The addition of cloned genes to increase gene expression may include maintenance of the cloned gene(s) in replicating plasmids or integration of the cloned gene(s) into the genome of the production organism. Furthermore, enhancing expression of desired cloned genes may include operatively linking the cloned gene(s) to heterologous or native transcriptional control elements.
[00123] When desired, the expression of one or more of the enzymes provided herein under the control of a regulatory sequence that directly or indirectly controls the expression of the enzyme in a time-dependent manner during the fermentation reaction.
In some embodiments, a microorganism is transformed or transfected with a genetic vehicle, such as an expression vector that comprises an exogenous polynucleotide sequence that encodes the enzymes provided herein.
[00125] Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may usually, but not always, comprise a replication system (i.e., vector) recognized by the host, including the desired polynucleotide fragment encoding the desired polypeptide, and it may, preferably, but not necessarily, also include regulatory sequences for initiation of transcription and translation operatively linked to the segment encoding the polypeptide. Expression systems (expression vectors) can include, for example, an origin of replication or autonomously replicating sequences (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription termination sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides can also be included where appropriate, preferably from secreted polypeptides of the same or related species which allow the protein to cross and/or be housed in cell membranes or be secreted from the cell.
Vectors can be constructed using standard methods (see, for example, Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, Co. NY, 1995).
The manipulation of polynucleotides of the present description including the polynucleotides that encode one or more of the enzymes described herein is typically carried out in recombinant vectors. A number of vectors are publicly available, including bacterial plasmids, bacteriophages, artificial chromosomes, episomal vectors and gene expression vectors, all of which can be employed. A vector for use in accordance with the description can be selected to accommodate a protein coding sequence of a desired size. A suitable host cell is transformed with the vector after in vitro cloning manipulations. Host cells can be prokaryotic, such as any one of a number of bacterial strains, or can be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, mammalian cells. rodent, simian or human. Each vector contains several functional components which generally include a cloning site, an origin of replication and at least one selectable marker gene. If a given vector is an expression vector, it additionally has one or more of the following: enhancer element, promoter, and transcription termination and signal sequences, each positioned in proximity to the cloning site so that they are operably linked to the gene encoding a member of the polypeptide repertoire according to the description.
Vectors, including cloning and expression vectors, may contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. For example, the sequence may be one that allows the vector to replicate independently of the host's chromosomal DNA and may include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the pBR322 plasmid origin of replication is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (eg, from SV 40, from adenovirus) are useful. for cloning vectors on. mammalian cells. Generally, the origin of replication is not required for mammalian expression vectors unless these are used in mammalian cells capable of replicating high levels of DNA, such as COS cells.
[00129] The cloning or expression vector may contain a selection gene also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene, therefore, will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, for example, ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, auxotrophic complement deficiencies, or provide critical nutrients not available in the growth medium.
[00130] Vector replication can be performed in E. coli (eg TB1 or TGI strain, DH5α, DHlOβ, JM110). A selectable E. coli marker, for example, the β-lactamase gene that confers resistance to the antibiotic ampicillin, may be of use. These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid, such as pUC18 or pUC19, or pUC119.
[00131] Expression vectors may contain a promoter that is recognized by the host organism. The promoter can be operably linked to a coding sequence of interest. Such a promoter can be constitutive or inducible. Polynucleotides are operably linked when the polynucleotides are in a relationship that allows them to function in their intended way.
Promoters suitable for use with prokaryotic hosts may include, for example, lactose and α-lactamase promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, the apramycin promoter, hygromycin promoter , methylenemycin promoter, and hybrid promoters such as the tac promoter. In addition, host constitutive or inducible promoters can be used. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.
Viral promoters obtained from virus genomes include promoters from polyoma viruses, avian poxviruses, adenoviruses (for example adenovirus 2 or 5), herpes simplex virus (thymidine kinase promoter), virus papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (eg, MoMLV, or RSV LTR), hepatitis-B virus, myeloproliferative sarcoma virus promoter (MPSV), VISNA, and Simian Virus 40 (SV40) . Promoters from heterologous mammals include, for example, actin promoter, immunoglobulin, heat shock protein promoters.
The SV40 virus early and late promoters are conveniently obtained as a restriction fragment which also contains the SV40 viral origin of replication (see, for example, Fiers et al., Nature, 273:113 (1978); Mulligan and Berg, Science, 209:1422-1427 (1980); and Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981. The immediate early promoter of the human cytomegalovirus (CMV) is conveniently obtained as a Hind III E restriction fragment (see, for example, Greenaway et al., Gene, 18:355-360 (1982)) A broad host range promoter, such as the SV40 early promoter, or the Rous sarcoma virus LTR, is suitable for use in the present expression vectors.
[00135] Generally, a strong promoter can be employed to provide high level transcription and expression of the desired product. Among the eukaryotic promoters that have been identified as strong promoters for high level expression are the SV40 early promoter, adenovirus major late promoter, metallothionein-I promoter, long terminal repeat in Rous sarcoma virus, and cytomegalovirus immediate early promoter human (CMV or CMV IE). In one embodiment, the promoter is an SV40 or a CMV early promoter.
