Microorganisms for producing insect pheromones and related compounds

Abstract

The present invention relates to a method for biosynthesizing unsaturated C-terminal polyols which can be used as insect pheromones, spices, flavoring agents and polymer intermediates. 6 ‑C 24 The recombinant microorganism can express the recombinant microorganism for producing unsaturated C 6 ‑C 24 The enzymes or enzyme variants that produce fatty alcohols, fatty aldehydes, and fatty acetates and / or can be modified to down-regulate the pathway to divert production toward unsaturated C 6 ‑C 24 Fatty alcohols, fatty aldehydes and fatty acetates. 6 ‑C 24 Fatty alcohols, fatty aldehydes and fatty acetates can be used as substrates for metathesis reactions to expand the library of target compounds and pheromones. Also provided is a method for producing unsaturated C 6 ‑C 24 Methods for producing fatty alcohols, fatty aldehydes and fatty acetates, and compositions comprising the recombinant microorganisms and / or optionally one or more of the product alcohols, aldehydes or acetates.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 507,654, filed May 17, 2017, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated into this specification by reference. The name of the text file containing the sequence listing is PRVI_020_01WO_SeqList_ST25.txt. The text file is approximately 240KB, was created on May 17, 2018, and was submitted electronically via EFS-Web. Technical Field

[0005] This application relates to the use of unsaturated C6-C 24 The present invention also relates to a recombinant microorganism for the biosynthesis of fatty alcohols, fatty aldehydes and fatty acetates, which can be used as insect pheromones, spices, flavorings and polymer intermediates. 24 Methods for producing fatty alcohols, fatty aldehydes, and fatty acetates, and compositions comprising one or more of these compounds and / or recombinant microorganisms. Background of the Invention

[0007] As global demand for food grows, the need for effective pest control is increasing. Conventional insecticides are one of the most popular chemical control agents because they are readily available, fast-acting, and highly reliable. However, overuse, misuse, and abuse of these chemicals have led to resistant pests, changes in natural ecosystems, and, in some cases, environmental damage.

[0008] The use of insect pheromones to control pest populations is gaining popularity as a viable, safe, and environmentally friendly alternative to conventional insecticides. Since their discovery in the late 1950s, these molecules have demonstrated efficacy in reducing insect populations through a variety of methods, including mass capture, attract-and-kill, and mating disruption. The latter approach, in particular, represents a non-toxic means of pest control and exploits the ability of synthetic pheromones to mask naturally occurring pheromones, causing confusion and mating disruption.

[0009] Although pheromones have significant potential for agricultural insect control, the cost of synthesizing pheromones using currently available technologies is very high, which prevents the widespread use of this sustainable technology outside of high-value crops. Therefore, there is a need to develop new technologies for the cost-effective production of insect pheromones and related fragrances, flavorings, and polymer intermediates. The present inventors have developed a novel method that can produce a wide range of unsaturated C6-C8 pheromones from low-cost raw materials. 24 Recombinant microorganisms that produce fatty alcohols, fatty aldehydes, and fatty acetates (including synthetic insect pheromones) address this need. SUMMARY OF THE INVENTION

[0011] The present application relates to a method for producing an unsaturated C6-C 24 The recombinant microorganisms described herein can be used to produce a biosynthetic pathway for one or more compounds selected from unsaturated C6-C 24 At least one compound of fatty alcohol, fatty aldehyde and fatty acetate, such as an insect pheromone, fragrance or flavoring.

[0012] In one embodiment, the recombinant microorganism comprises a method for producing an unsaturated C6-C 24 Therefore, in the first aspect, the present application relates to a recombinant microorganism capable of synthesizing a saturated C6-C 24 Endogenous or exogenous sources of fatty acyl-CoA generate unsaturated C6-C 24 Fatty aldehydes or fatty alcohols, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty acyl-CoA; and (b): at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty aldehyde, the fatty acyl reductase that catalyzes the conversion of the mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty aldehydes are insect pheromones. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty aldehydes are spices or flavorings. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24(c) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase to form a fatty alcohol, the fatty acyl reductase to form a fatty alcohol catalyzing the conversion of the mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are insect pheromones. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are spices or flavorings. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of a mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty acetate.

[0013] In some embodiments, the fatty acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0014] In some embodiments, the fatty acyl desaturase is capable of generating a double bond at the C5, C6, C7, C8, C9, C10, C11, C12, or C13 position of a fatty acid or a derivative thereof, such as, for example, a fatty acid CoA ester.

[0015] In an exemplary embodiment, the fatty acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase or the nucleic acid sequence encoding it can be isolated from an organism of the species Agrotissegetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosirapseudonana. Additional Z11 desaturases or nucleic acid sequences encoding them can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutellaxylostella, Bombyx mori, or Diaphania nitidalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase is codon-optimized and is derived from an organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosirapseudonana. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 18, 24, and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni.In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12, 17, and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 27, and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in GenBank Accession Nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, and NP001299594.1, Q9N9Z8, ABX71630.1, and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, all or part of the Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase is replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 28, and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 78, 79, and 80.

[0016] In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA to a mono- or di-unsaturated product selected from the group consisting of: Z11-13: acyl-CoA, E11-13: acyl-CoA, (Z,Z)-7,11-13: acyl-CoA, Z11-14: acyl-CoA, E11-14: acyl-CoA, (E,E)-9,11-14: acyl-CoA, (E,Z)-9,11-14: acyl-CoA, (Z,E)-9,11-14: acyl- CoA, (Z,Z)-9,11-14: acyl-CoA, (E,Z)-9,11-15: acyl-CoA, (Z,Z)-9,11-15: acyl-CoA, Z11-16: acyl-CoA, E11-16: acyl-CoA, (E,Z)-6,11-16: acyl-CoA, (E,Z)-7,11-16: acyl-CoA, (E,Z)-8,11-16: acyl-CoA, (E,E)-9,11-16: acyl-CoA, (E ,Z)-9,11-16: acyl-CoA, (Z,E)-9,11-16: acyl-CoA, (Z,Z)-9,11-16: acyl-CoA, (E,E)-11,13-16: acyl-CoA, (E,Z)-11,13-16: acyl-CoA, (Z,E)-11,13-16: acyl-CoA, (Z,Z)-11,13-16: acyl-CoA, (Z,E)-11,14-16: acyl-CoA, (E,E,Z)-4, 6,11-16: acyl-CoA, (Z,Z,E)-7,11,13-16: acyl-CoA, (E,E,Z,Z)-4,6,11,13-16: acyl-CoA, Z11-17: acyl-CoA, (Z,Z)-8,11-17: acyl-CoA, Z11-18: acyl-CoA, E11-18: acyl-CoA, (Z,Z)-11,13-18: acyl-CoA, (E,E)-11,14-18: acyl-CoA or a combination thereof.

[0017] In another exemplary embodiment, the fatty acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase or the nucleic acid sequence encoding it can be isolated from an organism of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In an exemplary embodiment, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon-optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence listed in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence listed in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence listed in SEQ ID NO: 60 from Helicoverpa zea. Other Z9 desaturases of the present disclosure include SEQ ID Nos: 95, 97, 99, 101, 103, and 105. In some embodiments, overexpression of a Z9-18 specific desaturase can increase membrane fluidity to improve diffusion of fatty alcohols into the supernatant.

[0018] In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA to a monounsaturated or polyunsaturated product selected from the group consisting of: Z9-11: acyl-CoA, Z9-12: acyl-CoA, E9-12: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, (Z,E)-7,9-12: acyl-CoA, (Z,Z)-7,9-12: acyl-CoA, Z9-13: acyl-CoA, E9-13: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, (Z,Z)-7,9-12: acyl-CoA, Z9-14: acyl-CoA, E9-14: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, Z)-5,9-13: acyl-CoA, (Z,E)-5,9-13: acyl-CoA, (Z,Z)-5,9-13: acyl-CoA, Z9-14: acyl-CoA, E9-14: acyl-CoA, (E,Z)-4,9-14: acyl-CoA, (E,E)-9,11-14: acyl-CoA, (E,Z)-9,11-14: acyl-CoA, (Z,E)-9,11-14: acyl-CoA, (Z,Z)-9,11-14: acyl-CoA, (E,E)-9,1 2-14: acyl-CoA, (Z,E)-9,12-14: acyl-CoA, (Z,Z)-9,12-14: acyl-CoA, Z9-15: acyl-CoA, E9-15: acyl-CoA, (Z,Z)-6,9-15: acyl-CoA, Z9-16: acyl-CoA, E9-16: acyl-CoA, (E,E)-9,11-16: acyl-CoA, (E,Z)-9,11-16: acyl-CoA, (Z,E)-9,11-16: acyl-CoA, (Z,Z)- 9,11-16: acyl-CoA, Z9-17: acyl-CoA, E9-18: acyl-CoA, Z9-18: acyl-CoA, (E,E)-5,9-18: acyl-CoA, (E,E)-9,12-18: acyl-CoA, (Z,Z)-9,12-18: acyl-CoA, (Z,Z,Z)-3,6,9-18: acyl-CoA, (E,E,E)-9,12,15-18: acyl-CoA, (Z,Z,Z)-9,12,15-18: acyl-CoA or a combination thereof.

[0019] In some embodiments, the recombinant microorganism can express a bifunctional desaturase capable of catalyzing the subsequent desaturation of two double bonds.

[0020] In some embodiments, the recombinant microorganism can express more than one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the desaturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24Fatty acyl-CoA. For example, the recombinant microorganism can express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.

[0021] In some embodiments, the recombinant microorganism can express a fatty acyl conjugating enzyme that functions independently or in conjunction with a fatty acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.

[0022] In one embodiment, the present disclosure provides a recombinant microorganism capable of converting saturated or monounsaturated C6-C 24 Endogenous or exogenous sources of fatty acyl-CoA generate polyunsaturated C6-C 24 Fatty aldehydes or fatty alcohols, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugating enzyme that catalyzes the conversion of a saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty aldehyde or fatty alcohol, the fatty acyl reductase that forms a fatty alcohol catalyzes the conversion of a polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty aldehydes or fatty alcohols.

[0023] In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty acyl conjugating enzymes that catalyze the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA.

[0024] In another embodiment, the present disclosure provides a recombinant microorganism capable of converting saturated or monounsaturated C6-C 24 Endogenous or exogenous sources of fatty acyl-CoA generate polyunsaturated C6-C 24 Fatty alcohols, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugating enzyme, the fatty acyl desaturase and the fatty acyl conjugating enzyme catalyzing the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24Fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, the fatty acyl reductase that catalyzes the conversion of a polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty alcohol.

[0025] In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty acyl conjugating enzymes that catalyze the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA.

[0026] In yet another embodiment, the fatty acyl conjugating enzyme is one that is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0027] In certain embodiments, the conjugating enzyme or the nucleic acid sequence encoding it can be isolated from the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowii, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana). In an exemplary embodiment, the conjugating enzyme comprises the following sequence, which is selected from GenBank Accession Nos. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

[0028] In various embodiments described herein, the fatty alcohol-forming acyl-CoA reductase, i.e., the fatty alcohol-forming fatty acyl reductase or the nucleic acid sequence encoding it, can be isolated from an organism of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus. In exemplary embodiments, the reductase comprises a sequence selected from GenBank Accession Nos. JX679210 and HG423128 and UniProt Accession No. I3PN86. In some embodiments, the nucleic acid sequence encoding the fatty acyl reductase is codon-optimized and is derived from an organism of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus. In some embodiments, the reductase comprises the nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the reductase comprises the nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the reductase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 32, 40, 72, 74, 76, and 81. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis.In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.

[0029] In some embodiments, the present disclosure teaches the use of multiple fatty acyl reductases. In some embodiments, the present disclosure teaches recombinant microorganisms comprising multiple copies of the same fatty acyl reductase. In other embodiments, the present disclosure teaches recombinant microorganisms comprising two or more different fatty acyl reductases. In some embodiments, the different fatty acyl reductases utilize different cofactors. For example, the fatty acyl reductase from Euglena gracilis (SEQ ID NO: 75) uses NADH as a reducing equivalent, rather than NADPH. In some embodiments, this allows for cofactor balancing using two or more different reductases.

[0030] In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.

[0031] In certain embodiments, the fatty alcohol-forming fatty acyl reductase catalyzes the conversion of a mono- or polyunsaturated fatty acyl-CoA to a fatty alcohol product selected from the group consisting of: (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (E)-9-dodecenol, (Z)-9-tetradecenol, (E)-9-tetradecenol, (Z)-9-hexadecenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-hexadecenol, (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or a combination thereof.

[0032] In some embodiments, the recombinant microorganism can express more than one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA to the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.

[0033] In additional embodiments, the present disclosure provides recombinant microorganisms capable of producing mono- or polyunsaturated ≤C18 fatty alcohols from saturated C6-C24 fatty acids from an endogenous or exogenous source, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or polyunsaturated C6-C24 fatty acyl-CoA. The mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) is converted into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA, wherein a given cycle produces a mono- or poly-unsaturated C4-C22 fatty acyl-CoA intermediate having a two-carbon truncation relative to the starting mono- or poly-unsaturated C6-C24 fatty acyl-CoA substrate in the cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from the group consisting of fatty acyl desaturases derived from red banded moth, Spodoptera litura, Sesamia inferens, Manduca hornworm, European corn borer, Zea mays, rose leaf roller, Drosophila melanogaster, Spodoptera litura, Red currant borer, Navel orange borer, Trichoplusia ni, Agrotissima serrata, Asian corn borer, and Thalassiosira pseudonana. In some embodiments, the fatty acyl desaturase is selected from the group consisting of SEQ ID NO:39, 49-54, 58-63, 78-80 and GenBank accession numbers AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1 have a fatty acyl desaturase with at least 99%, 98%, 97% or more of the fatty acyl desaturase. %, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity.In some embodiments, the acyl Co-A oxidase is selected from Table 5a. In other embodiments, the fatty alcohol-forming fatty acyl reductase is selected from the group consisting of fatty alcohol-forming fatty acyl reductases derived from Agrotissima exigua, Spodoptera litura, Euglena gracilis, Prunella vulgaris, and Helicoverpa armigera. In further embodiments, the fatty alcohol-forming fatty acyl reductase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% of the fatty alcohol-forming fatty acyl reductase selected from SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77, and 82. %, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity. In some embodiments, the recombinant microorganism is a yeast selected from Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswinii.

[0034] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C18 fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT), and diacylglycerol acyltransferase (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.

[0035] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, >C14, >C12, or >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.

[0036] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes that catalyze the production of mono- or polyunsaturated ≤C 18In another embodiment, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases; (ii) one or more acyltransferases; (iii) one or more acylglycerol lipases and / or sterol ester esterases; (iv) one or more (fatty) alcohol dehydrogenases; (v) one or more (fatty) alcohol oxidases; and (vi) one or more cytochrome P450 monooxygenases.

[0037] In some preferred embodiments, one or more genes encoding acyl-CoA oxidases of the microbial host are deleted or down-regulated to eliminate or reduce truncation of the desired fatty acyl-CoA beyond the desired chain length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acyl-CoA oxidases selected from the group consisting of Yarrowia lipolytica POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E27664g), Yarrowia lipolytica POX7 (YALI0E27665g), Yarrowia lipolytica POX8 (YALI0E276664g), Yarrowia lipolytica POX9 (YALI0E276665g), Yarrowia lipolytica POX10 (YALI0E276665g), Yarrowia lipolytica POX11 (YALI0E276665g), Yarrowia lipolytica POX12 (YALI0E276665g), Yarrowia lipolytica POX13 (YALI0E276665g), Yarrowia lipolytica POX14 (YALI0E276665g), Yarrowia lipolytica POX15 (YALI0E276665g), Yarrowia lipolytica POX16 (YALI0E276665g), Yarrowia lipoly Saccharomyces cerevisiae POX6 (YALI0E06567g); Saccharomyces cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160) and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

[0038] In some embodiments, a recombinant microorganism is provided that is capable of producing mono- or polyunsaturated ≤C18 fatty alcohols, fatty aldehydes, and / or fatty acetates from saturated C6-C24 fatty acids of endogenous or exogenous origin, wherein the recombinant microorganism expresses one or more acyl-CoA oxidases, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous acyl-CoA oxidases. In some embodiments, the one or more acyl-CoA oxidases expressed are different from the one or more endogenous acyl-CoA oxidases that are deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidases expressed regulate the production of mono- or polyunsaturated ≤C18 fatty alcohols, fatty aldehydes, and / or fatty acetates. 18Chain length of fatty alcohol, fatty aldehyde and / or fatty acetate. In other embodiments, the one or more acyl-CoA oxidases expressed are selected from Table 5a.

[0039] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acyltransferases selected from the group consisting of Yarrowia lipolytica YALI0C00209g, Yarrowia lipolytica YALI0E18964g, Yarrowia lipolytica YALI0F19514g, Yarrowia lipolytica YALI0C14014g, Yarrowia lipolytica YALI0E16797g, Yarrowia lipolytica YALI0E32769g, and Yarrowia lipolytica YALI0D07986g, Saccharomyces cerevisiae YBL011w, Saccharomyces cerevisiae YDL052c, Saccharomyces cerevisiae YOR175C, Saccharomyces cerevisiae YPR139C, Saccharomyces cerevisiae YNR008w, and Saccharomyces cerevisiae YOR245 c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203 and Candida CTRG_06209.

