ORGANISMS AND BIOSYNTHETIC PROCESSES FOR HYDROCARBON SYNTHESIS

Abstract

Methods for biosynthesising hydrocarbons from a gaseous substrate in non-naturally occurring acetogens as well as non-naturally occurring acetogens for production of hydrocarbons are provided.

Claims

1. A non-naturally occurring acetogen capable of producing hydrocarbons or derivatives thereof from a gaseous substrate, said acetogen comprising: (i) an alteration of at least one polynucleotide, the polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase or encoding a polypeptide having an activity of a lactate dehydrogenase, or (ii) an alteration of at least two polynucleotides, wherein a first polynucleotide encodes a polypeptide having an activity of an alpha-acetolactate decarboxylase and a second polynucleotide encodes a polypeptide having an activity of a lactate dehydrogenase, wherein the polynucleotide alteration eliminates the activity of the encoded polypeptide.

2. The non-naturally occurring acetogen of claim 1 (i) or (ii), further comprising an alteration of a polynucleotide encoding a polypeptide having an activity of one or more members selected from the group consisting of an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and a Nfn complex, wherein the polynucleotide alteration eliminates the activity of the encoded polypeptide.

3. The non-naturally occurring acetogen of claim 1 wherein at least three to five polynucleotides are altered.

4. The non-naturally occurring acetogen of claim 3, wherein said altered polynucleotides encode: polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase and a dihydrolipoylprotein:NAD+oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, an aldehyde dehydrogenase and a purine nucleoside phosphorylase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and a Nfn complex; or polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and a formate dehydrogenase.

5-10. (canceled)

11. The non-naturally occurring acetogen of claim 1, wherein said acetogen is a Clostridium species.

12. The non-naturally occurring acetogen of claim 11, wherein said Clostridium species is any one of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium coskatii or Clostridium ragsdalei.

13. A method for producing the non-naturally occurring acetogen of claim 1, said method comprising: (i) altering at least one polynucleotide, the polynucleotide encoding a polypeptide having the activity of an alpha-acetolactate decarboxylase or encoding a polypeptide having the activity of a lactate dehydrogenase, or (ii) altering at least two polynucleotides, wherein a first polynucleotide encodes a polypeptide having an activity of an alpha-acetolactate decarboxylase and a second polynucleotide encodes a polypeptide having an activity of a lactate dehydrogenase, wherein the polynucleotide alteration eliminates the activity of the encoded polypeptide.

14. The method of producing the non-naturally occurring acetogen of claim 13(i) or (ii) further comprising altering a polynucleotide encoding a polypeptide having an activity of one or more of a member selected from the group consisting of an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and a Nfn complex, wherein the polynucleotide alteration eliminates the activity of the encoded polypeptide.

15. The method of producing the non-naturally occurring acetogen of claim 13 wherein at least three to five polynucleotides are altered.

16. The method of producing the non-naturally occurring acetogen of claim 15, wherein said altered polynucleotides encode: polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase or a dihydrolipoylprotein:NAD+oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, an aldehyde dehydrogenase and a purine nucleoside phosphorylase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and a Nfn complex; or polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and a formate dehydrogenase.

17-22. (canceled)

23. The method of producing the non-naturally occurring acetogen of claim 13, wherein said acetogen is a Clostridium species.

24. The method of producing the non-naturally occurring acetogen of claim 23, wherein said Clostridium species is any one of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium coskatii or Clostridium ragsdalei.

25. A method for biosynthesizing a hydrocarbon or derivative thereof in a the non-naturally occurring acetogen of claim 1, said method comprising enzymatically producing the hydrocarbon from a gaseous substrate in a non-naturally occurring acetogen with (i) an alteration of at least one polynucleotide, the polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase or the polynucleotide encoding a polypeptide having an activity of a lactate dehydrogenase, or (ii) an alteration of at least two polynucleotides, wherein a first polynucleotide encodes a polypeptide having an activity of an alpha-acetolactate decarboxylase and a second polynucleotide encodes a polypeptide having an activity of a lactate dehydrogenase, wherein the polynucleotide alteration eliminates the activity of the encoded polypeptide.

26. The method of claim 25 wherein the hydrocarbon is a saturated or unsaturated 5 carbon branched structure derived from an isoprenoid.

27. The method of claim 25 wherein the hydrocarbon is isoprene.

28. The method of claim 27 wherein the isoprene is produced via a beta-ketothiolase route via pyruvate via ldh, a 2-hydroxyacyl-CoA dehydratase route via ldh, a 2-hydroxyacyl-CoA dehydratase route via mdd or a polyketide synthase route utilizing ldh.

29. The method of claim 25 wherein the non-naturally occurring acetogen further comprises an alteration of a polynucleotide encoding a polypeptide having an activity of one or more members selected from the group consisting of an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and a Nfn complex.

30. The method of claim 25 wherein the non-naturally occurring acetogen has at least three to five altered polynucleotides.

31. The method of claim 30, wherein said altered polynucleotides encode: polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase and a dihydrolipoylprotein:NAD+oxidoreductase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, an aldehyde dehydrogenase and a purine nucleoside phosphorylase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase; polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and a Nfn complex; or polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and a formate dehydrogenase.