[00136] The promoters used can be constitutive or adjustable, for example, inducible. Illustrative inducible promoters include jun, fos and metallothionein and heat shock promoters. One or both of the transcription unit promoters may be an inducible promoter. In one embodiment, GFP is expressed from a constitutive promoter, while an inducible promoter directs transcription of the gene encoding one or more enzymes as disclosed herein and/or the selectable amplifiable selectable marker.
[00137] The transcriptional regulatory region in higher eukaryotes may comprise an enhancer sequence. Many enhancer sequences from mammalian genes are known, for example, globin, elastase, albumin, α-fetoprotein and insulin genes. A suitable enhancer is an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus immediate early promoter enhancer (Boshart et al., Cell 41: 521 (1985)), the polyoma enhancer on the late side of the origin of replication, and adenovirus (see also, for example, Yaniv, Nature, 297:17-18 (1982) on enhancer elements for activation of eukaryotic promoters). Enhancer sequences can be introduced into the vector at a 5' or 3' position in the gene of interest, but is preferably located at a site 5' from the promoter.
[00138] Yeast and mammalian expression vectors may contain the prokaryotic sequences that facilitate the propagation of the vector in bacteria. Therefore, the vector may have other components such as an origin of replication (eg a nucleic acid sequence that allows the vector to replicate in one or more selected host cells), antibiotic resistant genes for selection in bacteria, and/or an amber stop codon that can allow translation for reading through the codon. Additional selectable eukaryotic gene(s) may be incorporated. Generally, in cloning vectors the origin of replication is one that allows the vector to replicate independently of the host's chromosomal DNA, and includes autonomous origins of replication or sequences of replication. Such sequences are well known, for example, the ColEL origin of replication in bacteria. Several viral sources (eg, SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, a eukaryotic replicon is not required for expression in mammalian cells unless extrachromosomal (episomal) replication is intended (eg, the SV40 origin can typically be used only because it contains the early promoter).
[00139] To facilitate the insertion and expression of different genes encoding enzymes as described herein from expression constructs and vectors, the constructs can be designed with at least one cloning site for insertion of a gene encoding any enzyme herein described. The cloning site can be a multiple cloning site, for example containing multiple restriction sites.
[00140] Plasmids can be propagated in bacterial host cells to prepare DNA stocks for steps of sub-cloning or introduction into eukaryotic host cells. Transfection of eukaryotic host cells can be any performed by any method well known in the art. Transfection methods include lipofection, electroporation, calcium phosphate coprecipitation, rubidium chloride or polycation mediated transfection, protoplast fusion and microinjection. Preferably, the transfection is a stable transfection. The transfection method that provides optimal frequency of transfection and expression of the construct in the particular host cell lineage type is favored. Appropriate methods can be determined by routine procedures. For stable transfectants, the constructs are integrated so that they are stably maintained within the host chromosome.
Vectors can be introduced into selected host cells by any of a number of suitable methods known to those skilled in the art. For example, vector constructs can be introduced into appropriate cells by any of a number of transformation methods for plasmid vectors. For example, standard calcium chloride-mediated bacterial transformation is still commonly used to introduce naked DNA into bacteria (see, eg, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but electroporation and conjugation can also be used (see, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY).
[00142] For the introduction of vector constructs into yeast or other fungal cells, chemical transformation methods can be used (eg Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Transformed cells can be isolated in appropriate selective medium by the selectable marker used. Alternatively, or in addition, plaques or filters lifted from the plaques can be checked for GFP fluorescence to identify transformed clones.
[00143] For the introduction of vectors comprising differentially expressed sequences into mammalian cells, the method used may depend on the vector form. Plasmid vectors can be introduced by any of a number of transfection methods including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran mediated transfection, electroporation, or calcium phosphate precipitation (see, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY).
[00144] Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and untransformed cells, or primary cells are widely available, making lipofection an attractive method of introducing constructs into eukaryotic cells, and in particular of mammals into culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies offering the reagents and methods for lipofection include Bio-Rad Laboratories, CLONTECH, Glen Research, InVitrogen, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.
[00145] The host cell may be able to express the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Transformation includes co- and post-translational modification such as leader peptide cleavage, GPI o linkage, glycosylation, ubiquitination and disulfide bond formation. Transfectable immortalized host cell cultures and in vitro cell culture of the type typically employed in genetic engineering are preferred. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 derivatives adapted for growth in suspension culture, Graham et al., J. Gen. Virol, 36:59 (1977), baby hamster kidney cells (BHK, ATCC CCL 10) Chinese hamster ovary cells-DHFR (ATCC CRL-9096); dp12.CHO cells, a derivative of CHO/DHFR- (EP 307,247 published March 15, 1989); rat sertoli cells (TM4, Mather, Biol Reprod, 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA) , ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); rat mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); human acute lymphoblastic cell line PEER (Ravid et al., Int. J. Cancer 25:705-710 (19 80)); MRC 5 cells; FS4 cells; human hepatoma lineage (Hep G2), human HT1080 cells, KB cells, JW-2 cells, detroit 6 cells, NIH-3T3 cells, myeloma and hybridoma cells. Embryonic cells used to generate transgenic animals are also suitable (eg zygotes and embryonic stem cells).