[0040] In some embodiments, saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 A recombinant microorganism of a fatty alcohol, fatty aldehyde and / or fatty acetate, wherein the recombinant microorganism expresses one or more acyltransferases, and wherein the recombinant microorganism is manipulated to delete, destroy, mutate one or more endogenous acyltransferases and / or reduce the activity of one or more endogenous acyltransferases. In some preferred embodiments, one or more genes encoding GPAT, LPAAT, GPLAT and / or DGAT of the microbial host are deleted or downregulated and replaced by one or more GPAT, LPAAT, GPLAT or DGAT that preferably stores short-chain fatty acyl-CoA. In some embodiments, the one or more acyltransferases expressed are different from the one or more endogenous acyltransferases deleted or downregulated. In other embodiments, the one or more acyltransferases expressed are selected from Table 5b.

[0041] In some preferred embodiments, one or more genes encoding acylglycerol lipase (mono-, di-, or triacylglycerol lipase) and sterol ester esterase in the microbial host are deleted or down-regulated and replaced with one or more acylglycerol lipases that prefer long-chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acylglycerol lipases and / or sterol ester esterases selected from the group consisting of Yarrowia lipolytica YALI0E32035g, Yarrowia lipolytica YALI0D17534g, Yarrowia lipolytica YALI0F10010g, Yarrowia lipolytica YALI0C14520g, and Yarrowia lipolytica YALI0E00528g, Saccharomyces cerevisiae YKL140w, Saccharomyces cerevisiae YMR313c, Saccharomyces cerevisiae YKR089c, Saccharomyces cerevisiae YOR081c, Saccharomyces cerevisiae YKL094W, Saccharomyces cerevisiae YLL012W, and Saccharomyces cerevisiae YLR020C, and Candida sp. CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683 and Candida CTRG_04630.

[0042] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Yarrowia lipolytica YALI0E25982g (ALK1), Yarrowia lipolytica YALI0F01320g (ALK2), Yarrowia lipolytica YALI0E23474g (ALK3), Yarrowia lipolytica YALI0B13816g (ALK4), Yarrowia lipolytica YALI0B1383 8g (ALK5), Yarrowia lipolytica YALI0B01848g (ALK6), Yarrowia lipolytica YALI0A15488g (ALK7), Yarrowia lipolytica YALI0C12122g (ALK8), Yarrowia lipolytica YAL IOB06248g (ALK9), Yarrowia lipolytica YALI0B20702g (ALK10), Yarrowia lipolytica YALIOC10054g (ALK11) Yarrowia lipolytica YALI0A20130g (ALK12).

[0043] In some embodiments, saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 A recombinant microorganism of a fatty alcohol, fatty aldehyde and / or fatty acetate, wherein the recombinant microorganism expresses one or more acylglycerol lipases and / or sterol ester esterases, and wherein the recombinant microorganism is manipulated to delete, destroy, mutate one or more endogenous acylglycerol lipases and / or sterol ester esterases and / or reduce the activity of one or more endogenous acylglycerol lipases and / or sterol ester esterases. In some embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are different from the one or more endogenous acylglycerol lipases and / or sterol ester esterases deleted or lowered. In some embodiments, the one or more endogenous or exogenous acylglycerol lipases and / or sterol ester esterases expressed preferably hydrolyze the ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are selected from Table 5c.

[0044] In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from the group consisting of E5-10: acyl-CoA, E7-12: acyl-CoA, E9-14: acyl-CoA, E11-16: acyl-CoA, E13-18: acyl-CoA, Z7-12: acyl-CoA, Z9-14: acyl-CoA, Z11-16: acyl-CoA, Z13-18: acyl-CoA, Z8-12: acyl-CoA, Z 10-14: acyl-CoA, Z12-16: acyl-CoA, Z14-18: acyl-CoA, Z7-10: acyl-CoA, Z9-12: acyl-CoA, Z11-14: acyl-CoA, Z13-16: acyl-CoA, Z15-18: acyl-CoA, E7-10: acyl-CoA, E9-12: acyl-CoA, E11-14: acyl-CoA, E13-16: acyl-CoA, E15-18: acyl-CoA, E5Z7-12: acyl- CoA, E7Z9-12: acyl-CoA, E9Z11-14: acyl-CoA, E11Z13-16: acyl-CoA, E13Z15-18: acyl-CoA, E6E8-10: acyl-CoA, E8E10-12: acyl-CoA, E10E12-14: acyl-CoA, E12E14-16: acyl-CoA, Z5E8-10: acyl-CoA, Z7E10-12: acyl-CoA, Z9E12-14: acyl-CoA, Z11E14- 16: acyl-CoA, Z13E16-18: acyl-CoA, Z3-10: acyl-CoA, Z5-12: acyl-CoA, Z7-14: acyl-CoA, Z9-16: acyl-CoA, Z11-18: acyl-CoA, Z3Z5-10: acyl-CoA, Z5Z7-12: acyl-CoA, Z7Z9-14: acyl-CoA, Z9Z11-16: acyl-CoA, Z11Z13-16: acyl-CoA and Z13Z15-18: acyl-CoA. In another embodiment, the mono- or polyunsaturated ≤C 18 The fatty alcohol is selected from E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH and Z13-18:OH.

[0045] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde-forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or polyunsaturated ≤C 18 Fatty acids are converted into corresponding ≤C 18 Fatty aldehyde. In some preferred embodiments, the aldehyde-forming fatty acyl-CoA reductase is selected from Acinetobacter calcoaceticus A0A1C4HN78, Acinetobacter calcoaceticus N9DA85, Acinetobacter calcoaceticus R8XW24, Acinetobacter calcoaceticus A0A1A0GGM5, ​​Acinetobacter calcoaceticus A0A117N158 and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 In some preferred embodiments, the ≤C 18 The fatty aldehyde is selected from Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0046] In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from alcohol oxidase and alcohol dehydrogenase, wherein the alcohol oxidase and alcohol dehydrogenase are capable of catalyzing the mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 Fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of the mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 In some preferred embodiments, the mono- or polyunsaturated fatty acid esters are 18 Fatty aldehydes and ≤C 18 The fatty acetate is selected from E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0047] In some embodiments, the present disclosure provides for engineering saturated C6-C24 Fatty acids produce mono- or polyunsaturated ≤C 18 A method for producing fatty alcohols in a microorganism, wherein the method comprises introducing into the microorganism: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the oxidation of a mono- or polyunsaturated C6-C6 fatty acyl-CoA fatty acid from (a) after one or more consecutive cycles of acyl-CoA oxidase activity. 24 Fatty acyl-CoA is converted to mono- or polyunsaturated ≤C18 fatty acyl-CoA, where a given cycle produces ≤C18 fatty acyl-CoA relative to the starting mono- or polyunsaturated C6-C 24 Fatty acyl-CoA substrates have two carbon-truncated mono- or polyunsaturated C4-C 22 fatty acyl-CoA intermediate; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of the mono- or polyunsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or polyunsaturated ≤C 18 In some embodiments, the microorganism is MATA ura3-302::SUC2Δpox1Δpox2Δpox3Δpox4Δpox5Δpox6ΔfadhΔadh1Δadh2Δadh3Δadh4Δadh5Δadh6Δadh7Δfao1::URA3.

[0048] In some embodiments, the present disclosure provides saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 The method of producing fatty alcohol, fatty aldehyde or fatty acetate comprises: culturing the recombinant microorganism described herein in a culture medium containing a raw material, the raw material providing a sufficient amount of the mono- or polyunsaturated ≤C 18 In some embodiments, the method further comprises recovering the mono- or polyunsaturated ≤C 18 In another embodiment, the step of recovering the fatty alcohol, fatty aldehyde or fatty acetate comprises a step of recovering the fatty alcohol, fatty aldehyde or fatty acetate. ...

[0049] In some embodiments, chemical methods are used to convert mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18In other embodiments, the chemical method is selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the chemical method is used to convert the mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 In another embodiment, the chemical method uses a chemical agent selected from acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propionyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to convert the mono- or polyunsaturated ≤C 18 Fatty alcohols are esterified to the corresponding ≤C 18 Fatty acetate.

[0050] In other embodiments, the present disclosure provides saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 A recombinant Yarrowia lipolytica microorganism producing a fatty alcohol, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 74%, 75%, 76%, 77%, 78%, 79 ... 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity; and (b) at least one nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of a mono- or polyunsaturated C6-C8 fatty acid to a mono- or polyunsaturated C6-C8 fatty acid from (a). 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 The invention relates to a fatty alcohol-forming fatty acyl reductase having 95% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75, and 77.

[0051] In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes that catalyze the production of mono- or polyunsaturated C6-C 24In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), and YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenases selected from the group consisting of: (ii) (fatty) alcohol oxidase YALI0B14014g (FAO1); (iii) one or more cytochrome P450 enzymes selected from the group consisting of YALI0E2, YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iv) one or more cytochrome P450 enzymes selected from the group consisting of YALI0E2 5982g(ALK1), YALI0F01320g(ALK2), YALI0E23474g(ALK3), YALI0B13816g(ALK4), YALI0B13838g(ALK5), YALI0B01848g(ALK6), YALI0A15488g(ALK7), (YALI0C12122g(ALK8), YALI0B06248g(ALK9), YALI0B20702g(ALK10), YALI0C10054g(ALK11) and YALI0A2013 Og (Alk12); YAS1 (YALI1C03349), Yas2 (YALI0E32417), Gsyl1 (YALI0F18502), HFD1 (YALI0F23793), HFD2 (YALI0E15400), HFD3 (YALI0A17875), HFD4 (YALI0B01298), SDR (YALI0A19536); and (v) one or more diacylglycerol acyltransferases selected from YALI0E32791g (DGA1) and YALI0D07986g (DGA2).In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6), YALI0E06567g (POX7), YALI0E06567g (POX8), YALI0E06567g (POX9), YALI0E06567g (POX10), YALI0E06567g (POX11), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6), YALI0E06567g (POX7), YALI0E06567g (POX8), YALI0E06567g (POX9), YALI0E06567g (POX12), YALI0F10857g (POX2), YALI0D24750g (POX3), (POX6); (ii) one or more (fatty) alcohol dehydrogenases selected from YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7), YALI0E15818g (ADH8), YALI0D02167g (ADH9), YALI0E15818g (ADH10), YALI0E15818g (ADH11), YALI0E15818g (ADH12), YALI0E15818g (ADH13), YALI0E15818g (ADH14), YALI0E15818g (ADH15), YALI0E15818g (ADH16), YALI0E15818g (ADH17), YALI0E15818g (ADH18), YALI0E15818g (ADH19), YALI0E15818g (ADH19), YALI0E15818g (ADH1 H7); (iii) (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzymes selected from YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YAL I0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferases selected from YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

[0052] In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from the group consisting of Z9-14: acyl-CoA, Z11-14: acyl-CoA, E11-14: acyl-CoA, Z9-16: acyl-CoA, and Z11-16: acyl-CoA. In other embodiments, the mono- or poly-unsaturated C6-C 24 The fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH and Z13-18:OH.

[0053] In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C6-C 24 The fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

[0054] In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 and at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty aldehydes and C6-C 24 The fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0055] In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66, and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from a desaturase derived from Aurochs borer, Spodoptera tinctoria, Agrotis cinerea, or Trichoplusia ni.

[0056] In some embodiments, the present disclosure provides an engineered saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24A method for producing fatty alcohols from a Yarrowia lipolytica microorganism, wherein the method comprises introducing into the Yarrowia lipolytica microorganism: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 a fatty acyl-CoA having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 74%, 75%, 76%, 77%, 78%, 79 ... %, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity; and (b) at least one nucleic acid molecule encoding a fatty acyl reductase to form a fatty alcohol that catalyzes the conversion of a mono- or polyunsaturated C6-C8 fatty acid to a mono- or polyunsaturated C6-C8 fatty acid from (a). 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 A fatty alcohol, the fatty alcohol-forming fatty acyl reductase having 95% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 55, 56, 57, 73, 75, and 77. In some embodiments, the microorganism is MATAura3-302::SUC2Δpox1Δpox2Δpox3Δpox4Δpox5Δpox6ΔfadhΔadh1Δadh2Δadh3Δadh4Δadh5Δadh6Δadh7Δfao1::URA3.

[0057] In some embodiments, the present disclosure provides saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 The method comprises: culturing the recombinant microorganism described herein in a culture medium containing a raw material, the raw material providing a sufficient amount of the mono- or polyunsaturated C6-C 24 In some embodiments, the method further comprises recovering the mono- or polyunsaturated C6-C 24In another embodiment, the step of recovering the fatty alcohol, fatty aldehyde or fatty acetate comprises a step of recovering the fatty alcohol, fatty aldehyde or fatty acetate. ...

[0058] In some embodiments, chemical methods are used to convert the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In other embodiments, the chemical method is selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In another embodiment, the chemical method uses a chemical agent selected from acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propionyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to convert the mono- or polyunsaturated C6-C8 fatty acid ester into fatty acid ester. 24 Esterification of fatty alcohols to the corresponding C6-C 24 Fatty acetate.

[0059] In addition to the biosynthetic pathway described in the first aspect above, the present application also provides a method for utilizing the biosynthetic pathway derived from C6-C 24 Saturated C6-C 24 Fatty acyl-ACP intermediates generate unsaturated C6-C 24 Therefore, in a second aspect, the present application relates to a recombinant microorganism capable of synthesizing fatty alcohols from C6-C 24 Fatty acids produce unsaturated C6-C 24 Fatty alcohols, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of C6-C 24 Fatty acids are converted to the corresponding saturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding a fatty acyl-ACP desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or polyunsaturated C6-C 24 The fatty acyl-ACP is converted to the corresponding mono- or polyunsaturated C6-C ... 24Fatty acyl-ACP; (d): at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty aldehyde, the fatty acyl reductase that catalyzes the conversion of a mono- or polyunsaturated C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of a mono- or polyunsaturated C6-C 24 Fatty aldehyde C6-C 24 Converted into the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are insect pheromones. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are spices or flavorings. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of a mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty acetate.

[0060] In some embodiments, the acyl-ACP synthetase is a synthetase capable of utilizing as a substrate a fatty acid having a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0061] In various embodiments described herein, the acyl-ACP synthetase or nucleic acid encoding it can be isolated from an organism of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.

[0062] In some embodiments, the fatty acyl-ACP desaturase is a soluble desaturase. In various embodiments described herein, the fatty acyl-ACP desaturase or nucleic acid encoding it can be isolated from an organism of the species Pelargonium hortorum, Asclepias syriaca, or Uncariatomentosa.

[0063] In some embodiments, the recombinant microorganism can express more than one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the desaturation of C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty acyl-ACP.

[0064] As described above, fatty acid elongases (ie, fatty acid synthase complexes) can be used to convert mono- or polyunsaturated C6-C 24 The chain length of the fatty acyl-ACP is extended by two additional carbons at the α-carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA. In one embodiment, the one or more nucleic acid molecules encoding the fatty acid synthase complex are endogenous nucleic acid molecules, i.e., the one or more nucleic acid molecules are native to the recombinant microorganism. In another embodiment, the one or more nucleic acid molecules encoding the fatty acid synthase complex are exogenous nucleic acid molecules.

[0065] In various embodiments described herein, the fatty aldehyde-forming acyl-ACP reductase (i.e., fatty aldehyde-forming fatty acyl reductase) or the nucleic acid sequence encoding the same can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa.

[0066] As described above, the recombinant microorganism according to the second aspect comprises at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase capable of catalyzing the mono- or polyunsaturated C6-C 24 Fatty aldehydes are converted into the corresponding mono- or polyunsaturated C6-C 24Fatty alcohols. In one embodiment, the dehydrogenase is encoded by an endogenous nucleic acid molecule. In another embodiment, the dehydrogenase is encoded by an exogenous nucleic acid molecule. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding the dehydrogenase is isolated from an organism of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.

[0067] In addition to the biosynthetic pathways described in the first and second aspects, the present application also provides a method for utilizing the biosynthetic pathways derived from C6-C 24 Saturated C6-C 24 Fatty acyl-ACP intermediates generate unsaturated C6-C 24 Therefore, in a third aspect, the present application relates to a recombinant microorganism capable of synthesizing fatty alcohols from endogenous or exogenous C6-C 24 Fatty acids produce unsaturated C6-C 24 Fatty alcohols, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of C6-C 24 Fatty acids are converted to the corresponding saturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding a fatty acyl-ACP desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 (c) at least one exogenous fatty acyl-ACP thioesterase that catalyzes the conversion of the mono- or polyunsaturated C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 (d) one or more endogenous or exogenous nucleic acid molecules encoding an elongase that catalyzes the elongation from the mono- or polyunsaturated C6-C 24 Mono- or polyunsaturated C6-C 24 The fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C ... 24 Fatty acyl-CoA; and (e): at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, the fatty acyl reductase catalyzing the conversion of the mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are insect pheromones. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are spices or flavorings. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of a mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty acetate

[0068] In some embodiments according to this third aspect, fatty acyl-ACP thioesterases can be used to convert mono- or polyunsaturated C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty acids. In some embodiments, soluble fatty acyl-ACP thioesterases can be used to release free fatty acids for reactivation to CoA thioesters. Fatty acyl-ACP thioesterases that can be included in the embodiments include, but are not limited to, Q41635, Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AAO77182, CAH09236, ACL08376, and homologs thereof. In some embodiments, mono- or polyunsaturated C6-C 24 Fatty acyl-CoA can serve as a substrate for elongases that can be used to convert mono- or polyunsaturated C6-C 24 The chain length of the fatty acyl-CoA is extended by two additional carbons at the α-carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA.

