Production of c6-c26 fatty acids from methanol

Abstract

The present invention provides a microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR). The present invention further provides methods for the manufacture of at least one C6-C26 free fatty acids or mixtures thereof and C6-C26 free fatty alcohols or mixtures thereof.

Description

[0001] Production of C6-C26 Faty Acids from Methanol

[0002] Technical field of the invention

[0003] The present invention relates to the field of biotechnology, microbiology, food ingredients, cosmetics ingredient and fatty acids. In particular the present invention relates to a microorganism capable of producing at least one C6-C26 free fatty acid from methanol.

[0004] Background

[0005] Fatty acids are carboxylic acids with aliphatic chains. Based on the number of carbon atoms in their molecules, they are divided into long-chain fatty acids (more than 12 C-atoms), medium-chain fatty acids (between 12 and 6 C-atoms) and short chain fatty acids (under 6 C-atoms). The length of the aliphatic chain and presence, location or absence of double bonds within the chain determines the physical properties and potential uses of the acid. Saturated fatty acids of lengths 12, 14 and 16 are commonly used in the chemical industry for detergents, cosmetics, food and various other market segments. Currently, they are primarily produced from palm kernel oil, the production of which has well known sustainability and environmental issues. Key market players are in search of alternative sources of this 70 billion USD annual market feedstock. Microbial production of fatty acids is a promising alternative pathway. Fatty acid synthesis in microbes is carried out by the FAS (fatty acid synthesis) pathway. The pathway has been well described in various model organisms such as Escherichia coli. In brief, the first committed step (point of no return) of fatty acid synthesis is the carboxylation of Acetyl-CoA by the enzyme Acetyl-CoA carboxylase (ACC), which yields Malonyl-CoA. Malonyl- CoA is the basic building block of fatty acid synthesis. It is first loaded onto an acyl carrier protein (AGP), releasing the CoA moiety. Synthesis is then initiated by condensation of the malonyl-ACP with acetyl-CoA, forming acetoacetyl-ACP, which serves as the foundation for a new fatty acid. The acetoacetyl-ACP enters the FAS cycle, and an additional malonyl-ACP is added in each cycle, increasing the chain length by 2 carbon atoms. The chain remains bound to the acyl carrier protein this entire time. Once it achieves the required length, the fatty acid is removed from the AGP, usually by a thioesterase enzyme, though enzymes of the FAS pathway itself can also carry out this function. Most microbes naturally produce primarily C16 and C18 fatty acids with their native biosynthetic pathways. However, they can be engineered to produce shorter fatty acids by introduction of additional enzymes from other organisms. Since microbial production of fatty acids typically uses plant sugar as a feedstock, it is not inherently more efficient or environmentally friendly than using fatty acid or alcohols derived from oil crops such as oil palm, sunflowers, soy, or rapeseed.

[0006] Thus, there is a need for sustainable and alternative sources of C6-C26 free fatty acids for the detergent, food and cosmetics industry. There is also a need for alternative sources of C6-C26 free fatty alcohols. Also, there is a need for such alternative sources which can be exploited in an environmental sound way, e.g. not requiring vast areas of agricultural land and sugars as raw materials.

[0007] In particular there is a need for a source of C6-C26 free fatty acids, such as C12, C14, or C16 free fatty acids, or a mixture thereof, as well as the corresponding free fatty alcohols being amenable for effective production on a large scale having acceptable environmental effects and climate footprint. Summary of the invention

[0008] The present inventors have developed recombinant Methylobacillus bacteria expressing a thioesterase and a fatty alcohol reductase which surprisingly enable the industrial production of C12, C14 and C16 free fatty acids and mixtures thereof from methanol.

[0009] Expressing a thioesterase in microorganisms may lead to the secretion of C12 and other free fatty acids. Expressing a thioesterase from Umbellularia californica (Uca_TE) in a methylotrophic host from the genus Methylobacillus leads to very high secretion of C12 free fatty acids. Fatty alcohol reductase (FAR) is a bifunctional enzyme that converts ACP-bound fatty acids into fatty alcohols. Surprisingly, when the Uca_TE enzyme was expressed in the host cell in combination with a FAR enzyme, this led to a surprising increase in C12 free fatty acid production. When expressed on its own, the FAR enzyme from Marinobacter hydrocarbonoclasticus (Mar-FAR) leads to moderate production of C16 and C14 fatty alcohols, but no C12 fatty acids. Expression of the FAR enzyme from Ostrinia nubilialis (Far_0nu_5) leads to modest production of C12 and C14 fatty alcohols, but again no free fatty acids. However, when expressed alongside Uca_TE, the FAR enzymes surprisingly lead to a 50-100% increase in C12 free fatty acid production, alongside the expected fatty alcohol production (Fig 4).

[0010] Methanol is a promising third generation feedstock that can be used for biotechnological production of various compounds. It can be produced from CO2 directly, bypassing agricultural crops and drastically decreasing the carbon footprint and land usage.

[0011] In a first aspect the present invention provides a microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR). The microorganism may be a recombinant microorganism. In various embodiments, the at least one gene enabling the expression of a thioesterase (TE) and / or the at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR) is heterologously expressed in said microorganism.

[0012] In various embodiments, said TE is from Umbellularia californica (Uca-TE). Said TE may, in various embodiments, be a polypeptide that has at least 80% sequence identity to SEQ ID NO:39, at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39. In various embodiments, said TE or Uca-TE comprises, consists essentially of or consists of SEQ ID NO:39.

[0013] In various embodiments, said TE is a truncated TE from Umbellularia californica (Uca-TE). Said TE may, in various embodiments, be a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to sequence identity to any one of SEQ ID NOs:61-73. In various embodiments, said TE or Uca-TE comprises, consists essentially of or consists of any one of SEQ ID NOs:61-73. In various embodiments, the at least one FAR is Marinobacter hydrocarbonoclasticus FAR (Mar-FAR) or Ostrinia nubiliatis FAR (Far_0nu_5).

[0014] In various embodiments, the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO:2, at least 85% sequence identity to SEQ ID NO:2, at least 90% sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2.

[0015] In various embodiments, the FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 14, 15, 16, 17, 18, and 19, at least 85% sequence identity to any one of SEQ ID NOs: 14-19, at least 90% sequence identity to any one of SEQ ID NOs: 14-19, at least 95% sequence identity to any one of SEQ ID NOs: 14-19, or at least 98% sequence identity to any one of SEQ ID NOs: 14-19.

[0016] In various embodiments, (1) said TE is a polypeptide that has at least 80% sequence identity to SEQ ID NO:39, at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39; and (2) said FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 85% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 90% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 95% sequence identity to any one of SEQ ID NOs:2 and 14-19, or at least 98% sequence identity to any one of SEQ ID NOs:2 and 14-19.

[0017] In various embodiments, (1) said TE is a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to sequence identity to any one of SEQ ID NOs:62-73; and (2) said FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 85% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 90% sequence identity to any one of SEQ ID NOs:2 and 14- 19, at least 95% sequence identity to any one of SEQ ID NOs:2 and 14-19, or at least 98% sequence identity to any one of SEQ ID NOs:2 and 14-19.

[0018] In various embodiments, said at least one C6-C26 free fatty acid or mixture thereof comprises at least one C12, C14 or C16 free fatty acid. In various embodiments, said at least one C6-C26 free fatty acid or a mixture thereof comprises or is dodecanoic acid (lauric acid). In various embodiments, said at least one C6-C26 free fatty acid or a mixture thereof comprises 1 -tetradecanoic acid (myristic acid).

[0019] In various embodiments, said microorganism is a bacterium, in particular Methylobacillus, in particular Methylobacillus flagellatus or Methylobacillus glycogenes.

[0020] In various embodiments, In a second aspect the present invention provides a method for the manufacture of at least one C6-C26 free fatty acid or a mixture thereof comprising growing a microorganism as defined herein, for example as defined in any one of items 1-57 below, in a suitable medium, and isolation of said at least one C6-C26 free fatty acid or a mixture thereof from the medium, such as the spent medium. In a third aspect the invention provides a free fatty acid composition obtained by the method for producing at least one C6-C26 free fatty acid or a mixture thereof.

[0021] In a fourth aspect the present invention provides a method for the manufacture of at least one C6-C26 free fatty alcohol comprising growing a microorganism as defined herein, for example as defined in any one of items 1 to 57 below, in a suitable medium, optionally isolating at least one C6-C26 free fatty acid from the medium, such as the spent medium, conversion of the C6-C26 free fatty acid into the corresponding C6-C26 free fatty alcohol, and isolation of said C6-C26 free fatty alcohol.

[0022] In a fifth aspect the invention provides a free fatty alcohol composition obtained by the method for producing at least one C6-C26 free fatty alcohol.

[0023] In a sixth aspect the present invention provides the use of a microorganism as defined herein for the manufacture of a C6-C26 free fatty acid or a mixture thereof, or a C6-C26 free fatty alcohol or a mixture thereof.

[0024] The present invention is further characterized by the following items:

[0025] 1. Microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR).

[0026] 2. The microorganism according to item 1 , wherein

[0027] (1) said microorganism is a recombinant microorganism;

[0028] (2) the at least one gene enabling the expression of a thioesterase (TE) and / or the at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR) is heterologously expressed in said microorganism; and / or

[0029] (3) said TE is from Umbellularia californica (Uca-TE).