Suitable host cells for cloning or expressing polynucleotides (eg, DNA) in vectors may include, for example, prokaryote, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, for example, E.coll, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella tiphimurium , Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710 published April 12, 1989), pseudomonas such as P. aeruginosa, and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31446), although other strains such as E. coli B, E. coli X1776 (ATCC 31.537), E. coli JM110 (ATCC 47013) e. coli W3110 (ATCC 27,325) are suitable.
[00147] In addition to prokaryotes, eukaryotic microbes such as filamentous fungus or yeast can be suitable cloning or expression hosts for vectors comprising the polynucleotides encoding one or more enzymes. Saccharomices cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are commonly available and are useful here, such as Schizosaccharomices pombe; Kluiveromices hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402.226); Pichia pastors (EP 183.070); Candida; Trichoderma reesia (EP 244.234); Gross neurospora; Schwanniomices such as Schwanniomices occidentalis; and filamentous fungi such as, for example, neurospora, penicillium, tolipocladium and aspergillus hosts such as A. nidulans and A. niger.
[00148] When the enzyme is glycosylated, suitable host cells for expression can be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified from hosts such as Spodoptera frugiperda (worm), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori (worm). silk) were identified. A variety of viral strains for translation are publicly available, e.g., the L1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus herein in accordance with the present description. , in particular for translation of Spodoptera frugiperda cells.
[00149] Plant cell cultures from cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be used as host cells.
Examples of useful mammalian host cells are Chinese hamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary/-DHFR cells (CHO, Urlaub et al. al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen. Virol 36:59, 1977) baby hamster kidney cells (BHK, ATCC CCL 10); rat sertoli cells (TM4, Mather, (Biol. Reprod. 23: 243-251, 1980), monkey kidney cells (CVI ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL -1587); human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138 , ATCC CCL 75); human liver cells (Hep G2, HB 8065); rat mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 ( 1982)); MRC 5 cells; FS4 cells; and a human hepatoma strain (Hep G2).
[00151] Host cells are transformed or transfected with the expression described above, or cloning vectors for the production of one or more enzymes as described herein or with polynucleotides encoding one or more enzymes as described herein and grown in nutrient medium modified as appropriate for inducing promoters, selector transformants, or amplifying the genes encoding the desired sequences.
Host cells that contain desired nucleic acid sequences encoding the described enzymes can be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells . Furthermore, any of the media described in Ham et al., Meth. Enz. 58: 44, (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Patent Re. No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or growth factor such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMICIN™), trace elements (defined as inorganic compounds normally present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the person skilled in the art. Polynucleotides and encoded enzymes
[00153] Any known polynucleotide (eg gene) that encodes an enzyme or a variant thereof that is capable of catalyzing enzymatic conversion including, for example, an enzyme as shown in any one of Tables 1 to 3 or Figures 1 to 3, is contemplated for use by the present description. Such polynucleotides can be modified (e.g., by genetic engineering) to modulate (e.g., increase or decrease) the substrate specificity of an encoded enzyme, or polynucleotides can be modified to alter the substrate specificity of the encoded enzyme (e.g. , a polynucleotide encoding an enzyme with a specificity for a substrate can be modified in such a way that the enzyme has specificity for an alternative substrate). Preferred microorganisms can include polynucleotides that encode one or more of the enzymes as shown in any one of Tables 1 to 3 and in Figures 1 to 3.
In some embodiments, the microorganism may comprise an oxidoreductase, such as hydroxyvaleryl-CoA dehydrogenase, a crotonyl-CoA reductase (bifunctional), a crotonaldehyde dehydrogenase, a crotonyl alcohol dehydrogenase, a 3,5-ketovaleryl-CoA dehydrogenase, or an oxidoreductase as described in SEQ ID NOs: 103-123. In some embodiments, the microorganism can comprise a transferase such as a pentenoyl-CoA transferase, a penta-2,4-dienoyl-CoA transferase, a formyl-CoA transferase, a 3-hydroxy-4-pentenoyl-CoA transferase, or a transferase as described in SEQ ID NOs: 1-28. In some embodiments, the microorganism can comprise a hydrolase, such as a pentenoyl-CoA hydrolase, a penta-2,4-dienoyl-CoA hydrolase, a 3-hydroxy-4-pentenoyl-CoA hydrolase, or a hydrolase as described in SEQ ID NOs: 29-33. In some embodiments, the microorganism can comprise a CoA synthetase such as a formyl-CoA synthase or a CoA synthase as described in SEQ ID NOs: 34-36. In some embodiments, the microorganism can comprise a ketothiolase, such as a thiolase, an acetyl-CoA:formate C-acetyltransferase, an acetoacetyl-CoA thiolase, a 3,5-ketovaleryl thiolase, or a ketothiolase as described in SEQ ID NOs: 58-78. In some embodiments, the microorganism can comprise a dehydrogenase, such as pentenoyl-CoA dehydrogenase, a formate dehydrogenase, or a dehydrogenase as described in SEQ ID NOs: 124-139. In some embodiments, the microorganism can comprise a dehydratase, such as a hydroxyvaleryl-CoA dehydratase, a crotonyl alcohol dehydratase, a 3,5-hydroxyvaleryl-CoA dehydratase, or a dehydratase as described in SEQ ID NOs: 37-55. In some embodiments, the microorganism can comprise an isomerase, such as a C-C linkage transposition, or an isomerase as described in SEQ ID NOs: 99-102. In some embodiments, the microorganism can comprise a decarboxylase such as 2-pentenoic acid decarboxylase, 4-pentenoic acid decarboxylase, a pent-2,4-dienoic acid decarboxylase, a 3-hydroxy-4-pentenoic acid decarboxylase, or such a decarboxylase. as defined in SEQ ID NOs: 79-98.
[00155] The enzymes to catalyze the conversions in Figures 1 to 3 are classified in Table 4 by an Enzyme Commission (EC) number, function, and the step in Figures 1 to 3 where they catalyze the conversion (Table 4) . Table 4: EC number for enzymes used