32-37. (canceled)

38. The method of claim 27 wherein said isoprene is produced via a beta-ketothiolase route via pyruvate via ldh or a 2-hydroxyacyl-CoA dehydratase route via ldh or a 2-hydroxyacyl-CoA dehydratase route via mdd or a polyketide synthase route utilizing ldh and the non-naturally occurring acetogen has at least one altered polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase.

39. The method of claim 27, wherein said isoprene is produced via a beta-ketothiolase route via pyruvate via ldh or a 2-hydroxyacyl-CoA dehydratase route via ldh or a 2-hydroxyacyl-CoA dehydratase route via mdd or a polyketide synthase route utilizing ldh and the non-naturally occurring acetogen has at least three to five altered polynucleotides.

40-43. (canceled)

44. The method of claim 39 wherein isoprene is produced via a 2-hydroxyacyl-CoA dehydratase route via ldh and the altered polynucleotides encode polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase.

45-46. (canceled)

47. The method of claim 39 wherein isoprene is produced via a 2-hydroxyacyl-CoA dehydratase route via mdd and said altered polynucleotides encode polypeptides having an activity of members selected from the group consisting an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase.

48. (canceled)

49. The method of claim 39 wherein isoprene is produced via a 2-hydroxyacyl-CoA dehydratase route via mdd and said altered polynucleotides encode polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and a Nfn complex.

50-51. (canceled)

52. The method of claim 39 wherein isoprene is produced via a polyketide synthase route utilizing ldh and wherein said altered polynucleotides encode polypeptides having an activity of members selected from the group consisting an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase.

53. (canceled)

54. The method of claim 39 wherein isoprene is produced via a polyketide synthase route utilizing ldh and wherein said altered polynucleotides encode polypeptides having an activity of members selected from the group consisting of an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and a formate dehydrogenase.

55. The method of claim 25 wherein the gaseous substrate comprises a mixture of CO, CO.sub.2 and H.sub.2.

56. The method of claim 25 wherein the gaseous substrate comprises CO.

57. The method of claim 25 wherein said acetogen is a Clostridium species.

58. The method of claim 57, wherein said Clostridium species is any one of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium coskatii or Clostridium ragsdalei.

59. A composition comprising a means for producing a hydrocarbon via a beta-ketothiolase route via pyruvate via ldh, a 2-hydroxyacyl-CoA dehydratase route via ldh, a 2-hydroxyacyl-CoA dehydratase route via mdd or a polyketide synthase route utilizing ldh.

60. A genetic construct comprising at least one polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase and/or encoding a polypeptide having an activity of a lactate dehydrogenase, wherein said polynucleotide is altered to eliminate activity of the encoded polypeptide.

61. (canceled)

62. The genetic construct of claim 60, further comprising a polynucleotide encoding a polypeptide having an activity of one or more members selected from the group consisting of an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and a Nfn complex, wherein the polynucleotide is altered to eliminate the activity of the encoded polypeptide.

63. A bio-derived hydrocarbon produced with an acetogen of claim 1.

64. A bio-derived, bio-based, or fermentation-derived product produced from an acetogen of claim 1, wherein said product comprises: (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound or any combination thereof; (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof; (iii) a bio-derived, bio-based, or fermentation-derived cis-polyisoprene rubber, trans-polyisoprene rubber, or liquid polyisoprene rubber, comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i), or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii), or any combination thereof; (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii), or the bio-derived, bio-based, or fermentation-derived rubber of (iii), or any combination thereof; (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), the bio-derived, bio-based, or fermentation-derived compound of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived rubber of (iii), or the bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof; or (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), the bio-derived, bio-based, or fermentation-derived compound of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived rubber of (iii), the bio-derived, bio-based, or fermentation-derived formulation of (iv), or the bio-derived, bio-based, or fermentation-derived molded substance of (v), or any combination thereof.

65. A bio-derived, bio-based, or fermentation-derived product or derivative thereof produced according to any of FIGS. 1-4.

Description

BRIEF DESCRIPTION OF FIGURES

[0042] FIG. 1 is a biosynthetic schematic of isoprene synthesis using via a beta-ketothiolase route utilizing pyruvate and ldh.

[0043] FIG. 2 is a biosynthetic schematic of isoprene synthesis via a 2-hydroxyacyl-CoA dehydratase route utilizing ldh.

[0044] FIG. 3 is a biosynthetic schematic of isoprene synthesis via a 2-hydroxyacyl-CoA dehydratase route utilizing mdd.

[0045] FIG. 4 is a biosynthetic schematic of isoprene synthesis via a polyketide synthase route utilizing ldh.

[0046] FIG. 5A and FIG. 5B show results simulated for suboptimal growth by MOMA and evolved growth-optimality by FBA for the reference pathway and the beta-ketothiolase route via pyruvate via ldh, the 2-hydroxyacyl-CoA dehydratase route via ldh, the 2-hydroxyacyl-CoA dehydratase route via mdd and the polyketide synthase route via ldh. In FIGS. 5A and 5B Reference refers to mevalonate pathway and isoprene synthase, 1 refers to Pathway 1--ketothiolaseroute via pyruvate via ldh, 2 refers to Pathway 2-2-hydroxyacyl-CoA dehydratase route via ldh, 3 refers to Pathway 3-2-hydroxyacyl-CoAdehydratase route via mdd and 4 refers to Pathway 4-Polyketide synthase route via ldh. Suboptimal growth results in FIG. 5A indicate that all supplied pathways are superior to the references. These range from 1.4 to 1.9 higher than the reference mevalonate pathway. Evolved growth-optimality depicted in FIG. 5B indicate that all supplied pathways also offer better growth-coupled yields than the reference during growth-optimal conditions. These growth-coupled predicted yields range from 1.7 to 9 higher than the reference mevalonate pathway.