[0030] 3. The microorganism according to item 1 or 2, wherein said TE or said Uca-TE

[0031] (1) has at least 80% sequence identity to SEQ ID NO:39, at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39; or

[0032] (2) has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to sequence identity to any one of SEQ ID NOs:62-73.

[0033] 4. The microorganism according to any one of items 1 to 3, wherein said TE or Uca-TE has

[0034] (1) at least 50% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 70% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 80% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 90% thioesterase activity as compared to the activity of SEQ ID NO:39, or at least 95% thioesterase activity as compared to the activity of SEQ ID NO:39; or

[0035] (2) at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% thioesterase activity as compared to the activity of any one of SEQ ID NOs:62-73.

[0036] 5. The microorganism according to any of the preceding items, wherein said TE or Uca-TE is a truncated version of SEQ ID NO: 39 having at least 85% sequence identity to the corresponding sequence of SEQ ID NO:39, at least 90% sequence identity to the corresponding sequence of SEQ ID NO:39, at least 95% sequence identity to the corresponding sequence of SEQ ID NO:39, wherein said truncated version optionally has at least 50% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 70% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 80% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 90% thioesterase activity as compared to the activity of SEQ ID NO:39, or at least 95% thioesterase activity as compared to the activity of SEQ ID NO:39 .

[0037] 6. The microorganism according to item 5, wherein said TE or Uca-TE is a truncated version of SEQ ID NO: 39, wherein said truncated version optionally has at least 50% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 70% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 80% thioesterase activity as compared to the activity of SEQ ID NO:39, at least 90% thioesterase activity as compared to the activity of SEQ ID NO:39, or at least 95% thioesterase activity as compared to the activity of SEQ ID NO:39.

[0038] 7. The microorganism according to item 6, wherein said TE or Uca-TE is SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, or SEQ ID NO:73.

[0039] 8. The microorganism according to any of the preceding items, wherein said TE or Uca-TE is SEQ ID NO:39 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, additions or deletions and retaining thioesterase activity.

[0040] 9. The microorganism according to any of the preceding items, wherein said TE or Uca-TE comprises, consists essentially of or consists of SEQ ID NO:39, SEQ ID NO:61 SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, or SEQ ID NO:73.

[0041] 10. The microorganism according to any of the preceding items, wherein said TE or Uca-TE is SEQ ID NO:39, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, or SEQ ID NO:73.

[0042] 11 . The microorganism according to any of the proceeding items, wherein said at least one FAR is Marinobacter hydrocarbonoclasticus FAR (Mar-FAR) or Ostrinia nubiliatis FAR (Far_Onu_5). 12. The microorganism according to item 11, wherein the FAR is a Mar-FAR having at least 80% sequence identity to SEQ ID NO:2, at least 85% sequence identity to SEQ ID NO:2, at least 90% sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2.

[0043] 13. The microorganism according to item 11 or 12, wherein the FAR is Mar-FAR.

[0044] 14. The microorganism according to any one of the preceding items, wherein the FAR is a Mar-FAR comprising, consisting essentially of or consisting of SEQ ID NO:2.

[0045] 15. The microorganism according to item 11, wherein the FAR is a Far_Onu_5.

[0046] 16. The microorganism according to item 11 or 15, wherein the FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 14-19, at least 85% sequence identity to any one of SEQ ID NOs: 14-19, at least 90% sequence identity to any one of SEQ ID NOs: 14-19, at least 95% sequence identity to any one of SEQ ID NOs: 14-19, or at least 98% sequence identity to any one of SEQ ID NOs: 14-19.

[0047] 17. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 14, at least 85% sequence identity to SEQ ID NO: 14, at least 90% sequence identity to SEQ ID NO: 14, at least 95% sequence identity to SEQ ID NO: 14, or at least 98% sequence identity to SEQ ID NO: 14.

[0048] 18. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 15, at least 85% sequence identity to SEQ ID NO: 15, at least 90% sequence identity to SEQ ID NO: 15, at least 95% sequence identity to SEQ ID NO: 15, or at least 98% sequence identity to SEQ ID NO: 15.

[0049] 19. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 16, at least 85% sequence identity to SEQ ID NO: 16, at least 90% sequence identity to SEQ ID NO: 16, at least 95% sequence identity to SEQ ID NO: 16, or at least 98% sequence identity to SEQ ID NO: 16.

[0050] 20. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 17, at least 85% sequence identity to SEQ ID NO: 17, at least 90% sequence identity to SEQ ID NO: 17, at least 95% sequence identity to SEQ ID NO: 17, or at least 98% sequence identity to SEQ ID NO: 17.

[0051] 21. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 18, at least 85% sequence identity to SEQ ID NO: 18, at least 90% sequence identity to SEQ ID NO: 18, at least 95% sequence identity to SEQ ID NO: 18, or at least 98% sequence identity to SEQ ID NO: 18.

[0052] 22. The microorganism according to item 16, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO: 19, at least 85% sequence identity to SEQ ID NO: 19, at least 90% sequence identity to SEQ ID NO: 19, at least 95% sequence identity to SEQ ID NO: 19, or at least 98% sequence identity to SEQ ID NO: 19. 23. The microorganism according to items 11 or 15, wherein the FAR is a polypeptide comprising, consisting essentially of or consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO:19.

[0053] 24. The microorganism according to any one of the preceding items, wherein the carboxylic group is in the primary (Ci) position of said at least one C6-C26 free fatty acid.

[0054] 25. The microorganism according to any one of the preceding items, wherein said at least one C6-C26 free fatty acid is a 06, C8, C10, C12, C13, C14, C15, C16, C17 or C18 free fatty acid.

[0055] 26. The microorganism according to any one of the preceding items, wherein said at least one C6-C26 free fatty acid is an unsaturated free fatty acid.

[0056] 27. The microorganism according to item 26, wherein said unsaturated free fatty acid is 010: 1 , 012:1 , 014: 1 , 016:1 or 018:1.

[0057] 28. The microorganism according to item 26 or 27, wherein said unsaturated free fatty acid is unsaturated at the omega-3, omega-6, omega-7 or omega-9 position.

[0058] 29. The microorganism according to item 26, wherein said unsaturated free fatty acid comprises a cis double bond.

[0059] 30. The microorganism according to any one of items 1 to 25, wherein said C6-C26 free fatty acid is a saturated free fatty acid.

[0060] 31 . The microorganism according to any one of the preceding items, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises at least one C12, C14 or C16 free fatty acid or a mixture thereof, preferably at least one C12, C14 or C16 saturated free fatty acid or a mixture thereof.

[0061] 32. The microorganism according to any of the preceding items, wherein said at least one C6-C26 free fatty acid or a mixture thereof is at least one C12, C14 or C16 free fatty acid or a mixture thereof, preferably at least one C12, C14 or C16 saturated free fatty acid or a mixture thereof.

[0062] 33. The microorganism according to item 31, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises a saturated C12 free fatty acid.

[0063] 34. The microorganism according to any of the preceding items, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises a saturated, unbranched C12 free fatty acid.

[0064] 35. The microorganism according to any of the preceding items, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises dodecanoic acid (lauric acid). 36. The microorganism according to any of the preceding items, wherein said at least one C6-C26 free fatty acid is dodecanoic acid (lauric acid).

[0065] 37. The microorganism according to any one of the items 1 to 35, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises a C14 free fatty acid.

[0066] 38. The microorganism according to item 37, wherein said at least one C6-C26 free fatty acid comprises a saturated C14 free fatty acid.

[0067] 39. The microorganism according to any one of items 1 to 35 and 37 to 38, wherein said at least one C6-C26 free fatty acid comprises a saturated, unbranched C14 free fatty acid.

[0068] 40. The microorganism according to any one of the items 37 to 39, wherein said at least one C6-C26 free fatty acid comprises 1 -tetradecanoic acid (myristic acid).

[0069] 41 . The microorganism according to any one of the items 37 to 40, wherein said at least one C6-C26 free fatty acid is 1 -tetradecanoic acid (myristic acid).

[0070] 42. The microorganism according to any one of the preceding items, wherein said microorganism is a bacterium.

[0071] 43. The microorganism according to item 42, wherein the bacterium is selected from the group consisting of Escherichia, Bacillus, Enterobacter Azotobacter, Erwinia, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla and Paracoccus, Methylobacillus, Methylobacterium, Methylococcus, Methylophilus, Methylibium, Hyphomicrobium, Methylocella, Methylocapsa, and Methylocystis.

[0072] 44. The microorganism according to any one of items 1 to 41 , which is a yeast.

[0073] 45. The microorganism according to item 44, wherein the yeast selected from the group consisting of Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Schwanniomyces occidentis, Schizosaccharomyces pombe, Trichoderma reesei and Yarrowia lipolytica.

[0074] 46. The microorganism according to any one of the preceding items, which does not express exopolysaccharides (EPS).

[0075] 47. The microorganism according to any of the preceding items, which has at least one EPS gene cluster inactivated.

[0076] 48. The microorganism according to any of the preceding items, which has both EPS gene clusters inactivated.

[0077] 49. The microorganism according to item 47 or 48, wherein said at least one EPS gene cluster or said both EPS gene clusters are entirely or partly deleted (absent). 50. The microorganism according to any one of the preceding items, which has a decreased activity of polyphosphate kinase (PPK), inactivated PPK or deleted PPK gene.