[00156] Steps D and I of Figure 1, and steps C and I in Figure 3 can be catalyzed by transferases in EC 2.8.3, including, for example, a transferase that catalyzes the reversible transfer of a CoA fraction from from one molecule to another. Any polynucleotide that encodes a known CoA transferase enzyme including, for example, those polynucleotides shown in Table 5 below, is contemplated for use in the present description. Table 5: Examples of genes encoding enzymes in EC 2.8.3



[00157] Steps D and I of Figure 1 and step I of Figure 3 can be catalyzed by hydrolases in EC 3.1.2 including, for example, hydrolases with broad substrate bands capable of hydrolyzing 2-petentenoyl-CoA, 2,4- pentenoyl-CoA, and 3-hydroxypentenoyl-CoA to their corresponding acids. Any known polynucleotide that encodes a hydrolase including, for example, those polynucleotides shown in Table 6 below, is contemplated for use in the present description. Table 6: Examples of genes encoding enzymes in EC 3.1.2.

[00158] Step C in Figure 3 can be catalyzed by a CoA synthetase in EC 6.2.1., including, for example, a CoA synthetase with. a wide range of substrate capable of activating formic acid to formyl-CoA. Any known polynucleotide that encodes a CoA synthetase, including, for example, those polynucleotides shown in Table 7 below, is contemplated for use in the present description. Table 7: Examples of genes encoding enzymes in EC 6.2.1.


The hydration of a double bond can be catalyzed by hydratase enzymes in EC 4.2.1 and the removal of water to form a double bond can be catalyzed by dehydratase enzymes in EC 4.2.1. Hydratase enzymes are sometimes reversible and can also catalyze dehydration. Likewise, dehydratase enzymes are sometimes reversible and can also catalyze hydration. The addition or removal of water from a given substrate is required for step C in Figure 1, step D in Figure 2, and step H in Figure 3. Any known polynucleotide that encodes a hydratase or dehydratase including, for example, the polynucleotides shown in Table 7 below, is contemplated for use in the present description. Table 8: Examples of genes encoding enzymes in EC 4.2.1.


[00160] The linalool dehydratase-isomerase from the 65Phen Castellaniella defragrans strain catalyzes the stereospecific hydration of beta-myrcene to (3S)-linalool, the isomerization of (3S)-linalool to geraniol, and is involved in the early stages of anaerobic degradation of the beta-myrcene monoterpene. Furthermore, this linalool dehydratase-isomerase catalyzes the reverse reactions, that is, the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. In this sense, the formation of myrcene in geraniol can be seen as a detoxification process from monoterpene alcohol. Other dehydratase isomerases include 4-hydroxybutyryl-CoA dehydrogenase/vinylacetyl-CoA-Delta isomerase. A dehydratase/isomerase can be manipulated by standard methods to accept crotonyl-alcohol as a substrate, thus representing a suitable candidate for this step D in Figure 2 below: Table 9: Examples of genes that can be manipulated in a crotonyl-alcohol dehydrase.

[00161] Step A of Figure 1, and steps C, D and E of Figure 3 require condensation of either acetyl-CoA or acetoacetyl-CoA with formyl-CoA or propionyl-CoA. Such a condensation can be catalyzed with a ketothiolase described in EC 2.3.1. However, any known polynucleotide that encodes a known one including, for example, those polynucleotides described in Table 10 below, is contemplated for use in the present description. Table 10: Examples of genes encoding enzymes in EC 2.3.1.


[00162] Steps E, G, J and in Figure 1, and step J in Figure 2 can be catalyzed by a decarboxylase enzyme as described in class EC 4.1.1 Several decarboxylases have been characterized and shown to decarboxylate structurally similar substrates to acid 2-pentenoic acid, 2,4-pentedienoic acid (Figure 1) and 3-hydroxypentenoic acid (Figure 3). Examples of enzymes for step J of Figure 1 include sorbic acid decarboxylase and aconitate decarboxylase as described in EC 4.1.1.16. Examples of enzymes for steps G and E of Figure 1 may include Jeotgalicoccus p450 fatty acid decarboxylase. Examples of enzymes for step J of Figure 3 may include those enzymes described in EC 4.1.1.33, such as diphosphomevalonate decarboxylase. However, any polynucleotide that encodes a known decarboxylase including, for example, those polynucleotides described in Table 11 below, is contemplated for use in the present specification. Table 11: Examples of genes encoding enzymes in EC 4.1.1.