[0047] FIG. 6 shows predicted yield of isoprene in Clostridium autoethanogenum in strains genetically engineered with different numbers of knock-outs (2 KO, 3-4 KO and 5-10 KO).

[0048] FIG. 7 shows isoprene yields of C. autoethanogenum LZ1561 across different gas uptake ratios.

DETAILED DESCRIPTION

[0049] The present invention provides organisms, such as non-naturally occurring acetogens and other means derived therefrom or related thereto capable of producing hydrocarbons, as well as methods for the production of these acetogens and methods for their use in production of hydrocarbons and derivatives thereof.

[0050] Accordingly, disclosed herein are acetogens genetically engineered by alteration of one or more polynucleotides to produce hydrocarbons from gaseous substrates, as well as methods for their production and their use in biosynthesis of hydrocarbons. The non-naturally occurring acetogens and methods disclosed herein provide low cost processes for conversion of industrial gases to chemicals in a fermenter. In the methods of the present invention, the non-naturally occurring acetogens are introduced into a fermenter, mixed with gas feedstocks which are enzymatically converted to a hydrocarbon by the non-naturally occurring acetogens, and the hydrocarbon is then separated from the off-gases from the fermenter.

[0051] By hydrocarbon or hydrocarbons as used herein, it is meant to encompass any organic compound comprised of carbons and hydrogens which can be enzymatically synthesized from a gas and is inclusive of saturated as well as unsaturated structures with double or triple bonds formed between carbon atoms, ring structures, salts and derivatives thereof. In one nonlimiting embodiment, the hydrocarbon comprises one or more isoprene units as depicted in Formula I

##STR00001##

or a salt or derivative thereof.

[0052] By the phrase one or more isoprene units as depicted in Formula I it is meant to encompass any saturated or unsaturated 5 carbon branched structure derived from an isoprenoid including, isoprene as well as other isoprenoids, terpenes and terpenoids and derivatives such as, but not limited to isoprenols, and salts thereof.

[0053] Nonlimiting examples of hydrocarbons comprising one or more isoprene units produced in accordance with the present invention include isoprene as well as other isoprenoids, terpenes or terpenoid derivatives of 5, including C5, C10, C15, C20, C25, C30, C35, C40, C45, C50, etc. Nonlimiting examples include hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, polyterpene, lycopene, abietadiene, amorphadiene, carene, alpha-farnesene, beta-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, beta-pinene, sabinene, gamma-terpinene, terpinolene and valencene, as well as derivatives and salts thereof.

[0054] The present invention provides non-naturally occurring acetogens capable of producing hydrocarbons from a gaseous substrate.

[0055] In one nonlimiting embodiment, the hydrocarbon comprises any saturated or unsaturated 5 carbon branched structure derived from an isoprenoid including, isoprene as well as other isoprenoids, terpenes and terpenoids and derivatives such as, but not limited to isoprenols, and salts thereof.

[0056] In one nonlimiting embodiment, the hydrocarbon is isoprene produced in a non-naturally occurring acetogen via a beta-ketothiolase route via pyruvate via ldh (see FIG. 1), a 2-hydroxyacyl-CoA dehydratase route via ldh (see FIG. 2), a 2-hydroxyacyl-CoA dehydratase route via mdd (see FIG. 3) or a polyketide synthase route utilizing ldh (FIG. 4) as these pathways to isoprene offer considerably higher maximum predicted yields than the reference pathway (mevalonate pathway with isoprene synthase). For example, the beta-ketothiolase route via pyruvate via ldh pathway offers a maximum predicted yield which is two times higher than the mevalonate pathway with isoprene synthase wherein maximum yield is defined as the maximum isoprene yield in C. autoethanogenum LZ1561 with no growth. C. autoethanogenum LZ1561 is deposited under DSMZ accession DSM23693. As shown in FIG. 7, C. autoethanogenum LZ1561 can grow on a mixture of CO, CO.sub.2 and H.sub.2 as a carbon and energy source. Growth on CO offers the highest yield.

[0057] The non-naturally occurring acetogens of the present invention have at least one altered polynucleotide. In one nonlimiting embodiment, the alteration in the polynucleotide eliminates an activity of a polypeptide encoded by the polynucleotide. In one nonlimiting embodiment, the alteration comprises knock-out of an endogenous polynucleotide of the acetogen. By knock-out it is meant replacement or disruption of an existing gene with an artificial piece of DNA. In one nonlimiting embodiment, at least two polynucleotides of the non-naturally occurring acetogen have been altered. In one nonlimiting embodiment, at least three polynucleotides of the non-naturally occurring acetogen have been altered. In one nonlimiting embodiment, at least five polynucleotides of the non-naturally occurring acetogen have been altered.