[0078] 51 . The microorganism according to any one of the preceding items, which has an inactivated PPK or a deleted PPK gene.

[0079] 52. The microorganism according to any one of the preceding items which has a decreased activity of Acyl- homoserine-lactone (AHL) synthase, inactivated AHL synthase or deleted AHL synthase.

[0080] 53. The microorganism according to any one of the preceding items which has an inactivated or deleted AHL synthase.

[0081] 54. The microorganism according to any one of items 1 to 43 and 46 to 53, wherein said microorganism is selected from the group consisting of Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics and Methylobacillus methilovorans.

[0082] 55. The microorganism according to item 54, wherein said microorganism is Methylobacillus flagellatus or Methylobacillus glycogenes.

[0083] 56. The microorganism according to item 55, wherein said microorganism is Methylobacillus flagellatus.

[0084] 57. The microorganism according to item 55, wherein said microorganism is Methylobacillus glycogenes.

[0085] 58. A method for the manufacture of a least one C6-C26 free fatty acid or a mixture thereof comprising growing a microorganism as defined in any one of items 1-57 in a suitable medium, and isolation of said at least one C6-C26 free fatty acid or a mixture thereof from the medium, such as the spent medium.

[0086] 59. The method according to item 58, wherein the carboxylic group is in the primary (Ci) position of said at least one C6-C26 free fatty acid.

[0087] 60. The method according to item 58 or 59, wherein said at least one C6-C26 free fatty acid is a C6, C8, C10, C12, C13, C14, C15, C16, C17 or C18 free fatty acid.

[0088] 61 . The method according to any one of items 58 to 60, wherein said at least one C6-C26 free fatty acid is an unsaturated free fatty acid.

[0089] 62. The method according to item 61 , wherein said unsaturated free fatty acid is C10: 1, C12: 1, C14:1 , C16:1 or C18: 1.

[0090] 63. The method according to item 61 or 62, wherein said unsaturated free fatty acid is unsaturated at the omega- 3, omega-6, omega-7 or omega-9 position. 64. The method according to item 61 , wherein said unsaturated free fatty acid comprises a cis double bond.

[0091] 65. The method according to any one of items 58 to 60, wherein said C6-C26 free fatty acid is a saturated free fatty acid.

[0092] 66. The method according to any one of items 58 to 65, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises at least one C12, C14 or C16 free fatty acid or a mixture thereof.

[0093] 67. The method according to any one of items 58 to 66, wherein said C6-C26 free fatty acid is a C12 free fatty acid.

[0094] 68. The method according to item 67, wherein said C12 free fatty acid is dodecanoic acid.

[0095] 69. The method according to any one of items 58 to 66, wherein said C12, C14 or C16 free fatty acid is a C14 free fatty acid.

[0096] 70. The method according to item 69, wherein said C14 free fatty acid is 1 -tetradecanoic acid (myristic acid).

[0097] 71 . Free fatty acid composition obtained by the method as defined in any one of items 58 to 70.

[0098] 72. A method for the manufacture of at least one C6-C26 free fatty alcohol comprising growing a microorganism as defined in any one of items 1 to 57 in a suitable medium, optionally isolating at least one C6-C26 free fatty acid from the (spent) medium, conversion of the C6-C26 free fatty acid into the corresponding C6-C26 free fatty alcohol, and isolation of said C6-C26 free fatty alcohol.

[0099] 73. The method according to item 72, wherein said C6-C26 free fatty alcohol is a C12 free fatty alcohol.

[0100] 74. The method according to item 72 or 73, wherein said C12 free fatty alcohol is 1-dodecanol (lauryl alcohol).

[0101] 75. The method according to item 72, wherein said C6-C26 free fatty alcohol is a C14 free fatty alcohol.

[0102] 76. The method according to item 72 or 75, wherein said C14 free fatty alcohol is 1 -tetradecanol (myristyl alcohol).

[0103] 77. Free fatty alcohol composition obtained by the method as defined in any one of items 58 to 76.

[0104] 78. Use of a microorganism as defined in any one of items 1 to 57 for the manufacture of a C6-C26 free fatty acid or a mixture thereof, or a C6-C26 free fatty alcohol or a mixture thereof.

[0105] 79. The use according to item 78, which is for the manufacture of a C12, C14 or C16 free fatty acid or a mixture thereof.

[0106] 80. The use according to item 78 or 79, wherein the use is for the manufacture of a C12 free fatty acid.

[0107] 81 . The use according to any one of items 78 to 80, wherein said C12 free fatty acid is dodecanoic acid.

[0108] 82. The use according to item 78 or 79, wherein the use is for the manufacture of a C14 free fatty acid. 83. The use according to any one of items 78, 79 and 82, wherein said C14 free fatty acid is tetradecanoic acid (myristic acid).

[0109] 84. The use according to item 78, wherein said C12 free fatty alcohol is 1-dodecanol.

[0110] 85. The use according to item 78, wherein said C14 free fatty alcohol is 1 -tetradecanol (myristyl alcohol).

[0111] Description of the figures

[0112] Figure 1: Phylogenetic trees constructed from sequence alignments of 161 FAR enzymes from C12 fatty alcohol producing organisms (left) and 67 FAR sequences from C14 fatty alcohol producing organisms (right). Rough groups were determined manually to screen more diverse enzymes.

[0113] Figure 2. Titers of C12, C14 and C16 fatty alcohols produced from methanol by M. flagellatus ABBAg_2 heterologously expressing different FAR enzymes.

[0114] Figure 3. Titers of C12, C14 and C16 fatty acids produced from methanol by M. flagellatus ABBAg_2 heterologously expressing different TE enzymes in combination with FAR_Onu_5 (SEQ ID NO:18).

[0115] Figure 4. Effect of MAR and Far_Onu_5 on titers of C12, C14 and C16 fatty acids produced from methanol in M. flagellatus heterologously expressing TE_Uca (SEQ ID NO:39) only or in combination with FAR_Onu_5 (SEQ ID NO:18) or Far_Mar (SEQ ID NO:2).

[0116] Figure 5. Effect of active and inactivated FAR on C12 fatty acid production.

[0117] Figure 6. Titers of C12, C14 and C16 fatty acids produced from methanol heterologously expressing Uca_TE sequences of different length in M. flagellatus ABBAg_2

[0118] Description

[0119] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of biochemistry, genetics, and molecular biology.

[0120] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0121] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, fermentation technology, recombinant DNA, and food science, which are within the skill of the art. Such techniques are explained fully in the literature. Saturated, unbranched fatty acids with 12 carbon atoms are currently primarily produced from palm kernel oil and have many uses in the food, cosmetics, and surfactants industries. Currently, most C12 fatty acids used in these industries are produced from palm kernel oil. Microbial production of C12 fatty acids from methanol would have a favourable environmental impact by replacing harmful oil palm farming. The present invention describes an enzyme and a combination of enzymes that facilitate a surprising effect on production of C12 free fatty acids from methanol. Expressing a thioesterase in a microorganism leads to secretion of C12 free fatty acids. In particular the thioesterase from Umbellularia californica (Uca_TE) in a methylotrophic host from the genus Methylobacillus has proven useful. The Uca_TE enzyme has been described before in the literature to preferentially cleave C12 fatty acids from AGP, compared to other lengths, so this result was to be expected.

[0122] Fatty alcohol reductase (FAR) is a bifunctional enzyme that converts ACP-bound fatty acids into fatty alcohols. Surprisingly, when the Uca_TE enzyme was expressed in the Methylobacillus host in combination with a FAR enzyme, specifically from Marinobacter hydrocarbonoclasticus (Mar_FAR) and Ostrinia nubilalis (Far_0nu_5), this led to a surprising increase in C12 free fatty acid production. When expressed on its own, Mar_FAR leads to moderate production of C16 and C14 fatty alcohols, but no C12 fatty acids. Expression of Onu_5_FAR leads to modest production of C12 and C14 fatty alcohols, but again no free fatty acids. However, when expressed alongside Uca_TE, they lead to a 50-100% increase in C12 free fatty acid production, alongside the expected fatty alcohol production. This is the first time such an effect has been reported and the mechanism of it is not known. Without wishing to be bound by theory, this effect seems to depend on the catalytic activity of the FAR enzymes. This is proven in the Examples by mutating only the active sites of the FAR enzymes, which removed their ability to produce fatty alcohols and at the same time removed the increase in C12 fatty acid production when expressed alongside Uca_TE.

[0123] In a first aspect the present invention provides a microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR). It is understood that the term "at least one gene enabling the expression of' means genes that include a nucleotide sequence that encodes the respective enzyme but may additionally comprise regulatory sequences that facilitate the expression of the coding sequence in the host organism. A "gene enabling the expression of” includes thus a gene that comprise a nucleotide sequence that encodes the respective enzyme and can be expressed by the microorganism. In some embodiments said gene may be a nucleotide sequence that encodes the respective enzyme and can be expressed by the microorganism.

[0124] The microorganism may be a recombinant microorganism in that it is genetically engineered to comprise and express a gene that it does not naturally comprise and expresses.

[0125] In various embodiments of this aspect, at least one of the at least one gene enabling the expression of a thioesterase (TE) and the at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR) is a heterologous gene. "Heterologous”, as used in this context, means that said gene does not naturally occur in the respective organism and may, for example, originate from a different species than the host organism or may be artificially generated or modified to differ from the genes naturally occurring in said organism. These heterologous genes are then heterologously expressed in the microorganisms of the invention.

