MICROBIAL ORGANISMS FOR CONVERTING ACETYL-COA INTO CROTYL ALCOHOL AND METHODS FOR PRODUCING CROTYL ALCOHOL

Abstract

The present invention provides microorganisms capable of converting acetyl-coA into crotyl alcohol as well as fermentation methods for producing crotyl alcohol, either alone, or in combination with acetone and/or isopropanol. The microorganisms may be genetically engineered to express and/or disrupt one or more of the following enzymes: acetaldehyde dehydrogenase, alcohol dehydrogenase, bifunctional acetaldehyde/alcohol dehydrogenase, aldehyde oxidoreductase, phosphotransacetylase, acetate kinase, CoA-transferase A, CoA-transferase B, acetoacetate decarboxylase, secondary alcohol dehydrogenase, butyryl-CoA dehydro genase (BCD), and/or trans-2-enoyl-CoA reductase (TER).

Claims

1. A non-naturally occurring microbial organism capable of converting acetyl-CoA into crotyl alcohol, wherein at least one of the following genes are deleted, disrupted or silenced and/or expression from at least one of the following genes is disrupted or silenced: i. Butyryl-CoA dehydrogenase (BCD); and/or ii. Trans-2-enoyl-CoA reductase (TER).

2, A microbial organism according to claim 1, comprising a disrupted, deleted, or mutated BCD and/or TER gene.

3. A microbial organism according to claim 1, wherein disruption or silencing of expression includes disruption or silencing of RNA transcription and/or protein translation.

4. A microbial organism according to claim 1, wherein disruption or silencing of expression comprises protein translation silencing using RNA interference.

5. A microbial organism according to claim 1, comprising at least one exogenous nucleic acid encoding one or more of the following enzymes fbr producing crotyl alcohol from crotonyl-CoA: A. Acetaldehyde dehydrogenase; B. Alcohol dehydrogenase; C, Bifunctional acetaldehyde/alcohol dehydrogenase; D. Aldehyde oxidoreductase; E. Pbosphotransacetylase; and/or F. Acetate kinase.

6. A microbial organism according to claim 5 for further producing acetone and/or isopronanol, comprising at least a second exogenous nucleic acid encoding one or more acetone pathway enzymes and/or one or more isopropanol pathway enzvmes, comprising G. CoA-transferase subunit A; H. CoA-trans (erase subunit B; I. Acctoacetate decarboxylase; and/or J. Secondary alcohol dehydrogenase.

7.-9. (canceled)

10. A microbial organism according to claim 6, comprising exogenous nucleic acids encoding each of the enzymes (i) A, B, G, H, and I; (ii) C, G, H, and I; (iii) B, D, E, F, G, H, and I; (iv) C, D, E, F, G, H, and I; (v) A, B, C, G, H, and I; (vi) A, B, C, D, E, F, G, H, and I; (vii) A, B, C, D, E, F, G, H, and I.

11.-16. (canceled)

17. A microbial organism according to claim 5, comprising exogenous nucleic acids encoding each of the enzymes (i) A, B, G, H, I, and J; (ii) C, G, H, I, and J; (iii) B, D, E, F, G, H, I, and J; (iv) C, D, E, F, G, H, I, and J; (v) A, B, C, G, H, I, and J; (vi) A, B, D, E, F, G, H, I, and J; or (vii) A, B, C, D, E, F, G, H, I, and J.

18.-23. (canceled)

24. A microbial organism according to claim 1, comprising two, three, four, five, six, seven, eight, nine, or ten exogenous nucleic acids.

25. (canceled)

26. A microbial organism according to claim 1, wherein said organism is an acetogenic bacterium.

27. A method of producing crotyl alcohol, acetone and/or isopropanol comprising culturing a microbial organism according to claim 1 on a growth substrate, optionally comprising a carbohydrate, under conditions to form a broth comprising crotyl alcohol, acetone and/or isopropanpl.

28. A method of producing crotyl alcohol and acetone, comprising culturing a microbial organism according to claim 6 on a growth substrate, under conditions to form a broth comprising crotyl alcohol and acetone, wherein the acetone to crotyl alcohol molar ratio in said broth is in the range from 0.1 to 0.95.

29. (canceled)

30. A method of producing crotyl alcohol and isopropanol, comprising culturing a microbial organism according to claim 17 on a growth substrate, under conditions to form a broth comprising crotyl alcohol and isopropanol, wherein the isopropanol to crotyl alcohol molar ratio in said broth is in the range from 0.1 to 0.95.

31.-35. (canceled)

36. A method according to claim 27, wherein said growth substrate comprises at least one gaseous compound, optionally exogenously added.

37. (canceled)

38. A method according to claim 36, wherein said at least one gaseous compound is selected from a group consisting of CO, CO2, H2 and combinations thereof.

39. A method according to claim 27, wherein said growth substrate comprises a carbohydrate in combination with at least one of a one-carbon molecule and/or a gaseous compound.

40.-41. (canceled)

42. A method according to claim 27, comprising supplementing pressurized CO2, pressurized CO, pressurized H2, or a combination thereof to said growth substrate.

43. A method according to claim 42, wherein said culturing is conducted at a pressure in the range between 1 atm and 5 atm.

44.-45. (canceled)

46. A method according to claim 27, comprising at least partially separating crotyl alcohol from said broth to form separated crotyl alcohol, at least partially separating acetone frortl said broth to form separated acetone and/or at least partially separating isopropanol from said broth to form separated isopropanol.

47.-48. (canceled)

49. A method according to claim 46, wherein said separating comprises liquid-liquid extraction.

50. A method according to claim 46, further comprising dehydrating said separated crotyl alcohol to form butadiene.

51.-105. (canceled)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0086] The particulars shown herein are by way of example and for purposes of illustrative discussion of the various embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0087] The present invention will now be described by reference to more detailed embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0089] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0090] As used herein, the term about, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/10%, more preferably +/5%, even more preferably, +/1%, and still more preferably +/0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0091] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth 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 testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0092] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0093] The presently claimed subject matter relates to novel microorganisms and biosynthesis methods for production of crotyl alcohol, acetone, and isopropanol. Unexpectedly superior levels of crotyl alcohol, acetone, and/or isopropanol production levels are achieved with microorganisms as described herein and by methods of their use.

I. MICROORGANISMS OF THE INVENTION

[0094] Microorganisms suitable for use in the present invention are not particularly limited as long as the native form of the microorganisms is capable of converting acetyl-CoA into crotonyl-CoA.

[0095] Host organisms suitable for use in the invention include bacteria, including acetogenic bacteria, yeast, fungi and/or other microorganisms known for use in fermentative processes.

[0096] Example organisms that are naturally capable of converting acetyl-CoA into crotonyl-CoA include bacteria such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium kluyveri, Clostridium saccharoperbutylacetonicum, Clostridium pasteurianum, Clostridium saccharobutylicum, Clostridium carboxidovorans, Clostridium butyricum, Clostridium tyrobutyricum, Clostridium cellulovorans, Clostridium bornimense, Clostridium scatologenes, Clostridium drakei, Clostridium tetani, Clostridium baratii, Clostridium perfringens, Clostridium botulinum, Clostridium novyi, Clostridium sporogenes, Clostridium sticklandii, Thermoanaerobacterium thermosaccharolyticum, Fervidobacterium pennivorans, Fervidobacterium nodosum, Thermoanaerobacter wiegelii, Thermoanaerobacter tengcongensis, Alkaliphilus metalliredigens, Alkaliphilus oremlandii, Eubacterium limosum, Eubacterium aggregans, Butyribacterium methylotrophicum, Peptoclostridium difficile, and Oxobacter pfennigii.

[0097] In an embodiment, the microorganism may be genetically modified to express one or more of the following crotyl alcohol pathway enzymes: acetaldehyde dehydrogenase (aldehyde forming enzyme), alcohol dehydrogenase (alcohol forming enzyme), bifunctional acetaldehyde/alcohol dehydrogenase (aldehyde & alcohol forming enzyme), aldehyde oxidoreductase (aldehyde forming enzyme), phosphotransacetylase (phosphate forming enzyme), and/or acetate kinase (carboxylic acid forming enzyme).

[0098] In an embodiment, the microorganism may be genetically modified to express one or more of the following acetone pathway enzymes: CoA-transferase subunit A, CoA-transferase subunit B, and/or acetoacetate decarboxylase.

[0099] In an embodiment, the microorganism may be genetically modified to express one or more of the following isopropanol pathway enzymes: CoA-transferase subunit A, CoA-transferase subunit B, acetoacetate decarboxylase, and/or secondary alcohol dehydrogenase.