[0058] In one nonlimiting embodiment, the non-naturally occurring acetogen capable of producing hydrocarbons from a gaseous substrate comprises either an alteration of at least one polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase or encoding a polypeptide having an activity of a lactate dehydrogenase; or an alteration of at least two polynucleotides, the first polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase and the second polynucleotide encoding a polypeptide having an activity of a lactate dehydrogenase.

[0059] In one nonlimiting embodiment, at least two polynucleotides of the non-naturally occurring acetogen have been altered. In one nonlimiting embodiment, at least one polynucleotide encoding a polypeptide having activity of a member selected from an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase and at least one polynucleotide encoding a polypeptide having activity of a member selected from an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and/or a Nfn complex have been altered.

[0060] In one nonlimiting embodiment, wherein at least three polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase. In another nonlimiting embodiment, wherein at least three polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase and/or a dihydrolipoylprotein:NAD+oxidoreductase.

[0061] In one nonlimiting embodiment wherein at least five polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, an aldehyde dehydrogenase and/or a purine nucleoside phosphorylase. In another nonlimiting embodiment wherein at least five polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and/or a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase. In yet another nonlimiting embodiment wherein at least five polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and/or a Nfn complex. In yet another nonlimiting embodiment wherein at least five polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of a member selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and/or a formate dehydrogenase.

[0062] In one nonlimiting embodiment, the non-naturally occurring acetogen is a Clostridium species. Examples of Clostridium species which can be used include, but are not limited to Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium coskatii or Clostridium ragsdalei.

[0063] Also provided by the present invention are methods for producing the above-described non-naturally occurring acetogens. While various methods known to those skilled in the art, in one nonlimiting embodiment, the non-naturally occurring acetogen is altered by a knock-out procedure such as described in the Examples herein.

[0064] The present invention also relates to compositions comprising a means for producing a hydrocarbon via a beta-ketothiolase route via pyruvate via ldh, a 2-hydroxyacyl-CoA dehydratase route via ldh, a 2-hydroxyacyl-CoA dehydratase route via mdd or a polyketide synthase route utilizing ldh. In one nonlimiting embodiment, the means is derived from or related to the non-naturally occurring acetogens disclosed herein. Nonlimiting examples include the non-naturally occurring acetogen, a cell lysate thereof or one or more polypeptides derived therefrom.

[0065] In addition, the present invention provides genetic constructs comprising at least one polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase or encoding a polypeptide having an activity of a lactate dehydrogenase, wherein said polynucleotide is altered to eliminate activity of the encoded polypeptide. In one nonlimiting embodiment, the genetic construct comprises at least two polynucleotides, wherein a first polynucleotide encodes a polypeptide having an activity of an alpha-acetolactate decarboxylase and a second polynucleotide encodes a polypeptide having an activity of a lactate dehydrogenase, wherein the polynucleotides are altered to eliminate the activity of the encoded polypeptides. In one nonlimiting embodiment, the genetic construct further comprises a polynucleotide encoding a polypeptide having an activity of one or more members selected from the group consisting of an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and a Nfn complex, wherein the polynucleotide is altered to eliminate the activity of the encoded polypeptide.

[0066] The present invention also provides methods for biosynthesising hydrocarbons from a gaseous substrate in a non-naturally occurring acetogen or other means as disclosed herein capable of producing hydrocarbons from a gaseous substrate.

[0067] In one nonlimiting embodiment, the hydrocarbon produced via the present invention comprises a saturated or unsaturated 5 carbon branched structure derived from an isoprenoid. Examples include, but are not limited to, isoprene as well as other isoprenoids, terpenes and terpenoids and derivatives such as, but not limited to isoprenols, and salts thereof.

[0068] In one nonlimiting embodiment, the hydrocarbon is isoprene produced in a non-naturally occurring acetogen via a beta-ketothiolase route via pyruvate via ldh (see FIG. 1), a 2-hydroxyacyl-CoA dehydratase route via ldh (see FIG. 2), a 2-hydroxyacyl-CoA dehydratase route via mdd (see FIG. 3) or a polyketide synthase route utilizing ldh (see FIG. 4).

[0069] In these methods of isoprene production, the non-naturally occurring acetogen has at least one altered polynucleotide. In one nonlimiting embodiment, at least two polynucleotides of the non-naturally occurring acetogen have been altered. In one nonlimiting embodiment, the non-naturally occurring acetogen has at least three altered polynucleotides. In another nonlimiting embodiment, the non-naturally occurring acetogen has at least five altered polynucleotides. In one nonlimiting embodiment, the alteration in the polynucleotide eliminates an activity of a polypeptide encoded by the polynucleotide.

[0070] In one nonlimiting embodiment, the non-naturally occurring acetogen used in this method comprises either an alteration of at least one polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase or encoding a polypeptide having an activity of a lactate dehydrogenase; or an alteration of at least two polynucleotides, the first polynucleotide encoding a polypeptide having an activity of an alpha-acetolactate decarboxylase and the second polynucleotide encoding a polypeptide having an activity of a lactate dehydrogenase.