[0069] As described above, in some embodiments, the recombinant microorganism according to the first, second or third aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted into the corresponding mono- or polyunsaturated C6-C 24Fatty aldehydes. In certain embodiments, the alcohol oxidase or the nucleic acid sequence encoding it can be isolated from an organism of the species Candida boidinii, Pichia pastoris, Tanacetum vulgare, Simmondsia chinensis, Arabidopsis thaliana, Lotus japonicas, or Candida tropicalis. In exemplary embodiments, the alcohol oxidase comprises a sequence selected from GenBank Accession Nos. Q00922, F2QY27, Q6QIR6, Q8LDP0, and L7VFV2.

[0070] As described above, in some embodiments, the recombinant microorganism according to the first or second aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In certain embodiments, the acetyltransferase or the nucleic acid sequence encoding it can be isolated from organisms of the species Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum, or Euonymus alatus. In exemplary embodiments, the acetyltransferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, and XP_505513.1.

[0071] In alternative embodiments, chemical methods can be used to convert fatty alcohols to fatty acetates, for example, by chemical catalysis using chemical reagents such as acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propionyl chloride, and propionic anhydride.

[0072] In some embodiments, the24 The recombinant microorganisms that have a biosynthetic pathway for fatty alcohols, fatty aldehydes or fatty acetates are further engineered to express one or more nucleic acids encoding proteins or polypeptides that are toxic to insects when expressed. Exemplary toxicant production genes suitable for the present disclosure can be obtained from insect pathogenic organisms such as Bacillus thuringiensis, Pseudomonas aeruginosa, Serratia marcescens and members of the genus Streptomyces. In an exemplary embodiment, the recombinant microorganisms comprising the enzyme for producing unsaturated C6-C 24 The recombinant microorganism comprising the biosynthetic pathway for fatty alcohols, fatty aldehydes or fatty acetates is further engineered to express a nucleic acid encoding a Bacillus thuringiensis ("Bt") toxin. In additional or alternative embodiments, the recombinant microorganism comprising the biosynthetic pathway for fatty alcohols, fatty aldehydes or fatty acetates may be further engineered to express a nucleic acid encoding a Bacillus thuringiensis ("Bt") toxin. 24 The recombinant microorganisms expressing the biosynthetic pathways for fatty alcohols, fatty aldehydes, or fatty acetates are further engineered to express nucleic acids encoding other toxic proteins such as spider venom.

[0073] In some embodiments, the 24 The recombinant microorganisms expressing the biosynthetic pathway for a fatty alcohol, fatty aldehyde, or fatty acetate are further engineered to express an RNAi molecule that, when expressed, produces an oligonucleotide that is toxic to insects.

[0074] In some embodiments, the 24 The recombinant microorganisms expressing the biosynthetic pathways for fatty alcohols, fatty aldehydes, or fatty acetates are further engineered to express metabolic pathways that, when expressed, produce small molecules toxic to insects. Non-limiting examples of toxic small molecules include azadirachtin, spinosad, avermectin, pyrethrins, and various terpenoids.

[0075] In various embodiments described herein, a method for producing an unsaturated C6-C 24The recombinant microorganism of the biosynthetic pathway of fatty alcohol, fatty aldehyde or fatty acetate can be a eukaryotic microorganism, such as yeast, filamentous fungi or algae, or alternatively a prokaryotic microorganism, such as bacteria. For example, a suitable host cell can include a cell of the genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas. The genus includes Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, and Streptomyces.

[0076] In some embodiments, the method comprises the steps of producing an unsaturated C6-C 24The recombinant microorganism of the biosynthetic pathway of fatty alcohol, fatty aldehyde or fatty acetate is yeast.The example of suitable yeast comprises the yeast of the genus selected from lower group, and this group is made up of the following: Ye Shi yeast belongs to (Yarrowia), Candida (Candida), Saccharomyces (Saccharomyces), Pichia (Pichia), Hansenula (Hansenula), Kluyveromyces (Kluyveromyces), Issatchenkia (Issatchenkia), Zygosaccharomyces (Zygosaccharomyces), Debaryomyces (Debaryomyces), Schizosaccharomyces (Schizosaccharomyces), Pachysolen (Pachysolen), Cryptococcus (Cryptococcus), Trichosporon (Trichosporon), Rhodotorula (Rhodotorula) or Myxozyma.In certain embodiments, this yeast is oleaginous yeast. Exemplary oleaginous yeast suitable for use in the present disclosure include members of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, including, but not limited to, species Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon spp. pullans), T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

[0077] As will be understood in the art, endogenous enzymes can convert unsaturated C6-C 24Key substrates and / or intermediates upstream or within the fatty alcohol, fatty aldehyde or fatty acetate biosynthetic pathway are converted into unwanted byproducts. Therefore, in some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate and / or reduce the activity of one or more endogenous enzymes that catalyze the synthesis of unsaturated C6-C 24 Reactions in pathways that compete with fatty alcohol, fatty aldehyde, or fatty acetate biosynthetic pathways.

[0078] In one embodiment, the recombinant microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous enzymes that catalyze the conversion of fatty acids to ω-hydroxy fatty acids. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more enzymes selected from the group consisting of: XP_504406, XP_504857, XP_504311, XP_500855, XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BA 957, XP_002546278, or a homologue thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate and / or reduce the activity of one or more enzymes selected from: BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967, WP_011027348 or a homolog thereof.

[0079] In another embodiment, the recombinant microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more enzymes selected from the group consisting of: CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, or a homolog thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate and / or reduce the activity of one or more enzymes selected from: AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657, WP_005293707, WP_005883960 or a homolog thereof.

[0080] In embodiments where the recombinant microorganism is a yeast microorganism, the recombinant microorganism is manipulated to delete, disrupt, mutate, and / or reduce the activity of one or more enzymes involved in peroxisome assembly and / or peroxidase import. The recombinant yeast microorganism is engineered to delete, disrupt, mutate and / or reduce the activity of one or more enzymes selected from the group consisting of: XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868, XP_002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743, EIW08286, or a homolog thereof.

[0081] In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate and / or reduce the activity of one or more endogenous reductases or desaturases that interfere with the unsaturated C6-C 24 fatty alcohols, fatty aldehydes, or fatty acetates, that is, catalyzes the conversion of pathway substrates or products into undesirable byproducts.

[0082] In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate and / or reduce the activity of one or more endogenous alcohol oxidases or alcohol dehydrogenases that catalyze the production of a desired product, such as an unsaturated C6-C 24 Fatty alcohols are converted into the corresponding unsaturated C6-C 24 Fatty aldehydes.

[0083] In another embodiment, the recombinant microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous enzymes that catalyze reactions in a pathway that competes with the biosynthetic pathway for one or more unsaturated fatty acyl-CoA intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g, and CTRG_06209, or homologs thereof. In another embodiment, the one or more endogenous enzymes comprise one or more glycerophospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homologs thereof. In another embodiment, the one or more endogenous enzymes comprise one or more acyl-CoA / sterol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more acyl-CoA / sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764, and CTRG_01765, or homologs thereof.

[0084] In another embodiment, the recombinant microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous enzymes that catalyze reactions in a pathway that oxidizes a fatty aldehyde intermediate. In one embodiment, the one or more endogenous enzymes comprise one or more fatty aldehyde dehydrogenases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more fatty aldehyde dehydrogenases selected from the group consisting of: YALI0A17875g, YALI0E15400g, YALI0B01298g, YALI0F23793g, CTRG_05010, and CTRG_04471, or homologs thereof.

[0085] In another embodiment, the recombinant microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous enzymes that catalyze reactions in a pathway that consumes fatty acetate products. In one embodiment, the one or more endogenous enzymes comprise one or more sterol esterases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more sterol esterases selected from the group consisting of: YALI0E32035g, YALI0E00528g, CTRG_01138, CTRG_01683, and CTRG_04630, or homologs thereof. In another embodiment, the one or more endogenous enzymes comprise one or more triacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more triacylglycerol lipases selected from the group consisting of YALI0D17534g, YALI0F10010g, CTRG_00057, and CTRG_06185, or homologs thereof. In another embodiment, the one or more endogenous enzymes comprise one or more monoacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and / or reduce the activity of one or more monoacylglycerol lipases selected from the group consisting of YALI0C14520g, CTRG_03360, and CTRG_05049, or homologs thereof. In another embodiment, the one or more endogenous enzymes comprise one or more extracellular lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate and / or reduce the activity of one or more extracellular lipases selected from the group consisting of: YALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG_05930, CTRG_04188, CTRG_02799, CTRG_03052 and CTRG_03885, or homologs thereof.

[0086] In embodiments where the recombinant microorganism is a yeast microorganism, the one or more exogenous unsaturated C6-C 24The fatty alcohol, fatty aldehyde, or fatty acetate pathway genes encode enzymes that localize to a yeast compartment selected from the group consisting of the cytosol, mitochondria, or endoplasmic reticulum. In an exemplary embodiment, one or more exogenous pathway genes encode enzymes that localize to the endoplasmic reticulum. In another embodiment, at least two exogenous pathway genes encode enzymes that localize to the endoplasmic reticulum. In yet another embodiment, all exogenous pathway genes encode enzymes that localize to the endoplasmic reticulum.

[0087] In another embodiment, the present application provides the use of the recombinant microorganisms described herein to produce unsaturated C6-C 24 In one embodiment, the method comprises culturing the recombinant microorganism in a medium containing a raw material providing a carbon source until an unsaturated C6-C 24 Fatty alcohol, fatty aldehyde or fatty acetate and optionally recover the unsaturated C6-C 24 Once produced, the unsaturated C6-C8 fatty alcohol, fatty aldehyde or fatty acetate can be separated from the fermentation medium using various methods known in the art. 24 Fatty alcohol, fatty aldehyde or fatty acetate, the method includes but is not limited to distillation, membrane-based separation stripping, solvent extraction and expanded bed adsorption.

[0088] In some embodiments, recombinant microorganisms, such as yeast, can be recovered and produced in the form of dry microparticles. In embodiments involving yeast, the yeast can be dried to produce powdered yeast. In some embodiments, the process for producing powdered yeast comprises spray drying a liquid yeast composition in air, optionally followed by further drying. In some embodiments, upon drying, the recombinant microorganism composition will contain unsaturated C6-C 24 Fatty alcohol, fatty aldehyde or fatty acetate.

[0089] As described herein, the preferred recombinant microorganisms of the present disclosure will have the ability to utilize alkanes and fatty acids as carbon sources. However, as will be appreciated in the art, a variety of carbon sources can be utilized, including but not limited to various sugars (e.g., glucose, fructose, or sucrose), glycerol, alcohols (e.g., ethanol), organic acids, lignocellulose, proteins, carbon dioxide, carbon monoxide, and the above-mentioned alkanes and fatty acids. In some embodiments, the recombinant microorganism will convert the carbon source into unsaturated C6-C8 hydrocarbons under aerobic conditions. 24 Fatty alcohol, fatty aldehyde or fatty acetate.

[0090] As emphasized above, the present application provides methods for producing one or more unsaturated C6-C 24In some embodiments, the product is an insect pheromone. As will be appreciated by those skilled in the art equipped with this disclosure, a variety of different exogenous and endogenous enzymes can be expressed in a recombinant host microorganism to produce the desired insect pheromone. Exemplary insect pheromones in the form of fatty alcohols, fatty aldehydes, or fatty acetates that can be produced using the recombinant microorganisms and methods described herein include, but are not limited to, (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadecadienal, (E,E)-8,10-dodecenal Dien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecen-1-ol acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecen-1-ol, (Z)-8-dodecen-1-ol, (Z)-9-dodecen-1-ol, (Z)-9-dodecen-1-ol, (Z)-9-tetradecen-1-ol, (Z)-11-tetradecen-1-ol, (Z)-11-tetradecen-1-ol, (E)-11-tetradecen-1-ol, (E)-11-tetradecen-1-ol, ( (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, (Z)-13-hexadecenyl acetate and (Z)-13-octadecenyl acetate and (Z)-13-octadecenal.

[0091] In another embodiment of the present invention, a composition comprising one or more recombinant microorganisms producing insect pheromones as described herein may be provided. In certain embodiments, the composition may further comprise one or more insect pheromones produced by the recombinant microorganism. In other embodiments, the composition may further comprise one or more toxic proteins or polypeptides produced by the recombinant microorganism. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The accompanying drawings show illustrative embodiments of the present disclosure, in which:

[0094] Figure 1 The conversion of saturated fatty acyl-CoA to unsaturated fatty alcohols is shown.

[0095] Figure 2The conversion of saturated fatty acids to mono- or polyunsaturated fatty aldehydes, fatty alcohols or fatty acetates is shown.

[0096] Figure 3 Alternative pathways for the conversion of saturated fatty acids to mono- or polyunsaturated fatty aldehydes, fatty alcohols, or fatty acetates are shown.

[0097] Figure 4 The pathways for the conversion of saturated fatty acids to various trienes, dienes, epoxides, and odd-numbered pheromones are shown.

[0098] Figure 5 Shown are the production of Z11-hexadecenol from W303A and BY4742ΔPOX1. Strains expressing empty vector (EV), S. littoralis reductase (FAR-SL), H. amigera reductase (FAR-HA), and A. segetum reductase (FAR-AS). Error bars represent standard deviations derived from N = 2 biologically independent samples.

[0099] Figure 6 A- Figure 6 B shows the expression of Saccharomyces cerevisiae using an empty vector ( Figure 6 A) or alcohol-forming reductase from Helicoverpa armigera ( Figure 6 B) Chromatogram of the resulting sample of the bioconversion product of Z11-hexadecenoic acid. Black line: no substrate added. Purple line: Z11-hexadecenoic acid added as substrate.

[0100] Figure 7 A- Figure 7 B shows the Z11-hexadecenol credible compound ( Figure 7 A) and bio-derived Z11-hexadecenol ( Figure 7 B) Comparison of GC-MS fragmentation patterns.

[0101] Figure 8 Biomass at harvest is shown for product analysis of W303A (wild type) and BY4742ΔPOX1 (β-oxidation deletion mutant). Strains expressing empty vector (EV), S. littoralis reductase (FAR-SL), H. amigera reductase (FAR-HA), and A. segetum reductase (FAR-AS). Error bars represent standard deviation from N = 2 biologically independent samples.

[0102] Figure 9A Z11-hexadecenol calibration curve constructed using authentic standards is shown. Samples were generated using the extraction and analytical methods described in the Materials and Methods of Example 3. Error bars represent standard deviations derived from N=3 samples.

[0103] Figure 10 Shown is a pOLE1 cassette comprising an extended OLE1 promoter sequence (light yellow), OLE1 promoter (orange), OLE1 leader sequence (dark grey), a synthon such as an insect desaturase sequence (light grey), and a VSP13 terminator sequence (blue).

[0104] Figure 11 A- Figure 11 E shows the validation and complementation assay of the pOLE1 cassette. Figure 11 A: YPD+palmitoleic acid; Figure 11 B: YPD-palmitoleic acid; Figure 11 C: CM-Ura glucose + palmitoleic acid; Figure 11 D: CM-Ura glucose-palmitoleic acid; Figure 11 E: Figure 11 A- Figure 11 Diagram of the strain in D. Dasher = GFP synthon.

[0105] Figure 12 A shows complementation of ΔOLE1 growth in the absence of UFA on YPD.

[0106] Figure 12 B shows complementation of ΔOLE1 growth in the absence of UFA on CM-Ura glucose.

[0107] Figure 13 A shows the complete fatty acid profile of a ΔOLE1 strain expressing: S. cerevisiae OLE1 desaturase (blue), chimeric T. ni desaturase (red).

[0108] Figure 13 B shows the focused fatty acid profile of a S. cerevisiae ΔOLE1 strain expressing the S. cerevisiae OLE1 desaturase (red) and the chimeric Trichoplusia ni desaturase (blue) at retention times of 5.5 min to 8 min.

[0109] Figure 14 A- Figure 14 B shows the ( Figure 14 A) and bioderived ( Figure 14 B) Comparison of GC-MS fragmentation patterns of (Z)-11-hexadecenoic acid.

[0110] Figure 15Shown is the production of C16 fatty alcohols from ΔOLE1 expressing various fatty alcohol pathway variants in media supplemented with palmitic acid and palmitoleic acid. Error bars represent 5% uncertainty in metabolite quantification accuracy.

[0111] Figure 16 Shown are representative chromatograms of bioconversion products C16 fatty acids obtained using Saccharomyces cerevisiae expressing the fatty alcohol pathway TN_desat-HA_reduc when palmitic acid is added (black) and when palmitic acid and palmitoleic acid are added (orange). The curve for the negative control strain (containing an empty vector) to which palmitic acid is added is shown (purple).

[0112] Figure 17 Shown is the detection of (Z)-11-hexadecenoic acid in the pellet of Saccharomyces cerevisiae cells expressing the fatty alcohol pathway TN_desat-SL_reduc (blue), SC_desat-HA_reduc (red), TN_desat-HA_reduc (green), SC_desat-SL_reduc (pink).