[0126] In various embodiments, the expression of both genes provides for a higher production of at least one C6-C26 free fatty acid or a mixture thereof, in particular C12, C14 and / or C16 fatty acid, more preferably C12 fatty acid, from methanol relative to the same microorganism that expresses none or only one of these genes. The production yield is preferably at least 5, 10, 15, 20, 25 or 30 % higher than the production achieved relative to the same microorganism that expresses none or only one of these genes (in particular the reference that provides for the highest production, typically the microorganism only expressing the TE but not the FAR) under identical production conditions. In various embodiments, the invention thus refers to a microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, in particular C12, C14 and / or C16 fatty acid, more preferably C12 fatty acid, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR), wherein said microorganism provides for a higher production yield, preferably an at least 10 % higher production yield, relative to the same microorganism that lacks one or both of the TE and the FAR genes, in particular to a microorganism that lacks the FAR gene.

[0127] Recombinant enzymes

[0128] All recombinant enzymes referred to herein may be derived from a naturally occurring nucleotide sequence which has been codon-optimized for expression in the microorganism of choice. The respective enzymes are thus generally defined by reference to their amino acid sequence.

[0129] Unless otherwise stated, percent sequence identity (% sequence identity) between two sequences is determined from a pairwise sequence alignment created by using program "Needle” (as implemented in the European Molecular Biology Open Software Suite (EMBOSS), version 6.3.1.2 or later (Trends in Genetics (2000) 16 (6), p. 276-277) with applying a gap open penalty of 10, a gap extension penalty of 0.5, the matrix EBLOSUM62 (which is the "Needle” EMBOSS version of matrix BLOSUM62) for protein sequences, and the matrix EDNAFULL (which is the "Needle” EMBOSS version of matrix DNAFULL) for nucleotide sequences, by the following calculation: % sequence identity = (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment), which is also reported as “Longest_ldentity” in the output of "Needle” EMBOSS when parameter "-nobrief” is applied. Program "Needle” EMBOSS has implemented the algorithm of Needleman & Wunsch (J. Mol. Biol. (1979) 48, p. 443-453) for aligning of two sequences.

[0130] The thioesterase (TE) may in one embodiment be a thioesterase from Umbellularia californica (UCa TE).

[0131] In various embodiments, the Uca-TE has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to SEQ ID NO:39, such as at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39. It is understood that in an enumeration such as "at least 80, 81 the "at least” refers to all listed values. In various embodiments, the Uca-TE has at least 50% thioesterase activity as compared to the activity of SEQ ID NO:39, preferably at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39. Said activity can be determined in a suitable enzymatic activity assay, which are routinely known to those skilled in the art.

[0132] The above definitions may be combined such that the TE has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39.

[0133] In various embodiments, the Uca-TE is a truncated version of SEQ ID NO: 39, for example having at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to the corresponding sequence of SEQ ID NO:39, for example at least 90% sequence identity to the corresponding sequence of SEQ ID NO:39, at least 95% sequence identity to the corresponding sequence of SEQ ID NO:39.

[0134] If the N-terminus is truncated, the amino acid sequence may additionally comprise an N-terminal M residue. In such truncated versions, the enzymatic activity may be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39.

[0135] In various embodiments, the Uca-TE comprises or is SEQ ID NO: 61 , which is a truncated 287 amino acid long C- terminal fragment of SEQ ID NO:39 (aa 97-382) that comprises an N-terminal methionine residue and has the following amino acid sequence:

[0136] MDDHFGLHGLVFRRTFAIRSYEVGPDRSTSILAVMNHMQEATLNHAKSVGILGDGFGTTLEMSKRDLMWWRRTHV AVERYPTWGDTVEVECWIGASGNNGMRRDFLVRDCKTGEILTRCTSLSVLMNTRTRRLSTIPDEVRGEIGPAFIDNVA VKDDEIKKLQKLNDSTADYIQGGLTPRWNDLDVNQHVNNLKYVAWVFETVPDSIFESHHISSFTLEYRRECTRDSVLR SLTTVSGGSSEAGLVCDHLLQLEGGSEVLRARTEWRPKLTDSFRGISVIPAEPRV

[0137] In various embodiments, the Uca-TE has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to any one of SEQ ID NO: 61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, or SEQ ID NO:73.

[0138] In various embodiments, the Uca-TE has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to any one of SEQ ID NO: 61 , SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NQ:70, SEQ ID NO:71 , SEQ ID NO:72, or SEQ ID NO:73 and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39.

[0139] In various embodiments, the Uca-TE is SEQ ID NO:39 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, additions or deletions and retaining thioesterase activity. The activity may be as defined above, i.e. at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39. In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:39. In various embodiments, the Uca-TE is SEQ ID NO:39. "Consists essentially of”, as used in relation to a given sequence, means that the respective enzyme consists of more than 50%, preferably more than 60, 70, 80, or 90 % of the given sequence, i.e. may comprise <50%, <40%, <30%, <20%, or <10% of additional amino acids in addition to the recited sequence, typically in form of flanking sequences that are extending the N- and / or C-terminus of the given sequence.

[0140] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:61. In various embodiments, the Uca-TE is SEQ ID NO:61.

[0141] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:62. In various embodiments, the Uca-TE is SEQ ID NO:62.

[0142] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:63. In various embodiments, the Uca-TE is SEQ ID NO:63.

[0143] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:64. In various embodiments, the Uca-TE is SEQ ID NO:64.

[0144] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:65. In various embodiments, the Uca-TE is SEQ ID NO:65.

[0145] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:66. In various embodiments, the Uca-TE is SEQ ID NO:66.

[0146] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:67. In various embodiments, the Uca-TE is SEQ ID NO:67.

[0147] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:68. In various embodiments, the Uca-TE is SEQ ID NO:68.

[0148] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:69. In various embodiments, the Uca-TE is SEQ ID NO:69.

[0149] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NQ:70. In various embodiments, the Uca-TE is SEQ ID NQ:70.

[0150] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:71. In various embodiments, the Uca-TE is SEQ ID NO:71.

[0151] In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:72. In various embodiments, the Uca-TE is SEQ ID NO:72. In various embodiments the Uca-TE comprises, consists essentially of, or consists of SEQ ID NO:73. In various embodiments, the Uca-TE is SEQ ID NO:73.

[0152] In various embodiments, the at least one FAR is Marinobacter hydrocarbonoclasticus FAR (Mar-FAR) or Ostrinia nubiliatis FAR (Far_0nu_5).

[0153] In various embodiments, the FAR is Mar-FAR.

[0154] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO:2, for example at least 85% sequence identity to SEQ ID NO:2, at least 90% sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2.

[0155] In various embodiments, the FAR has at least 50% activity as compared to the activity of SEQ ID NO:2, preferably at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of SEQ ID NO:2. Said activity can be determined in a suitable enzymatic activity assay, which are routinely known to those skilled in the art.

[0156] The above definitions may be combined such that the FAR has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:2 and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO:2.

[0157] In yet another embodiment the FAR is a truncated version of SEQ ID NO:2, for example having at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to the corresponding sequence of SEQ ID NO:2, for example at least 90% sequence identity to the corresponding sequence of SEQ ID NO:2, at least 95% sequence identity to the corresponding sequence of SEQ ID NO:2. If the N-terminus is truncated, the amino acid sequence may additionally comprise an N-terminal M residue. In such truncated versions, the enzymatic activity may be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of SEQ ID NO:2.

[0158] In various embodiments, the FAR is SEQ ID NO:2 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, additions or deletions and retaining its enzymatic activity. The activity may be as defined above, i.e. at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of SEQ ID NO:2.

[0159] In various embodiments the FAR comprises, consists essentially of, or consists of SEQ ID NO:2. In various embodiments, the FAR is SEQ ID NO:2.

[0160] In various embodiments, the FAR is a Far_Onu_5.

[0161] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID NOs: 14-19, for example at least 85% sequence identity to any one of SEQ ID NOs: 14-19, at least 90% sequence identity to any one of SEQ ID NOs: 14-19, at least 95% sequence identity to any one of SEQ ID NOs: 14-19, or at least 98% sequence identity to any one of SEQ ID NOs: 14-19. In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 14, for example at least 85% sequence identity to SEQ ID NO: 14, at least 90% sequence identity to SEQ ID NO: 14, at least 95% sequence identity to SEQ ID NO: 14, or at least 98% sequence identity to SEQ ID NO: 14.

[0162] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 15, for example at least 85% sequence identity to SEQ ID NO: 15, at least 90% sequence identity to SEQ ID NO: 15, at least 95% sequence identity to SEQ ID NO: 15, or at least 98% sequence identity to SEQ ID NO: 15.

[0163] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 16, for example at least 85% sequence identity to SEQ ID NO: 16, at least 90% sequence identity to SEQ ID NO: 16, at least 95% sequence identity to SEQ ID NO: 16, or at least 98% sequence identity to SEQ ID NO: 16.

[0164] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 17, for example at least 85% sequence identity to SEQ ID NO: 17, at least 90% sequence identity to SEQ ID NO: 17, at least 95% sequence identity to SEQ ID NO: 17, or at least 98% sequence identity to SEQ ID NO: 17.