[0071] In one nonlimiting embodiment, at least two polynucleotides of the non-naturally occurring acetogen have been altered. In one nonlimiting embodiment, at least one polynucleotide encoding a polypeptide having activity of members selected from an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase and at least one polynucleotide encoding a polypeptide having activity of members selected from an aldehyde:ferredoxin oxidoreductase, a purine nucleoside phosphorylase, a dihydrolipoylprotein:NAD+oxidoreductase, an L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase, an N2-Acetyl-L-ornithine amidohydrolase, a formate dehydrogenase and/or a Nfn complex have been altered.

[0072] In one nonlimiting embodiment, wherein the method comprises use of a non-naturally occurring acetogen with at least three altered polynucleotides, the polynucleotides may encode polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase and an aldehyde:ferredoxin oxidoreductase. In another nonlimiting embodiment, wherein the method comprises use of a non-naturally occurring acetogen with at least three altered polynucleotides, the polynucleotides may encode polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase and/or a dihydrolipoylprotein:NAD+oxidoreductase.

[0073] In one nonlimiting embodiment, wherein the method comprises use of a non-naturally occurring acetogen with at least five altered polynucleotide, the altered polynucleotides, the polynucleotides may encode polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, an aldehyde dehydrogenase and/or a purine nucleoside phosphorylase. In another nonlimiting embodiment, wherein the method comprises use of a non-naturally occurring acetogen with at least five altered polynucleotides, the polynucleotides may encode polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase. In yet another nonlimiting embodiment, wherein the method comprises use of a non-naturally occurring acetogen with at least five altered polynucleotides, the polynucleotides may encode polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and/or a Nfn complex. In yet another nonlimiting embodiment wherein at least five polynucleotides are altered, the polynucleotides may encode polypeptides having an activity of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine amidohydrolase and/or a formate dehydrogenase.

[0074] For nonlimiting embodiments of the present invention where isoprene is produced via a beta-ketothiolase route via pyruvate via ldh, nonlimiting examples of useful non-naturally occurring acetogens include those having at least one altered polynucleotide encoding a polypeptide in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase, those having at least two polynucleotides altered in polypeptides in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase and those having at least 3 or at least 5 altered polynucleotides.

[0075] For nonlimiting embodiments of the present invention where isoprene is produced via a 2-hydroxyacyl-CoA dehydratase route via ldh, nonlimiting examples of useful non-naturally occurring acetogens include those having at least one altered polynucleotide encoding a polypeptide in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase, those having at least two polynucleotides altered in polypeptides in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase and those having at least 3 or at least 5 altered polynucleotides. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least three altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase and/or a dihydrolipoylprotein:NAD+oxidoreductase altered. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least five altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a L-Aspartate ammonia-lyase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase and/or a N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase altered.

[0076] For nonlimiting embodiments of the present invention where isoprene is produced via a 2-hydroxyacyl-CoA dehydratase route via mdd, nonlimiting examples of useful non-naturally occurring acetogens include those having one altered polynucleotide encoding a polypeptide in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase, those having at least two polynucleotides altered in polypeptides in an alpha-acetolactate decarboxylase and a lactate dehydrogenase and those having at least 3 or at least 5 altered polynucleotides. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least three altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, and an aldehyde:ferredoxin oxidoreductase altered. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least five altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a pyruvate formate lyase, a glutamate synthase, an L-Threonine acetaldehyde-lyase, an 2,6-Diaminoheptanedioate: 2-oxoglutarate aminotransferase, an N2-Acetyl-L-ornithine:L-glutamate N-acetyltransferase/Acetyl-CoA:L-glutamate N-acetyltransferase and/or a Nfn complex altered.

[0077] For nonlimiting embodiments of the present invention where isoprene is produced via a polyketide synthase route utilizing ldh, nonlimiting examples of useful non-naturally occurring acetogens include those having at least one altered polynucleotide encoding a polypeptide in an alpha-acetolactate decarboxylase and/or a lactate dehydrogenase, those having at least two polynucleotides altered in polypeptides in an alpha-acetolactate decarboxylase and a lactate dehydrogenase and those having at least 3 or at least 5 altered polynucleotides. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least three altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, and an aldehyde:ferredoxin oxidoreductase altered. A nonlimiting example of a non-naturally occurring acetogen useful in this embodiment with at least five altered polynucleotides is that having polynucleotides encoding polypeptides having activities of members selected from an alpha-acetolactate decarboxylase, a lactate dehydrogenase, a 2,6-Diaminoheptanedioate:2-oxoglutarate aminotransferase, a N2-Acetyl-L-ornithine amidohydrolase and/or a formate dehydrogenase altered.

[0078] In one nonlimiting embodiment, the non-naturally occurring acetogen used in the method of the present invention is a Clostridium species. Examples of Clostridium species which can be used include, but are not limited to, Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium coskatii and Clostridium ragsdalei.

[0079] In any the methods described herein, a fermentation strategy can be used that entails anaerobic, micro-aerobic or aerobic cultivation. A fermentation strategy can entail nutrient limitation such as nitrogen, phosphate or oxygen limitation. A cell retention strategy using a ceramic hollow fiber membrane can be employed to achieve and maintain a high cell density during fermentation. The principal gaseous substrate fed to the fermentation can derive from a biological or non-biological feedstock. The biological feedstock can be, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles or municipal waste. The non-biological feedstock can be, or can derive from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue (NVR) a caustic wash waste stream from cyclohexane oxidation processes or waste stream from a chemical industry such as, but not limited to a carbon black industry or a hydrogen-refining industry, or petrochemical industry.