[0113] Figure 18 Shown is the production of C16 fatty alcohols from ΔOLE1 expressing various fatty alcohol pathway variants in cultures supplemented with palmitic acid alone. Error bars represent 5% uncertainty in metabolite quantification accuracy.

[0114] Figure 19 A- Figure 19 C shows the detection of (Z)-11-hexadecenol. Figure 19 A: Fragmentation pattern of an authentic standard. m / z 297.3 was used in subsequent experiments to selectively detect this alcohol. To also detect an internal standard, masses 208 and 387.3 were also included. Figure 19 B: In addition to the detection of the specific mass fragment, the retention time was used as a second level of confirmation. The retention time was 6.22. Figure 19 C: Comparison of two different regioisomers 9Z- and 11Z-hexadecenol when detected using the same method in SIM mode (297.3).

[0115] Figure 20 The pXICL expression cassette architecture is shown. Also shown is the Candida albicans OLE1 leader sequence-A. segetum desaturase fusion.

[0116] Figure 21 A- Figure 21 D shows mCherry control integration. Figure 21 A: Negative (water only) control transformation plate. Figure 21 B: pPV0137 mCherry transformation plate. Figure 21C: Plaque plate from negative control clone. Figure 21 D: Plaque plate from pPV0137 clone.

[0117] Figure 22 Integration efficiency as a function of total observed colonies is shown. The control plate to which no DNA was added for transformation had 350 colonies (indicated by the orange line). The fraction of clones confirmed as positive integrants was positively correlated with the total colony count. A sharp increase was observed over 6,000 total colonies. The data indicate that the presence of positive integrants increased the observed background growth. For some transformations, the efficiency was high enough that the background population was smaller relative to the positive integration population.

[0118] Figure 23 Shown is a chromatogram overlay for Candida tropicalis strain SPV053. Compared to the mCherry control (red), clear peaks for the desaturases from A. transitella (blue) and H. zea (green) are observed at 6.22 minutes. Therefore, the formation of Z-11-hexadecenoic acid is only observed in strains expressing active Z-11-desaturases.

[0119] Figure 24 A- Figure 24 E shows the confirmation of the 11Z-regioisomer. Figure 24 A: A specific peak having an ion fragment of 245 m / z was observed only in C. tropicalis SPV053 expressing the Z11-desaturase from A. transitella or H. zea. Figure 24 B: Fragmentation diagram of authentic standards. Figure 24 D: The fragmentation pattern of the newly formed compound in the sample expressing the desaturase from H. zea matches the fragmentation pattern of the standard. Figure 24 E: The fragmentation pattern of the newly formed compound in the sample expressing the desaturase from D. citrinum matches the fragmentation pattern of the standard. Figure 24 C: Fragmentation diagram of mCherry control and Figure 24 B. Figure 24 D and Figure 24 The fragmentation pattern of E is significantly different.

[0120] Figure 25 A- Figure 25 B shows GC-FID chromatograms of different Candida tropicalis SPV053 strains incubated with methyl myristate. Figure 25 A: Overall spectrum: For strains expressing the Z11-desaturases from C. zea and C. zea, the appearance of the Z11-C16:1 peak was observed. Figure 25B: Zoom-in of the C14 to C18 region. A new peak is visible at 4.8 min, which may correspond to Z11-C14:1. Another peak near Z9-C18:1 is also visible, which may correspond to Z11-C18:1.

[0121] Figure 26 It is shown that only the codon-optimized H. zea desaturase variants produced detectable Z11-hexadecenoic acid in the SPV140 screen. Control = pPV101 integrant of SPV140, native T. niger = T. zea Z11 desaturase with native codon usage (pPV195), T. niger HS opt = T. zea Z11 desaturase with H. sapiens codon optimization (pPV196), T. zi HS opt Y1 leader = T. zi Z11 desaturase with H. sapiens codon optimization and exchanged Yarrowia lipolytica OLE1 leader (pPV197), native T. zea = T. zea Z11 desaturase with native codon usage (pPV198), T. zea HS opt = T. zea Z11 desaturase with H. sapiens codon optimization (pPV199), T. zea HS opt Y1 leader = H. zea Z11 desaturase with H. sapiens codon-optimized and exchanged Yarrowia lipolytica OLE1 leader (pPV200), native A. zea Z11 desaturase = A. zea Z11 desaturase with native codon usage (pPV201). All data are averages of three biological replicates. Error bars represent standard deviations.

[0122] Figure 27 This analysis shows that only the codon-optimized H. zea desaturase variants produced detectable Z11-hexadecenoic acid in the SPV300 screen. Markers indicate the parental strain and the plasmids containing the desaturase expression cassettes. pPV101 = hrGFP control, pPV198 = H. zea Z11 desaturase with native codon usage, pPV199 = H. zea Z11 desaturase with codon optimization, pPV200 = H. zea Z11 desaturase with H. zea codon optimization and a swapped Yarrowia lipolytica OLE1 leader, and pPV201 = H. zea Z11 desaturase with native codon usage.

[0123] Figure 28 Final cell densities for desaturase screening in the SPV140 and SPV300 backgrounds are shown. The SPV300 strain with the integrated desaturase cassette was grown to a higher cell density.

[0124] Figure 29 Shown are Z11-hexadecenoic acid titers of individual isolates of strains SPV140 and SPV300 expressing the H. zea Z11 desaturase with a codon-optimized H. sapiens .

[0125] Figure 30 Shown is an overlay of chromatograms of extracted metabolites of a Z11-16OH-producing strain (SPV0490) and a control strain (SPV0488) of Candida viswanathii (Candida tropicalis).

[0126] Figure 31 The following pathways are shown, which can be deleted or disrupted to reduce or eliminate the production of mono- or polyunsaturated C6-C 24 Competition for the biosynthetic pathways of fatty alcohols, fatty aldehydes, or fatty acetates.

[0127] Figure 32 A- Figure 32 B shows the pEXP clones in YPD ( Figure 32 A) and semi-limited C:N=80( Figure 32 B) Z9-16OH and Z11-16OH titers in culture. Ten isolates expressing the H. zea desaturase under the TEF promoter and the H. armigera reductase under the EXP promoter from two independent competent cell preparations (Competent Cell Preparation 1, Competent Cell Preparation 2) were compared to a parental negative control (SPV300) and a desaturase-only negative control (SPV459Hz_desat alone). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). *One replicate from clone 5 and clone 18 was lost during workup under the semi-defined C:N=80 condition; therefore, the titer for this condition is from a single data point (N=1, Competent Cell Preparation 1 clone 18 and Competent Cell Preparation 2 clone 5).

[0128] Figure 33 A- Figure 33 B shows the pEXP1 clone in YPD ( Figure 33 A) and semi-defined C:N=80( Figure 33 B) 16-carbon fatty acid species profile in the culture medium. The 16-carbon lipid profiles of five selected clones expressing the H. zea desaturase under the TEF promoter and the H. armigera reductase under the EXP promoter were compared to the parental negative control (SPV300) and the desaturase-only negative control (SPV459Hz_desat alone). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

[0129] Figure 34Shown are Z9-16OH and Z11-16OH titers in semi-defined C:N=80 medium for pTAL1 clones. Nine isolates expressing the H. zea desaturase under the TEF promoter and the H. armigera reductase under the TAL promoter were compared to a parental negative control (SPV300) and positive Bdr pathway controls (SPV575, SPV578) using the EXP promoter to drive H. armigera FAR expression. Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

[0130] Figure 35 Shown are the 16-carbon fatty acid species profiles in semi-defined C:N=80 medium for pTAL1 clones. The 16-carbon lipid profiles of five selected clones expressing the H. zea desaturase under the TEF promoter and the H. armigera reductase under the EXP promoter were compared to a parental negative control (SPV300) and a positive Bdr pathway control (SPV575, SPV578) using the EXP promoter to drive H. armigera FAR expression. Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). * indicates a clone lost in one replicate during sample processing, N=1.

[0131] Figure 36 Shown are the full lipid profiles of the complete Bdr pathway pTAL1 screen (strains expressing the Z11 desaturase (pTEF) of the zea larvae and the FAR of the cotton bollworm) in semi-defined C:N=80 medium after 48 hours of biotransformation. Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). *Indicates a clone lost in one of the replicates during sample processing, N=1.

[0132] Figure 37 A- Figure 37 B shows the expression of SPV471 (expressing native Yarrowia lipolytica OLE1 and H222ΔPΔAΔF of H. armigera FAR) Z9-16OH ( Figure 37 A) and fatty acids ( Figure 37 B) Titer. Error bars represent SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

[0133] Figure 38 Shown are the full lipid profiles of SPV471 (H222ΔPΔAΔF expressing native Yarrowia lipolytica OLE1 and H. armigera FAR) in semi-defined C:N=80 medium after 24 hours of bioconversion.

[0134] Figure 39 A- Figure 39 B shows SPV471 (expressing native Yarrowia lipolytica OLE1 and H222ΔPΔAΔF of H. armigera FAR) Z9-16OH ( Figure 39 A) and Z9-16 acid ( Figure 39 B) Titer time course. Biotransformation of 16-acid was performed using methyl palmitate (16-acid) substrate in a semi-defined C:N=80 medium.

[0135] Figure 40 Examples of acyl-CoA intermediates generated by selective β-oxidation controlled by acyl-CoA oxidase activity are shown.

[0136] Figure 41 Shown are the Z11-14 acid (myristate methyl ester feed - 14ME) and Z11-16 acid (palmitate methyl ester feed - 16ME) titers of the characterized Δ11 desaturases. SPV300 = Desaturase library integration parent. SPV298 = Prototrophic parent of SPV300, negative control. SPV459 = SPV300 with the best desaturase to date (Hymenoptera zea, SEQ ID NO: 54), positive control. The desaturase in DST006 is genetically identical to the Hymenoptera zea desaturase expressed in SPV459 and serves as an internal library control.

[0137] Figure 42 Shown are the C14 and C18 product profiles of SPV298 (negative control, parental strain) and SPV459 (SPV298 lineage with the H. zea desaturase (SEQ ID NO: 54)) fed with either methyl palmitate (16ME) or methyl myristate (14ME).

[0138] Figure 43 Bioinformatics analysis of potential serine, threonine and tyrosine phosphorylation sites of H. amigera FAR enzyme (SEQ ID NO: 41) is shown. The horizontal line resembles the threshold value of potential phosphorylation.

[0139] Figure 44Bioinformatics analysis of potential serine and threonine phosphorylation sites of the cotton bollworm-derived FAR enzyme once expressed in yeast is shown. The server used (world wide web address: cbs.dtu.dk / services / NetPhosYeast / ; Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites 1. J. Mol. Biol. 294, 1351–1362 (1999)) Predict specific phosphorylated amino acids in yeast The horizontal line resembles the threshold for possible phosphorylation sites.

[0140] Figure 45 Shown is analysis of the Z9 / Z11-16OH titers of a library of HaFAR mutants once expressed in Yarrowia lipolytica SPV603. *Indicates inclusion of a second copy of the HaFAR enzyme in addition to the existing copy of the parent strain.

[0141] Figure 46 Shown is an analysis of the Z9 / Z11-16 acid titers of a library of HaFAR mutants once expressed in Yarrowia lipolytica SPV603. *Indicates the inclusion of a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain.

[0142] Figure 47 Shown is an analysis of fatty alcohol titers in selected strains expressing HaFAR and derived mutants. Strains were cultured in shake flasks for 72 hours after addition of 10 g / L methyl palmitate. * Indicates the presence of a second copy of the HaFAR enzyme in addition to the existing copy in the parent strain. This analysis was based on technical quadruplicates.

[0143] Figure 48 Shown is an analysis of fatty acid titers in selected strains expressing HaFAR and derived mutants. Strains were cultured in shake flasks for 72 hours after addition of 10 g / L methyl palmitate. * Indicates the presence of a second copy of the HaFAR enzyme in addition to the existing copy in the parent strain. This analysis was based on technical quadruplicates.

[0144] Figure 49 Analysis of fatty alcohol titers of selected strains expressing HaFAR and derived mutants is shown. Strains were cultured in shake flasks for 20 hours after addition of 10 g / L methyl palmitate. The analysis was based on technical quadruplicates.

[0145] Figure 50Shown is an analysis of fatty alcohol titers of selected strains from a time-course experiment in shake flasks. One copy of the enzyme HaFAR or HaS195A was introduced into strains SPV1053 (Δdga1ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed in biological triplicate in shake flasks. After addition of 10 g / L methyl palmitate, the strains were cultured in shake flasks for 72 hours.

[0146] Figure 51 Shown is an analysis of fatty acid titers for selected strains from a time-course experiment in shake flasks. One copy of the enzyme HaFAR or HaS195A was introduced into strains SPV1053 (Δdga1ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed in biological triplicate in shake flasks. Following the addition of 10 g / L methyl palmitate, the strains were cultured in shake flasks for 72 hours.

[0147] Figure 52 Analysis of fatty alcohol titers of novel strains in a 24-well plate FAR library screen is shown. One copy of each FAR enzyme from Table 24 was introduced into strain SPV1054 (Δdga2ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultures were performed in quadruplicate in 24-well plates. Following addition of 10 g / L methyl palmitate, the strains were cultured for 96 hours.

[0148] Figure 53 Analysis of fatty acid titers of novel strains in a 24-well plate FAR library screen is shown. A single copy of each individual FAR enzyme from Table 24 was introduced into strain SPV1054 (Δdga2ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultures were performed in biological quadruplicate in 24-well plates. Following the addition of 10 g / L methyl palmitate, the strains were cultured for 96 hours.

[0149] Figure 54A biosynthetic pathway is shown that can use tetradecyl-ACP (14:ACP) input to produce a mixture of E- and Z-tetradecenyl acetate (E11-14:OAc and Z11-14:OAC) pheromones in the recombinant microorganisms of the present disclosure.

[0150] sequence

[0151] The sequence listing of SEQ ID NO: 1 to SEQ ID NO: 105 is part of the present application and is incorporated herein by reference.A sequence listing is provided at the end of this document and is provided separately in a computer readable format. Detailed Description of the Invention

[0153] definition

[0154] The following definitions and abbreviations are used to explain this disclosure.

[0155] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pheromone" includes a plurality of such pheromones, and reference to "a microorganism" includes reference to one or more microorganisms, etc.

[0156] As used herein, the terms "comprises / comprising," "includes / including," "has / having," "contains / containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Additionally, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or.

[0157] The terms "about" and "around" used herein to modify a numerical value indicate a close range around that explicit value. If "X" is a stated value, then "about X" or "around X" will indicate a value of 0.9X to 1.1X, or in some embodiments, a value of 0.95X to 1.05X. Any reference to "about X" or "around X" specifically indicates at least X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "around X" are intended to teach and provide written descriptive support for a claim limitation of, for example, "0.98X."

[0158] As used herein, the terms "microbial," "microbial organism," and "microorganism" include any organism that exists as a microscopic cell within the domains of archaea, bacteria, or eukaryotes, the latter including yeast and filamentous fungi, protozoa, algae, or higher protists. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size and includes all species of bacteria, archaea, and eubacteria, as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of chemicals.

[0159] As described herein, in some embodiments, the recombinant microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium. "Bacteria" or "eukaryotes" refers to the domain of prokaryotes. Bacteria include at least eleven different groups, as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, etc.) 2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria (Proteobacteria) teria), such as purple photosynthetic + non-photosynthetic Gram-negative bacteria (including most "common" Gram-negative bacteria); (3) Cyanobacteria, such as oxygenic phototrophs; (4) Spirochaetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant Micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

[0160] "Gram-negative bacteria" include cocci, non-enteric rods, and enterobacteriaceae. Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, and io), Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0161] "Gram-positive bacteria" include cocci, non-sporing bacilli, and sporing bacilli. Genera of gram-positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0162] The terms "recombinant microorganism" and "recombinant host cell" are used interchangeably herein and refer to a microorganism that has been genetically modified to express or overexpress an endogenous enzyme in an integration construct, to express a heterologous enzyme (such as those included in a vector), or to an altered expression of its endogenous gene. By "alteration" is meant that the expression of a gene, or the level of an RNA molecule encoding one or more polypeptides or polypeptide subunits, or equivalent RNA molecules, or the activity of one or more polypeptides or polypeptide subunits is raised or lowered so that expression, level, or activity is greater or less than that observed without the alteration. For example, the term "alteration" can mean "inhibition," but the use of the word "alteration" is not limited to this definition. It should be understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to specific recombinant microorganisms, but also to the offspring or potential offspring of such a microorganism. Because some changes can occur in offspring due to sudden changes or environmental influences, such progeny may in fact be different from the parental cell, but are still included within the scope of the term as used herein.

[0163] In terms of gene sequences, the term "expression" refers to the transcription of the gene and, as the case may be, to the translation of the resulting mRNA transcript into protein. Therefore, it is clear from the context that protein expression is caused by the transcription and translation of the open reading frame sequence. The expression level of the desired product in the host cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantified by qRT-PCR or Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by measuring the biological activity of the protein, or by adopting an assay independent of such activity (such as Western blotting or radioimmunoassay), using antibodies that recognize and bind to the protein. See Sambrook et al., 1989, supra.

