Compositions and methods for biological production of fatty acid derivatives
The present disclosure provides compositions and methods for biologically producing fatty acid derivatives, such as fatty alcohols, from recombinant C 1 metabolizing microorganisms that utilize C 1 substrates such as methane or natural gas as a feedstock.
1. A alpha-proteobacterial methanotroph, comprising a heterologous nucleic acid molecule encoding a fatty acid converting enzyme, wherein the alpha-proteobacterial methanotroph comprising the heterologous nucleic acid molecule encoding the fatty acid converting enzyme is capable of converting a C 1 substrate into a C 8 -C 24 fatty aldehyde, fatty alcohol, fatty ester wax, a hydroxy fatty acid, dicarboxylic acid, or a combination thereof, and wherein the encoded fatty acid converting enzyme comprises:
(a) a fatty acyl-CoA reductase capable of forming a fatty alcohol; or
(b) a fatty acyl-CoA reductase capable of forming a fatty aldehyde; or
(c) a carboxylic acid reductase; and
(d) a thioesterase; and/or
(e) an acyl-CoA synthetase.
2. The alpha-proteobacterial methanotroph according to claim 1 , wherein the host alpha-proteobacterial methanotroph is selected from Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylobacterium organophilum, Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocella daltona, Methylocystis bryophila, Methylocapsa aurea , or high growth variants thereof.
3. The alpha-proteobacterial methanotroph according to claim 1 , wherein the C 1 substrate is methane, natural gas, or unconventional natural gas.
4. The alpha-proteobacterial methanotroph according to claim 1 , wherein:
(a) the fatty acyl-CoA reductase capable of forming a fatty alcohol is FAR, CER4, or Maqu_2220; or
(b) the fatty acyl-CoA reductase capable of forming a fatty aldehyde is acr1.
5. The alpha-proteobacterial methanotroph according to claim 4 , wherein:
(a) the thioesterase is a tesA lacking a signal peptide, UcFatB or BTE; and/or
(b) the acyl-CoA synthetase is a FadD, yng1, or FAA2.
6. The alpha-proteobacterial methanotroph according to claim 5 , wherein:
(a) endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity; and/or
(b) endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.
7. The alpha-proteobacterial methanotroph according to claim 5 , wherein the alpha-proteobacterial methanotroph further comprises a heterologous nucleic acid molecule encoding a P450 enzyme or monoxygenase enzyme to produce ω-hydroxy fatty acid.
8. The alpha-proteobacterial methanotroph according to claim 7 , wherein endogenous alcohol dehydrogenase activity is inhibited as compared to unaltered endogenous alcohol dehydrogenase activity.
9. The alpha-proteobacterial methanotroph according to claim 5 , wherein endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.
10. The alpha-proteobacterial methanotroph according to claim 5 , wherein the alpha-proteobacterial methanotroph produces fatty alcohol comprising:
(a) one or more of C 8 -C 14 or C 10 -C 16 or C 14 -C 24 fatty alcohols;
(b) C 10 to C 18 fatty alcohol and the C 10 to C 18 fatty alcohols comprise at least 70% of the total fatty alcohol; or
(c) a branched chain fatty alcohol.
11. The alpha-proteobacterial methanotroph according to claim 1 , comprising:
(a) a heterologous nucleic acid molecule encoding an acyl-CoA independent fatty acyl-CoA reductase, and a heterologous nucleic acid molecule encoding a thioesterase, wherein the methanotroph is capable of converting a C 1 substrate into a C 8 -C 24 fatty alcohol;
(b) a heterologous nucleic acid molecule encoding an acyl-CoA dependent fatty acyl-CoA reductase, a heterologous nucleic acid molecule encoding a thioesterase, and a heterologous nucleic acid molecule encoding an acyl-CoA synthetase, wherein the methanotroph is capable of converting a C 1 substrate into a C 8 -C 24 fatty alcohol;
(c) a heterologous nucleic acid molecule encoding a carboxylic acid reductase, a heterologous nucleic acid molecule encoding a phosphopantetheinyl tranferase, and a heterologous nucleic acid molecule encoding an alcohol dehydrogenase, wherein the methanotroph is capable of converting a C 1 substrate into a C 8 -C 24 fatty alcohol;
(d) a heterologous nucleic acid molecule encoding a fatty acyl-CoA reductase, a heterologous nucleic acid molecule encoding a thioesterase, and a heterologous nucleic acid molecule encoding a P450 or monooxygenase, wherein the native alcohol dehydrogenase is inhibited, and wherein the methanotroph is capable of converting a C 1 substrate into a C 8 -C 24 ω-hydroxy fatty acid; or
(e) a heterologous nucleic acid molecule encoding a fatty acyl-CoA reductase, and a heterologous nucleic acid molecule encoding a thioesterase, wherein the methanotroph over-expresses native alcohol dehydrogenase as compared to the normal expression level of native alcohol dehydrogenase or comprises a heterologous nucleic acid molecule encoding an alcohol dehydrogenase or both, and wherein the methanotroph is capable of converting a C 1 substrate into a C 8 -C 24 dicarboxylic acid alcohol.
12. The alpha-proteobacterial methanotroph according to claim 11 , wherein the alpha-proteobacterial methanotroph is selected from Methylosinus trichosporium OB3b, Methylosinus sporium, Methylocystis parvus , or a high growth variant thereof.