[0100] In an embodiment, the microorganism may have decreased expression of butyryl-CoA dehydrogenase (BCD) or BCD expression may be silenced.

[0101] In an embodiment, the microorganism may have decreased expression of trans-2-enoyl-CoA reductase (TER) or TER expression may be silenced.

[0102] Of course, the above genetic modifications are not particularly limited and one or more genes may be inserted into the genome of the host microorganism in combination. Additionally, one or more genes may be disrupted or silenced while others have increased expression.

[0103] Depending on the host microorganism selected for production of crotyl alcohol, acetone, and/or isopropanol, nucleic acids for some or all of a particular biosynthetic pathway can be expressed. For example, if a selected microorganism is deficient in a desired biosynthetic pathway, then exogenous nucleic acids encoding the enzymes for the desired pathway may be introduced into the microbial host. Alternatively, if the selected microorganism expresses some pathway enzymes/genes, but is deficient in others, an exogenous nucleic acid may be introduced into the host to compensate only for those pathway enzymes that are not endogenously expressed in the host microorganism.

[0104] In an embodiment, the microorganism comprises a native butanoate pathway. For example, the microorganism may comprise one or more genes encoding enzymes and/or substrates necessary for the production or metabolism of butanoate (also known as butyrate). In an embodiment, the microorganism may endogenously express one or more of the following butanoate pathway enzymes: acetyl-CoA acetyltransferase (also known as thiolase), 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase (also known as crotonase), butyryl-CoA dehydrogenase, trans-2-enoyl-CoA reductase, CoA-transferase subunit A, CoA-transferase subunit B, acetaldehyde/alcohol dehydrogenase, butanol dehydrogenase, aldehyde:ferredwdn oxidoreductase, acetoacetate decarboxylase, and/or secondary alcohol dehydrogenase.

[0105] In an embodiment, the microorganism is genetically engineered to inhibit native production of butanoate and to thereby force increased expression of a bioproduct of interest such as crotyl alcohol. For example, crotonyl-CoA production in the microorganism host may be enhanced by disruption of butyryl-CoA dehydrogenase (BCD) expression of the butanoate pathway.

[0106] While a genomic deletion is a preferred embodiment for decreasing or silencing gene expression, any genomic mutation resulting in inactivation of the enzyme would be sufficient, including but not limited to partial gene deletion, nonsense mutation, transcriptional promoter deletion, etc. In another embodiment, the transcriptional expression of this gene can be reduced by using antisense RNA.

[0107] In an embodiment, the microorganism may be a bacteria or yeast or fungus capable of metabolizing CO.sub.2. The organism may be autotrophic. In an embodiment, the organism may be capable of assimilating CO, CO.sub.2, methanol, etc., for growth. The organism may also be capable of utilizing glycolysis for growth. In certain embodiments, the microorganism may be mixotrophic such that it is capable of assimilating CO, CO.sub.2, methanol, etc., for growth and also capable of utilizing glycolysis for growth, either concurrently or at various stages of growth or fermentation. According to an embodiment, said organism is acetogenic. For example, said organism may be acetogenic Clostridia. Mixotrophic fermentation methods and microorganisms for use in such methods are described in detail in PCT International Application No. PCT/US2016/019760 as well as U.S. patent application Ser. No. 15/055,045.

[0108] In an embodiment, the microorganism may comprise a native butanoate metabolic pathway and have a genetic deletion of the BCD and/or TER genes. BCD and/or TER expression may alternatively be disrupted or silenced by other mechanisms. Examples of microorganism comprising a native butanoate metabolic pathway include Clostridium carboxidovorans, Eubacterium limosum, Butyribacterium methylotrophicum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium kluyveri, Clostridium butyricum, Clostridium tyrobutyricum, Clostridium cellulovorans, Clostridium pasteurianum, Clostridium saccharoperbutylacetonicum, and Clostridium saccharobutylicum. In an embodiment, such a microorganism may be capable of redirecting crotonyl-CoA into crotyl alcohol. Such a microorganism may also be capable of producing acetone and/or isopropanol. Such a microorganism may be mixotrophic or non-mixotrophic.

[0109] In an embodiment, the microorganism may be mixotrophic and comprise a native butanoate metabolic pathway and have a genetic deletion of the BCD and/or TER genes. BCD and/or TER expression may alternatively be disrupted or silenced by other mechanisms. In an embodiment, such a microorganism may be capable of redirecting crotonyl-CoA into crotyl alcohol. Such a microorganism may also be capable of producing acetone and/or isopropanol.

II. EXEMPLARY POLYNUCLEOTIDE AND AMINO ACIDS SEQUENCES OF THE INVENTION

[0110] An exemplary acetaldehyde dehydrogenase (ALDH) for use in the present invention catalyzes a CoA-acylating reaction in which crotonyl-CoA is converted into crotonaldehyde. Any similar substrates can also be used, such as acetyl-CoA into acetaldehyde, butyryl-CoA into butyraldehyde, and others. This reaction typically requires a coenzyme, such as NADH or NADPH. Exemplary nucleic acid and amino acid sequences are set forth below:

[0111] EC number: 1.2.1.10 or 1.2.1.57

[0112] Example nucleic acid sequence:

TABLE-US-00001 ATGAATAAAGACACACTAATACCTACAACTAAAGATTTAAAAGTAAAAAC AAATGGTGAAAACATTAATTTAAAGAACTACAAGGATAATTCTTCATGTT TCGGAGTATTCGAAAATGTTGAAAATGCTATAAGCAGCGCTGTACACGCA CAAAAGATATTATCCCTTCATTATACAAAAGAGCAAAGAGAAAAAATCAT AACTGAGATAAGAAAGGCCGCATTACAAAATAAAGAGGTCTTGGCTACAA TGATTCTAGAAGAAACACATATGGGAAGATATGAGGATAAAATATTAAAA CATGAATTGGTAGCTAAATATACTCCTGGTACAGAAGATTTAACTACTAC TGCTTGGTCAGGTGATAATGGTCTTACAGTTGTAGAAATGTCTCCATATG GTGTTATAGGTGCAATAACTCCTTCTACGAATCCAACTGAAACTGTAATA TGTAATAGCATAGGCATGATAGCTGCTGGAAATGCTGTAGTATTTAACGG ACACCCATGCGCTAAAAAATGTGTTGCCTTTGCTGTTGAAATGATAAATA AGGCAATTATTTCATGTGGCGGTCCTGAAAATCTAGTAACAACTATAAAA AATCCAACTATGGAGTCTCTAGATGCAATTATTAAGCATCCTTCAATAAA ACTTCTTTGCGGAACTGGGGGTCCAGGAATGGTAAAAACCCTCTTAAATT CTGGTAAGAAAGCTATAGGTGCTGGTGCTGGAAATCCACCAGTTATTGTA GATGATACTGCTGATATAGAAAAGGCTGGTAGGAGCATCATTGAAGGCTG TTCTTTTGATAATAATTTACCTTGTATTGCAGAAAAAGAAGTATTTGTTT TTGAGAATGTTGCAGATGATTTAATATCTAACATGCTAAAAAATAATGCT GTAATTATAAATGAAGATCAAGTATCAAAATTAATAGATTTAGTATTACA AAAAAATAATGAAACTCAAGAATACTTTATAAACAAAAAATGGGTAGGAA AAGATGCAAAATTATTCTTAGATGAAATAGATGTTGAGTCTCCTTCAAAT GTTAAATGCATAATCTGCGAAGTAAATGCAAATCATCCATTTGTTATGAC AGAACTCATGATGCCAATATTGCCAATTGTAAGAGTTAAAGATATAGATG AAGCTATTAAATATGCAAAGATAGCAGAACAAAATAGAAAACATAGTGCC TATATTTATTCTAAAAATATAGACAACCTAAATAGATTTGAAAGAGAAAT AGATACTACTATTTTTGTAAAGAATGCTAAATCTTTTGCTGGTGTTGGTT ATGAAGCAGAAGGATTTACAACTTTCACTATTGCTGGATCTACTGGTGAG GGAATAACCTCTGCAAGGAATTTTACAAGACAAAGAAGATGTGTACTTGC CGGCTAA

[0113] Example amino acid sequence:

TABLE-US-00002 MNKDTLIPTTKDLKVKINGENINLKNYKDNSSCFGVFENVENAISSAVHA QKILSLHYTKEQREKIITEIRKAALQNKEVLATMILEETHMGRYEDKILK HELVAKYTPGTEDLTTTAWSGDNGLTVVEMSPYGVIGAITPSTNPTETVI CNSIGMIAAGNAVVFNGHPCAKKCVAFAVEMINKAIISCGGPENLVTTIK NPTMESLDAIIKHPSIKLLCGTGGPGMVKTLLNSGKKAIGAGAGNPPVIV DDTADIEKAGRSIIEGCSFDNNLPCIAEKEVFVFENVADDLISNMLKNNA VIINEDQVSKLIDLVLQKNNETQEYFINKKWVGKDAKLFLDEIDVESPSN VKCIICEVNANHPFVMTELMMPILPIVRVKDIDEAIKYAKIAEQNRKHSA YIYSKNIDNLNRFEREIDTTIFVKNAKSFAGVGYEAEGFTTFTIAGSTGE GITSARNFTRQRRCVLAG

[0114] An exemplary alcohol dehydrogenase (ADH) for use in the present invention catalyzes the dehydrogenation of an aldehyde into an alcohol, particularly crotonaldehyde into crotyl alcohol, though any aldehyde can be a substrate. This reaction typically requires a coenzyme, such as NADH or NADPH. This enzyme can also be known as a butanol dehydrogenase (BDH). Exemplary nucleic acid and amino acid sequences are set forth below:

[0115] EC number: 1.1.1.1

[0116] Example nucleic acid sequence:

TABLE-US-00003 GTGGTTGATTTCGAATATTCAATACCAACTAGAATTTTTTTCGGTAAAGA TAAGATAAATGTACTTGGAAGAGAGCTTAAAAAATATGGTTCTAAAGTGC TTATAGTTTATGGTGGAGGAAGTATAAAGAGAAATGGAATATATGATAAA GCTGTAAGTATACTTGAAAAAAACAGTATTAAATTTTATGAACTTGCAGG AGTAGAGCCAAATCCAAGAGTAACTACAGTTGAAAAAGGAGTTAAAATAT GTAGAGAAAATGGAGTTGAAGTAGTACTAGCTATAGGTGGAGGAAGTGCA ATAGATTGCGCAAAGGTTATAGCAGCAGCATGTGAATATGATGGAAATCC ATGGGATATTGTGTTAGATGGCTCAAAAATAAAAAGGGTGCTTCCTATAG CTAGTATATTAACCATTGCTGCAACAGGATCAGAAATGGATACGTGGGCA GTAATAAATAATATGGATACAAACGAAAAACTAATTGCGGCACATCCAGA TATGGCTCCTAAGTTTTCTATATTAGATCCAACGTATACGTATACCGTAC CTACCAATCAAACAGCAGCAGGAACAGCTGATATTATGAGTCATATATTT GAGGTGTATTTTAGTAATACAAAAACAGCATATTTGCAGGATAGAATGGC AGAAGCGTTATTAAGAACTTGTATTAAATATGGAGGAATAGCTCTTGAGA AGCCGGATGATTATGAGGCAAGAGCCAATCTAATGTGGGCTTCAAGTCTT GCGATAAATGGACTTTTAACATATGGTAAAGACACTAATTGGAGTGTACA CTTAATGGAACATGAATTAAGTGCTTATTACGACATAACACACGGCGTAG GGCTTGCAATTTTAACACCTAATTGGATGGAGTATATTTTAAATAATGAT ACAGTGTACAAGTTTGTTGAATATGGTGTAAATGTTTGGGGAATAGACAA AGAAAAAAATCACTATGACATAGCACATCAAGCAATACAAAAAACAAGAG ATTACTTTGTAAATGTACTAGGTTTACCATCTAGACTGAGAGATGTTGGA ATTGAAGAAGAAAAATTGGACATAATGGCAAAGGAATCAGTAAAGCTTAC AGGAGGAACCATAGGAAACCTAAGACCAGTAAACGCCTCCGAAGTCCTAC AAATATTCAAAAAATCTGTGTAA

[0117] Example amino acid sequence:

TABLE-US-00004 MVDFEYSIPTRIFFGKDKINVLGRELKKYGSKVLIVYGGGSIKRNGIYDK AVSILEKNSIKFYELAGVEPNPRVTTVEKGVKICRENGVEVVLAIGGGSA IDCAKVIAAACEYDGNPWDIVLDGSKIKRVLPIASILTIAATGSEMDTWA VINNMDTNEKLIAAHPDMAPKFSILDPTYTYTVPTNQTAAGTADIMSHIF EVYFSNIKTAYLQDRMAEALLRICIKYGGIALEKPDDYEARANLMWASSL AINGLLTYGKDTNWSVHLMEHELSAYYDITHGVGLAILTPNWMEYILNND TVYKFVEYGVNVWGIDKEKNHYDIAHQAIQKTRDYFVNVLGLPSRLRDVG IEEEKLDIMAKESVKLTGGTIGNLRPVNASEVLQIFKKSV

[0118] An exemplary bifunctional acetaldehyde/alcohol dehydrogenase (ADHE) for use in the present invention is a bifunctional enzyme that catalyzes two reactions sequentially. The first reaction is a CoA-acylating reaction in which crotonyl-CoA is converted into crotonaldehyde. The second reaction is a dehydrogenase reaction in which crotonaldehyde is converted into crotyl alcohol. Any similar substrates can also be used, such as acetyl-CoA, butyryl-CoA, and others. This reaction typically requires a coenzyme, such as NADH or NADPH. Exemplary nucleic acid and amino acid sequences are set forth below:

[0119] EC number: For the first reaction (1.2.1.10 or 1.2.1.57); for the second reaction (1.1.1.1)

[0120] Example nucleic acid sequence:

TABLE-US-00005 ATGAAAGTCACAACAGTAAAGGAATTAGATGAAAAACTCAAGGTAATTAA AGAAGCTCAAAAAAAATTCTCTTGTTACTCGCAAGAAATGGTTGATGAAA TCTTTAGAAATGCAGCAATGGCAGCAATCGACGCAAGGATAGAGCTAGCA AAAGCAGCTGTTTTGGAAACCGGTATGGGCTTAGTTGAAGACAAGGTTAT AAAAAATCATTTTGCAGGCGAATACATCTATAACAAATATAAGGATGAAA AAACCTGCGGTATAATTGAACGAAATGAACCCTACGGAATTACAAAAATA GCAGAACCTATAGGAGTTGTAGCTGCTATAATCCCTGTAACAAACCCCAC ATCAACAACAATATTTAAATCCTTAATATCCCTTAAAACTAGAAATGGAA TTTTCTTTTCGCCTCACCCAAGGGCAAAAAAATCCACAATACTAGCAGCT AAAACAATACTTGATGCAGCCGTTAAGAGTGGTGCCCCGGAAAATATAAT AGGTTGGATAGATGAACCTTCAATTGAACTAACTCAATATTTAATGCAAA AAGCAGATATAACCCTTGCAACTGGTGGTCCCTCACTAGTTAAATCTGCT TATTCTTCCGGAAAACCAGCAATAGGTGTTGGTCCGGGTAACACCCCAGT AATAATTGATGAATCTGCTCATATAAAAATGGCAGTAAGTTCAATTATAT TATCCAAAACCTATGATAATGGTGTTATATGTGCTTCTGAACAATCTGTA ATAGTCTTAAAATCCATATATAACAAGGTAAAAGATGAGTTCCAAGAAAG AGGAGCTTATATAATAAAGAAAAACGAATTGGATAAAGTCCGTGAAGTGA TTTTTAAAGATGGATCCGTAAACCCTAAAATAGTCGGACAGTCAGCTTAT ACTATAGCAGCTATGGCTGGCATAAAAGTACCTAAAACCACAAGAATATT AATAGGAGAAGTTACCTCCTTAGGTGAAGAAGAACCTTTTGCCCACGAAA AACTATCTCCTGTTTTGGCTATGTATGAGGCTGACAATTTTGATGATGCT TTAAAAAAAGCAGTAACTCTAATAAACTTAGGAGGCCTCGGCCATACCTC AGGAATATATGCAGATGAAATAAAAGCACGAGATAAAATAGATAGATTTA GTAGTGCCATGAAAACCGTAAGAACCTTTGTAAATATCCCAACCTCACAA GGTGCAAGTGGAGATCTATATAATTTTAGAATACCACCTTCTTTCACGCT TGGCTGCGGATTTTGGGGAGGAAATTCTGTTTCCGAGAATGTTGGTCCAA AACATCTTTTGAATATTAAAACCGTAGCTGAAAGGAGAGAAAACATGCTT TGGTTTAGAGTTCCACATAAAGTATATTTTAAGTTCGGTTGTCTTCAATT TGCTTTAAAAGATTTAAAAGATCTAAAGAAAAAAAGAGCCTTTATAGTTA CTGATAGTGACCCCTATAATTTAAACTATGTTGATTCAATAATAAAAATA CTTGAGCACCTAGATATTGATTTTAAAGTATTTAATAAGGTTGGAAGAGA AGCTGATCTTAAAACCATAAAAAAAGCAACTGAAGAAATGTCCTCCTTTA TGCCAGACACTATAATAGCTTTAGGTGGTACCCCTGAAATGAGCTCTGCA AAGCTAATGTGGGTACTATATGAACATCCAGAAGTAAAATTTGAAGATCT TGCAATAAAATTTATGGACATAAGAAAGAGAATATATACTTTCCCAAAAC TCGGTAAAAAGGCTATGTTAGTTGCAATTACAACTTCTGCTGGTTCCGGT TCTGAGGTTACTCCTTTTGCTTTAGTAACTGACAATAACACTGGAAATAA GTACATGTTAGCAGATTATGAAATGACACCAAATATGGCAATTGTAGATG CAGAACTTATGATGAAAATGCCAAAGGGATTAACCGCTTATTCAGGTATA GATGCACTAGTAAATAGTATAGAAGCATACACATCCGTATATGCTTCAGA ATACACAAACGGACTAGCACTAGAGGCAATACGATTAATATTTAAATATT TGCCTGAGGCTTACAAAAACGGAAGAACCAATGAAAAAGCAAGAGAGAAA ATGGCTCACGCTTCAACTATGGCAGGTATGGCATCCGCTAATGCATTTCT AGGTCTATGTCATTCCATGGCAATAAAATTAAGTTCAGAACACAATATTC CTAGTGGCATTGCCAATGCATTACTAATAGAAGAAGTAATAAAATTTAAC GCAGTTGATAATCCTGTAAAACAAGCCCCTTGCCCACAATATAAGTATCC AAACACCATATTTAGATATGCTCGAATTGCAGATTATATAAAGCTTGGAG GAAATACTGATGAGGAAAAGGTAGATCTCTTAATTAACAAAATACATGAA CTAAAAAAAGCTTTAAATATACCAACTTCAATAAAGGATGCAGGTGTTTT GGAGGAAAACTTCTATTCCTCCCTTGATAGAATATCTGAACTTGCACTAG ATGATCAATGCACAGGCGCTAATCCTAGATTTCCTCTTACAAGTGAGATA AAAGAAATGTATATAAATTGTTTTAAAAAACAACCTTAA