[00163] Step F of Figure 1 involves an isomerase enzyme as described in EC 5.3.3. Examples of enzymes for the step include isopentenyl-diphosphate delta-isomerase. However, any known polynucleotide that encodes an isomerase including, for example, those polynucleotides described in Table 12 below, are contemplated for use in the present description. Table 12: Examples of genes encoding enzymes in EC 5.3.3.

[00164] Step B of Figure 1, steps A, B and C of Figure 2, and steps F and G of Figure 3 involve the reduction of a ketone to an alcohol and can be catalyzed by oxidoreductase enzymes in class EC 1.1.1. However, any known polynucleotide that encodes an oxidoreductase including, for example, those polynucleotides described in Table 13 below, are contemplated for use in the present specification. Table 13: Examples of genes encoding enzymes in EC 1.1.1.


[00165] Step I of Figure 1, and step A of Figure 3 involve a dehydrogenase as described in EC 1.3.1 or 1.2.99. However, any known polynucleotide that encodes a dehydrogenase including, for example, those polynucleotides described in Table 14 below, is contemplated for use in the present description. Table 14: Examples of genes encoding enzymes in EC 1.3.1 or 1.2.99.

Methods for the production of butadiene
[00166] Butadiene (eg fermentation product) can be produced by contacting one or more genetically modified microorganisms provided herein with a source of fermentable carbon. Such methods may preferably comprise contacting a fermentable carbon source with a microorganism comprising one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to any of the intermediates described in Tables 1 to 3 or Figures 1 to 3 is one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of one or more intermediates described in Tables 1 to 3 or Figures 1 to 3 to butadiene in a fermentation medium including and under sufficient conditions by a suitable period of time; and expressing one or more polynucleotides encoding the enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates provided in Tables 1 to 3 or Figures 1 to 3 and one or more polynucleotides encoding the enzymes in a pathway which catalyze the conversion of one or more intermediates given in Tables 1 to 3 or Figures 1 to 3 to butadiene in the microorganism for the production of butadiene. In some embodiments, the conversion of the fermentable carbon source to butadiene is ATP positive (eg generates a flux of ATP per mole of butadiene produced; produces ATP as a by-product) and when combined with an NADH consumption pathway can provide an anaerobic process for the production of butadiene. For example, conversion of a fermentable carbon source such as glucose or fructose to butadiene can produce a flux of 1 mole of ATP per mole of butadiene produced.
[00167] Examples of fermentable carbon sources may include, but are not limited to, sugarcane juice, sugarcane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any shape or mixture of these. In some embodiments, the carbon source is a monosaccharide, oligosaccharide, or polysaccharide.
[00168] Metabolic pathways leading to the production of industrially important compounds such as butadiene involve oxidation-reduction (redox) reactions. For example, during fermentation, glucose is oxidized in a series of enzymatic reactions to smaller molecules with the concomitant release of energy. Released electrons are transferred from one reaction to another through electron transporters such as Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Phosphate (NAD(P)), which act as cofactors of the oxidoreductase enzymes. In microbial catabolism, glucose is oxidized by enzymes using the oxidized form of the cofactors (NAD(P)+ and/or NAD +) as cofactors, thus generating reducing equivalents in the form of the reduced cofactor (NAD(P)H and NADH) . In order for fermentation to continue, balanced redox metabolism is necessary, ie, cofactors must be regenerated by the reduction of metabolic compounds from the microbial cells. In some embodiments, the novel pathways described herein are advantageous in that they provide for the conversion of a fermentable carbon source to butadiene via a pathway that redistributes end products to achieve a redox balance.
[00169] Some key parameters for the efficient fermentation of a fermentable carbon source by one or more modified microorganisms as described herein include: the ability to grow microorganisms at a higher cell density, increase the yield of desired products, quantity increased volumetric productivity, removal of unwanted co-metabolites, better use of low-cost carbon and nitrogen sources, adaptation to different fermentation conditions, increased production of a major metabolite, increased production of secondary metabolites, greater tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions, and increased tolerance to high or low temperatures. Inefficiencies in any of these parameters can result in high production costs, inability to capture or maintain market share, and/or failure to bring finished fermented products to market.
[00170] The methods of the present description can be adapted to conventional fermentation bioreactors (e.g., batch, semi-batch, cell recycling, and continuous fermentation). In some embodiments, a microorganism (e.g., a genetically modified microorganism) as provided herein is cultivated in a liquid fermentation medium (i.e., a submerged culture) which drives the excretion of the fermented product(s) into the fermentation medium. Fermentation can take place in a bioreactor configured as a stir tank, a bubble column, an airlift reactor or any other suitable configuration known in the art. In one embodiment, the final fermented product(s) can be isolated from the fermentation medium using any suitable method known in the art.
[00171] In some embodiments, the formation of the fermented product can occur during a period of initial and rapid growth of the microorganism. In one embodiment, formation of the fermented product can occur during a second period during which the culture is maintained in a slow-growing or non-growing state. In one embodiment, formation of the fermented product can occur over more than one period of growth of the microorganism. In such modalities, the amount of fermented product formed per unit of time is generally a function of the metabolic activity of the microorganisms, the physiological conditions of culture (for example, pH, temperature, composition of the medium), and the amount of microorganisms present in the process of fermentation.