[0164] The term "polynucleotide" is used interchangeably with the term "nucleic acid" herein and refers to an organic polymer composed of two or more monomers (including nucleotides, nucleosides, or their analogs), including but not limited to single-stranded or double-stranded sense or antisense deoxyribonucleic acids (DNA) of any length, and single-stranded or double-stranded sense or antisense ribonucleic acids (RNA) of any length, including siRNA, when appropriate. The term "nucleotide" refers to any of several compounds that are composed of ribose or deoxyribose sugars connected to a purine or pyrimidine base and a phosphate group and are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (such as guanosine or adenosine) composed of a purine or pyrimidine base combined with deoxyribose or ribose and found particularly in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers to a nucleotide or nucleoside, respectively, in which one or more individual atoms have been replaced by different atoms or different functional groups. Therefore, the term polynucleotide includes nucleic acids, DNA, RNA, analogs, and fragments thereof of any length. Polynucleotides of three or more nucleotides are also referred to as nucleotide oligomers or oligonucleotides.

[0165] It is understood that the polynucleotides described herein include "genes" and the nucleic acid molecules described herein include "vectors" or "plasmids". Thus, the term "gene", also referred to as "structural gene", refers to a polynucleotide that encodes a specific amino acid sequence, which contains all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, the conditions for expression of the gene. The transcribed region of the gene may include untranslated regions, including introns, 5' untranslated regions (UTRs) and 3'-UTRs, as well as coding sequences.

[0166] As used herein, the term "enzyme" refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, and generally includes enzymes composed entirely or in part of one or more polypeptides, but may include enzymes composed of different molecules, including polynucleotides.

[0167] As used herein, the term "non-naturally occurring" in reference to a microbial organism or enzyme activity of the present disclosure is intended to mean that the microbial organism or enzyme has at least one alteration not normally present in naturally occurring strains of the referenced species (including wild-type strains of the referenced species). Genetic alterations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions, and / or other functional disruptions of the microbial genetic material. Such modifications include, for example, coding regions for heterologous, homologous, or heterologous and homologous polypeptides of the referenced species, and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions, wherein the modifications alter the expression of a gene or operon. Exemplary non-naturally occurring microbial or enzyme activities include the hydroxylation activity described above.

[0168] The term "exogenous" as used herein with respect to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) refers to molecules that are not normally present or naturally found in and / or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0169] On the other hand, the terms "endogenous" or "native" as used herein with respect to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) refer to molecules that are normally or naturally present in and / or produced by a given yeast, bacterium, organism, microorganism or cell in nature.

[0170] As used herein, the term "saturated C6-C24 fatty acids of endogenous or exogenous origin" refers to a source of saturated C6-C24 fatty acids that originates from within a microorganism (such as when the saturated C6-C24 fatty acids are produced or synthesized within the microorganism) (endogenous) or a source of saturated C6-C24 fatty acids that originates from outside the microorganism (such as when the saturated C6-C24 fatty acids are provided to the microorganism during the process of culturing or cultivating the microorganism in a culture medium in a shake flask or other container (exogenous).

[0171] As used herein, the term "heterologous" in the context of modified host cells refers to various molecules (e.g., polynucleotides, polypeptides, enzymes, etc.) where at least one of the following is true: (a) the molecule(s) are foreign ("exogenous") to the host cell (i.e., not naturally occurring); (b) the molecule(s) are naturally occurring (e.g., "endogenous") to a given host microorganism or host cell, but are produced in a non-natural location or in a non-natural amount in the cell; and / or (c) the molecule(s) differ in nucleotide or amino acid sequence from an endogenous nucleotide or amino acid sequence, such that the molecule(s) differ in nucleotide or amino acid sequence from an endogenous nucleotide or amino acid found endogenously are produced in a non-natural amount (e.g., greater than that found in nature) in the cell.

[0172] As used herein, the terms "homologous sequence," "homologs," or "orthologs" refer to related sequences (nucleic acids or amino acids) that are functionally related to a reference sequence. A functional relationship can be indicated in one of a number of ways, including, but not limited to: (a) degree of sequence identity, and / or (b) the same or similar biological function. The use of the term "homolog" in this disclosure refers to situations where both (a) and (b) are indicated. The degree of sequence identity can vary, but in one embodiment, is the same when using standard sequence alignment programs known in the art (e.g., Clustal using default parameters). Omega alignment), the degree of sequence identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987) Annex 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA). Therefore, reference to homologs in this disclosure will be understood as reference to related sequences having the same or similar biological function and a high degree of sequence identity as described above.

[0173] As used herein, the term "fatty acid" refers to a compound having the structure R-COOH, wherein R is a C6-C24 saturated, unsaturated, linear, branched, or cyclic hydrocarbon and the carboxyl group is at position 1. In a specific embodiment, R is a C6-C24 saturated or unsaturated linear hydrocarbon and the carboxyl group is at position 1.

[0174] As used herein, the term "fatty alcohol" refers to an aliphatic alcohol having the formula R-OH, wherein R is C6-C 24 In a specific embodiment, R is C6-C 24 Saturated or unsaturated straight-chain hydrocarbons.

[0175] The term "fatty acyl-CoA" refers to a compound having the structure R-(CO)-S-R1, wherein R1 is coenzyme A, and the term "fatty acyl-ACP" refers to a compound having the structure R-(CO)-S-R1, wherein R1 is acyl carrier protein ACP.

[0176] The term "short chain" or "short-chain" refers to fatty alcohols, fatty aldehydes and / or fatty acetates with a carbon chain length shorter than or equal to C18, including pheromones, spices, flavorings and polymer intermediates.

[0177] introduce

[0178] The present disclosure relates to the need for new technologies for efficiently producing valuable products from low-cost raw materials. Specifically, the present inventors have developed a novel method for producing a wide range of unsaturated C6-C8 24 Recombinant microorganisms that produce fatty alcohols, fatty aldehydes, and fatty acetates, including synthetic insect pheromones, fragrances, flavorings, and polymer intermediates, address this need. Accordingly, aspects of the present disclosure are based on the inventors' discovery that recombinant microorganisms can be engineered to produce valuable products from low-cost feedstocks that circumvent conventional synthetic methods for producing valuable products.

[0179] As mentioned above, recombinant microorganisms can be engineered to synthesize mono- or polyunsaturated C6-C 24 Fatty alcohols. Mono- or polyunsaturated C6-C 24 Fatty alcohols can be further converted into corresponding aldehydes or acetates. Thus, various embodiments of the present disclosure can be used to synthesize a variety of insect pheromones selected from fatty alcohols, fatty aldehydes, and fatty acetates. In addition, the embodiments described herein can also be used to synthesize fragrances, flavorings, and polymer intermediates.

[0180] Engineering of a microbial host requires expression of a non-native pheromone biosynthetic pathway consisting of, but not limited to, one or more fatty acyl desaturases and a fatty alcohol-forming or fatty aldehyde-forming reductase. The fatty acids produced by the desaturation reaction can be stored intracellularly as triacylglycerides or enzymatically reduced by reductases to form fatty alcohols or fatty aldehydes. Triacylglycerides containing unsaturated fatty acids can be extracted, esterified, and chemically reduced to produce unsaturated fatty alcohols. The fatty alcohols produced via the described pathway can be further converted to fatty aldehyde pheromones and fatty acetate pheromones via subsequent chemical oxidation and esterification methods, respectively. Methods for chemical oxidation and esterification are known in the art. The fatty alcohols produced via the described pheromone biosynthetic pathway can also be further converted to fatty aldehyde pheromones and fatty acetate pheromones using enzymatic conversions such as alcohol dehydrogenases and acetyltransferases, respectively. Similarly, fatty acyl-CoA or fatty acyl-ACP formed as intermediates in the pheromone biosynthetic pathway can be released as free fatty acids by native or heterologously derived thioesterases, thereby becoming substrates for pheromone synthesis using metathesis.

[0181] pheromones

[0182] As described above, embodiments of the present disclosure provide for the synthesis of one or more insect pheromones using recombinant microorganisms. Pheromones are volatile chemical compounds secreted by specific insects that function to facilitate chemical communication within that species. In other words, pheromones are secreted or excreted chemical factors that trigger social responses in members of the same species. Examples include alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, dispersal pheromones, release pheromones, primer pheromones, and territorial pheromones, among others, that influence behavior or physiology.

[0183] Non-limiting examples of insect pheromones that can be synthesized using the recombinant microorganisms and methods disclosed herein include the linear alcohols, aldehydes, and acetates listed in Table 1.

[0184] Table 1. C6-C 20 Straight pheromone

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] In some aspects, pheromones synthesized as taught by the present disclosure include at least one pheromone listed in Table 2a to modulate the behavior of insects listed in Table 2a. In other aspects, non-limiting examples of insect pheromones that can be synthesized using the recombinant microorganisms and methods disclosed herein include the alcohols, aldehydes, and acetates listed in Table 2a. However, the microorganisms described herein are not limited to the C6-C6 pheromones listed in Table 1 and Table 2a. 20 In contrast, the disclosed microorganisms can also be used to synthesize various C6-C 24 Mono- and polyunsaturated fatty alcohols, fatty aldehydes and fatty acetates, including fragrances, flavorings and polymer intermediates.

[0193] Table 2a. Exemplary pheromones that can be synthesized according to the methods described in this disclosure.

[0194]

[0195]

[0196]

[0197]

[0198] Most pheromones contain a hydrocarbon backbone with terminal hydrogens replaced by functional groups (Ryan MF (2002). Insect Chemoreception. Fundamental and Applied. Kluwer Academic Publishers). Table 2b shows some common functional groups and their chemical formulas, prefixes, and suffixes. The presence of one or more double bonds, resulting from the loss of hydrogens from adjacent carbon atoms, determines the degree of unsaturation of the molecule and changes the name from alkane (no multiple bonds) to alkene. The presence of two and three double bonds is indicated by ending the nomenclature with -diene and -triene, respectively. The position of each double bond is represented by a number corresponding to the position of the carbon at which it originates, and each carbon atom is numbered by the carbon to which the functional group is attached. The carbon to which the functional group is attached is referred to as -1-. Pheromones can have hydrocarbon chain lengths numbered, but not limited to, 10 (decene), 12 (dodecaproide), 14 (tetradecaproide), 16 (hexadecaproide), or 18 (octadecenoide). The presence of double bonds has another effect. The molecule is fixed in one of two possible configurations, each representing a different geometric isomer. When the carbon chains are attached on opposite (trans) or the same (cis) sides of the double bond, they are designated E (from the German word Entgegen, meaning opposite) or Z (Zusammen, meaning together), respectively.

[0199] Table 2b. Common functional group prefixes and suffixes

[0200]

[0201] From Howse, PE, Stevens, IDR and Jones, OT (1998). Insect pheromones and their use in pest management. London: Chapman and Hall.

[0202] The pheromones described herein may be referred to using IUPAC nomenclature or various abbreviations or variations known to those skilled in the art. For example, (11Z)-hexadecene-1-aldehyde may also be written as Z-11-hexadecene-1-aldehyde, Z-11-hexadecenal, or Zxy:Ald, where x represents the position of the double bond and y represents the number of carbons in the hydrocarbon backbone. Abbreviations used herein and known to those skilled in the art for identifying functional groups on the hydrocarbon backbone include "Ald" for aldehyde, "OH" for alcohol, and "Ac" for acetyl. Additionally, the number of carbons in the chain may be indicated using a number rather than a written name. Thus, as used herein, an unsaturated carbon chain consisting of sixteen carbons may be written as hexadecene or I6.

[0203] Similar abbreviations and derivatives are used herein to describe pheromone precursors. For example, the fatty acyl-CoA precursor of (11Z)-hexadecen-1-aldehyde can be identified as (11Z)-hexadecenyl-CoA or Z-11-16:acyl-CoA.

[0204] The present disclosure relates to the synthesis of mono- or polyunsaturated C6-C8 monounsaturated fatty acids using recombinant microorganisms comprising one or more heterologous enzymes. 24 Fatty alcohols, fatty aldehydes, and fatty acetates, the one or more heterologous enzymes catalyze the conversion of substrates to products of one or more steps of the synthetic process.

[0205] Desaturase

[0206] This disclosure describes enzymes that desaturate fatty acyl substrates to the corresponding unsaturated fatty acyl substrates.

[0207] In some embodiments, desaturases are used to catalyze the conversion of fatty acyl-CoA or acyl-ACP into the corresponding unsaturated fatty acyl-CoA or acyl-ACP. Desaturases are enzymes that catalyze the formation of carbon-carbon double bonds in saturated fatty acids or fatty acid derivatives (e.g., fatty acyl-CoA or fatty acyl-ACP (collectively referred to herein as "fatty acyl")) by removing at least two hydrogen atoms to produce the corresponding unsaturated fatty acid / acyl group. Desaturases are classified according to the ability of the enzyme to selectively catalyze the formation of double bonds at the near-terminal carbon relative to the methyl terminus of the fatty acid / acyl group or at the near-terminal carbon relative to the carbonyl terminus of the fatty acid / acyl group. Omega (ω) desaturases catalyze the formation of carbon-carbon double bonds at a fixed near-terminal carbon relative to the methyl terminus of the fatty acid / acyl group. For example, ω 3 Desaturases catalyze the formation of a double bond between the third and fourth carbons relative to the methyl terminus of the fatty acid / acyl group. Delta (Δ) desaturases catalyze the formation of a carbon-carbon double bond at a specific position relative to the carboxyl group of a fatty acid or the carbonyl group of a fatty acyl CoA. For example, Δ 9 Desaturases catalyze the conversion of C to C-terminus of fatty acids or to the carbonyl group of fatty acyl-CoA. 9 and C 10 Double bonds are formed between carbons.

[0208] As used herein, desaturases can be described with reference to the position of double bond formation catalyzed by the desaturase and the resulting geometry (i.e., E / Z) of the unsaturated hydrocarbon. Thus, as used herein, a Z9 desaturase refers to a delta desaturase that catalyzes the formation of a double bond between C9 and C1 relative to the carbonyl terminus of the fatty acid / acyl group. 10 The formation of a double bond between carbon atoms, thereby orienting the two hydrocarbons on opposite sides of the carbon-carbon double bond in a cis or Z configuration. Similarly, as used herein, a Z11 desaturase refers to a delta desaturase that catalyzes the desaturation of C relative to the carbonyl terminus of a fatty acid / acyl group. 11 and C 12 The formation of double bonds between carbon atoms.

[0209] Desaturases have a conserved structural motif. This sequence motif for transmembrane desaturases is characterized by [HX3–4HX7–41(3 non-His)HX2–3(1 non-His)HHX61–189(40 non-His)HX2–3(1 non-His)HH]. Soluble desaturases have a sequence motif characterized by two occurrences of [D / EEXXH].

[0210] In some embodiments, the desaturase is a fatty acyl-CoA desaturase that catalyzes the formation of a double bond in a fatty acyl-CoA. In some such embodiments, the fatty acyl-CoA desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate having a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.

[0211] In some embodiments, the fatty acyl desaturases described herein are capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal CoA on an unsaturated fatty acyl-CoA. Thus, in some embodiments, a desaturase can be selected for use in a recombinant microorganism that catalyzes the insertion of a double bond at position 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 relative to the carbonyl group on the fatty acyl-CoA.

[0212] In some embodiments, the fatty acyl desaturases described herein are capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resulting unsaturated fatty acyl-CoA has a cis or trans (ie, Z or E) geometry.

[0213] In some embodiments, the desaturase is a fatty acyl-ACP desaturase that catalyzes the formation of a double bond in a fatty acyl-ACP. In some embodiments, the fatty acyl-ACP desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate having a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.

[0214] In some embodiments, the fatty acyl-ACP desaturases described herein are capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal carbonyl group on an unsaturated fatty acyl-ACP. Thus, in some embodiments, a desaturase can be selected for use in a recombinant microorganism that catalyzes the insertion of a double bond at position 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 relative to the carbonyl group on the fatty acyl-ACP.

[0215] In some embodiments, the fatty acyl desaturases described herein are capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resulting unsaturated fatty acyl-ACP has a cis or trans (ie, Z or E) geometry.

[0216] In one embodiment, the fatty acyl desaturase is a Z11 desaturase. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase is codon-optimized and is derived from an organism of the species Agrotissegetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosirapseudonana. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 18, 24, and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10 and 16 from Agrotissegetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 53 from Agrotissegetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12, 17, and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 27, and 31 from Helicoverpazea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpazea.In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO:39 from Sesamia inferens. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in GenBank Accession Nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, and NP001299594.1, Q9N9Z8, ABX71630.1, and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, all or part of the Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase is replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 28, and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 78, 79, and 80.

[0217] In one embodiment, the fatty acyl desaturase is a Z9 desaturase. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence listed in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence listed in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence listed in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 60 from Hippocampus zea.

[0218] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a sequence selected from the group consisting of SEQ ID NOs: 39, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 78, 79, 80, 95, 97, 99, 101, 103, and 105. or Z9 desaturases exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51% or 50% sequence identity to any of No.

[0219] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding a Z11 or Z9 desaturase, wherein the nucleic acid molecule is identical to a sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 68, 69, 70, 71, 94, 96, 98, 100, 102, and 104. %, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity.

[0220] In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA and has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 82%, 81%, 8 %, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51% or 50% sequence identity.

[0221] Fatty acyl reductase

[0222] This disclosure describes enzymes that reduce fatty acyl substrates to the corresponding fatty alcohols or aldehydes.