[0121] Example amino acid sequence:

TABLE-US-00006 MKVTTVKELDEKLKVIKEAQKKFSCYSQEMVDEIFRNAAMAAIDARIELA KAAVLETGMGLVEDKVIKNHFAGEYIYNKYKDEKTCGIIERNEPYGITKI AEPIGVVAAIIPVTNPTSTTIFKSLISLKTRNGIFFSPHPRAKKSTILAA KTILDAAVKSGAPENIIGWIDEPSIELTQYLMQKADITLATGGPSLVKSA YSSGKPAIGVGPGNTPVIIDESAHIKMAVSSIILSKTYDNGVICASEQSV IVLKSIYNKVKDEFQERGAYIIKKNELDKVREVIFKDGSVNPKIVGQSAY TIAAMAGIKVPKTTRILIGEVTSLGEEEPFAHEKLSPVLAMYEADNFDDA LKKAVTLINLGGLGHTSGIYADEIKARDKIDRFSSAMKTVRTFVNIPTSQ GASGDLYNFRIPPSFTLGCGFWGGNSVSENVGPKHLLNIKTVAERRENML WERVPHKVYFKFGCLQFALKDLKDLKKKRAFIVTDSDPYNLNYVDSIIKI LEHLDIDEKVENKVGREADLKTIKKATEEMSSFMPDTIIALGGTPEMSSA KLMWVLYEHPEVKFEDLAIKFMDIRKRIYTFPKLGKKAMLVAITTSAGSG SEVTPFALVTDNNTGNKYMLADYEMTPNMAIVDAELMMKMPKGLTAYSGI DALVNSIEAYTSVYASEYTNGLALEAIRLIFKYLPEAYKNGRTNEKAREK MAHASTMAGMASANAFLGLCHSMAIKLSSEHNIPSGIANALLIEEVIKFN AVDNPVKQAPCPQYKYPNTIFRYARIADYIKLGGNTDEEKVDLLINKIHE LKKALNIPTSIKDAGVLEENFYSSLDRISELALDDQCTGANPRFPLTSEI KEMYINCFKKQP

[0122] An exemplary aldehyde oxidoreductase (AOR), also known as an aldehyde:ferredoxin oxidoreductase, for use in the present invention catalyzes the reduction of a carboxylic acid into its corresponding aldehyde. For example, crotonic acid into crotonaldehyde. This reaction typically requires a coenzyme, such as ferredoxin. Exemplary nucleic acid and amino acid sequences are set forth below:

[0123] EC number: 1.2.7.5

[0124] Example nucleic acid sequence:

TABLE-US-00007 ATGTACGGATATAAGGGTAAGGTATTAAGAATTAATCTAAGTAGTAAAAC TTATATAGTGGAAGAATTGAAAATTGACAAAGCTAAAAAATTTATAGGTG CAAGAGGGTTAGGCGTAAAAACCTTATTTGACGAAGTAGATCCAAAGGTA GATCCATTATCACCTGATAACAAATTTATTATAGCAGCGGGACCACTTAC AGGTGCACCTGTTCCAACAAGCGGAAGATTCATGGTAGTTACTAAATCAC CTTTAACAGGAACTATTGCTATTGCAAATTCAGGTGGAAAATGGGGAGCA GAATTCAAAGCAGCTGGATACGATATGATAATCGTTGAAGGTAAATCTGA TAAAGAAGTTTATGTAAATATAGTAGATGATAAAGTAGAATTTAGGGATG CTTCTCATGTTTGGGGAAAACTAACAGAAGAAACTACAAAAATGCTTCAA CAGGAAACAGATTCGAGAGCTAAGGTTTTATGCATAGGACCAGCTGGGGA AAAGTTATCACTTATGGCAGCAGTTATGAATGATGTTGATAGAACAGCAG GACGTGGTGGTGTTGGAGCTGTTATGGGTTCAAAGAACTTAAAAGCTATT GTAGTTAAAGGAAGCGGAAAAGTAAAATTATTTGATGAACAAAAAGTGAA GGAAGTAGCACTTGAGAAAACAAATATTTTAAGAAAAGATCCAGTAGCTG GTGGAGGACTTCCAACATACGGAACAGCTGTACTTGTTAATATTATAAAT GAAAATGGTGTACATCCAGTAAAGAATTTTCAAAAATCTTATACAGATCA AGCAGATAAGATCAGTGGAGAAACTTTAACTAAAGATTGCTTAGTTAGAA AAAATCCTTGCTATAGGTGTCCAATTGCCTGTGGAAGATGGGTAAAACTT GATGATGGAACTGAATGTGGAGGACCAGAATATGAAACATTATGGTCATT TGGATCTGATTGTGATGTATACGATATAAATGCTGTAAATACAGCAAATA TGTTGTGTAATGAATATGGATTAGATACCATTACAGCAGGATGTACTATT GCAGCAGCTATGGAACTTTATCAAAGAGGTTATATTAAGGATGAAGAAAT AGCAGCAGATGGATTGTCACTTAATTGGGGAGATGCTAAGTCCATGGTTG AATGGGTAAAGAAAATGGGACTTAGAGAAGGATTTGGAGACAAGATGGCA GATGGTTCATACAGACTTTGTGACTCATACGGTGTACCTGAGTATTCAAT GACTGTAAAAAAACAGGAACTTCCAGCATATGACCCAAGAGGAATACAGG GACATGGTATTACTTATGCTGTTAACAATAGGGGAGGATGTCACATTAAG GGATATATGGTAAGTCCTGAAATACTTGGCTATCCAGAAAAACTTGATAG ACTTGCAGTGGAAGGAAAAGCAGGATATGCTAGAGTATTCCATGATTTAA CAGCTGTTATAGATTCACTTGGATTATGTATTTTTACAACATTTGGTCTT GGTGCACAGGATTATGTTGATATGTATAATGCAGTAGTTGGTGGAGAATT ACATGATGTAAATTCTTTAATGTTAGCTGGAGATAGAATATGGACTTTAG AAAAAATATTTAACTTAAAGGCAGGCATAGATAGTTCACAGGATACTCTT CCAAAGAGATTGCTTGAAGAACAAATTCCAGAAGGACCATCAAAAGGAGA AGTTCATAAGTTAGATGTACTACTACCTGAATATTATTCAGTACGTGGAT GGGATAAAAATGGTATTCCTACAGAGGAAACGTTAAAGAAATTAGGATTA GATGAATACGTAGGTAAGCTTTAG