[00172] In some embodiments, the fermentation product is recovered from the periplasm or culture medium as a secreted metabolite. In one embodiment, the fermentation product is extracted from the microorganism, for example, when the microorganism does not show a secretion signal that corresponds to the fermentation product. In one embodiment, microorganisms are disrupted and the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions can then be separated if necessary. The fermentation product of interest can then be purified from the remaining supernatant solution or suspension by, for example, distillation, fractional distillation, chromatography, precipitation, filtration and the like. In one embodiment, fermentation products are extracted by one or more of: distillation, reactive distillation, azeotropic distillation and extractive distillation.
The methods of the present description are preferably preformed under anaerobic conditions. Both the degree of reduction of a product and the ATP requirement of its synthesis determine whether a production process is capable of proceeding aerobically or anaerobically. To produce butadiene via microbial anaerobic conversion, or at least through a process with reduced oxygen consumption, redox imbalance must be avoided. Several types of metabolic conversion steps involve redox reactions including some of the conversions as described in Table 1 to 3 or Figures 1 to 3. Such redox reactions involve electron transfer mediated by the participation of redox cofactors such as NADH, NADPH and ferrodoxine. Since the amounts of redox cofactors in cells are limited in order to allow the metabolic processes to proceed, the cofactors must be regenerated. In order to avoid such redox imbalances, alternative forms of cofactor regeneration can be manipulated, and in some cases other sources of ATP generation can be provided. Alternatively, oxidation and reduction processes can be spatially separated in bioelectrochemical systems (Rabaey and Rozendal, 2010, Nature reviews, Microbology, vol 8:706-716).
[00174] In some embodiments, redox imbalances can be avoided by using substrates (eg, fermentable carbon sources) that are more oxidized or more reduced. For example, if the use of a substrate results in a deficit or excess of electrons, a requirement for oxygen can be circumvented by using substrates that are more reduced or oxidized, respectively. For example, glycerol, which is an important by-product of biodiesel production, is more reduced than sugars, and is therefore more suitable for the synthesis of compounds where sugar production results in cofactor oxidation, such as succinic acid. In some embodiments, if the conversion of a substrate to a product results in an electron deficit, co-substrates can be added in roles as electron donors (Babel 2009, Eng. Life Sci. 9,285-290). An important criterion for anaerobic use of co-substrates is that their redox potential is greater than that of NADH (Geertman et al., 2006, FEMS Yeast Res.6, 1193-1203). If substrate conversion to produce an excess of electrons results, co-substrates that can be added in functions such as electron takers. Methods for making polybutadiene and other compounds from butadiene
[00175] Butadiene is gaseous at room temperature or under fermentation conditions (20 to 45°C), and its production from a fermentation process results in a gas that can accumulate on top of a fermentation tank, and be siphoned and concentrated. Butadiene can be purified from the fermentation of gases including gaseous alcohol, CO2 and other compounds by solvent extraction, cryogenic processes, distillation, fractionation, chromatography, precipitation, filtration and the like.
[00176] Butadiene produced by any of the processes or methods described herein can be converted to polybutadiene. Alternatively, butadiene produced via the methods described herein can be polymerized with other olefins to form copolymers, such as acrylonitrile-butadiene-styrene (ABS), acrylonitrile-butadiene (ABR), or styrene-butadiene (SBR) copolymers. BR (RB), polybutadiene rubber (PBR), nitrile rubber and polychloroprene (Neoprene). Those synthetic rubber or plastic elastomer applications include tire production, plastic materials, soles, shoe heels, technical products, appliances, neoprene, paper coatings, gloves, gaskets and seals.
[00177] Without further description, it is believed that a person skilled in the art can, using the foregoing description and the illustrative examples below, make and use the agents of the present disclosure and practice the claimed methods. The following working examples are provided to facilitate the practice of the present description, and are not to be construed as limiting the remainder of the description in any way. EXAMPLES Example 1: Modification of the microorganism for the production of butadiene.
[00178] A microorganism such as a bacterium can be genetically modified to produce butadiene from a fermentable carbon source including, for example, glucose.
[00179] In an example method, a microorganism can be genetically manipulated by any methods known in the art to comprise: i.) one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the fermentable carbon source to acetyl- CoA and propionyl-CoA and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of acetyl-CoA and propionyl-CoA to butadiene; ii.) one or more polynucleotides that encode enzymes that catalyze in a pathway a conversion of the fermentable carbon source to crotonyl-CoA and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of crotonyl-CoA to butadiene; or iii.) one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of the fermentable carbon source to formic acid and/or one or more polynucleotides that encode enzymes in a pathway that catalyze the conversion of formic acid to butadiene .