13. A method for making a fatty acid derivative, comprising culturing a alpha-proteobacterial methanotroph with a C 1 substrate feedstock and recovering the fatty acid derivative,
wherein the alpha-proteobacterial methanotroph comprises a heterologous nucleic acid molecule encoding a fatty acid converting enzyme,
wherein the alpha-proteobacterial methanotroph converts the C 1 substrate into a C 8 -C 24 fatty acid derivative comprising a fatty aldehyde, a fatty alcohol, fatty ester wax, a hydroxy fatty acid, a dicarboxylic acid, or a combination thereof, and
wherein the encoded fatty acid converting enzyme comprises:
(a) a fatty acyl-CoA reductase capable of forming a fatty alcohol; or
(b) a fatty acyl-CoA reductase capable of forming a fatty aldehyde; or
(c) a carboxylic acid reductase; and
(d) a thioesterase; and/or
(e) an acyl-CoA synthetase.
14. The method according to claim 13 , wherein the alpha-proteobacterial methanotroph is selected from a Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylobacterium organophilum, Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocella daltona, Methylocystis bryophila, Methylocapsa aurea , or high growth variants thereof.
15. The method according to claim 13 , wherein the culture further comprises a heterologous bacterium.
16. The method according to claim 13 , wherein:
(a) the fatty acyl-CoA reductase capable of forming a fatty alcohol is FAR, CER4, or Maqu_2220; or
(b) the fatty acyl-CoA reductase capable of forming a fatty aldehyde is acr1.
17. The method according to claim 13 , wherein the thioesterase is a tesA lacking a signal peptide, UcFatB or BTE.
18. The method according to claim 17 , wherein endogenous thioesterase activity is reduced, minimal or abolished as compared to unaltered endogenous thioesterase activity.
19. The method according to claim 16 , wherein the acyl-CoA synthetase is FadD, yng1, or FAA2.
20. The method according to claim 16 , wherein endogenous acyl-CoA synthetase activity is reduced, minimal or abolished as compared to unaltered endogenous acyl-CoA synthetase activity.
21. The method according to claim 13 , further comprising a heterologous nucleic acid molecule encoding a P450 enzyme or monoxygenase enzyme to produce w-hydroxy fatty acid.
22. The method according to claim 21 , wherein endogenous alcohol dehydrogenase activity is reduced, minimal or abolished as compared to unaltered endogenous alcohol dehydrogenase activity.
23. The method according to claim 13 , wherein endogenous alcohol dehydrogenase activity is increased or elevated as compared to unaltered endogenous alcohol dehydrogenase activity to produce dicarboxylic acid.
24. The method according to claim 13 , wherein the alpha-proteobacterial methanotroph produces fatty alcohol comprising one or more of C 8 -C 14 or C 10 -C 16 or C 12 -C 14 or C 14 -C 18 or C 14 -C 24 fatty alcohols.
25. The method according to claim 13 , wherein the alpha-proteobacterial methanotroph produces fatty alcohol comprising C 10 to C 18 fatty alcohol and the C 10 to C 18 fatty alcohols comprise at least 70% of the total fatty alcohol.
26. The method according to claim 13 , wherein the alpha-proteobacterial methanotroph produces fatty alcohols comprising a branched chain fatty alcohol.
27. The method according to claim 13 , wherein the C 1 substrate is methane, natural gas, or unconventional natural gas.
28. The method according to claim 13 , wherein the C 1 substrate is methane, and the alpha-proteobacterial methanotrophs are cultured under aerobic conditions.
29. The method according to claim 13 , wherein the culturing is in a fermentor or bioreactor.
30. An aerobic, facultative methanotrophic bacteria, comprising a heterologous nucleic acid molecule encoding a fatty acid converting enzyme, wherein the facultative methanotrophic bacteria comprising the heterologous nucleic acid molecule encoding the fatty acid converting enzyme is capable of converting a C 1 substrate under aerobic conditions into a C 8 -C 24 fatty aldehyde, fatty alcohol, fatty ester wax, a hydroxy fatty acid, dicarboxylic acid, or a combination thereof, and wherein the encoded fatty acid converting enzyme comprises:
(a) a fatty acyl-CoA reductase capable of forming a fatty alcohol; or
(b) a fatty acyl-CoA reductase capable of forming a fatty aldehyde; or
(c) a carboxylic acid reductase; and
(d) a thioesterase; and/or
(e) an acyl-CoA synthetase.
31. The facultative methanotrophic bacteria according to claim 30 , wherein the host facultative methanotrophic bacteria is selected from Methylobacterium organophilum, Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocella daltona, Methylocystis bryophila, Methylocapsa aurea , or high growth variants thereof.
32. A methylotrophic bacteria, comprising a heterologous nucleic acid molecule encoding a fatty acid converting enzyme, wherein the methylotrophic bacteria comprising the heterologous nucleic acid molecule encoding the fatty acid converting enzyme is capable of converting a C 1 substrate under aerobic conditions into a C 8 -C 24 fatty aldehyde, fatty alcohol, fatty ester wax, a hydroxy fatty acid, dicarboxylic acid, or a combination thereof, and wherein the encoded fatty acid converting enzyme comprises:
(a) a fatty acyl-CoA reductase capable of forming a fatty alcohol; or
(b) a fatty acyl-CoA reductase capable of forming a fatty aldehyde; or
(c) a carboxylic acid reductase; and
(d) a thioesterase; and/or
(e) an acyl-CoA synthetase.
33. The methylotrophic bacteria according to claim 32 , wherein the host methylotrophic bacteria is selected from Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans , or high growth variants thereof.