Methods of producing 6-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage

1. A method for biosynthesizing one or more products selected from the group consisting of adipic acid, 6-aminohexanoic acid, hexamethylenediamine, caprolactam, and 1,6-hexanediol, said method comprising enzymatically synthesizing hexanoyl-CoA and enzymatically forming, in one or more steps, two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in hexanoyl-CoA to produce the one or more products.

2. The method of claim 1 , wherein said two terminal functional groups are the same.

3. The method of claim 1 , wherein said two terminal functional groups are different.

4. The method of claim 1 , wherein hexanoyl-CoA is enzymatically synthesized from acetyl-CoA via two cycles of CoA-dependent carbon chain elongation using either NADH or NADPH dependent enzymes.

5. The method of claim 1 , comprising forming a first terminal carboxyl group in hexanoyl CoA using a thioesterase to produce hexanoate.

6. The method of claim 5 , wherein said thioesterase is encoded by YciA, tesB or Acot13.

7. The method of claim 1 , comprising converting hexanoyl-CoA to hexanal by a butanal dehydrogenase classified under EC 1.2.1.57 before forming a first terminal carboxyl group in hexanal.

8. The method of claim 7 , comprising producing hexanoate by forming a first terminal carboxyl group in hexanal by an aldehyde dehydrogenase classified under EC 1.2.1.4.

9. The method of claim 5 , further comprising converting hexanoate to adipic acid, 6-aminohexanoic acid, hexamethylenediamine, εcaprolactam or 1,6 hexanediol with one or more enzymatic conversions, wherein one of said enzymatic conversions introduces the second terminal functional group.

10. The method of claim 9 , comprising converting hexanoate to 6-hydroxyhexanoate using a cytochrome P450/ω-hydroxylase.

11. The method of claim 10 , wherein said cytochrome P450/ω-hydroxylase is from the family CYP153 such as CYP153A6.

12. The method of claim 10 , comprising converting 6-hydroxyhexanoate to adipate semialdehyde using (i) an alcohol dehydrogenase, (ii) a 6-hydroxyhexanoate dehydrogenase or (iii) a cytochrome P450/ω-hydroxylase.

13. The method of claim 12 , wherein said alcohol dehydrogenase is encoded by YMR318C.

14. The method of claim 12 , wherein said 6-hydroxyhexanoate dehydrogenase is encoded by ChnD.

15. The method of claim 12 , comprising producing adipic acid by forming the second terminal functional group in adipate semialdehyde using (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, (ii) a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63 or (iii) a cytochrome P450/ω-hydroxylase.

16. The method of claim 15 , wherein said 6-oxohexanoate dehydrogenase is encoded by ChnE.

17. The method of claim 9 , comprising producing 6-aminohexanoic acid by forming the second terminal functional group in adipate semialdehyde using an ω-transaminase classified under EC 2.61.18, EC 2.6.1.19 or EC 2.6.1.48.

18. The method of claim 1 , comprising producing ε-caprolactam from 6-aminohexanoic acid using a lactamase classified under EC 3.5.2.

19. The method of claim 18 , comprising converting 6-aminohexanoic acid to 6-aminohexanal using a carboxylate reductase classified under EC 1.2.99.6.

20. The method of claim 1 , comprising producing hexamethylenediamine by forming a second terminal functional group in 6-aminohexanal using a diamine transaminase classified under EC 2.6.1.29 or EC 2.6.1.82.

21. The method of claim 19 , wherein said carboxylate reductase is encoded by car alongside the gene product of npt.

22. The method of claim 19 , wherein said carboxylate reductase is encoded by GriC & GriD.

23. The method of claim 1 , comprising producing 1,6 hexanediol by forming the second terminal functional group in 6-hydroxyhexanal using an alcohol dehydrogenase classified under EC 1.1.1.-(1,2,21,184).

24. The method of claim 1 , wherein said method is performed in a recombinant host.

25. The method of claim 24 , wherein said host is cultured under anaerobic, aerobic or micro-aerobic cultivation conditions.

26. The method of claim 24 , wherein said host is subjected to a cyclical cultivation strategy to alternate said host between anaerobic cultivation conditions and aerobic cultivation conditions.

27. The method of claim 24 , wherein said host is cultured under conditions of nutrient limitation either via nitrogen, phosphate or oxygen limitation.

28. The method of claim 24 , wherein the C6 building block is produced by a single microorganism strain via a non-cyclical or cyclical fermentation strategy.

29. The method of claim 24 , wherein the C6 building block is produced by co-culturing two or more microorganism strains via a non-cyclical or cyclical fermentation strategy.

30. The method of claim 24 , wherein the C6 building block is produced by successive fermentations, wherein the broth or centrate from the preceding fermentation is fed to a succession of fermentations as a source of feedstock, central metabolite, or central precursor; finally producing the C6 building block.

31. The method of claim 24 , wherein said recombinant host cells are retained in ceramic hollow fiber membranes to maintain a high cell density during fermentation.