[0165] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 18, for example at least 85% sequence identity to SEQ ID NO: 18, at least 90% sequence identity to SEQ ID NO: 18, at least 95% sequence identity to SEQ ID NO: 18, or at least 98% sequence identity to SEQ ID NO: 18.

[0166] In various embodiments, the FAR is a polypeptide having at least 80, 81 , 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 19, for example at least 85% sequence identity to SEQ ID NO: 19, at least 90% sequence identity to SEQ ID NO: 19, at least 95% sequence identity to SEQ ID NO: 19, or at least 98% sequence identity to SEQ ID NO: 19.

[0167] In various embodiments, the FAR has at least 50% activity as compared to the activity of any one of SEQ ID NOs: 14- 19, preferably at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of any one of SEQ ID NOs: 14-19. Said activity can be determined in a suitable enzymatic activity assay, which are routinely known to those skilled in the art.

[0168] The above definitions may be combined such that the FAR has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to of any one of SEQ ID NOs: 14-19 and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of any one of SEQ ID NOs: 14-19.

[0169] In yet another embodiment the FAR is a truncated version of any one of SEQ ID NOs: 14-19, for example having at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to the corresponding sequence of any one of SEQ ID NOs: 14-19. If the N-terminus is truncated, the amino acid sequence may additionally comprise an N-terminal M residue. In such truncated versions, the enzymatic activity may be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of any one of SEQ ID NOs: 14-19.

[0170] In various embodiments, the FAR is any one of SEQ ID NOs:14-19 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, additions or deletions and retaining its enzymatic activity. The activity may be as defined above, i.e. at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % activity as compared to the activity of any one of SEQ ID NOs: 14-19.

[0171] In various embodiments, the FAR is a polypeptide comprising, consisting essentially of or consisting of SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0172] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:2 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO:2. Also included in such embodiments are all variants of said TE and said FAR detailed above. In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 14 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 14. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0173] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 15 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 15. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0174] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 16 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 16. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0175] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 17 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 17. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0176] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 18 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 18. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0177] In various embodiments, (1) the TE is a TE as defined herein above, e.g. Uca-TE that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO:39 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % thioesterase activity as compared to the activity of SEQ ID NO:39; and (2) the FAR is a FAR as defined herein above, e.g. a FAR that has at least 80%, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to SEQ ID NO: 19 and / or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 % enzymatic activity as compared to the activity of SEQ ID NO: 19. Also included in such embodiments are all variants of said TE and said FAR detailed above.

[0178] In various embodiments, the following combinations of enzymes are encompassed as specific embodiments of the present invention, where "variants” refers to the above enzymes that have at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity to the reference sequence and optionally retain the activity of the reference sequence to at least 50% or more as defined herein above. n all the above embodiments, it is preferred that the respective enzymes meet the sequence identity and activity definition given.

[0179] In various embodiments, the carboxylic group is in the primary (Ci) position of said at least one C6-C26 free fatty acid.

[0180] In various embodiments, the at least one C6-C26 free fatty acid is a 06, C8, C10, C12, C13, C14, C15, C16, C17 or C18 free fatty acid.

[0181] In various embodiments, the at least one C6-C26 free fatty acid is an unsaturated free fatty acid.

[0182] In various embodiments, the unsaturated free fatty acid is C10: 1, C12:1, C14:1, 016:1 or C18:1.

[0183] In various embodiments, the unsaturated free fatty acid is unsaturated at the omega-3, omega-6, omega-7 or omega- 9 position. In various embodiments, the unsaturated free fatty acid comprises a cis double bond.

[0184] In various embodiments, the C6-C26 free fatty acid is a saturated free fatty acid. In various embodiments, the at least one C6-C26 free fatty acid or a mixture thereof comprises at least one C12, C14 or C16 free fatty acid or a mixture thereof.

[0185] In various embodiments, the at least one C6-C26 free fatty acid or a mixture thereof is at least one C12, C14 or C16 free fatty acid or a mixture thereof.

[0186] In yet another embodiment the at least one C6-C26 free fatty acid is a saturated C12 free fatty acid.

[0187] In various embodiments, the at least one C12 free fatty acid is a saturated, unbranched C12 free fatty acid.

[0188] In various embodiments, the at least one C12 free fatty acid comprises dodecanoic acid (lauric acid).

[0189] In various embodiments, the at least one C12 free fatty acid is dodecanoic acid (lauric acid).

[0190] In various embodiments, the at least one C12 or C14 free fatty acid comprises a C14 free fatty acid.

[0191] In various embodiments, the at least one C14 free fatty acid comprises a saturated C14 free fatty acid.

[0192] In various embodiments, the at least one C14 free fatty acid comprises a saturated, unbranched C14 free fatty acid.

[0193] In various embodiments, the at least one C14 free fatty acid comprises 1 -tetradecanoic acid (myristic acid).

[0194] In various embodiments, the at least one C14 free fatty acid is 1 -tetradecanoic acid (myristic acid).

[0195] Host cell

[0196] The host cell is a microorganism. In an embodiment the microorganism is a bacterium.

[0197] In an embodiment the bacterium is selected from the group consisting of Escherichia, Bacillus, Enterobacter Azotobacter, Erwinia, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla and Paracoccus, Methylobacillus, Methylobacterium, Methylococcus, Methylophilus, Methylibium, Hyphomicrobium, Methylocella, Methylocapsa, and Methylocystis.

[0198] In various embodiments, the microorganism is a yeast.

[0199] In various embodiments, the yeast is selected from the group consisting of Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces lactis, Schwanniomyces occidentis, Schizosaccharomyces pombe, Trichoderma reesei and Yarrowia lipolytica.

[0200] In various embodiments, the microorganism is a Methylobacillus bacterium. This may be a Methylobacillus bacterium selected from the group consisting of Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacillus pratensis, Methylobacillus rhizosphaerae, Methylobacillus gramineus, Methylobacillus arboreus, Methylobacillus caricics and Methylobacillus methilovorans. In one embodiment the Methylobacillus bacterium is Methylobacillus flagellatus or Methylobacillus glycogenes. In a further embodiment the Methylobacillus bacterium is Methylobacillus flagellatus. In a further embodiment the Methylobacillus bacterium is Methylobacillus glycogenes.

[0201] In one embodiment the Methylobacillus bacterium does not express exopolysaccharides (EPS). In a further embodiment the Methylobacillus bacterium has at least one EPS gene cluster inactivated. In yet another embodiment the Methylobacillus has both EPS gene clusters inactivated. In a further embodiment the at least one EPS gene cluster or said both EPS gene clusters are entirely or partly deleted (absent).

[0202] In various embodiments,, the Methylobacillus bacterium has a decreased activity of polyphosphate kinase (PKK), inactivated PPK or deleted PPK gene. In various embodiments,, the Methylobacillus bacterium has an inactivated PPK or a deleted PPK gene.

[0203] In various embodiments, the Methylobacillus bacterium has a decreased activity of Acyl-homoserine-lactone (AHL) synthase, inactivated AHL synthase or deleted AHL synthase.

[0204] In various embodiments, the Methylobacillus bacterium has an inactivated or deleted AHL synthase.

[0205] Methylobacillus strains having a decreased, inactivated or deleted polyphosphate kinase (PPK) and / or a decreased, inactivated or deleted Acyl-homoserine-lactone (AHL) synthase are disclosed in e.g. WO2023 / 139255.

[0206] Method of manufacture

[0207] In a further aspect the invention provides a method for producing a least one C6-C26 free fatty acid or a mixture thereof comprising growing a microorganism as defined in any one of items 1-53 in a suitable medium, and isolation of said at least one C6-C26 free fatty acid or a mixture thereof from the spent medium.

[0208] In one embodiment the carboxylic group is in the primary (Ci) position of said at least one C6-C26 free fatty acid.

[0209] In various embodiments, the at least one C6-C26 free fatty acid is a C6, C8, C10, C12, C13, C14, C15, C16, C17 or C18 free fatty acid.

[0210] In various embodiments, the at least one C6-C26 free fatty acid is an unsaturated free fatty acid.

[0211] In various embodiments, the unsaturated free fatty acid is C10:1, C12:1 , C14:1 , C16:1 or C18:1 .

[0212] In various embodiments, the unsaturated free fatty acid is unsaturated at the omega-3, omega-6, omega-7 or omega- 9 position.

[0213] In various embodiments, the unsaturated free fatty acid comprises a cis double bond.

[0214] In various embodiments, the C6-C26 free fatty acid is a saturated free fatty acid.

[0215] In various embodiments, the at least one C6-C26 free fatty acid or a mixture thereof comprises at least one C12, C14 or C16 free fatty acid or a mixture thereof. In a yet further aspect the invention provides a free fatty acid composition obtained by the method for producing at least one C6-C26 free fatty acid or a mixture thereof.

[0216] In yet a further aspect the invention provides a method for the manufacture of at least one C6-C26 free fatty alcohol comprising growing a microorganism as defined in any one of items 1 to 57 in a suitable medium, optionally isolating at least one C6-C26 free fatty acid from the spent medium, conversion of the C6-C26 free fatty acid into the corresponding C6-C26 free fatty alcohol, and isolation of said C6-C26 free fatty alcohol.

[0217] In various embodiments, the C6-C26 free fatty alcohol is a C12 free fatty alcohol.

[0218] In various embodiments, the C12 free fatty alcohol is 1-dodecanol (lauryl alcohol).