[0125] Example amino acid sequence:

TABLE-US-00008 MYGYKGKVLRINLSSKTYIVEELKIDKAKKFIGARGLGVKTLFDEVDPKV DPLSPDNKFIIAAGPLTGAPVPTSGREMVVIKSPLIGTIAIANSGGKWGA EFKAAGYDMIIVEGKSDKEVYVNIVDDKVEFRDASHVWGKLTEETTKMLQ QETDSRAKVLCIGPAGEKLSLMAAVMNDVDRTAGRGGVGAVMGSKNLKAI VVKGSGKVKLFDEQKVKEVALEKTNILRKDPVAGGGLPTYGTAVLVNIIN ENGVHPVKNFQKSYTDQADKISGETLTKDCLVRKNPCYRCPIACGRWVKL DDGTECGGPEYETLWSFGSDCDVYDINAVNTANMLCNEYGLDTITAGCTI AAAMELYQRGYIKDEEIAADGLSLNWGDAKSMVEWVKKMGLREGFGDKMA DGSYRLCDSYGVPEYSMTVKKQELPAYDPRGIQGHGITYAVNNRGGCHIK GYMVSPEILGYPEKLDRLAVEGKAGYARVFHDLTAVIDSLGLCIFTTFGL GAQDYVDMYNAVVGGELHDVNSLMLAGDRIWTLEKIFNLKAGIDSSQDTL PKRLLEEQIPEGPSKGEVHKLDVLLPEYYSVRGWDKNGIPTEETLKKLGL DEYVGKL

[0126] An exemplary phosphotransacetylase (PTA) for use in the present invention catalyzes the conversion of crotonyl-CoA into crotonyl phosphate. This reaction requires a phosphate group to transfer onto the crotonyl substrate and releases a CoA group. Exemplary nucleic acid and amino acid sequences are set forth below:

[0127] EC number: 2.3.1.19

[0128] Example nucleic acid sequence:

TABLE-US-00009 GTGATTAAGAGTTTTAATGAAATTATCATGAAGGTAAAGAGCAAAGAAAT GAAAAAAGTTGCTGTTGCTGTAGCACAAGACGAGCCAGTACTTGAAGCAG TAAGAGATGCTAAGAAAAATGGTATTGCAGATGCTATTCTTGTTGGAGAC CATGACGAAATCGTGTCAATCGCGCTTAAAATAGGAATGGATGTAAATGA TTTTGAAATAGTAAACGAGCCTAACGTTAAGAAAGCTGCTTTAAAGGCAG TAGAGCTTGTATCAACTGGAAAAGCTGATATGGTAATGAAGGGACTTGTA AATACAGCAACTTTCTTAAGATCTGTATTAAACAAAGAAGTTGGACTTAG AACAGGAAAAACTATGTCTCACGTTGCAGTATTTGAAACTGAGAAATTTG ATAGACTATTATTTTTAACAGATGTTGCTTTCAATACTTATCCTGAATTA AAGGAAAAAATTGATATAGTAAACAATTCAGTTAAGGTTGCACATGCAAT AGGAATTGAAAATCCAAAGGTTGCTCCAATTTGTGCAGTTGAGGTTATAA ACCCTAAAATGCCATCAACACTTGATGCAGCAATGCTTTCAAAAATGAGT GACAGAGGACAAATTAAAGGTTGTGTAGTTGACGGACCTTTAGCACTTGA TATAGCTTTATCAGAAGAAGCAGCACATCATAAGGGAGTAACAGGAGAAG TTGCTGGAAAAGCTGATATCTTCTTAATGCCAAACATAGAAACAGGAAAT GTAATGTATAAGACTTTAACATATACAACTGATTCAAAAAATGGAGGAAT CTTAGTTGGAACTTCTGCACCAGTTGTTTTAACTTCAAGAGCTGACAGCC ATGAAACAAAAATGAACTCTATAGCACTTGCAGCTTTAGTTGCAGGCAAT AAATAA

[0129] Example amino acid sequence:

TABLE-US-00010 MIKSFNEIIMKVKSKEMKKVAVAVAQDEPVLEAVRDAKKNGIADAILVG DHDEIVSIALKIGMDVNEFEIVNEPNVKKAALKAVELVSTGKADMVMKG LVNTATFLRSVLNKEVGLRIGKTMSHVAVFETEKFDRLLFLTDVAENTY PELKEKIDIVNNSVKVAHAIGIENPKVAPICAVEVINPKMPSTLDAAML SKMSDRGQIKGCVVDGPLALDIALSEEAAHHKGVTGEVAGKADIFLMPN IETGNVMYKTLTYTTDSKNGGILVGTSAPVVLTSRADSHETKMNSIALA ALVAGNK

[0130] An exemplary acetate kinase (ACK) for use in the present invention catalyzes the conversion of crotonyl phosphate into crotonate while simultaneously generating a molecule of ATP. This reaction requires an ADP (adenosine diphosphate) onto which the phosphate from crotonyl phosphate is transferred to in order to generate the ATP (adenosine triphosphate). Exemplary nucleic acid and amino acid sequences are set forth below:

[0131] EC number: 2.7.2.7

[0132] Example nucleic acid sequence:

TABLE-US-00011 ATGTATAGATTACTAATAATCAATCCTGGCTCGACCTCAACTAAAATTGG TATTTATGACGATGAAAAAGAGATATTTGAGAAGACTTTAAGACATTCAG CTGAAGAGATAGAAAAATATAACACTATATTTGATCAATTTCAATTCAGA AAGAATGTAATTTTAGATGCGTTAAAAGAAGCAAACATAGAAGTAAGTTC TTTAAATGCTGTAGTTGGAAGAGGCGGACTCTTAAAGCCAATAGTAAGTG GAACTTATGCAGTAAATCAAAAAATGCTTGAAGACCTTAAAGTAGGAGTT CAAGGTCAGCATGCGTCAAATCTTGGTGGAATTATTGCAAATGAAATAGC AAAAGAAATAAATGTTCCAGCATACATAGTTGATCCAGTTGTTGTGGATG AGCTTGATGAAGTTTCAAGAATATCAGGAATGGCTGACATTCCAAGAAAA AGTATATTCCATGCATTAAATCAAAAAGCAGTTGCTAGAAGATATGCAAA AGAAGTTGGAAAAAAATACGAAGATCTTAATTTAATCGTAGTCCACATGG GTGGAGGTACTTCAGTAGGTACTCATAAAGATGGTAGAGTAATAGAAGTT AATAATACACTTGATGGAGAAGGTCCATTCTCACCAGAAAGAAGTGGTGG AGTTCCAATAGGAGATCTTGTAAGATTGTGCTTCAGCAACAAATATACTT ATGAAGAAGTAATGAAAAAGATAAACGGCAAAGGCGGAGTTGTTAGTTAC TTAAATACTATCGATTTTAAGGCTGTAGTTGATAAAGCTCTTGAAGGAGA TAAGAAATGTGCACTTATATATGAAGCTTTCACATTCCAGGTAGCAAAAG AGATAGGAAAATGTTCAACCGTTTTAAAAGGAAATGTAGATGCAATAATC TTAACAGGCGGAATTGCGTACAACGAGCATGTATGTAATGCCATAGAGGA TAGAGTAAAATTCATAGCACCTGTAGTTAGATATGGTGGAGAAGATGAAC TTCTTGCACTTGCAGAAGGTGGACTTAGAGTTTTAAGAGGAGAAGAAAAA GCTAAGGAATACAAATAA

[0133] Example amino acid sequence:

TABLE-US-00012 MYRLLIINPGSTSTKIGIYDDEKEIFEKTLRHSAEEIEKYNTIFDQFQFR KNVILDALKEANIEVSSLNAVVGRGGLLKPIVSGTYAVNQKMLEDLKVGV QGQHASNLGGIIANEIAKEINVPAYIVDPVVVDELDEVSRISGMADIPRK SIFHALNQKAVARRYAKEVGKKYEDLNLIVVHMGGGTSVGTHKDGRVIEV NNTLDGEGPFSPERSGGVPIGDLVRLCFSNKYTYEEVMKKINGKGGVVSY LNTIDFKAVVDKALEGDKKCALIYEAFTFQVAKEIGKCSTVLKGNVDAII LTGGIAYNEHVCNAIEDRVKFIAPVVRYGGEDELLALAEGGLRVLRGEEK AKEYK

[0134] An exemplary CoA-transferase subunit A (COAT-A) for use in the present invention catalyzes the transfer of coenzyme-A (CoA) between two molecules. For example, from acetoacetyl-CoA to acetate to form acetoacetate and acetyl-CoA or from acetoacetyl-CoA to crotonate to form acetoacetate and crotonyl-CoA. Exemplary subunit A nucleic acid and amino acid sequences are set forth below:

[0135] EC number: 2.8.3.8 or 2.8.3.9 or other related enzymes

[0136] Example nucleic acid sequence:

TABLE-US-00013 ATGAACTCTAAAATAATTAGATTTGAAAATTTAAGGTCATTCTTTAAAGA TGGGATGACAATTATGATTGGAGGTTTTTTAAACTGTGGCACTCCAACCA AATTAATTGATTTTTTAGTTAATTTAAATATAAAGAATTTAACGATTATA AGTAATGATACATGTTATCCTAATACAGGTATTGGTAAGTTAATATCAAA TAATCAAGTAAAAAAGCTTATTGCTTCATATATAGGCAGCAACCCAGATA CTGGCAAAAAACTTTTTAATAATGAACTTGAAGTAGAGCTCTCTCCCCAA GGAACTCTAGTGGAAAGAATACGTGCAGGCGGATCTGGCTTAGGTGGTGT ACTAACTAAAACAGGTTTAGGAACTTTGATTGAAAAAGGAAAGAAAAAAA TATCTATAAATGGAACGGAATATTTGTTAGAGCTACCTCTTACAGCCGAT GTAGCATTAATTAAAGGTAGTATTGTAGATGAGGCCGGAAACACCTTCTA TAAAGGTACTACTAAAAACTTTAATCCCTATATGGCAATGGCAGCTAAAA CCGTAATAGTTGAAGCTGAAAATTTAGTTAGCTGTGAAAAACTAGAAAAG GAAAAAGCAATGACCCCCGGAGTTCTTATAAATTATATAGTAAAGGAGCC TGCATAA

[0137] Example amino acid sequence:

TABLE-US-00014 MNSKIIRFENLRSFFKDGMTIMIGGFLNCGTPTKLIDFLVNLNIKNLTII SNDTCYPNTGIGKLISNNQVKKLIASYIGSNPDTGKKLFNNELEVELSPQ GTLVERIRAGGSGLGGVLTKTGLGTLIEKGKKKISINGTEYLLELPLTAD VALIKGSIVDEAGNTFYKGTTKNENPYMAMAAKTVIVEAENLVSCEKLEK EKAMTPGVLINYIVKEPA

[0138] An exemplary CoA-transferase subunit B (COAT-B) for use in the present invention catalyzes the transfer of coenzyme-A (CoA) between two molecules. For example, from acetoacetyl-CoA to acetate to form acetoacetate and acetyl-CoA or from acetoacetyl-CoA to crotonate to form acetoacetate and crotonyl-CoA. Exemplary subunit B nucleic acid and amino acid sequences are set forth below:

[0139] EC number: 2.8.3.8 or 2.8.3.9 or other related enzymes

[0140] Example nucleic acid sequence:

TABLE-US-00015 ATGATTAATGATAAAAACCTAGCGAAAGAAATAATAGCCAAAAGAGTTGC AAGAGAATTAAAAAATGGTCAACTTGTAAACTTAGGTGTAGGTCTTCCTA CCATGGTTGCAGATTATATACCAAAAAATTTCAAAATTACTTTCCAATCA GAAAACGGAATAGTTGGAATGGGCGCTAGTCCTAAAATAAATGAGGCAGA TAAAGATGTAGTAAATGCAGGAGGAGACTATACAACAGTACTTCCTGACG GCACATTTTTCGATAGCTCAGTTTCGTTTTCACTAATCCGTGGTGGTCAC GTAGATGTTACTGTTTTAGGGGCTCTCCAGGTAGATGAAAAGGGTAATAT AGCCAATTGGATTGTTCCTGGAAAAATGCTCTCTGGTATGGGTGGAGCTA TGGATTTAGTAAATGGAGCTAAGAAAGTAATAATTGCAATGAGACATACA AATAAAGGTCAACCTAAAATTTTAAAAAAATGTACACTTCCCCTCACGGC AAAGTCTCAAGCAAATCTAATTGTAACAGAACTTGGAGTAATTGAGGTTA TTAATGATGGTTTACTTCTCACTGAAATTAATAAAAACACAACCATTGAT GAAATAAGGTCTTTAACTGCTGCAGATTTACTCATATCCAATGAACTTAG ACCCATGGCTGTTTAG

[0141] Example amino acid sequence:

TABLE-US-00016 MINDKNLAKEIIAKRVARELKNGQLVNLGVGLPTMVADYIPKNFKITFQS ENGIVGMGASPKINEADKDVVNAGGDYTTVLPDGTFFDSSVSFSLIRGGH VDVTVLGALQVDEKGNIANWIVPGKMLSGMGGAMDLVNGAKKVIIAMRHT NKGQPKILKKCTLPLTAKSQANLIVTELGVIEVINDGLLLTEINKNTTID EIRSLTAADLLISNELRPMAV

[0142] An exemplary acetoacetate decarboxylase (ADC) for use in the present invention catalyzes the decarboxylation of acetoacetate into acetone and CO.sub.2. Exemplary nucleic acid and amino acid sequences are set forth below:

[0143] EC number: 4.1.1.4

[0144] Example nucleic acid sequence:

TABLE-US-00017 ATGTTAAAGGATGAAGTAATTAAACAAATTAGCACGCCATTAACTTCGCC TGCATTTCCTAGAGGACCCTATAAATTTCATAATCGTGAGTATTTTAACA TTGTATATCGTACAGATATGGATGCACTTCGTAAAGTTGTGCCAGAGCCT TTAGAAATTGATGAGCCCTTAGTCAGGTTTGAAATTATGGCAATGCATGA TACGAGTGGACTTGGTTGTTATACAGAAAGCGGACAGGCTATTCCCGTAA GCTTTAATGGAGTTAAGGGAGATTATCTTCATATGATGTATTTAGATAAT GAGCCTGCAATTGCAGTAGGAAGGGAATTAAGTGCATATCCTAAAAAGCT CGGGTATCCAAAGCTTTTTGTGGATTCAGATACTTTAGTAGGAACTTTAG ACTATGGAAAACTTAGAGTTGCGACAGCTACAATGGGGTACAAACATAAA GCCTTAGATGCTAATGAAGCAAAGGATCAAATTTGTCGCCCTAATTATAT GTTGAAAATAATACCCAATTATGATGGAAGCCCTAGAATATGTGAGCTTA TAAATGCGAAAATCACAGATGTTACCGTACATGAAGCTTGGACAGGACCA ACTCGACTGCAGTTATTTGATCACGCTATGGCGCCACTTAATGATTTGCC AGTAAAAGAGATTGTTTCTAGCTCTCACATTCTTGCAGATATAATATTGC CTAGAGCTGAAGTTATATATGATTATCTTAAGTAA

[0145] Example amino acid sequence:

TABLE-US-00018 MLKDEVIKQISTPLTSPAFPRGPYKEHNREYFNIVYRTDMDALRKVVPEP LEIDEPLVRFEIMAMHDTSGLGCYTESGQAIPVSFNGVKGDYLHMMYLDN EPAIAVGRELSAYPKKLGYPKLFVDSDTLVGTLDYGKLRVATATMGYKHK ALDANEAKDQICRPNYMLKIIPNYDGSPRICELINAKITDVTVHEAWTGP TRLQLFDHAMAPLNDLPVKEIVSSSHILADIILPRAEVIYDYLK

[0146] An exemplary secondary alcohol dehydrogenase (SADH) for use in the present invention catalyzes the reduction of a ketone into a secondary alcohol. For example, acetone into 2-propanol (a.k.a. isopropanol). Exemplary nucleic acid and amino acid sequences are set forth below:

[0147] EC number: 1.1.1.1

[0148] Example nucleic acid sequence:

TABLE-US-00019 ATGAAAGGTTTTGCAATGTTAGGTATTAACAAATTAGGATGGATTGAAAA GAAAAACCCAGTGCCAGGTCCTTATGATGCGATTGTACATCCTCTAGCTG TATCCCCATGTACATCAGATATACATACGGTTTTTGAAGGAGCACTTGGT AATAGGGAAAATATGATTTTAGGCCATGAAGCTGTAGGTGAAATAGCCGA AGTTGGCAGCGAAGTTAAAGATTTTAAAGTTGGCGATAGAGTTATCGTAC CATGCACAACACCTGACTGGAGATCTTTAGAAGTCCAAGCTGGTTTTCAG CAGCATTCAAACGGTATGCTTGCAGGATGGAAGTTTTCCAATTTTAAAGA TGGTGTATTTGCAGATTACTTTCATGTAAACGATGCAGATATGAATCTTG CCATACTCCCAGATGAAATACCTTTAGAAAGTGCAGTTATGATGACAGAC ATGATGACTACTGGTTTTCATGGAGCAGAACTTGCAGACATAAAAATGGG CTCCAGCGTTGTAGTAATTGGTATAGGAGCTGTTGGATTAATGGGAATAG CCGGTTCCAAACTTCGAGGAGCAGGCAGAATTATCGGTGTTGGAAGCAGA CCTGTTTGTGTTGAAACAGCTAAATTTTATGGAGCAACTGATATTGTAAA TTATAAAAATGGTGATATAGTTGAACAAATCATGGACTTAACTCATGGTA AAGGTGTAGACCGTGTAATCATGGCAGGCGGTGGTGCTGAAACACTAGCA CAAGCAGTAACTATGGTTAAACCTGGCGGCGTAATTTCTAACATCAACTA CCATGGAAGCGGTGATACTTTACCAATACCTCGTGTTCAATGGGGCTGCG GCATGGCTCACAAAACTATAAGAGGAGGATTATGCCCCGGCGGACGTCTT AGAATGGAAATGCTAAGAGATCTTGTTCTATATAAACGTGTTGATTTGAG TAAACTTGTTACTCATGTATTTGATGGTGCAGAAAATATTGAAAAGGCCC TTTTGCTTATGAAAAATAAGCCAAAAGATTTAATTAAATCAGTAGTTACA TTCTAA

[0149] Example amino acid sequence:

TABLE-US-00020 MKGFAMLGINKLGWIEKKNPVPGPYDAIVHPLAVSPCTSDIHTVFEGAL GNRENMILGHEAVGEIAEVGSEVKDFKVGDRVIVPCTTPDWRSLEVQAG FQQHSNGMLAGWKESNEKDGVFADYFHVNDADMNLAILPDEIPLESAVM MTDMMTTGFHGAELADIKMGSSVVVIGIGAVGLMGIAGSKLRGAGRIIG VGSRPVCVETAKFYGATDIVNYKNGDIVEQIMDLTHGKGVDRVIMAGGG AETLAQAVTMVKPGGVISNINYHGSGDTLPIPRVQWGCGMAHKTIRGGL CPGGRLRMEMLRDLVLYKRVDLSKLVTHVEDGAENIEKALLLMKNKPKD LIKSVVTF

[0150] An exemplary butyryl-CoA dehydrogenase (BCD) for use in the present invention catalyzes the reduction of crotonyl-CoA into butyryl-CoA by reducing the carbon-carbon double bond in crotonyl-CoA. This enzyme requires an electron-transfer flavoprotein. Exemplary nucleic acid and amino acid sequences are set forth below:

[0151] EC number: 1.3.8.1

[0152] Example nucleic acid sequence:

TABLE-US-00021 ATGGATTTTAATTTAACAAGAGAACAAGAATTAGTAAGACAGATGGTTAG AGAATTTGCTGAAAATGAAGTTAAACCTATAGCAGCAGAAATTGATGAAA CAGAAAGATTTCCAATGGAAAATGTAAAGAAAATGGGTCAGTATGGTATG ATGGGAATTCCATTTTCAAAAGAGTATGGTGGCGCAGGTGGAGATGTATT ATCTTATATAATCGCCGTTGAGGAATTATCAAAGGTTTGCGGTACTACAG GAGTTATTCTTTCAGCACATACATCACTTTGTGCTTCATTAATAAATGAA CATGGTACAGAAGAACAAAAACAAAAATATTTAGTACCTTTAGCTAAAGG TGAAAAAATAGGTGCTTATGGATTGACTGAGCCAAATGCAGGAACAGATT CTGGAGCACAACAAACAGTAGCTGTACTTGAAGGAGATCATTATGTAATT AATGGTTCAAAAATATTCATAACTAATGGAGGAGTTGCAGATACTTTTGT TATATTTGCAATGACTGACAGAACTAAAGGAACAAAAGGTATATCAGCAT TTATAATAGAAAAAGGCTTCAAAGGTTTCTCTATTGGTAAAGTTGAACAA AAGCTTGGAATAAGAGCTTCATCAACAACTGAACTTGTATTTGAAGATAT GATAGTACCAGTAGAAAACATGATTGGTAAAGAAGGAAAAGGCTTCCCTA TAGCAATGAAAACTCTTGATGGAGGAAGAATTGGTATAGCAGCTCAAGCT TTAGGTATAGCTGAAGGTGCTTTCAACGAAGCAAGAGCTTACATGAAGGA GAGAAAACAATTTGGAAGAAGCCTTGACAAATTCCAAGGTCTTGCATGGA TGATGGCAGATATGGATGTAGCTATAGAATCAGCTAGATATTTAGTATAT AAAGCAGCATATCTTAAACAAGCAGGACTTCCATACACAGTTGATGCTGC AAGAGCTAAGCTTCATGCTGCAAATGTAGCAATGGATGTAACAACTAAGG CAGTACAATTATTTGGTGGATACGGATATACAAAAGATTATCCAGTTGAA AGAATGATGAGAGATGCTAAGATAACTGAAATATATGAAGGAACTTCAGA AGTTCAGAAATTAGTTATTTCAGGAAAAATTTTTAGATAA

[0153] Example amino acid sequence:

TABLE-US-00022 MDFNLTREQELVRQMVREFAENEVKPIAAEIDETERFPMENVKKMGQYGM MGIPFSKEYGGAGGDVLSYIIAVEELSKVCGTTGVILSAHTSLCASLINE HGTEEQKQKYLVPLAKGEKIGAYGLTEPNAGTDSGAQQTVAVLEGDHYVI NGSKIFITNGGVADTEVIFAMTDRTKGTKGISAFIIEKGFKGESIGKVEQ KLGIRASSTTELVFEDMIVPVENMIGKEGKGFPIAMKTLEGGRIGIAAQA LGIAEGAFNEARAYMKERKQFGRSLDKFQGLAWMMADMDVAIESARYLVY KAAYLKQAGLPYTVDAARAKLHAANVAMDVITKAVQLEGGYGYTKEYPVE RMMRDAKITEIYEGTSEVQKLVISGKIFR

[0154] An exemplary trans-2-enoyl-CoA reductase (TER) for use in the present invention catalyzes the reduction of crotonyl-CoA into butyryl-CoA by reducing the carbon-carbon double bond in crotonyl-CoA. Exemplary nucleic acid and amino acid sequences are set forth below:

[0155] EC number: 1.3.1.44

[0156] Example nucleic acid sequence:

TABLE-US-00023 ATGATAGTAAAAGCAAAGTTTGTAAAAGGATTTATCAGAGATGTACATCC TTATGGTTGCAGAAGGGAAGTACTAAATCAAATAGATTATTGTAAGAAGG CTATTGGGTTTAGGGGACCAAAGAAGGTTTTAATTGTTGGAGCCTCATCT GGGTTTGGTCTTGCTACTAGAATTTCAGTTGCATTTGGAGGTCCAGAAGC TCACACAATTGGAGTATCCTATGAAACAGGAGCTACAGATAGAAGAATAG GAACAGCGGGATGGTATAATAACATATTTTTTAAAGAATTTGCTAAAAAA AAAGGATTAGTTGCAAAAAACTTCATTGAGGATGCCTTTTCTAATGAAAC CAAAGATAAAGTTATTAAGTATATAAAGGATGAATTTGGTAAAATAGATT TATTTGTTTATAGTTTAGCTGCGCCTAGGAGAAAGGACTATAAAACTGGA AATGTTTATACTTCAAGAATAAAAACAATTTTAGGAGATTTTGAGGGACC GACTATTGATGTTGAAAGAGACGAGATTACTTTAAAAAAGGTTAGTAGTG CTAGCATTGAAGAAATTGAAGAAACTAGAAAGGTAATGGGTGGAGAGGAT TGGCAAGAGTGGTGTGAAGAGCTGCTTTATGAAGATTGTTTTTCGGATAA AGCAACTACCATAGCATACTCGTATATAGGATCCCCAAGAACCTACAAGA TATATAGAGAAGGTACTATAGGAATAGCTAAAAAGGATCTTGAAGATAAG GCTAAGCTTATAAATGAAAAACTTAACAGAGTTATAGGTGGTAGAGCCTT TGTGTCTGTGAATAAAGCATTAGTTACAAAAGCAAGTGCATATATTCCAA CTTTTCCTCTTTATGCAGCTATTTTATATAAGGTCATGAAAGAAAAAAAT ATTCATGAAAATTGTATTATGCAAATTGAGAGAATGTTTTCTGAAAAAAT ATATTCAAATGAAAAAATACAATTTGATGACAAGGGAAGATTAAGGATGG ACGATTTAGAGCTTAGAAAAGACGTTCAAGACGAAGTTGATAGAATATGG AGTAATATTACTCCTGAAAATTTTAAGGAATTATCTGATTATAAGGGATA CAAAAAAGAATTCATGAACTTAAACGGTTTTGATCTAGATGGGGTTGATT ATAGTAAAGACCTGGATATAGAATTATTAAGAAAATTAGAACCTTAA

[0157] Example amino acid sequence:

TABLE-US-00024 MIVKAKFVKGFIRDVHPYGCRREVLNQIDYCKKAIGFRGPKKVLIVGASS GFGLATRISVAFGGPEAHTIGVSYETGATDRRIGTAGWYNNIFFKEFAKK KGLVAKNFIEDAFSNETKDKVIKYIKDEFGKIDLFVYSLAAPRRKDYKTG NVYTSRIKTILGDFEGPTIDVERDEITLKKVSSASIEEIEETRKVMGGED WQEWCEELLYEDCFSDKATTIAYSYIGSPRTYKIYREGTIGIAKKDLEDK AKLINEKLNRVIGGRAFVSVNKALVTKASAYIPTFPLYAAILYKVMKEKN IHENCIMQIERMFSEKIYSNEKIQFDDKGRLRMDDLELRKDVQDEVDRIW SNITPENFKELSDYKGYKKEFMNLNGFDLDGVDYSKDLDIELLRKLEP

[0158] The nucleotide sequence contained in the nucleic acid of the present invention may include a nucleotide sequence having an identity of at least 70% with one or more of the exemplary ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, and TER nucleotide sequences set forth above and having one or more of the respective activities described above (e.g., an activity of catalyzing the reduction of crotonyl-CoA into butyryl-CoA by reducing the carbon-carbon double bond in crotonyl-CoA). Preferably, for example, the nucleic acid comprises a nucleotide sequence having an identity of at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98%, or 99% or more) with one or more of the exemplary ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, and TER nucleotide sequences set forth above. The nucleotide sequences of the invention may have one or more nucleotide deletions, substitutions, or insertions relative to an exemplary nucleic acid sequence of the invention. For example, 1-300, 1-200, 1-100, 2-90, 3-80, 4-70, 5-50, 40, 30, 20, 10, 9, 8, 7, or 6 modifications may be made relative to one or more of the above ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, and TER nucleotide sequences.

[0159] Similarly, the protein encoded by a nucleic acid of the present invention may be any protein having an identity of at least 70% with one or more of the exemplary ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, and TER amino acid sequences set forth above, and having one or more of the respective activities described above. Specific examples of an amino acid sequence of the protein encoded by the nucleic acid of the present invention include an amino acid sequence having an identity of 75% or more, preferably 80% or more, more preferably 85% or more, and most preferably 90% or more (e.g., 95% or more, furthermore 98% or more) with the exemplary ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, or TER amino acid sequence set forth above. The polypeptide sequences of the invention may have one or more amino acid deletions, substitutions, or insertions relative to an exemplary amino acid sequence of the invention. For example, 1-100, 1-90, 2-80, 3-70, 4-60, 5-50, 40, 30, 20, 10, 9, 8, 7, or 6 amino acid modifications may be made relative to an exemplary ALDH, ADH, ADHE, AOR, PTA, ACK, COAT-A, COAT-B, ADC, SADH, BCD, and TER amino acid sequences insofar as the encoded protein retains ALDH-, ADH-, ADHE-, AOR-, PTA-, ACK-, COAT-A-, COAT-B-, ADC-, SADH-, BCD-, and/or TER-activity.

III. CULTURE AND FERMENTATION CONDITIONS

[0160] Culture and/or fermentation conditions for growth of microorganisms as described herein or for use in methods as set forth herein are not particularly limited, and may be selected as appropriate depending on the microorganism to be cultured as well as the bioproduct or bioproducts to be generated. For example, strains may be grown in clostridial growth medium (CGM).

[0161] In an embodiment, CGM consists of the following:

[0162] KH.sub.2PO.sub.4: 0.75 g/l

[0163] K.sub.2HPO.sub.4.3H.sub.2O: 0.98 g/l

[0164] NaCl: 1.0 g/l

[0165] MgSO.sub.4: 0.35 g/l

[0166] MnSO.sub.4H.sub.2O: 0.01 g/l

[0167] FeSO.sub.4.7H.sub.2O: 0.01 g/l

[0168] 4-Aminobenzoic acid: 0.004 g/l

[0169] Asparagine: 2.0 g/l

[0170] Yeast extract: 5.0 g/l

[0171] (NH.sub.4).sub.2SO.sub.4: 2.0 g/l

[0172] Sodium acetate: 2.46 g/l; and

[0173] Glucose: 80.0 g/l.

[0174] Certain strains may be grown under aerobic or anaerobic conditions, as would be known to those of skill in the art. Other strains may require anaerobic growth conditions. Gas mixtures for anaerobic growth conditions may comprise, for example, 10% CO.sub.2-5% H.sub.2-85% N.sub.2, or 80% H.sub.2 20% CO.sub.2, or 80% N.sub.2-20% CO.sub.2, or 80% N.sub.2-10% CO.sub.2-10% H.sub.2.

IV. EXAMPLES

[0175] All strains were cultivated in an anaerobic chamber with an atmosphere of 10% CO.sub.2, 5% H.sub.2, and the balance of N.sub.2 at 37 C. Individual colonies were selected from a solid agar plate and placed in the indicated liquid medium with appropriate antibiotics: 5 g/ml thiamphenicol for deletion strains and 5 pg/ml clarithromycin for plasmid-harboring strains. Solid agar plates for C. acetobutylicum were 2xYTG (pH 5.8) with 15 g/l of agar. The medium 2xYTG consists of:

[0176] NaCl: 10 g/l

[0177] Tryptone: 10 g/l

[0178] Yeast extract:

EXAMPLE 1

Crotyl Alchohol Production in C. acetobutylicum

[0179] C. acetobutylicum was genetically engineered to produce more crotyl alcohol. The bcd gene (CA_C2711) was deleted from the chromosome to generate the strain Abcd. In addition, a plasmid, called pTHCA, over expressing the genes thl (CA_C2783), hbd (CA_C2708), crt (CA_C2712), and adhE1 (CA_P0162), was introduced into the Abcd strain.

[0180] A total of three strains were tested: C. acetobutylicum ATCC 824 [WT], C. acetobutylicum bcd [BCD], and C. acetobutylicum bcd (pTHCA) [BCD (pTHCA)]. Each strain was grown in 10 ml of a clostridial growth medium (CGM) anaerobically at 37 C. Endpoint samples were taken after 5 days of growth. Metabolite concentrations are presented in Table 1.

TABLE-US-00025 TABLE 1 End point metabolite concentrations of crotyl alcohol producing strains of C. acetobutylicum. Concentration of crotyl alcohol Strain (mg/l) WT 20.3 BCD 41.1 BCD (pTHCA) 78.6

[0181] As can be seen from Table 1, the concentration of crotyl alcohol was increased in the C. acetobutylicum strain in which the bcd gene was deleted. The highest concentration of crotyl alcohol was obtained with the C. acetobutylicum strain in which the bcd gene was deleted and in which the thl, hbd, crt, and adhE2 genes were overexpressed.