[0223] In some embodiments, a fatty acyl reductase that forms fatty alcohols is used to catalyze the conversion of fatty acyl-CoA into the corresponding fatty alcohol. In some embodiments, a fatty acyl reductase that forms fatty aldehydes is used to catalyze the conversion of fatty acyl-ACP into the corresponding fatty aldehyde. Fatty acyl reductases are enzymes that catalyze the reduction of fatty acyl-CoA to the corresponding fatty alcohol or fatty acyl-ACP to the corresponding fatty aldehyde. Fatty acyl-CoA and fatty acyl-ACP have the structure of R-(CO)-S-R1, wherein R is a C6-C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon, and R1 represents CoA or ACP. In a specific embodiment, R is C6-C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon, and R1 represents CoA or ACP. 24 Saturated or unsaturated straight-chain hydrocarbons. "CoA" is a non-proteinaceous acyl carrier group involved in fatty acid synthesis and oxidation. "ACP" is the acyl carrier protein, a polypeptide or protein subunit, of fatty acid synthase, which is used to synthesize fatty acids.

[0224] Thus, in some embodiments, the present disclosure provides fatty alcohol-forming fatty acyl reductases that catalyze the reduction of fatty acyl-CoA to the corresponding fatty alcohol. For example, when two NAD(P)H molecules are oxidized to NAD(P) +When R-(CO)-S-CoA is converted to R-CH2OH and CoA-SH. Thus, in some such embodiments, the recombinant microorganisms described herein can include a heterologous fatty alcohol-forming fatty acyl reductase that catalyzes the reduction of fatty acyl-CoA to the corresponding fatty alcohol. In an exemplary embodiment, the recombinant microorganisms disclosed herein include at least one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the reduction of mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty alcohol.

[0225] In other embodiments, the present disclosure provides fatty aldehyde-forming fatty acyl reductases that catalyze the reduction of fatty acyl-ACP to the corresponding fatty aldehyde. For example, when one NAD(P)H molecule is oxidized to NAD(P) + When R-(CO)-S-ACP is converted to R-(CO)-H and ACP-SH. In some such embodiments, the recombinant microorganisms described herein can include a heterologous fatty aldehyde-forming fatty acyl reductase that catalyzes the reduction of fatty acyl-ACP to the corresponding fatty aldehyde. In an exemplary embodiment, the recombinant microorganisms disclosed herein include at least one exogenous nucleic acid molecule encoding a fatty aldehyde-forming fatty acyl reductase that catalyzes the reduction of mono- or polyunsaturated C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 Fatty aldehydes.

[0226] In some insect species, the respective alcohol-forming fatty acyl reductases (FARs) are activated via site-specific dephosphorylation (Jurenka, R. & Rafaeli, A. Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths. Front. Endocrinol. (Lausanne). 2:46 (2011); Gilbert, LI Insect Endocrinology. (Academic Press)). Without being bound by any theory, phosphorylation of heterologously expressed FAR enzymes in yeasts such as Yarrowia lipolytica may lead to inactivation and result in low fatty alcohol titers. In some embodiments, bioinformatics methods can be used to predict phosphorylated residues in FARs. It has been shown that substitution of serine and threonine residues with alanine can eliminate phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acci. mBio 5 (2014)). Therefore, the effect of alanine substitution on preventing phosphorylation of serine residues and its effect on fatty alcohol titer can be tested. In addition to alanine substitution, improvement of FAR activity can also be achieved by substitution of other amino acids.

[0227] In some embodiments, methods are provided for identifying beneficial mutations of FAR based on selection and alteration of phosphorylation-sensitive residues following FAR expression in a host microorganism. In preferred embodiments, the host microorganism is a yeast selected from the genera Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, and Kluyveromyces.

[0228] Other references on protein phosphorylation sites include: Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1. J. Mol. Biol. 294, 1351–1362 (1999); Ingrell, CR, Miller, ML, Jensen, ON & Blom, N. Net Phos Yeast: prediction of protein phosphorylationsites in yeast. Bioinforma. 23: 895–897 (2007); Miller, WT Tyrosine kinase signaling and the emergence of multicellularity. Biochim. Biophys. Acta 1823, 1053–1057 (2012), each of which is incorporated herein by reference in its entirety.

[0229] In some embodiments, a nucleic acid sequence encoding a fatty acyl reductase is codon-optimized and is derived from an organism selected from the species Agrotis segetum, Spodoptera exigua, Spodopteralittoralis, Euglena gracilis, Yponomeuta evonymellus, and Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises the nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises the nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 32, 40, 72, 74, 76, and 81. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO:55 from Agrotissima lutea. In other embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO:56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO:75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO:77 from Prunella sphaeroides.

[0230] In some embodiments, the production of unsaturated fatty alcohols in the recombinant microorganism comprises expression of one or more mutant FARs. In certain embodiments, variants of Helicoverpa armigera fatty acyl-CoA reductase (HaFAR) are provided that have increased enzymatic activity relative to the wild-type Helicoverpa armigera fatty acyl-CoA reductase encoded by the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the increased enzymatic activity is a net increase in the amount of fatty alcohol produced relative to the amount of fatty alcohol produced by the enzymatic activity of the wild-type HaFAR encoded by the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the wild-type HaFAR comprises the nucleotide sequence set forth in SEQ ID NO:90. In some embodiments, the variant of the wild-type HaFAR encoded by the amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises an amino acid selected from the group consisting of F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, and R. In some embodiments, the variant of the wild-type HaFAR encoded by the amino acid sequence set forth in SEQ ID NO: 41 comprises a combination of point mutations selected from the group consisting of mutations at the following amino acid positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises an amino acid selected from the group consisting of F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, and R. In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42- 48. In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.

[0231] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 73, 75, 77, and 82. or a fatty acyl reductase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to any of the fatty acyl reductases of No.

[0232] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding a fatty acyl reductase, wherein the nucleic acid molecule is identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 32, 37, 40, 72, 74, 76, 81, 83, 84, 85, 86, 87, 88, 89, and 90. exhibit at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51% or 50% sequence identity.

[0233] In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl reductase that catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol, wherein the fatty acyl reductase is identical to a nucleic acid molecule selected from the group consisting of SEQ ID NO: The fatty acyl reductases of NOs:41-48, 57, 73, 75 and 77 have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51% or 50% sequence identity.

[0234] Acyl-ACP synthetase

[0235] This disclosure describes enzymes that link fatty acids to the corresponding fatty acyl-ACPs.

[0236] In some embodiments, acyl-ACP synthetase is used to catalyze the conversion of fatty acids into corresponding fatty acyl-ACP. Acyl-ACP synthetase is an enzyme that can connect fatty acids to ACP to produce fatty acid acyl-ACP. In some embodiments, acyl-ACP synthetase can be used to catalyze the conversion of fatty acids into corresponding fatty acyl-ACP. In some embodiments, this acyl-ACP synthetase is a synthetase that can utilize following fatty acids as substrates, and this fatty acid has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms. In such an embodiment, recombinant microorganisms as herein described can include heterologous acyl-ACP synthetase, which catalyzes the conversion of fatty acids into corresponding fatty acyl-ACP. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the synthesis of a saturated C6-C 24 Fatty acids are converted to the corresponding saturated C6-C 24 Fatty acyl-ACP.

[0237] fatty acid synthase complex

[0238] This disclosure describes enzymes that catalyze carbon chain elongation in fatty acids.

[0239] In some embodiments, the fatty acid synthase complex is used to catalyze the initiation and extension of carbon chains in the fatty acid." fatty acid synthase complex" refers to the enzyme that initiates and extends the carbon chains on one group of catalysis fatty acids. The ACP in the fatty acid synthase (FAS) pathway controls the length, saturation and branching of the fatty acid produced together with the enzyme. The steps in this pathway are catalyzed by enzymes of fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families. Depending on desired product, one or more of these genes can be weakened, expressed or overexpressed. In exemplary embodiments, one or more of these genes are overexpressed.

[0240] There are two main classes of fatty acid synthases. Type I (FAS I) systems utilize a single, large, multifunctional polypeptide and are common to both mammals and fungi (although the structural arrangement of the fungal and mammalian synthases differs). Type I FAS systems are also found in the CMN bacterial group (Corynebacteria, Mycobacteria, and Nocardia). Type II FAS (FAS II) is characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis and is found in archaea and bacteria.

[0241] The mechanisms of elongation and reduction of FAS I and FAS II are essentially similar, as the domain structures of the FAS I multienzyme polypeptide and the FAS II enzyme are largely conserved.

[0242] Fatty acid is synthesized by a series of decarboxylation Claisen condensation reactions from acetyl-CoA and malonyl-CoA. The steps in this approach are catalyzed by enzymes of fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families. For the description of this approach, see, for example, Heath et al., Prog.Lipid Res.40:467,2001, which is incorporated herein by reference in its entirety. Not limited by theory, in bacterium, acetyl-CoA is carboxylated to form malonyl-CoA by acetyl-CoA carboxylase (Acc, a multi-subunit enzyme encoded by four independent genes accABCD). In yeast, acetyl-CoA is carboxylated by the yeast equivalent of the acetyl-CoA carboxylase encoded by ACC1 and ACC2. In bacterium, the malonate group is transferred to ACP to form malonyl-ACP by malonyl-CoA:ACP transacylase (FabD). In yeast, the malonyl-palmitoyltransferase domain adds the malonyl group from malonyl-CoA to the ACP domain of the FAS complex. A condensation reaction then occurs, where malonyl-ACP combines with acyl-CoA to yield β-ketoacyl-ACP. In this way, the hydrocarbon substrate is elongated by two carbon atoms.

[0243] After elongation, the β-keto group is reduced to a fully saturated carbon chain through the sequential action of ketoreductase (KR), dehydratase (DH) and enol reductase (ER). The extended fatty acid chain is located between these active sites and is covalently attached to the phosphopantetheine prosthetic group of ACP. First, β-ketoacyl-ACP is reduced by NADPH to form β-hydroxyacyl-ACP. In bacteria, this step is catalyzed by β-ketoacyl-ACP reductase (FabG). The equivalent yeast reaction is catalyzed by the ketoreductase (KR) domain of FAS. β-hydroxyacyl-ACP is then dehydrated to form trans-2-enoyl-ACP, which is catalyzed by β-hydroxyacyl-ACP dehydratase / isomerase (FabA) or β-hydroxyacyl-ACP dehydratase (FabZ) in bacteria or the dehydratase (DH) domain of FAS in yeast. NADPH-dependent trans-2-enoyl-ACP reductases I, II, or III (Fab1, FabK, and FabL, respectively) in bacteria and the enol reductase (ER) domain of FAS in yeast reduce trans-2-enoyl-ACP to form acyl-ACP. The subsequent cycle begins with the condensation of malonyl-ACP with acyl-ACP by β-ketoacyl-ACP synthase I or β-ketoacyl-ACP synthase II (FabB and FabF, respectively, in bacteria, or β-ketoacyl synthase (KS) domain in yeast).

[0244] In some embodiments, a fatty acid synthase complex can be used to catalyze the elongation of a fatty acyl-ACP to a corresponding fatty acyl-ACP having a two-carbon extension relative to the substrate.

[0245] Dehydrogenase

[0246] The present disclosure describes enzymes that catalyze the conversion of fatty aldehydes to fatty alcohols. In some embodiments, alcohol dehydrogenases (ADHs, Table 3 and Table 3a) are used to catalyze the conversion of fatty aldehydes to fatty alcohols. A number of ADHs identified from alkanotrophic organisms, namely Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butyricum ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have been shown to convert short-chain to medium-chain alkyl alcohols (C2 to C4). 14 ) are active. In addition, commercially available ADH from Sigma, horse liver ADH and baker's yeast ADH have a length of C 10 Detectable activity was observed with substrates of 100 or longer. The reported activity with longer fatty alcohols may be affected by difficulties in solubilizing the substrates. For yeast ADH from Sigma, little activity was observed with C 12 -C14 activity on aldehydes (Tani et al. 2000), however, no 12 and C 16 The activity of hydroxy-ω-fatty acids (Lu et al. 2010). Recently, the degradation of C 15 -C 36 The alkane-producing organism, Geobacillus thermodenitrificans NG80-2, was characterized to produce two ADHs from methanol to 1-triacontanol (C 30 ) assay for activity against ADH, where 1-octanol is the preferred substrate for ADH2 and ethanol is the preferred substrate for ADH1 (Liu et al. 2009).

[0247] The use of ADH in whole-cell bioconversion has primarily focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3-hydroxybutyrate from methyl acetoacetate, with a space-time yield of 29 g / L / h (Schroer et al. 2007). Examples of fatty alcohol oxidation in whole-cell conversions have been reported using commercially available Saccharomyces cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).

[0248] Table 3. Exemplary alcohol dehydrogenases.

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] Table 3a. Additional exemplary alcohol dehydrogenases

[0262]

[0263]

[0264] In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation and / or reduction in activity of one or more endogenous (fatty) alcohol dehydrogenases selected from YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7).

[0265] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion or other disruption of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the proteins encoded by YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), and YALI0E07766g (ADH7).

[0266] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91% or 90% sequence identity to uniprot database IDs Q6C297 (FADH), Q6C7T0 (ADH1), F2Z678 (ADH2), Q6CGT5 (ADH3), Q6C5R5 (ADH4), Q6CAT5 (ADH5), Q6CGX5 (ADH6), and Q6C7K3 (ADH7).

[0267] alcohol oxidase

[0268] This disclosure describes enzymes that oxidize fatty alcohols to fatty aldehydes.

[0269] In some embodiments, alcohol oxidase (AOX) is used to catalyze the conversion of fatty alcohols to fatty aldehydes. Alcohol oxidase catalyzes the conversion of alcohols to the corresponding aldehydes (or ketones) by electron transfer using molecular oxygen, forming hydrogen peroxide as a byproduct. The AOX enzyme utilizes flavin adenine dinucleotide (FAD) as an essential cofactor and is regenerated with the help of oxygen in the reaction medium. Catalase can be coupled to AOX to prevent the accumulation of hydrogen peroxide by catalyzing the conversion to water and oxygen.

[0270] Based on substrate specificity, AOX can be divided into four groups: (a) short-chain alcohol oxidases, (b) long-chain alcohol oxidases, (c) aromatic alcohol oxidases, and (d) secondary alcohol oxidases (Goswami et al. 2013). Depending on the chain length of the desired substrate, some members of these four groups are more suitable candidates for evaluation than others.

[0271] Short-chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 4) catalyze the oxidation of low-chain alcohol substrates in the C1-C8 carbon range (van der Klei et al. 1991) (Ozimek et al. 2005). Fatty alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Pichia pastoris (formerly known as Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes, with preference for unbranched, short-chain fatty alcohols. The broadest substrate specificity was found for the alcohol oxidase from Pichia pastoris (including propargyl alcohol, 2-chloroethanol, and 2-cyanoethanol) (Dienys et al. 2003). A major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilizing a two-liquid phase system (water / solvent), the aldehyde product can be removed in situ from the reaction phase before further conversion to the acid. For example, the production of hexanal from hexanol using a Pichia pastoris alcohol oxidase coupled to bovine liver catalase was achieved in a biphasic system using the presence of a stable alcohol oxidase in the aqueous phase (Karra-Chaabouni et al. 2003). For example, an alcohol oxidase from Pichia pastoris is capable of oxidizing C6 to C11 aliphatic alcohols when a biphasic organic reaction system is used (Murray and Duff 1990). Methods for using alcohol oxidases in biphasic systems according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.

[0272] Long-chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 5) include fatty alcohol oxidases, long-chain fatty acid oxidases, and long-chain fatty alcohol oxidases, which oxidize alcohol substrates with carbon chain lengths greater than six (Goswami et al. 2013). Banthorpee et al. reported long-chain alcohol oxidases purified from the leaves of Tanacetum vulgare, which were able to oxidize saturated and unsaturated long-chain alcohol substrates, including hexane-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Jojoba (Moreau, RA, Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and lotus (Zhao et al. 2008), have also been reported as sources of long-chain alcohol oxidases. Fatty alcohol oxidases are primarily reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990), and these enzymes play an important role in long-chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species, which degrade and grow on long-chain alkanes and fatty acids, catalyze the oxidation of fatty alcohols. Fatty alcohol oxidases from Candida tropicalis have been isolated as microsomal cell fractions and characterized against a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). For C8 to C8 fatty acid oxidases, the enzymes are oxidized to form fatty alcohols. 16 Significant activity was observed for primary alcohols, with the reported K M In the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases can be used to convert medium-chain fatty alcohols to aldehydes, for example, as described for whole-cell Candida boidinii (Gabelman and Luzio 1997) and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long-chain alcohol oxidases are produced from filamentous fungi during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). A long-chain fatty alcohol oxidase from Nelumbo nucifera (LjFAO1) has been heterologously expressed in Escherichia coli and exhibits broad substrate specificity for alcohol oxidation, including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).

[0273] Table 4. Alcohol oxidases capable of oxidizing short-chain alcohols (EC 1.1.3.13)

[0274]

[0275]

[0276]

[0277]

[0278] Table 5. Alcohol oxidases (EC 1.1.3.20) capable of oxidizing long-chain alcohols including fatty alcohols

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous (fatty) alcohol oxidases YALI0B14014g (FAO1).

[0302] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion or other disruption of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the protein encoded by (fatty) alcohol oxidase YALI0B14014g (FAO1).

[0303] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to uniprot database ID Q6CEP8 (FAO1).

[0304] Acetyltransferase

[0305] This disclosure describes enzymes that convert alcohols to fatty acetates.