32. The method of claim 25 , wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

33. The method of claim 25 , wherein the host is a prokaryote or a eukaryote.

34. The method of claim 33 , wherein the host is a prokaryote from the genus Escherichia such as Escherichia coli ; from the genus Clostridia such as Clostridium ljungdahlii , Clostridium autoethanogenum or Clostridium kluyveri ; from the genus Corynebacteria such as Corynebacterium glutamicum ; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans ; from the genus Pseudomonas such as Pseudomonas fluorescens , Pseudomonas putida or Pseudomonas oleavorans ; from the genus Delftia such as Delftia acidovorans ; from the genus Bacillus such as Bacillus subtillis ; from the genus Lactobacillus such as Lactobacillus delbrueckii ; or from the genus Lactococcus such as Lactococcus lactis.

35. The method of claim 33 , wherein the host is a eukaryote from the genus Aspergillus such as Aspergillus niger ; from the genus Saccharomyces such as Saccharomyces cerevisiae ; from the genus Pichia such as Pichia pastoris ; from the genus Yarrowia such as Yarrowia lipolytica ; from the genus Issatchenkia such as Issathenkia orientalis ; from the genus Debaryomyces such as Debaryomyces hansenii ; from the genus Arxula such as Arxula adenoinivorans ; or from the genus Kluyveromyces such as Kluyveromyces lactis.

36. The method of claim 33 , wherein the host's tolerance to high concentrations of a C6 building block is improved through continuous cultivation in a selective environment.

37. The method of claim 33 , wherein the host naturally accumulates polyhydroxyalkanoates, and wherein the polymer synthase enzymes are attenuated in the host strain.

38. The method of claim 27 , wherein the intracellular concentration of acetyl-CoA for biosynthesis of C6 building blocks is increased in the host by attenuating genes degrading acetyl-CoA.

39. The method of claim 24 , wherein an imbalance in NADH is generated that can only be balanced via the formation of a C6 building block.

40. The method of claim 39 , wherein a gene encoding a formate dehydrogenase is overexpressed, a gene encoding a pyruvate formate lyase is overexpressed, 2-hydroxybutyrate oxidoreductase activity is attenuated, DL glycerol-3-phosphatase activity is attenuated, and/or malate dehydrogenase activity is attenuated.

41. The method of claim 39 , wherein carbon flux is directed into the pentose phosphate cycle in the host by attenuating a glucose-6-phosphate isomerase classified under EC 5.3.1.9.

42. The method of claim 38 , wherein a gene encoding an acetate kinase, such as ack, a gene encoding an enzyme that mediates the degradation of pyruvate to lactate such as ldhA, genes encoding enzymes mediating the degradation of phophoenolpyruvate to succinate such as frdBC, a gene encoding an enzyme mediating the degradation of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded by adhE, a gene encoding an enzyme mediating the degradation of pyruvate to ethanol such as pyruvate decarboxylase, or a gene encoding an enzyme mediating the generation of isobutanol such as a 2-oxoacid decarboxylase is attenuated in the host.

43. The method of claim 24 , wherein the intracellular concentration of acetyl-CoA for biosynthesis of a C6 building block is increased in the host by overexpressing genes forming acetyl-CoA.

44. The method of claim 43 , wherein an acetyl-CoA synthetase such as the gene product of acs is overexpressed in the host.

45. The method of claim 24 , wherein an imbalance in NADPH is generated in the host that can only be balanced via the formation of a C6 building block.

46. The method of claim 45 , wherein a puridine nucleotide transhydrogenase gene such as UdhA or a glucose dehydrogenase such as the gene product of gdh is overexpressed in the host.

47. The method of claim 45 , wherein a glyceraldehyde-3P-dehydrogenase gene such as GapN is overexpressed in the host, a malic enzyme gene such as maeA or maeB is overexpressed in the host, a glucose-6-phosphate dehydrogenase gene such as zwf is overexpressed in the host, a fructose 1,6 diphosphatase gene such as fbp is overexpressed in the host, activity of a triose phosphate isomerase is attenuated in the host, and/or activity of malate dehydrogenase is attenuated.

48. The method of claim 45 , wherein membrane-bound cytochrome P450/ω-hydroxylases are solubilized in the host via truncation of the N-terminal region that anchors the P450 to the endoplasmic reticulum.

49. The method of claim 45 , wherein enzymes facilitating the conversion of NADPH to NADH are attenuated in the host or transport of hexanoate to the extracellular media is attenuated.

50. The method of claim 45 , wherein the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases in EC 1.4.1.2(NADH-specific) and EC 1.4.1.4(NADPH-specific) is attenuated in the host.

51. The method of claim 45 , wherein a glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors is attenuated in the host.

52. The method of claim 24 , wherein the degradation of central metabolites leading to the formation of the hexanoyl-CoA is attenuated in the host.

53. The method of claim 52 , wherein a propionyl-CoA synthetase such as the gene product of PrpE-RS is overexpressed in the host.

54. The method of claim 52 , wherein β-oxidation enzymes degrading central metabolites and central precursors leading to and including C6 building blocks are attenuated in the host.

55. The method of claim 52 , wherein enzymes activating C6 building blocks via Coenzyme A esterification such as CoA-ligases are attenuated.

56. The method of claim 24 , wherein the efflux of a C6 building block across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C6 building block.