[0080] In one nonlimiting embodiment, at least one of the enzymatic conversions of the hydrocarbon production method comprises fermentation of the gaseous substrate within the non-naturally occurring acetogen. In this embodiment, the gaseous substrate fermentation may comprise at least one of natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue, caustic wash from cyclohexane oxidation processes, or waste stream from a chemical industry such as, but not limited to a carbon black industry or a hydrogen-refining industry, or petrochemical industry. In one nonlimiting embodiment, the gas substrate comprises a mixture of CO, CO.sub.2 and H.sub.2. In one nonlimiting embodiment, the gas substrate comprises CO.

[0081] The methods of the present invention may further comprise recovering produced hydrocarbons from the non-naturally occurring host.

[0082] Once produced, any method can be used to isolate hydrocarbons. For example, hydrocarbons can be recovered from the fermenter off-gas stream as a volatile product as the boiling point of isoprene is 34.1 C. At a typical fermentation temperature of approximately 30 C., hydrocarbons have a high vapor pressure and can be stripped by the gas flow rate through the broth for recovery from the off-gas. Hydrocarbons can be selectively adsorbed onto, for example, an adsorbent and separated from the other off-gas components. Membrane separation technology may also be employed to separate hydrocarbons from the other off-gas compounds. Hydrocarbons may be desorbed from the adsorbent using, for example, nitrogen and condensed at low temperature and high pressure.

[0083] Because of the gaseous nature of isoprene, in embodiments of the present invention wherein the hydrocarbon produced is isoprene, an advantage is easy separation of the product.

[0084] Also provided by the present invention are hydrocarbons bioderived from a non-naturally occurring acetogen or other means according to any of the methods described herein.

[0085] In addition, the present invention provides bio-derived, bio-based, or fermentation-derived product produced using the methods and/or compositions disclosed herein. Examples of such products include, but are not limited to, compositions comprising at least one bio-derived, bio-based, or fermentation-derived compound or any combination thereof, as well as polymers, rubbers such as cis-polyisoprene rubber, trans-polyisoprene rubber, or liquid polyisoprene rubber, molded substances, formulations and semi-solid or non-semi-solid streams comprising one or more of the bio-derived, bio-based, or fermentation-derived compounds or compositions, combinations or products thereof.

[0086] Although specific advantages have been enumerated above, various embodiments may include some, none, or all of the enumerated advantages. Further, other technical advantages may become readily apparent to one of ordinary skill in the art after review of the figures and description herein. It should be understood at the outset that, although exemplary embodiments are described herein, the principles of the present disclosure may be implemented using any number of techniques, whether currently known or not. The present disclosure should in no way be limited to the exemplary implementations and techniques described herein.

[0087] Modifications, additions, or omissions may be made to the compositions, systems, apparatuses, and methods described herein without departing from the scope of the disclosure. For example, the components of the systems and apparatuses may be integrated or separated. Moreover, the operations of the systems and apparatuses disclosed herein may be performed by more, fewer, or other components and the methods described may include more, fewer, or other steps. Additionally, steps may be performed in any suitable order. As used in this document, each refers to each member of a set or each member of a subset of a set.

[0088] To aid the Patent Office and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims or claim elements to invoke 35 U.S.C. 112(f) unless the words means for or step for are explicitly used in the particular claim.

[0089] The following section provides further illustration of the methods and compositions of the present invention. These working examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1: Isoprene Yields

[0090] Pathways to isoprene, e.g., a beta-ketothiolase route via pyruvate via ldh, a 2-hydroxyacyl-CoA dehydratase route via ldh, a 2-hydroxyacyl-CoA dehydratase route via mdd and a polyketide synthase route via ldh, as depicted in FIGS. 1-4 were subjected to a strain optimisation pipeline to calculate predicted yields for various genetic variants (e.g., gene knock-out) strain designs that are predicted to favour isoprene production in Clostridium.

[0091] Genome scale model (GSM) simulations and flux balance analysis (FBA) were carried out to identify strategies to increase isoprene production from CO, CO.sub.2, and H.sub.2 containing substrate in acetogens via beta-ketothiolase route via pyruvate via ldh, 2-hydroxyacyl-CoA dehydratase route via ldh and 2-hydroxyacyl-CoA dehydratase route via mdd). Genome scale models exist for a number of acetogens including Clostridium ljungdahlii (Nagarajan et al. Microb. Cell Fact. 2013 12:118 doi:10.1186/1475-2859-12-118), Clostridium autoethanogenum (Marcellin et al. Green Chem. 2016 doi:10.1039/C5GC02708J; Valgepea et al. Metab. Eng. 2017 41: 202-211. doi:10.1016/j.ymben.2017.04.007; Valgepea et al. Cell Syst. 2017 4:505-515.e5. doi:10.1016/j.cels.2017.04.008) or Moorella thermoacetica (Islam et al. Integr. Biol. 2015 doi:10.1039/C5IB00095E).