[0306] In some embodiments, an acetyltransferase is used to catalyze the conversion of fatty alcohols to fatty acetates. An acetyltransferase is an enzyme that has the ability to produce acetate by transferring an acetyl group from acetyl-CoA to an alcohol. In some embodiments, the acetyltransferase may have an EC number of 2.3.1.84.

[0307] The acetyltransferase or the nucleic acid sequence encoding it can be isolated from various organisms, including but not limited to organisms of the species Candida glabrata, Saccharomyces cerevisiae, Danaus plexippus, Heliothis virescens, Bombyx mori, Agrotis ipsilon, Agrotis fulva, Euonymus alatus, Homo sapiens, Kluyveromyces thermotolerans, and Yarrowia lipolytica. In an exemplary embodiment, the acetyltransferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1, and XP_505513.1. Exemplary acetyltransferases are listed in Table 5d. Additional exemplary acetyltransferase peptides can be found in US 2010 / 0199548, which is incorporated herein by reference.

[0308] Table 5d. Exemplary acetyltransferases

[0309]

[0310] Fatty acyl-ACP thioesterase

[0311] Acyl-ACP thioesterases release free fatty acids from acyl-ACPs (synthesized by de novo fatty acid biosynthesis). The reaction terminates fatty acid biosynthesis. In plants, fatty acid biosynthesis occurs in the plastids, and therefore requires plastid-localized acyl-ACP thioesterases. In the vegetative tissues of all plants, the major product of acyl-ACP thioesterases is oleate (C18:0) and, to a lesser extent, palmitate (C16:0). The released free fatty acids are re-esterified to coenzyme A in the plastid envelope and exported from the plastids.

[0312] There are two acyl-ACP thioesterase isoforms, FatA and FatB. The substrate specificity of these isoforms determines the chain length and level of saturated fatty acids in plants. FatA has the highest activity towards C18:1-ACP. Compared to C18:1-ACP, FatA has very low activity towards other acyl-ACPs. FatB has the highest activity towards C16:0-ACP. It also has significantly higher activity towards C18:1-ACP, followed by C18:0-ACP and C16:1-ACP. Kinetic studies of FatA and FatB indicate that their substrate specificity for different acyl-ACPs comes from their Kcat values, rather than from their Km values. The Km values ​​for the two isoforms for different substrates are similar on the micromolar scale. Domain swapping of FatA and FatB indicates that the N-termini of the isoforms determine their substrate specificity (Salas JJ and Ohlrogge JB (2002) Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Arch BioChem Biophys 403(1):25-34). For those plants that accumulate primarily medium-chain saturated fatty acids in seeds, they have evolved FatB and / or FatA thioesterases (Voelker T and Kinney AJ (2001) Variations in the biosynthesis of seed-storage lipids. Annu Rev Plant Physiol Plant Mol Biol 52:335-361). For example, laurate (12:0) is the major seed oil in coconut. Accordingly, medium-chain specific acyl-ACP thioesterase activity has been detected in coconut seeds.

[0313] In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and / or reduction in activity of YALI0E16016g (FAT1).

[0314] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion or other disruption of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the protein encoded by YALI0E16016g (FAT1).

[0315] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to uniprot database ID Q6C5Q8 (FAO1).

[0316] Acyl-CoA oxidase

[0317] Acyl-CoA oxidases (ACOs) act on CoA derivatives of fatty acids with chain lengths of 8 to 18. They are flavoenzymes that contain a non-covalently bound FAD per subunit and belong to the same superfamily as mitochondrial acyl-CoA dehydrogenases. Like mitochondrial fatty acyl-CoA dehydrogenases, peroxisomal acyl-CoA oxidases catalyze the initiation and rate-limiting step of the peroxisomal fatty acid β-oxidation pathway (i.e., α,β-dehydrogenation of acyl-CoA), which produces trans-2-enoyl-CoA in the reductive half-reaction. In the oxidative half-reaction of peroxisomal acyl-CoA oxidases, the reduced FAD is reoxidized by molecular oxygen to produce hydrogen peroxide.

[0318] Acyl-CoA oxidase is a homodimer, and the polypeptide chain of the subunit folds into an N-terminal α domain, a β domain, and a C-terminal α domain. The functional differences between peroxisomal acyl-CoA oxidase and mitochondrial acyl-CoA dehydrogenase are attributed to structural differences in the FAD environment.

[0319] In some embodiments, recombinant microorganisms and methods for producing short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates are provided. In certain embodiments, the carbon chain length of the short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates is less than or equal to C16. In some embodiments, the short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates are produced from long-chain fatty acids. In some preferred embodiments of the methods for producing short-chain pheromones, a selected enzyme capable of shortening fatty acyl-CoAs in the pheromone biosynthetic pathway is co-expressed with the pheromone biosynthetic pathway enzymes. Examples of suitable chain-shortening enzymes include FAD-dependent acyl-CoA oxidases. In the case of fatty acid molecules with an even number of carbon atoms, the chain-shortening enzyme produces acetyl-CoA molecules and fatty acyl-CoAs shortened by two carbon atoms. During each round of oxidation, fatty acid molecules with odd numbers of carbon atoms are oxidized in a similar manner to produce acetyl-CoA molecules until the chain length is reduced to 5 carbon atoms. In the final round of oxidation, the 5-carbon acyl-CoA is oxidized to produce acetyl-CoA and propionyl-CoA.

[0320] Acyl-CoA oxidases are known to exhibit different specificities for substrates with different chain lengths ( Figure 40). Therefore, controlling the degree of fatty acyl-CoA truncation depends on engineering or selecting suitable enzyme variants. Examples of acyl-CoA oxidases suitable for this purpose are listed in Table 5a.

[0321] In another embodiment, the present disclosure provides a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 The recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the oxidation of a mono- or polyunsaturated C6-C6 fatty acyl-CoA fatty acid from (a) after one or more consecutive cycles of acyl-CoA oxidase activity. 24 Fatty acyl-CoA is converted to mono- or polyunsaturated ≤C 18 Fatty acyl-CoA, where a given cycle produces a mono- or polyunsaturated C6-C 24 Fatty acyl-CoA substrates have two carbon-truncated mono- or polyunsaturated C4-C 22a fatty acyl-CoA intermediate; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of the mono- or polyunsaturated ≤C18 fatty acyl-CoA from (b) to the corresponding mono- or polyunsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from the group consisting of fatty acyl desaturases derived from Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera litura, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana. In some embodiments, the fatty acyl desaturase is selected from the group consisting of SEQ ID NO: NO:39, 49-54, 58-63, 78-80 and GenBank accession numbers AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1 have a fatty acyl desaturase with at least 99%, 98%, 97% or more of the fatty acyl desaturase. %, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity. In some embodiments, the acyl-CoA oxidase is selected from Table 5a.In other embodiments, the fatty alcohol-forming fatty acyl reductase is selected from the group consisting of fatty alcohol-forming fatty acyl reductases derived from Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus, and Helicoverpa armigera. In yet another embodiment, the fatty alcohol-forming fatty acyl reductase is selected from the group consisting of SEQ ID Nos. 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82 have a fatty alcohol-forming fatty acyl reductase of at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%. %, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50% or 50% sequence identity. In some embodiments, the recombinant microorganism is a yeast selected from Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.

[0322] In some preferred embodiments of the method for producing fatty alcohols, fatty aldehydes and / or fatty acetates, one or more genes encoding acyl-CoA oxidases of the microbial host are deleted or down-regulated to eliminate or reduce the desired truncation of fatty acyl-CoA beyond the desired chain length. Such deletion or down-regulation targets include, but are not limited to, Yarrowia lipolytica POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E06567g); Mother POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

[0323] In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and / or reduction in activity of one or more acyl-CoA oxidases selected from the group consisting of POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E06567g).

[0324] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion or other disruption of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the protein encoded by POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E06567g).

[0325] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to uniprot database ID POX1 (074934), Yarrowia lipolytica POX2 (074935), Yarrowia lipolytica POX3 (074936), Yarrowia lipolytica POX4 (F2Z627), Yarrowia lipolytica POX5 (F2Z630), Yarrowia lipolytica POX6 (Q6C6T0).

[0326] In some embodiments, saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 The invention relates to a recombinant microorganism that produces fatty alcohols, fatty aldehydes, and / or fatty acetates, wherein the recombinant microorganism expresses one or more acyl-CoA oxidases, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and / or reduce the activity of one or more endogenous acyl-CoA oxidases. In some embodiments, the one or more acyl-CoA oxidases expressed are different from the one or more endogenous acyl-CoA oxidases that are deleted or downregulated. In some embodiments, the recombinant microorganism further expresses a pheromone biosynthetic pathway enzyme. In other embodiments, the pheromone biosynthetic pathway enzyme comprises one or more fatty acyl desaturases and / or fatty acyl ligases. In yet another embodiment, the pheromone biosynthetic pathway enzyme comprises one or more fatty acyl reductases that form fatty alcohols. In some embodiments, the one or more acyl-CoA oxidases expressed are selected from Table 5a. In other embodiments, the one or more acyl-CoA oxidases expressed regulate the production of mono- or polyunsaturated ≤C 18In some embodiments, the one or more endogenous acyl-CoA oxidases that are deleted, disrupted, mutated, or downregulated are selected from the group consisting of Yarrowia lipolytica POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E32835g), Yarrowia lipolytica POX7 (YALI0E32835g), Yarrowia lipolytica POX8 (YALI0E32835g), Yarrowia lipolytica POX9 (YALI0E32835g), Yarrowia lipolytica POX10 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E32835g), Yarrowia lipolytica POX7 (YALI0E32835g), Yarrowia lipolytica POX8 (YALI0E32835g), Yarrowia lipolytica POX9 (YALI0E32835g), Yarrowia lipolytica POX11 (YALI0E32835g), Yarrowia lipolytica POX1 E06567g); Saccharomyces cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, one or more endogenous acyl-CoA oxidases that are deleted, disrupted, mutated, or downregulated control the production of mono- or polyunsaturated ≤C 18 Chain length of the fatty alcohol, fatty aldehyde and / or fatty acetate.

[0327] In some embodiments, saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18A method for producing fatty alcohols, fatty aldehydes, and / or fatty acetates, wherein the method comprises introducing into a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase, and introducing a deletion, insertion, or loss-of-function mutation in one or more genes encoding an acyl-CoA oxidase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase that is introduced or expressed is different from the one or more genes encoding an acyl-CoA oxidase that is deleted or down-regulated. In some embodiments, the method further comprises introducing into a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase. In other embodiments, the method further comprises introducing into the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl reductase that forms fatty alcohols or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl reductase that forms fatty alcohols. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase selected from Table 5a. In other embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase that regulates the production of mono- or polyunsaturated C6-C 24 In some embodiments, the one or more genes deleted or downregulated encode an acyl-CoA oxidase selected from the group consisting of Yarrowia lipolytica POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E27664g), Yarrowia lipolytica POX7 (YALI0E276657g), Yarrowia lipolytica POX8 (YALI0E276664g), Yarrowia lipolytica POX9 (YALI0E276665g), Yarrowia lipolytica POX10 (YALI0E276665g), Yarrowia lipolytica POX11 (YALI0E276665g), Yarrowia lipolytica POX12 (YALI0E276665g), Yarrowia lipolytica POX13 (YALI0E276665g), Yarrowia lipolytica POX14 (YALI0E276665g), Yarrowia lipolytica POX15 (YALI0E276665g), Yarrowia lipolytica POX16 (YALI0E 06567g); Saccharomyces cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160) and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, one or more genes that are deleted or downregulated encode acyl-CoA oxidases that regulate the production of mono- or polyunsaturated C6-C 24 Chain length of the fatty alcohol, fatty aldehyde and / or fatty acetate.

[0328] Table 5a. Exemplary acyl-CoA oxidases

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339] Acyltransferase

[0340] In some embodiments, recombinant microorganisms and methods for producing short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates are provided. In certain embodiments, the carbon chain length of the short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates is less than or equal to C18. In some preferred embodiments of the methods for producing short-chain pheromones, a selective enzyme for storing short-chain fatty acyl-CoA is preferably co-expressed with one or more fatty acyl desaturases. Suitable acyltransferases are exemplified by heterologous or engineered glycerol-3-phosphate acyltransferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerophospholipid acyltransferases (GPLATs), and / or diacylglycerol acyltransferases (DGATs). Examples of acyltransferases suitable for this purpose are listed in Table 5b.

[0341] In some preferred embodiments of the methods for producing fatty alcohols, fatty aldehydes, and / or fatty acetates, one or more genes encoding glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT), and / or diacylglycerol acyltransferase (DGAT) of the microbial host are deleted or downregulated and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs that preferentially store short-chain fatty acyl-CoAs. Such deletion or down-regulation targets include, but are not limited to, Yarrowia lipolytica YALI0C00209g, Yarrowia lipolytica YALI0E18964g, Yarrowia lipolytica YALI0F19514g, Yarrowia lipolytica YALI0C14014g, Yarrowia lipolytica YALI0E16797g, Yarrowia lipolytica YALI0E32769g, Yarrowia lipolytica YALI0D07986g, Saccharomyces cerevisiae YBL011w, Saccharomyces cerevisiae YDL052c, Saccharomyces cerevisiae YOR175C, Saccharomyces cerevisiae YPR139C, Saccharomyces cerevisiae YNR008w, Saccharomyces cerevisiae YOR245c, Candida albicans I503_02577 , Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In other embodiments, the acyltransferase is inserted at the AXP acid extracellular protease site (YALI0B05654g).

[0342] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising an acyltransferase that exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to any one of SEQ ID Nos. selected from 92.

[0343] Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding an acyltransferase, wherein the nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to any one of SEQ ID Nos. selected from 91.

[0344] In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding an acyltransferase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to an acyltransferase selected from SEQ ID NO: 92.

[0345] In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and / or reduced activity of one or more acyltransferases selected from YALI0E32791g (DGA1) and / or YALI0D07986g (DGA2).

[0346] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion or other disruption of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the proteins encoded by YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

[0347] Thus, in some embodiments, the recombinant microorganisms of the present disclosure will comprise a deletion of an endogenous gene encoding a protein that exhibits at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91% or 90% sequence identity to uniprot database ID Q6C3R1 (DGA1) and / or Q6C9V5 (DGA2).

[0348] Glycerol-3-phosphate acyltransferase (GPAT)

[0349] The present disclosure describes enzymes that catalyze the acylation reaction at the sn-1 position of glycerol 3-phosphate, the reaction being as follows:

[0350] Long-chain acyl-CoA + sn-glycerol 3-phosphate → 1-acyl-sn-glycerol 3-phosphate + coenzyme A.

[0351] Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation reaction at the sn-1 position of glycerol 3-phosphate. Plant cells contain 3 types of GPAT, which are located in chloroplasts, mitochondria and cytoplasm. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as acyl donor, while the enzyme in mitochondria or cytoplasm is bound to membrane and uses acyl-CoA as acyl donor (Nishida I et al. (1993) The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana. Plant Mol Biol. 21 (2): 267-77; Murata N and Tasaka Y (1997) Glycerol-3-phosphate acyltransferase in plants. Biochim Biophys Acta. 1348 (1-2): 10-16).

[0352] Eight GPAT genes have been identified in Arabidopsis (Zheng Z et al. (2003) Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphateacyltransferase gene family, is essential for tapetum differentiation and male fertility. Plant Cell 15(8):1872-87). GPAT1 has been shown to encode a mitochondrial enzyme (Zheng et al. 2003). It has been shown that GPAT4, GPAT5 and GPAT8 are essential for cutin biosynthesis (Beisson F et al. (2007) The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19(1):351-368; Li, Y et al. (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci USA 104(46):18339-18344). GPAT2, GPAT3, GPAT6 and GPAT7 have not yet been characterized.

[0353] Cytoplasmic GPAT is responsible for the synthesis of triacylglycerol and non-chloroplast membrane phospholipids. Since these two fatty acids are the most common fatty acids found at the sn-1 position of plant triacylglycerol, it is expected that there is a substrate preference for palmitate (C16:0) and oleate (C18:1). Cytoplasmic GPAT was partially purified from avocado (Eccleston VS and Harwood JL (1995) Solubilisation, partial purification and properties of acyl-CoA:glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp. Biochim Biophys Acta 1257 (1): 1-10).

[0354] The membrane-bound glycerol-3-phosphate acyltransferase (PlsB) from Escherichia coli (E. coli) catalyzes the first key step in phospholipid biosynthesis and is thought to function in close proximity to the subsequent enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC) (Kessels JM et al. (1983) Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli. Biochim Biophys Acta 753(2):227-235). It is specific for acylation at the 1-position of sn-glycerol 3-phosphate and can utilize either fatty acyl-acyl carrier protein (acyl-ACP) or fatty acyl-coenzyme A (acyl-CoA) thioester as an acyl donor to form 1-acyl-sn-glycerol 3-phosphate. The fatty acids synthesized by the endogenous source are attached to ACP, and the fatty acids added by the exogenous source are attached to CoA. In E. coli phospholipids, the sn 1 position is mainly occupied by palmitic acid or cis-vaccenic acid, while the sn 2 position is mainly occupied by palmitic acid or cis-vaccenic acid. This is believed to be caused by the substrate preference of the PlsB and PlsC enzymes.