[0219] In various embodiments, the C6-C26 free fatty alcohol is a C14 free fatty alcohol.

[0220] In various embodiments, the C14 free fatty alcohol is 1 -tetradecanol (myristyl alcohol).

[0221] In a yet further aspect, the invention provides a free fatty alcohol composition obtained by the method for producing at least one C6-C26 free fatty alcohol.

[0222] In various embodiments, the C6-C26 free fatty acid is a C12 free fatty acid.

[0223] In an embodiment the C12 free fatty acid is dodecanoic acid.

[0224] In various embodiments, the C12 free fatty alcohol is 1-dodecanol.

[0225] In various embodiments, the C6-C26 free fatty acid is a C14 free fatty acid.

[0226] In various embodiments, the C14 free fatty acid is 1 -tetradecanoic acid (myristic acid).

[0227] In a further aspect the present invention provides the Use of a microorganism as defined in the present invention for the manufacture of a C6-C26 free fatty acid or a mixture thereof, or a C6-C26 free fatty alcohol or a mixture thereof.

[0228] In one embodiment the use is for the manufacture of a C12 free fatty acid.

[0229] In various embodiments, the use is for the manufacture of a free fatty acid which is dodecanoic acid.

[0230] In various embodiments, the use is for the manufacture of a C12 free fatty alcohol which is 1-dodecanol.

[0231] In various embodiments, the use is for the manufacture of a C14 free fatty acid.

[0232] In various embodiments, the use is for the manufacture of a C14 free fatty acid which is tetradecanoic acid (myristic acid).

[0233] In various embodiments, the use is for the manufacture of a C14 free fatty alcohol which is 1 -tetradecanol (myristyl alcohol). Culture medium

[0234] The culture medium for growing microorganisms typically comprise carbon- and nitrogen sources as well as different salts to supply phosphorous, magnesium, potassium, sulphate, and some vitamins. Such culture media are well known in the art for many different microorganisms.

[0235] For instance, the culture medium employed for growing the Methylobacillus bacterium of the present invention may be any conventional medium suitable for culturing a bacterium cell in question, and may be composed according to the principles of the prior art. The medium will usually contain all nutrients necessary for the growth and survival of the respective bacterium, such as carbon and nitrogen sources and various inorganic salts. Suitable media, e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains. Non-limiting standard medium well known to the skilled person include Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BMMY, GM MY, or Yeast Malt Extract (YM) broth, which are all commercially available. A non-limiting example of suitable media tor culturing bacterial cells, such as Methylobacillus, including minimal media and rich media such as Luria Broth (LB), M9 media, M17 media, SA media, MOPS media, Terrific Broth, YT and others.

[0236] The carbon source is preferably methanol but may be any suitable carbon substrate known in the art, and in particularly any carbon substrate commonly used in the cultivation of bacteria and / or fermentation. Non-limiting examples of suitable fermentable carbon substrates include carbohydrates (e.g., C5 sugars such as arabinose or xylose, or C6 sugars such as glucose), glycerol, glycerine, acetate, dihydroxyacetone, one-carbon source, methanol, methane, oils, animal fats, animal oils, plant oils, fatty acids, lipids, phospholipids, glycerolipids, monoglycerides, diglycerides, triglycerides, renewable carbon sources, polypeptides (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, peptone, casaminoacids or any combination of two or more of the foregoing.

[0237] According to some embodiments, the carbon substrate is selected from the group consisting of 05 sugars (such as arabinose or xylose), 06 sugars (such as glucose or fructose), lactose, sucrose, glycerol, glycerine, acetate, Corn steep liquor, yeast extract, component from a yeast extract, peptone, casaminoacids or combinations thereof.

[0238] In the present case where the bacterium is a methylotrophic bacterium, the culture medium preferably comprises a reduced one-carbon compound, such as methanol or methylamine, or a multi-carbon compound that contains no carbon-carbon bonds, such as dimethylamine. Thus, according to some embodiments, the culture medium comprises methanol as a carbon source. The concentration of methanol in the culture medium may generally be in the range from about 0,5% w / v to about 4 % w / v, such as from about 2% w / v to about 4 % w / v. According to some embodiments, the concentration of methanol in the culture medium is in the range from about 2,5% w / v to about 3,5% w / v.

[0239] As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybeanhydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. Suitably, the bacterium is cultivated under suitable conditions for growing the bacterium. Suitable conditions for culturing the respective bacterium are well known to the skilled person. Typically, a bacterium is cultured at a temperature ranging from about 20 to about 45°C, such as from about 30 to about 38°C, such as at about 37°C. The cultivation can be preferably performed under aerobic conditions, such as by a shaking culture, by a stirring culture or in a bioreactor with aeration, at a temperature of about 20 to about 45 °C, such as about 30 to 38 °C, preferably at about 37°C. The pH of the culture is usually above 5, such as in a range from about 6 to about 8, preferably from about 6.5 to about 7.5, more preferably from about 6.8 to about 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. The cultivation may be carried out for a period in the range from 10 to 70 h, preferably in a range from 24 to 60 h, more preferably in a range from 36 to 50 h.

[0240] After cultivation, solids such as bacterial biomass can be removed from the culture medium by centrifugation or membrane filtration. C12 free fatty acids may be recovered from the spent fermentation medium and optionally purified according to the intended application.

[0241] Definitions

[0242] A thioesterases (TE) according to this invention is an enzyme, which can hydrolyse a thioester bond, preferably the thioester bond of an acyl-ACP (Acyl Carrier Protein) complex to cleave free fatty acids from acyl-ACP / CoA.

[0243] A carboxylic acid reductase (CAR) according to this invention is an enzyme, which can catalyse the ATP- and NADPH- dependent reduction of acids, preferably fatty acids to the corresponding aldehydes.

[0244] An aldehyde reductase (ALDR) according to this invention is an enzyme, which can catalyze the NADPH-dependent reduction of aldehydes to alcohols, preferably fatty aldehydes to the corresponding alcohols.

[0245] Fatty acyl reductase (FAR) according to this invention is an enzyme, which catalyzes the NADPH-dependent reduction of native fatty acyl-CoA or acyl-ACP metabolites to primary fatty alcohols.

[0246] EPS according to this invention refers to exopolysaccharides produced by the bacteria used in this invention. EPS in a bioprocess increase viscosity of the broth and lead to increased emulsion formation, so removal of EPS production improves bioprocess performance, as described in WO2022069644A1 .

[0247] AHL according to this invention refers to the enzyme Acyl homoserine lactone synthase. The enzyme is used by some bacteria to synthesize cell signaling or quorum sensing molecules (acyl homoserine lactones). The signaling molecules can lead to a variety of responses, including programmed cell death. Removal of AHL activity from bacteria can prevent programmed cell death, which prevents premature bioprocess termination, as described in WO2023139255A1.

[0248] PPK according to this invention refers to the enzyme polyphosphate kinase. The enzyme is involved in programmed cell death through an unknown mechanism programmed cell death. Removal of PPK activity from bacteria can prevent programmed cell death, which prevents premature bioprocess termination, as described in WO2023139255A1.

[0249] Examples Example 1 : Selection of fatty alcohol reductases and thioesterases.

[0250] Fatty alcohols are common in nature as animal pheromones. To identify fatty alcohol reductase (FAR) enzymes with the potential to produce C12 and C14 fatty alcohols, the Pherobase database (El-Sayed AM 2024. The Pherobase: Database of Pheromones and Semiochemicals.) was screened for animal species known to produce dodecanol and tetradecanol. A list of these species was compiled and used as a taxonomy-based filter to query the UniProt database for fatty alcohol reductase genes (EC: 1 .2.1.84). The search resulted in over 200 protein sequences, which were used to generate a similarity-based phylogenetic tree. The tree was then roughly divided into different groups based on similarity and 9-10 representatives of each group were selected for screening (SEQ ID Nos: 1 to 28) (Fig. 1).

[0251] Thioesterase enzymes with reported selectivity for C12 and C14 acyl-ACP were selected from published literature. Measured production of C12 or C14 fatty acids by overexpression of the enzyme was the primary criterion. Functional parameters such as low Km values for ACP-bound C12 or C14 fatty acids were also considered in the selection process. See SEQ ID Nos: 29 to 43 for amino acid sequences. Additionally, 8 thioesterase enzymes from Cocos nucifera (SEQ ID Nos: 44 to 51) and 8 thioesterase enzymes from Elaeis guineensis were tested since both organisms are known to produce C12 and C14 fatty acids (SEQ ID Nos: 52 to 59).

[0252] Example 2: Preparation of expression constructs.

[0253] To achieve C12 and C14 fatty alcohol or fatty acid production, heterologous C12 and C14 chain length specific acyl- ACP thioesterases (TE), or fatty alcohol reductase (FAR) must be functionally expressed in the host organism.

[0254] To test different FAR enzymes, each of 28 FAR genes was codon harmonized for expression in Methylobacillus flagellatus ABBAg2 and cloned into the expression vector under I PTG-inducible lacO / Trc promoter using the NEB Hi- Fi cloning kit according to manufacturer instructions (pABBA_1-pABBA_28).