[00180] Alternatively, a microorganism that is devoid of one or more enzymes (for example, one or more functional enzymes that are catalytically active) for the conversion of a fermentable carbon source to butadiene can be genetically modified to comprise one or more polynucleotides which encode the enzymes (e.g., functional enzymes, including, for example, any enzyme described herein) in a pathway in which the microorganism is deprived of catalyzing the conversion of the fermentable carbon source to butadiene. Example 2: Fermentation of a carbon source by a genetically modified microorganism to produce butadiene.
[00181] A genetically modified microorganism, as produced in Example 1 above, can be used to ferment a carbon source for the production of butadiene.
[00182] In an example method, a previously sterilized culture medium comprising a fermentable carbon source (eg glucose 9 g/1, 1 g/1 KH2PO4, 2 g/1 (NH4)2HPO4, 5 mg /1 FeSO4*7H2O, 10 mg/1 MgSO4* 7H2O, 2.5 mg/1 MnSO4*H2O, 10 mg/1 CaCl*6H2O, 10 mg/1 COC12*6H20, and 10 g/1 of yeast extract) is loaded into a bioreactor.
[00183] During fermentation, anaerobic conditions are maintained by, for example, spraying nitrogen through the culture medium. A suitable temperature for fermentation (for example, about 30°C) is maintained by any method known in the art. A close physiological pH (eg around 6.5) is maintained, for example, by the automatic addition of sodium hydroxide. The bioreactor is stirred at, for example, about 50 rpm. Fermentation is allowed to proceed to completion.
[00184] The butadiene produced is then recovered from the culture medium using conventional methods. When fermentation products are recovered through distillation, the butadiene fraction may optionally be polymerized to form polybutadiene. Fractions from the distillation that contain other intermediates along the butadiene pathway (if any) can be subjected to a subsequent fermentation in a bioreactor to produce additional butadiene.
[00185] Unless otherwise indicated, all numbers expressing amounts of ingredients, properties such as molecular weight, reaction conditions, and so on used in the report and claims are to be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters presented in the report and appended claims are approximations which may vary depending on the desired properties intended to be obtained by the present description. At the very least, and not in an attempt to limit the application of the equivalents doctrine to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of significant figures described and by applying common rounding techniques.
[00186] Although the numerical ranges and parameters that establish the broad scope of the description are approximations, the numerical values described in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective test measurements.
[00187] The terms "a", "an", "the" and similar referents used in the context of describing the description (especially in the context of the following claims) are to be understood to cover both the singular and the plural, unless otherwise indicated here or clearly contradicted by the context. Recitation of ranges of values here is only intended to serve as an abbreviated method of referring individually to each separate value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated into the report as if it were described individually herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or example language (eg, "how") provided herein is intended only to further clarify the description and does not represent a limitation on the scope of the description otherwise claimed. No language in the report should be understood to indicate any essential element not claimed for the practice of the description.
[00188] Alternative element groups or description modalities described herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members or other elements found herein. It is anticipated that one or more members of a group may be included in, or excluded from a group, for reasons of convenience and/or patentability. When any inclusion or exclusion occurs, the report is deemed to contain the group as modified thus complying with the written description of all Markush groups used in the appended claims.
[00189] Certain embodiments of this description are described herein, including the best way known to the inventors to carry out the description. Of course, variations in these described embodiments will be evident to those skilled in the art upon reading the foregoing description. The inventor expects persons of skill to employ such variations, as the case may be, and the inventors intend the description to be practiced otherwise than specifically described herein. Thus, this description includes all modifications and equivalents of matter recited in the appended claims, as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is included by the description, unless otherwise indicated herein or otherwise clearly contradicted by the context.
[00190] Specific modalities described herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added by amendment, the transitional term "consisting of" excludes any non-specific element, step or ingredient in the claims. The transition term "consisting essentially of" limits the scope of a claim to specific materials or steps and those that do not significantly affect the new, basic feature(s). Modalities of description so claimed are either inherently or expressly described herein and permitted.
[00191] It is to be understood that the embodiments of the description described herein are illustrative of the principles of the present description. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not limitation, the alternative configurations of the present description can be used in accordance with the teachings contained herein. Therefore, the present description is not limited to this precisely as shown and described.
[00192] While the present description has been described herein and illustrated by references to various specific materials, procedures and examples, it is understood that the description is not restricted to particular combinations of materials and procedures selected for that purpose. Several variations of such details can be applied as will be appreciated by those skilled in the art. It is intended that the report and the examples be considered as examples only, with the true scope and spirit of the description being indicated by the following claims. All references, patents and patent applications mentioned in the present application are hereby incorporated by reference in their entirety.