[0092] A genome-scale metabolic model of C. autoethanogenum similar to the one described by Marcellin et al. (Green Chem. 2016 doi:10.1039/C5GC02708J) was utilized. Growth was simulated by flux balance analysis (FBA), using scripts from the COBRA Toolbox v2.0 in MATLAB R2014a (The Mathworks, Inc.) with Gurobi version 6.0.4 as the solver (Gurobi Optimization, Inc.). Maximum theoretical yield of isoprene was calculated using FBA.

[0093] Strain optimizations were obtained using OptFluxlibrary running on a high performance computing cluster with IBM ILOG CPLEX (version 12.6). Ten million simulations for each pathway option were assessed using Strength Pareto Evolutionary Algorithm 2. Simulation methods used were ParsimoniousFBA (pFBA), minimization of metabolic adjustment (MOMA), LMOMA, ROOM (Pereira et al. Metab. Eng. Commun. 2016 3:153-163 doi:10.1016/j.meteno. 2016.05.002) and for each strain three classes of strain designs (1-2 gene knock-outs, 3-4 gene KO, 5-10 gene KO) were simulated. Results simulated by MOMA and FBA for the reference pathway and the beta-ketothiolase route via pyruvate via ldh, the 2-hydroxyacyl-CoA dehydratase route via ldh, the 2-hydroxyacyl-CoA dehydratase route via mdd and the polyketide synthase route via ldh are depicted in FIG. 5A and FIG. 5B, respectively.

[0094] Reactions and associated genes that are predicted to improve isoprene in acetogens, when knocked-out, are listed in Tables 1 through 12 for a representative beta-ketothiolase route via pyruvate via ldh, a 2-hydroxyacyl-CoA dehydratase route via ldh, a 2-hydroxyacyl-CoA dehydratase route via mdd pathways and a polyketide synthase route utilizing ldh. Genbank gene locus tags are provided in the tables and can be accessed via ncbi with the extension .nlm.nih.gov/gene of the world wide web.

TABLE-US-00001 TABLE 1 Reaction/gene knock-outs in acetogens to improve isoprene production via beta- ketothiolase route via pyruvate via ldh, incorporates 1-2 reaction knock-outs Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8)

TABLE-US-00002 TABLE 2 Reaction/gene knock-outs in acetogens to improve isoprene production via beta- ketothiolase route via pyruvate via ldh, incorporates 3-4 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Aldehyde: CAETHG_RS00440 CLJU_c20110 CLRAG_29650 ferredoxin (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) oxidoreductase CAETHG_RS00490 CLJU_c20210 (AOR) (SEQ ID NO: 12) (SEQ ID NO: 13)

TABLE-US-00003 TABLE 3 Reaction/gene knock-outs in acetogens to improve isoprene production via beta- ketothiolase route via pyruvate via ldh, incorporates 5-10 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdah1ii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Pyruvate CAETHG_RS08855 CLJU_c39820 CLCOS_22680 CLRAG_22070 formate lyase (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (Acetyl- CAETHG_RS03170 CLJU_c25980 CLCOS_16780 CLRAG_04120 CoA:formate C- (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) acetyltransferase) CAETHG_RS16075 CLJU_c11830 CLCOS_41080 (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) Aldehyde CAETHG_RS08810 CLJU_c39730 CLCOS_24220 CLRAG_21980 dehydrogenase (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO: 28) (CoA CAETHG_RS16140 acetylating) (SEQ ID NO: 29) CAETHG_RS08865 CLJU_c39840 (SEQ ID NO: 30) (SEQ ID NO: 31) purine CAETHG_RS00760 CLJU_c20750 CLCOS_19750 CLRAG_19250 nucleoside (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35) phosphorylase

TABLE-US-00004 TABLE 4 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via ldh, incorporates 1-2 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8)

TABLE-US-00005 TABLE 5 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via ldh, incorporates 3-4 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Pyruvate CAETHG_RS08855 CLJU_c39820 CLCOS_22680 CLRAG_22070 formate lyase (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (Acetyl- CAETHG_RS03170 CLJU_c25980 CLCOS_16780 CLRAG_04120 CoA:formate C- (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) acetyltransferase) CAETHG_RS16075 CLJU_C11830 CLCOS_41080 (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) Dihydrolipoylprotein: CAETHG_RS07795 NAD + (SEQ ID NO: 36) oxidoreductase CAETHG_RS07825 CLJU_c37600 CLCOS_09450 CLRAG_37010 (SEQ ID NO: 37) (SEQ ID NO: 38) (SEQ ID NO: 39) (SEQ ID NO: 40)

TABLE-US-00006 TABLE 6 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via ldh, incorporates 5-10 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Pyruvate CAETHG_RS08855 CLJU_c39820 CLCOS_22680 CLRAG_22070 formate lyase (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (Acetyl- CAETHG_RS03170 CLJU_c25980 CLCOS_16780 CLRAG_04120 CoA:formate C- (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) acetyltransfer CAETHG_RS16075 CLJU_C11830 CLCOS_41080 ase) (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) L-Aspartate CAETHG_RS10085 CLJU_c42370 CLCOS_38200 CLRAG_05490 ammonia- (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NOL43) (SEQ ID NO: 44) lyase/Fumarase CAETHG_RS12205 CLJU_c04170 CLCOS_14400 CLRAG_26890 (SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) 2,6- CAETHG_RS17235 CLJU_c14280 CLCOS_27270 CLRAG_09600 Diaminoheptanedioate: (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) 2-oxoglutarate aminotransferase N2-Acetyl-L- CAETHG_RS01140 CLJU_c21530 CLCOS_33330 CLRAG_31090 ornithine:L- (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56) glutamate N- acetyltransferase/ Acetyl-CoA:L- glutamate N- acetyltransferase