[0255] For the first screen of thioesterases, FAR_Onu_5 (SEQ ID NO: 18) was combined with different thioesterase genes from Cinnamomum camphora, Cuphea palustris, Cuphea viscosissima, Escherichia coli, Umbellularia californica and Zea mays (SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 39 and SEQ ID NO: 40). All sequences were codon harmonized for expression in Methylobacillus flagellatus ABBAg2 and cloned into expression vector containing the FAR Onub gene and TE gene, each under I PTG-inducible lacO / Trc promoter. NEB Hi-Fi cloning kit was used for the purpose of plasmid construction according to manufacturer instructions (pABBA_47, pABBA_53, pABBA_42, pABBA_169, pABBA_54, pABBA_52, and pABBA_46). All plasmids carry the broad host range ori pBR322.

[0256] For the second screen of thioesterases, each of 31 TE genes was codon harmonized for expression in Methylobacillus flagellatus ABBAg2 and cloned into the expression vector under I PTG-inducible lacO / Trc promoter using the NEB Hi- Fi cloning kit according to manufacturer instructions (pABBA_171, pABBA_641, pABBA_642, pABBA_643, pABBA_644, pABBA_645, pABBA_646, pABBA_647, , pABBA_648, pABBA_649, pABBA_650, pABBA_651, pABBA_652, pABBA_653, pABBA_654, pABBA_655, pABBA_656, pABBA_713, pABBA_714, pABBA_715, pABBA_716, pABBA_717, pABBA_718, pABBA_719, pABBA_720, pABBA_721, pABBA_722, pABBA_723, pABBA_724, pABBA_725 and pABBA_726). Plasmid pABBA_536 contains a different codon optimisation of the TE_Uca (SEQ ID NO: 39). It is cloned into the expression vector under IPTG-inducible lacO / Trc promoter using the NEB Hi-Fi cloning kit according to manufacturer instructions.

[0257] List of constructed plasmids is in Table 1.

[0258] Table 1 : Plasmids constructed to express FAR and TE enzymes in M. flagellatus.

[0259] Example 3: Preparation of strains.

[0260] The prepared plasmids pABBA_1, pABBA_2, pABBA_3, pABBA_4, pABBA_6, pABBA_7, pABBA_11 , pABBA_12, pABBA_13, pABBA_15, pABBA_16, pABBA_17, pABBA_19, pABBA_20, pABBA_21, pABBA_23, pABBA_24, pABBA_25, pABBA_27, pABBA_26, pABBA_22, pABBA_18, pABBA_14, pABBA_5, pABBA_8, pABBA_9, pABBAJO, pABBA_28, pABBA_47, pABBA_53, pABBA_42, pABBA_169, pABBA_54, pABBA_52, pABBA_46, pABBA_171 and pABBA_536 were checked by sequencing and transformed into M. flagellatus ABBAg_2 via electroporation to create strains ABBAg_3, ABBAg_4, ABBAg_5, ABBAg_6, ABBAg_7, ABBAg_8, ABBAg_10, ABBAg_11 , ABBAg_12, ABBAg_13, ABBAg_14, ABBAg_15, ABBAg_16, ABBAg_17, ABBAg_18, ABBAg_19,

[0261] ABBAg_20, ABBAg_21, ABBAg_22, ABBAg_23, ABBAg_24, ABBAg_25, ABBAg_26, ABBAg_27, ABBAg_28,

[0262] ABBAg_29, ABBAg_30, ABBAg_57, ABBAg_103, ABBAg_104, ABBAg_102, ABBAg_146, ABBAg_105, ABBAg_133, ABBAg_135 and ABBAg_209 and ABBAg_539.

[0263] Strain ABBAg_226 was first transformed with linear DNA fragment endcoding for Far_Onu_5 (SEQ ID NO: 18) flanked by genome homology regions to insert Far_Onu_5 (SEQ ID NO: 18) on the genome and consecutively transformed with TEJJca (SEQ ID NO: 39) expressed from a plasmid vector - pABBA_171 , while ABBAg_231 was first transformed with linear DNA fragment encoding for Far_Mar (SEQ ID NO: 2) flanked by genome homology regions to insert Far_Mar (SEQ ID NO: 2) on the genome and consecutively transformed with a DNA fragment encoding for TE_Uca (SEQ ID NO: 39) on the plasmid - pABBA_171.

[0264] ABBAg_745 was first transformed with a linear DNA fragment encoding for Far_Mar (SEQ_ID_2) flanked by genome homology regions to insert Far_Mar (SEQ ID NO: 2) into the genome and consecutively transformed with a DNA fragment encoding for TE_Uca (SEQ_ID_39) on a plasmid - pABBA_536. ABBAg_748 was first transformed with the linear DNA fragment encoding for inactivated Far_Mar (SEQ ID NO: 60) flanked by genome homology regions to insert inactivated Far_Mar (SEQ ID NO: 60) on the genome and consecutively transformed with TE_Uca (SEQ ID NO: 39) on the plasmid - pABBA_536.

[0265] For the list of strains, see Table 2. Table 2: Plasmids constructed to test whether C12 and C14 fatty alcohols can be produced in M. flagellatus, employing different TE and FAR combinations.

[0266] Example 4: Shaker-scale production assay set-up with overlay.

[0267] To test fatty alcohol and / or fatty acid production, the strains ABBAg_2, ABBAg_3, ABBAg_4, ABBAg_5, ABBAg_6, ABBAg_7, ABBAgJ, ABBAgJ 0, ABBAgJ 1, ABBAgJ 2, ABBAgJ 3, ABBAgJ 4, ABBAgJ 5, ABBAgJ 6, ABBAgJ 7, ABBAgJ 8, ABBAgJ 9, ABBAgJO, ABBAg_21, ABBAg_22, ABBAg_23, ABBAg_24, ABBAg_25, ABBAg_26, ABBAg_27, ABBAg_28, ABBAg_29, ABBAgJO, ABBAg_57, ABBAgJ 03, ABBAgJ 04, ABBAgJ 02,

[0268] ABBAgJ 46, ABBAgJ 05, ABBAgJ 33, ABBAgJ 35, ABBAg_209, ABBAg_226, ABBAg_231, ABBAg_539, ABBAg_745 and ABBAg_748 were cultivated in 250 mL shake flasks in a mineral medium containing methanol, KH2PO4, Na2HPO4, MgSO4, NH4SO4, and trace elements. A dodecane overlay (20 %VA / ) was used to extract the products during fermentation. The shake flasks were inoculated with 10 %V / liquid overnight culture. After 4 h of growth, 0.5 g / L IPTG was added to induce gene expression. After 24 hours 20 g / L of methanol was fed to the cultures.

[0269] The cultures were sampled after 48 h of growth. Fatty alcohol content in the dodecane overlay was measured by GC- FID. Example 5: Screening of FAR enzymes.

[0270] Strains ABBAg_2, ABBAg_3, ABBAg_4, ABBAg_5, ABBAg_6, ABBAg_7, ABBAg_8, ABBAg_10, ABBAg_11 , ABBAg_12, ABBAg_13, ABBAg_14, ABBAg_15, ABBAg_16, ABBAg_17, ABBAg_18, ABBAg_19, ABBAg_20, ABBAg_21 , ABBAg_22, ABBAg_23, ABBAg_24, ABBAg_25, ABBAg_26, ABBAg_27, ABBAg_28, ABBAg_29, ABBAg_30 and ABBAg_57 each containing one FAR enzyme were tested for fatty alcohol production. ABBAg_6 produced 12 mg / L C14 fatty alcohols while ABBAg_21 produced 4 mg / L C14 fatty alcohols. Only these two FARs were expressing 014 fatty alcohols only. 014 together with 016 fatty alcohol production was observed in strains ABBAg_30 (38 mg / L 014 fatty alcohols) and ABBAg_57 (56 mg / L 014 fatty alcohols). Notably, ABBAg_57 achieved by far the highest total fatty alcohol titer of all tested FAR enzymes, reaching 643 mg / L of total fatty alcohols. Under the same conditions, the control strain ABBAg_2, that did not contain the plasmid produced 0 mg / L 012 and 0 014 fatty alcohols (Figure 2).

[0271] Example 6: Screening of thioesterase enzymes in combination with FAR Onub

[0272] Titers of C12, C14 and C16 fatty acids produced from methanol by M. flagellatus ABBAg_2 heterologously expressing different TE enzymes in combination with FAR_Onu_5 (SEQ ID NO: 18). The results are shown in Figure 3.

[0273] Example 7: Effect of MAR and Onub FAR on C12 fatty acid production.

[0274] Surprisingly, the addition of the gene encoding for Far_Onu_5 (SEQ ID NO: 18) and Far_Mar (SEQ ID NO: 2) in the genome, increased C12 fatty acid titer to 182 mg / L (ABBAg_226) and 264 mg / L (ABBAg_231), compared to the strain containing a gene encoding for TE_Uca only (125 mg / L) (Fig. 4). When expressed alone, without a TE enzyme, these two enzymes produced no C12 fatty acids.

[0275] Example 8: Effect of inactivated FAR on C12 fatty acid production.