权利要求:
Claims (19)
[0001]
1. Method of production of butadiene from a source of fermentable carbon, characterized in that the method comprises: a) providing a source of fermentable carbon; b) contacting the fermentable carbon source with a microorganism comprising one or more polynucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos: 103-123 and 34-36, or its degenerate sequences, which encode the enzymes in a pathway that catalyzes the conversion of the fermentable carbon source to one or more intermediates in a pathway for the production of butadiene comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 159-181, and one or more polynucleotides comprising a sequence of nucleotides selected from the group consisting of SEQ ID Nos: 37-55, or degenerate sequences thereof, which encode the enzymes in a pathway that catalyzes the conversion of one or more intermediates to butadiene in a fermentation medium comprising an amino acid sequence selected from group consisting of SEQ ID Nos: 140-158; and c) expressing said one or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of the fermentable carbon source into one or more intermediates in a pathway for the production of butadiene and one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of one or more intermediates to butadiene in the microorganism for the production of butadiene, where the one or more intermediates in a pathway for the production of butadiene is crotonyl-CoA, and where all enzymes are required from ( i) crotonyl-CoA reductase (bifunctional) (EC 1.1.1) and crotonyl alcohol dehydratase (EC 4.2.1, 4.2.1.127), or (ii) crotonaldehyde dehydrogenase (EC 1.2.1), crotonyl alcohol dehydrogenase (EC 1.1. 1, 1.1.1.1) and crotonyl alcohol dehydratase (EC 4.2.1, 4.2.1.127).
[0002]
2. Method according to claim 1, characterized in that the microorganism is a bacterium selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter or Lactobacillus.
[0003]
3. Method according to claim 1, characterized in that the microorganism is a eukaryote and is a yeast, filamentous fungus, protozoan or alga.
[0004]
4. Method according to claim 3, characterized in that the yeast is Saccharomices cerevisiae, Zimomonas mobilis, or Pichia pastoris.
[0005]
5. Method according to claim 1, characterized in that the carbon source is sugar cane juice, sugar cane molasses, hydrolyzed starch, hydrolyzed lignocellulosic materials, glucose, sucrose, fructose, lactate, lactose, xylose, pyruvate, or glycerol in any form or a mixture thereof.
[0006]
6. Method according to claim 1, characterized in that the carbon source is a monosaccharide, oligosaccharide or polysaccharide.
[0007]
7. Method according to claim 1, characterized in that butadiene is secreted by the microorganism in the fermentation medium.
[0008]
8. Method according to claim 7, characterized in that it further comprises the recovery of butadiene from the fermentation medium.
[0009]
9. Method according to claim 1, characterized in that the conversion of the fermentable carbon source to butadiene is carried out under anaerobic conditions.
[0010]
10. Method according to claim 1, characterized in that the enzymes are crotonyl-CoA reductase (bifunctional) (E.C. 1.1.1) and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).
[0011]
11. Method according to claim 1, characterized in that the enzymes are crotonaldehyde dehydrogenase (EC 1.2.1), crotonyl alcohol dehydrogenase (EC 1.1.1, 1.1.1.1) and crotonyl alcohol dehydrase (EC 4.2.1 , 4.2.1.127).
[0012]
12. Method according to claim 1, characterized in that said crotonyl alcohol dehydratase is a linalool dehydratase -isomerase of strain 65Phen of Castellaniella defragrans represented by SEQ ID No. 158.
[0013]
13. Genetically modified microorganism, characterized in that it comprises one or more polynucleotides comprising a heterologous nucleotide sequence selected from the group consisting of SEQ ID Nos: 103-123 and 34-36, or their degenerate sequences, which encode the enzymes in a pathway that catalyzes the conversion of a fermentable carbon source to one or more intermediates in a pathway for the production of butadiene, comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 159-181, and one or more polynucleotides that comprise a heterologous nucleotide sequence selected from the group consisting of SEQ ID Nos: 37-55, or degenerate sequences thereof, which encode the enzymes in a pathway that catalyzes the conversion of one or more intermediates to butadiene, comprising an amino acid sequence selected from group consisting of SEQ ID Nos: 140-158, wherein the one or more intermediates in a pathway for the production of butadiene is crotonyl-C oA, and in which all enzymes are required from (i) crotonyl-CoA reductase (bifunctional) (E.C. 1.1.1) and crotonyl alcohol dehydrogenase (EC 4.2.1, 4.2.1.127), or (ii) crotonaldehyde dehydrogenase (EC 1.2.1), crotonyl alcohol dehydrogenase (EC 1.1.1, 1.1.1.1) and crotonyl alcohol dehydrase ( EC 4.2.1, 4.2.1.127).
[0014]
14. Microorganism according to claim 13, characterized in that the microorganism is a bacterium selected from the genera consisting of: Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter or Lactobacillus.
[0015]
15. Microorganism according to claim 13, characterized in that the microorganism is a eukaryote and is a yeast, filamentous fungus, protozoan or alga.
[0016]
16. Microorganism according to claim 14, characterized in that the yeast is Saccharomices cerevisiae, Zimomonas mobilis, or Pichia pastoris.
[0017]
17. Microorganism according to claim 13, characterized in that the enzymes are crotonyl-CoA reductase (bifunctional) (E.C. 1.1.1) and crotonyl alcohol dehydratase (E.C. 4.2.1, 4.2.1.127).
[0018]
18. Microorganism according to claim 13, characterized in that the enzymes are crotonaldehyde dehydrogenase (EC 1.2.1), crotonyl alcohol dehydrogenase (EC 1.1.1, 1.1.1.1) and crotonyl alcohol dehydrase (EC 4.2.1 , 4.2.1.127).
[0019]
19. Microorganism according to claim 13, characterized in that said crotonyl alcohol dehydratase is a linalool dehydratase (4.2.1.127).

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-02| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-06-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-13| 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 17/12/2012, 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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US201161576788P| true| 2011-12-16|2011-12-16|

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US61/576,788|2011-12-16|

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US201261606035P| true| 2012-03-02|2012-03-02|

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US61/606,035|2012-03-02|

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PCT/US2012/070161|WO2013090915A1|2011-12-16|2012-12-17|Modified microorganisms and methods of making butadiene using same|

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