TABLE-US-00007 TABLE 7 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via mdd, incorporates 1-2 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8)

TABLE-US-00008 TABLE 8 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via mdd, incorporates 3-4 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Aldehyde: CAETHG_RS00440 CLJU_c20110 CLRAG_29650 ferredoxin (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) oxidoreductase CAETHG_RS00490 CLJU_c20210 (AOR) (SEQ ID NO: 12) (SEQ ID NO: 13)

TABLE-US-00009 TABLE 9 Reaction/gene knock-outs in acetogens to improve isoprene production via 2-hydroxyacyl- CoA dehydratase route via mdd, incorporates 5-10 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Pyruvate CAETHG_RS08855 CLJU_c39820 CLCOS_22680 CLRAG_22070 formate lyase (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (Acetyl- CAETHG_RS03170 CLJU_c25980 CLCOS_16780 CLRAG_04120 CoA:formate C- (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) acetyltransferase) CAETHG_RS16075 CLJU_c11830 CLCOS_41080 (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) Glutamate CAETHG_ RS18885 CLJU_c17370 CLCOS_13150 CLRAG_29210 synthase (SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59) (SEQ ID NO: 60) CAETHG_RS18890 CLJU_c17380 CLCOS_13160 CLRAG_29200 (SEQ ID NO: 61) (SEQ ID NO: 62) (SEQ ID NO: 63) (SEQ ID NO: 64) CAETHG_RS02275 CLJU_c24190 CLCOS_32470 CLRAG_24880 (SEQ ID NO: 65) (SEQ ID NO: 66) (SEQ ID NO: 67) (SEQ ID NO: 68) L-Threonine CAETHG_RS03265 CLJU_c26170 CLCOS_16980 CLRAG_04250 acetaldehyde- (SEQ ID NO: 69) (SEQ ID NO: 70) (SEQ ID NO: 71) (SEQ ID NO: 72) lyase 2,6- CAETHG_RS17235 CLJU_c14280 CLCOS_27270 CLRAG_09600 Diaminoheptanedioate: (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) 2-oxoglutarate aminotransferase N2-Acetyl-L- CAETHG_RS01140 CLJU_c21530 CLCOS_33330 CLRAG_31090 ornithine:L- (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55) (SEQ ID NO: 56) glutamate N- acetyltransfer ase/Acetyl- CoA:L- glutamate N- acetyltransferase Nfn complex CAETHG_RS07665 CLJU_c37240 CLCOS_09810 CLRAG_36680 (SEQ ID NO: 73) (SEQ ID NO: 74) (SEQ ID NO: 75) (SEQ ID NO: 76)

TABLE-US-00010 TABLE 10 Reaction/gene knock-outs in acetogens to improve isoprene production via polyketide synthase route, incorporates 1-2 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdah1ii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8)

TABLE-US-00011 TABLE 11 Reaction/gene knock-outs in acetogens to improve isoprene production via polyketide synthase route, incorporates 3-4 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Aldehyde: CAETHG_RS00440 CLJU_c20110 CLRAG_29650 ferredoxin (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) oxidoreductase CAETHG_RS00490 CLJU_c20210 (AOR) (SEQ ID NO: 12) (SEQ ID NO: 13)

TABLE-US-00012 TABLE 12 Reaction/gene knock-outs in acetogens to improve isoprene production via polyketide synthase route, incorporates 5-10 reaction knock-outs. Gene in C. Gene in C. Gene in C. Gene in C. Reaction autoethanogenum ljungdahlii coskatii ragsdalei Alpha- CAETHG_RS14410 CLJU_c08380 CLCOS_42470 CLRAG_08070 acetolactate (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) decarboxylase Lactate CAETHG_RS05500 CLJU_c32190 CLCOS_24090 CLRAG_02820 dehydrogenase (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) 2,6- CAETHG_RS17235 CLJU_c14280 CLCOS_27270 CLRAG_09600 Diaminoheptanedioate: (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) 2-oxoglutarate aminotransferase N2-Acetyl-L- CAETHG_RS04740 CLJU_c29950 CLRAG_35950 ornithine (SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) amidohydrolase CAETHG_RS02125 CLJU_c23810 CLCOS_28660 CLRAG_17360 (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) (SEQ ID NO: 83) Formate CAETHG_RS14690 CLJU_c20040 CLCOS_13030 CLRAG_29330 dehydrogenase (SEQ ID NO: 84) (SEQ ID NO: 85) (SEQ ID NO: 86) (SEQ ID NO: 87) CAETHG_RS13725 CLJU_c08930 CLCOS_19340 CLRAG_18840 (SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91)
FIG. 6 shows predicted yield of isoprene in Clostridium autoethanogenum in strains genetically engineered with different numbers of knock-outs (2 KO, 3-4 KO and 5-10 KO). It can be seen that the 5-10 knockout strains give the highest predicted yield of isoprene.