[0276] To test whether an active form of Far_Mar (SEQ ID NO: 2) is needed for its effect on fatty acid titer, a strain containing active Far_Mar (SEQ ID NO: 2) on the genome and strain containing an inactive FAR_Mar where the catalytic site was mutated (SEQ_ID_60) were transformed with TE_Uca (SEQ ID NO: 39) on the plasmid (ABBAg_745 and ABBAg_748, respectively). A codon-optimized version of the TE gene was used, which led to higher C12 fatty acid production. Strain ABBAg_745 produced 447 mg / L C12 fatty acids while ABBAg_748 produced 338 mg / L C12 fatty acids. Under the same conditions, the control strain ABBAg_539, that did not contain any form of FAR gene on the genome and contained only the plasmid expressing TE_Uca (SEQ ID NO: 39) produced 328 mg / L C12 fatty acids. The fact that only strain ABBAg_745 containing active form of Far_Mar increased fatty acid titer upon coexpression with TE_Uca (SEQ ID NO: 39) demonstrates, that an active form of FAR is needed to see the effect (Figure 5).

[0277] Example 9: Thioesterases truncation and preparation of expression constructs Expressing a thioesterase in a microorganism leads to secretion of C12 free fatty acids. In particular the thioesterase from Umbellularia californica (Uca_TE) in a methylotrophic host from the genus Methylobacillus has proven useful. The Uca_TE enzyme has been described before in literature to preferentially cleave C12 fatty acids from AGP, compared to other lengths, so this result was to be expected. It was also published that truncation of 30-50 amino acids at N- terminal demonstrates even higher enzyme activity. Thioesterases typically have an N-terminal region that includes a plastidial transit peptide, which is necessary for targeting the enzyme to plastids in plant cells but is not required (and can even be detrimental) for activity in bacterial expression systems. For this reason, we tried truncating Uca_TE at different N-terminal positions resulting in Uca_TE sequences of different length. To test different N-truncations of Uca_TE, 12 different N-truncations of Uca_TE gene (SEQ ID NOs:62-73) codon harmonized for expression in Methylobacillus flagellatus using a proprietary Python algorithm, each containing different N-teriminus have been cloned into the expression vector under IPTG-inducible lacO / Trc promoter using the NEB Hi-Fi cloning kit according to manufacturer instructions (pABBA_753, pABBA_752, pABBA_747, pABBA_746, pABBA_744, pABBA_743, pABBA_742, pABBA_741, pABBA_632, pABBA_732, pABBA_734, pABBA_737) as demonstrated in table 3. All plasmids carry the broad host range ori pBR322. Table 3: Plasmids constructed to express truncated TEJJca in M. flagellatus

[0278] Example 10: Preparation of strains. The prepared plasmids pABBA_753, pABBA_752, pABBA_747, pABBA_746, pABBA_744, pABBA_743, pABBA_742, pABBA_741 , pABBA_632, pABBA_732, pABBA_734 and pABBA_737 were checked by sequencing and transformed into M. flagellatus ABBAg_2 via electroporation to create strains ABBAg_975, ABBAg_948, ABBAg_940, ABBAg_929, ABBAg_928, ABBAg_936, ABBAg_933, ABBAg_932, ABBAg_786, ABBAg_968, ABBAg_945 and ABBAg_972. For the list of strains, see T able 4.

[0279] Table 4: Strains constructed to test different Uca_TE truncations

[0280] Example 11 : Shaker-scale production assay set-up with overlay

[0281] To test fatty acid production, the strains ABBAg_975, ABBAg_948, ABBAg_940, ABBAg_929, ABBAg_928, ABBAg_936, ABBAg_933, ABBAg_932, ABBAg_786, ABBAg_968, ABBAg_945 and ABBAg_972 were cultivated in 250 mL shake flasks in a mineral medium containing methanol, KH2PO4, Na2HPO4, MgSO4, NH4SO4, and trace elements. A dodecane overlay (20% VA / ) was used to extract the products during fermentation. The shake flasks were inoculated with 10% (V / V) liquid overnight culture. After 4 h of growth, two different concentrations of IPTG (0,05 mM and 0,5 mM) were added to induce gene expression. The cultures were sampled after 24 h of growth. Fatty alcohol content in the dodecane overlay was measured by GC-FID.

[0282] Example 12: Screening of different Uca_TE truncations

[0283] The strains ABBAg_975, ABBAg_948, ABBAg_940, ABBAg_929, ABBAg_928, ABBAg_936, ABBAg_933, ABBAg_932, ABBAg_786, ABBAg_968, ABBAg_945 and ABBAg_972 heterologously expressing N-terminally truncated Uca_TE sequences of different length were tested for fatty acid production. Two different concentrations of IPTG (0,05 mM and 0,5 mM) used to induce heterologous gene expression resulted in significant differences in product titers. However, at 0,05 mM concentration, all variants demonstrated high fatty acid production (and higher than that of non-truncated version - see ABBAg_539 in Table 2 and Example 6). The results are shown in Figure 6.

Claims

Claims1. Microorganism capable of producing at least one C6-C26 free fatty acid or a mixture thereof from methanol, said microorganism comprising at least one gene enabling the expression of a thioesterase (TE) and at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR).

2. The microorganism according to claim 1 , wherein the microorganism is a recombinant microorganism.

3. The microorganism according to claim 1 or 2, wherein the at least one gene enabling the expression of a thioesterase (TE) and / or the at least one gene enabling the expression of at least one Fatty Alcohol Reductase (FAR) is heterologously expressed by said microorganism.

4. The microorganism according to any one of the preceding claims wherein said microorganism provides for a higher production yield of the at least one C6-C26 free fatty acid or a mixture thereof, preferably an at least 10 % higher production yield, relative to the same microorganism that lacks one or both of the TE and the FAR genes, in particular to a microorganism that lacks the FAR gene.

5. The microorganism according to any one of the preceding claims, wherein said TE is from Umbellularia californica (Uca-TE).

6. The microorganism according to any one of the preceding claims, wherein(1) said TE is a polypeptide that has at least 80% sequence identity to SEQ ID NO:39, at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39; or(2) said TE is a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to sequence identity to any one of SEQ ID NOs:61-73.

7. The microorganism according to any of the preceding claims, wherein(1) said TE or Uca-TE comprises, consists essentially of or consists of SEQ ID NO:39; or(2) said TE or Uca-TE comprises, consists essentially of or consists of any one of SEQ ID NOs:61-73.

8. The microorganism according to any one of the preceding claims, wherein said at least one FAR is Marinobacter hydrocarbonoclasticus FAR (Mar-FAR) or Ostrinia nubiliatis FAR (Far_Onu_5).

9. The microorganism according to claim 8, wherein the FAR is a polypeptide having at least 80% sequence identity to SEQ ID NO:2, at least 85% sequence identity to SEQ ID NO:2, at least 90% sequence identity to SEQ ID NO:2, at least 95% sequence identity to SEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2.

10. The microorganism according to claim 8, wherein the FAR is a Far_Onu_5.11 . The microorganism according to claim 8 or 10, wherein the FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 14-19, at least 85% sequence identity to any one of SEQ ID NOs: 14-19, at least 90% sequence identity to any one of SEQ ID NOs: 14-19, at least 95% sequence identity to any one of SEQ ID NOs: 14-19, or at least 98% sequence identity to any one of SEQ ID NOs: 14-19.

12. The microorganism according to any one of the preceding claims, wherein (1) said TE is a polypeptide that has at least 80% sequence identity to SEQ ID NO:39, at least 85% sequence identity to SEQ ID NO:39, at least 90% sequence identity to SEQ ID NO:39, at least 95% sequence identity to SEQ ID NO:39, or at least 98% sequence identity to SEQ ID NO:39; and (2) said FAR is a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 85% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 90% sequence identity to any one of SEQ ID NOs:2 and 14-19, at least 95% sequence identity to any one of SEQ ID NOs:2 and 14- 19, or at least 98% sequence identity to any one of SEQ ID NOs:2 and 14-19.

13. The microorganism according to any of the preceding claims, wherein said at least one C6-C26 free fatty acid or mixture thereof comprises at least one C12, C14 or C16 free fatty acid.

14. The microorganism according to any of the preceding claims, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises dodecanoic acid (lauric acid).

15. The microorganism according to any one of the preceding claims, wherein said at least one C6-C26 free fatty acid or a mixture thereof comprises 1 -tetradecanoic acid (myristic acid).

16. The microorganism according to any one of the preceding claims, wherein said microorganism is a bacterium, in particular Methylobacillus, in particular Methylobacillus flagellatus or Methylobacillus glycogenes.

17. A method for the manufacture of a least one C6-C26 free fatty acid or a mixture thereof comprising growing a microorganism as defined in any one of claims 1 to 16 in a suitable medium, and isolation of said at least one C6-C26 free fatty acid or a mixture thereof from the spent medium.

18. The method according to claim 17, wherein said at least one C6-C26 free fatty acid is C12, C14 or C16 free fatty acid, preferably dodecanoic acid.

19. A free fatty acid composition obtained by the method according to claim 17 or 18.

20. A method for the manufacture of at least one C6-C26 free fatty alcohol comprising growing a microorganism as defined in any one of claims 1 to 16 in a suitable medium, optionally isolating at least one C6-C26 free fatty acid from the spent medium, conversion of the C6-C26 free fatty acid into the corresponding C6-C26 free fatty alcohol, and isolation of said C6-C26 free fatty alcohol.21 . The method according to claim 20, wherein said C6-C26 free fatty alcohol is 1-dodecanol (lauryl alcohol).

22. A free fatty alcohol composition obtained by the method according to claim 20 or 21.

23. Use of a microorganism as defined in any one of claims 1 to 16 for the manufacture of a C6-C26 free fatty acid or a mixture thereof, or a C6-C26 free fatty alcohol or a mixture thereof.

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