[0355] The plsB gene has been shown to be regulated by stress response regulators such as RNA polymerase, σ24 (σE) factor, and ppGpp (Wahl A et al. (2011) Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol 80(5):1260-75). PlsB is part of a protein network for phospholipid synthesis and interacts with holo-[acyl-carrier protein] (ACP), esterase / thioesterase (YbgC), and phosphatidylserine synthase (PssA) to form a complex at the cytoplasmic side of the inner membrane.

[0356] plsB is essential for growth (Baba T et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2:2006-2008; Yoshimura M et al. (2007) Involvement of the YneS / YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli. BMC Microbiol 7:69).

[0357] Site-directed mutagenesis and chemical modification studies have identified catalytically important amino acid residues in plsB, including an invariant histidine residue essential for catalysis (Lewin TM et al. (1999) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38(18):5764-5771). Genetic studies have identified the plsB locus as involved in the formation of multidrug-resistant persister cells.

[0358] In early work, the properties of the Escherichia coli (E. coli) B enzyme were studied (Kito M et al. (1972) Inhibition of L-glycerol 3-phosphate acyltransferase from Escherichia coli by cis-9,10-methylenehexadecanoic acid. J Biochem 71(1):99-105; Okuyama H and Wakil SJ (1973) Positional specificities of acyl coenzyme A:glycerophosphate and acylcoenzyme A:monoacylglycerophosphate acyltransferases in Escherichia coli. J Biol Chem 248(14):5197-5205; Kito M et al. (1978) Function of phospholipids on thegulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli. Biochim Biophys Acta 529(2):237-2499).

[0359] The glycerol-3-phosphate / dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferase is localized in lipid particles and the ER and is involved in the stepwise acylation of glycerol-3-phosphate and dihydroxyacetone in lipid biosynthesis. The most conserved motifs and functionally relevant residues are oriented toward the ER lumen.

[0360] A gene (SCT1) encoding a diglycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) was identified, cloned, and biochemically characterized from the yeast Saccharomyces cerevisiae. Overexpression of SCT1 from a plasmid vector in a yeast Δgpt1 mutant, which exhibits very low GAT / DHAT activity, showed increased GAT / DHAT activity, highlighting the proposed molecule's function as a glycerol-3-phosphate O-acyltransferase / dihydroxyacetone phosphate acyltransferase. GAT / DHAT activity towards the acyl donor was highest with palmitoyl-CoA, followed by palmitoyl-CoA, oleoyl-CoA, and stearoyl-CoA. SCT1p was localized to membranes in the cytosol, most likely to the endoplasmic reticulum. In vivo studies of the Δsct1 mutant did reveal effects on all four phospholipids, but the observed decrease in 16:0 fatty acids in the phosphatidylethanolamine class was offset by an increase in other fatty acids, particularly 18:0 species. Null mutants of SCT1 and GPT2 are synthetically lethal in yeast (Zheng Z and Zou J (2001) The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate / dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae. J Biol Chem 276(45):417104-41716).

[0361] The gene (GPT2) encoding diglycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) from the yeast Saccharomyces cerevisiae was identified, cloned, and biochemically characterized. GPT2 was recombinantly expressed in Escherichia coli in a ΔplsB background lacking GAT / DHAT activity and exhibited increased GAT activity, but the mutant could not be rescued due to mis-intercalation of GPT2 into the membrane. Overexpression of GPT2 from a plasmid vector in the yeast Δgpt1 mutant, which exhibits very low GAT / DHAT activity, showed increased GAT / DHAT activity, highlighting the proposed molecule's function as a glycerol-3-phosphate O-acyltransferase / dihydroxyacetone phosphate acyltransferase. GAT / DHAT activity towards the acyl donor was highest with oleoyl-CoA, followed by palmitoleoyl-CoA, palmitoyl-CoA, and stearoyl-CoA.

[0362] GPT2p is located in the membrane in the cytosol. In vivo studies of the Δgpt2 mutant did not reveal any significant effects on the overall fatty acid profile, but a decrease in 16:1 fatty acids of the phosphatidylethanolamine class was observed, which was compensated by an increase in 16:0 and 18:1 molecular species. Analysis of the yeast mutant TTA1, known to be defective in GAT activity, revealed that the TTA1 GPT2 gene harbors a missense mutation with a single nucleotide change in the conserved motif III of the acyltransferase. Null mutants of SCT1 and GPT2 are synthetically lethal in yeast (Zheng and Zou 2001).

[0363] In some embodiments, the glycerol-3-phosphate acyltransferase is GPAT (At1g02390) from Arabidopsis thaliana. In some embodiments, the glycerol-3-phosphate acyltransferase is PlsB (Gene ID EG10740) from Escherichia coli. In some embodiments, the glycerol-3-phosphate acyltransferase is diglycerol-3-phosphate O-acyltransferase (GAT) / dihydroacetonephosphate acyltransferase (DHAT) SCT1 (YBL011w) from Saccharomyces cerevisiae. In some embodiments, the glycerol-3-phosphate acyltransferase is YALI0C00209g from Yarrowia lipolytica. In some embodiments, the glycerol-3-phosphate acyltransferase is I503_02577 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG_02630 from Candida tropicalis. In some embodiments, the glycerol-3-phosphate acyltransferase is diglycerol-3-phosphate O-acyltransferase (GAT) / dihydroacetonephosphate acyltransferase (DHAT) GPT2 (YKR067w) from Saccharomyces cerevisiae. In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.5815 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.13237 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG_02630 from Candida tropicalis.

[0364] Lysophosphatidic acid acyltransferase (LPAAT)

[0365] The present disclosure describes enzymes that catalyze the acylation of the sn-2 position of triacylglycerols.

[0366] The membrane-bound 1-acylglycerol 3-phosphate O-acyltransferase, encoded by the gene plsC, catalyzes the second step in phospholipid biosynthesis and is thought to function in close proximity to the preceding enzyme, glycerol-3-phosphate acyltransferase, encoded by the gene plsB (Kessels JM et al. 1983). This is specific for the acylation reaction at the sn-2 position of 1-acyl-sn-glycerol 3-phosphate and can utilize either acyl-acyl carrier protein (acyl-ACP) or acyl-coenzyme A (acyl-CoA) as a fatty acyl donor to form 1,2-diacyl-sn-glycerol 3-phosphate (phosphatidate, phosphatidic acid). Endogenously synthesized fatty acids are attached to ACP, and exogenously added fatty acids are attached to CoA (Greenway DL and Silbert DF (1983) Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase. J Biol Chem 258(21):13034-13042). In Escherichia coli (E. coli), the phospholipids at the sn 1 position are mainly occupied by palmitic acid or cis-vaccenic acid, while the sn 2 position is mainly occupied by palmitoleic acid or cis-vaccenic acid. This is thought to be due to the substrate preferences of the PlsB and PlsC enzymes (Rock CO et al. (1981) Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate. J Biol Chem 256(2):736-742; Goelz SE and Cronan JE (1980) The positional distribution of fatty acids in Escherichia coliphospholipidsis not regulated by sn-glycerol 3-phosphate levels. J Bacteriol 144(1):462-464).

[0367] Site-directed mutagenesis studies have shown that changing threonine-122 to alanine or leucine results in a change in acyl-CoA substrate specificity (Morand LZ et al. (1998) Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis. Biochem Biophys Res Commun 244(1):79-84).

[0368] In engineered Escherichia coli (E. coli) strains, overexpression of PlsC and GalU resulted in increased production of glyceroglycolipids (Mora-Buye N et al. (2012). An engineered E. coli strain for the production of glycoglycerolipids. Metab Eng 14(5):551-559).

[0369] The plsC gene of Streptococcus pneumoniae encodes a 1-acylglycerol-3-phosphate acyltransferase homologous to the E. coli enzyme. The gene was cloned and expressed in E. coli and showed the expected function of membrane catalysis (Lu YJ et al. (2006) Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Mol Cell 23(5):765-772).

[0370] Plant lysophosphatidic acid acyltransferases (LPAATs) catalyze the acylation of the sn-2 position of triacylglycerols. In a given plant species, the substrate specificity of the LPAAT generally determines which fatty acid species are incorporated at the sn-2 position. LPAATs have been cloned from corn and meadow foam. There are two LPAAT genes in meadow foam, while there is only one in corn. The enzymatic activity of both LAT1 and LAT2 in meadow foam has been confirmed by in vitro assays. In addition, LAT2 has been shown to functionally complement LPAAT-deficient strains of Escherichia coli (E. coli) (Brown AP et al. (2002) Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene. Biochem J 364 (Pt 3): 795-805).

[0371] LAT1 is a highly selective acyltransferase that uses only 18:1-CoA as a substrate. LAT2 has lower selectivity. It exhibits the highest activity towards 22:1-CoA, followed by 16:0- and 18:1-CoA. The substrate specificities of LAT1 and LAT2 are consistent with their proposed roles: LAT1 in membrane lipid biosynthesis and LAT2 in storage lipid biosynthesis. Plant cell membranes primarily contain C16 and C18 unsaturated fatty acids, while storage lipids contain a wide range of fatty acids, including saturated fatty acids and very long-chain unsaturated fatty acids. Antibodies were used to measure LAT1 and LAT2 protein levels in various plant tissues. LAT1 was found in leaves and developing seeds, while LAT2 was only detected in developing seeds. This is again consistent with its proposed role. The role of LAT2 in triacylglycerol biosynthesis was further demonstrated by its conversion in rapeseed, which normally does not contain 22:1-CoA at the sn-2 position. Transformation of meadow foam LAT2 inserts 22:1-CoA at the sn-2 position (Lassner MW et al. (1995) Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol 109(4):1389-1394).

[0372] Using a viable mutant yeast strain deficient in sphingolipid biosynthesis, the gene SLC1 was isolated and shown to encode an acyl-CoA:lysophosphatidic acid acyltransferase. Sequence homology to the PLSC protein of E. coli, which is classified as a 1-acyl-sn-glycerol-3-phosphate acyltransferase, suggests similar functions. This putative molecular function of SLC1p was confirmed by its ability to rescue the ΔplsC mutant of E. coli. It could be shown that a single nucleotide change at position 131, changing L-glutamine to L-leucine, shifts the substrate preference from C16 and C18 fatty acids to C26 fatty acids, which is reflected in vivo in the corresponding fatty acid composition of the wild type (SLC1) versus the mutant (SLC1-1) (Nagiec MM et al. (1993) A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J Biol Chem 268(29):22156-22163).

[0373] In vitro assays with SLC1p recombinantly expressed and purified in Escherichia coli revealed a substrate preference for lysophosphatidic acid and oleoyl-CoA, but also accepted 1-palmitoylglycerol 3-phosphate and 1-stearoyl-sn-glycerol 3-phosphate. In vivo studies of mutants such as Δslc1, Δslc4 (another potential acyl-CoA:phosphatidyl acyltransferase) and a double mutant of Δslc1Δslc4 (which carries a plasmid with the SLC1 or SLC4 gene, designated 2.ΔSLC1 (or 2.ΔSLC4)) showed that SLC1 promoted the biosynthesis of phosphatidylate as well as phosphatidylinositol and diacylglycerol. This suggests that SLC1 participates in phospholipid remodeling by exchanging fatty acids on glycerophospholipids in vivo (Benghezal M et al. (2007) SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast. J Biol Chem 282(42):30845-30855).

[0374] The yeast genome was screened using candidate open reading frames (ORFs) of known acyltransferases and related deletion strains were tested to identify a gene encoding an acyl-CoA-dependent lysophosphatidyltransferase (ALE1). In the Δale1 strain, a sharp decrease in lysophosphatidylethanolamine acyltransferase (LPEAT) activity was observed, but it was also demonstrated that ALE1p can provide excess lysophosphatidyl acyltransferase (LPAAT) activity when the main LPAAT in Saccharomyces cerevisiae (i.e., SLC1p) is absent or inactivated. ALE1p preferentially attaches unsaturated acyl chains of varying lengths to the sn-2 position of lysophospholipids. The enzyme was localized to microsomal and mitochondrial membranes using high-purity cell fractionation. It has been proposed that ALE1 may be the major LPEAT in the exogenous lysolipid metabolism (ELM) pathway in yeast, but it is also required for the efficient function of the endogenous Kennedy pathway (Riekhof WR et al. (2007) Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(39):28344-28352).

[0375] In a concurrent study, LPT1 (synonymous with ALE1) was identified and shown to possess lysophosphatidylcholine acyltransferase activity using synthetic genetic array analysis. In this study, the optimal substrate for LPT1 (=ALE1) was lysophosphatidylcholine, thus acting as a lysophosphatidylcholine acyltransferase (LPCAT). Residual activity of the previously reported LPAAT was also demonstrated using a single Δlpt1 mutant and a double Δscl1Δlpt1 mutant (the latter being nonviable). The proportion of oleic acid incorporation into phosphatidylcholine was determined to be 70% de novo and 30% remodeled (Jain S et al. (2007) Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(42):30562-30569).

[0376] In another study, which simultaneously monitored the incorporation of radiolabeled lysophosphatidylcholine and / or palmitoyl-CoA into phosphatidylcholine (PC), the molecular function of ALE1 (also known as LCA1 or SLC4) as a lysophosphatidylcholine acyltransferase (LPCAT) was confirmed. This study demonstrated that ALE1p (in this study = LCA1p) accepts a variety of acyl donors, but regardless of the acyl chain of the lysophosphatidylcholine species (16:0 or 18:1), ALE1 exhibited the highest LPCAT activity. Furthermore, a high sensitivity to Zn2+ was observed, with inhibition at concentrations above 0.1 mM and activation at lower concentrations (10 to 25 μM). The high PC turnover rate measured for ALE1p (=LCA1p) emphasizes that this enzyme is a key catalyst involved in PC reacylation (Chen Q et al. (2007) The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover." FEBS Lett 581(28):5511-5516).

[0377] A search for genes that contribute to abnormal lipid droplet (LD) formation in Saccharomyces cerevisiae identified the gene LOA1 (formerly known as VPS66), encoding an acyl-CoA-dependent lysophosphatidic acid acyltransferase. Comparison of the lipidomes of wild-type and Δloa1 yeast strains was used to determine the in vivo molecular function of LOA1p. Analysis revealed that the percentage of oleic acid-containing phosphatidic acid molecular species was significantly reduced in the LOA1-deficient mutant (Δloa1), and the content of triacylglycerols (TGA) was reduced by 20%. The protein was recombinantly expressed in Escherichia coli and partially purified by obtaining a highly enriched lipid droplet fraction and affinity chromatography with LOA1p still attached to matrix beads. The purified LOA1p was characterized in an in vitro assay that demonstrated that LOA1p is specific for lysophosphatidic acid and oleoyl-CoA, thus acting as an oleoyl-CoA:lysophosphatidic acid acyltransferase in yeast. Based on these results, it is believed that LOA1p is significantly involved in directing excess oleic acid-containing phosphatidic acid species into TAG biosynthesis and the proper development of lipid droplets (LDs). Using genomic tagging constructs, subcellular fractionation, immunohistochemistry, and fluorescence microscopy, LOA1 can be localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) (Ayciriex S et al. (2012) YPR139c / LOA1 encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis. Mol Biol Cell 23(2):233-246).

[0378] In some embodiments, the lysophosphatidic acid acyltransferase is plsC from Escherichia coli (MetaCyc Accession ID EG11377). In other embodiments, the lysophosphatidic acid acyltransferase is plsC from Streptococcus pneumoniae (S. pneumoniae) (MetaCyc Accession ID G-10763). In some embodiments, the lysophosphatidic acid acyltransferase is LAT1 from Limnanthes douglasii. In some embodiments, the lysophosphatidic acid acyltransferase is LAT2 from Limnanthes douglasii (MetaCyc Accession ID G-9398). In some embodiments, the lysophosphatidic acid acyltransferase is SLC1 (YDL052c) from Saccharomyces cerevisiae. In some embodiments, the lysophosphatidic acid acyltransferase is YALI0E18964g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.250 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.7881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_02437 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is ALE1 (YOR175C) from Saccharomyces cerevisiae. In some embodiments, the lysophosphatidic acid acyltransferase is YALI0F19514g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.9437 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_01687 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is LOA1 (YPR139C) from Saccharomyces cerevisiae. In some embodiments, the lysophosphatidic acid acyltransferase is YALI0C14014g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1043 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.8645 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_04750 from Candida tropicalis.

[0379] Glycerol phospholipid acyltransferase (GPLAT)

[0380] The present disclosure describes enzymes that catalyze the following reactions:

[0381] GPLAT enzymes catalyze the transfer of fatty acids from intact glycerophospholipids containing choline or ethanolamine to the sn-2 position of lyso-glycerophospholipids. The organic group on the sn-1 position of the donor or acceptor molecule can be an alkyl, acyl, or alk-1-enyl group. The term "radyl" is sometimes used to refer to such substituents. This enzyme requires coenzyme A and is not conducive to the transfer of polyunsaturated acyl groups.

[0382] Diacylglycerol acyltransferase (DGAT)

[0383] The present disclosure describes enzymes that add an acyl group to the sn-3 position of diacylglycerol (DAG) to form triacylglycerol (TAG).

[0384] Diacylglycerol acyltransferase (DGAT) catalyzes the only unique reaction in triacylglycerol biosynthesis. It adds an acyl group to the sn-3 position of diacylglycerol (DAG) and forms triacylglycerol (TAG) as shown below:

[0385] Acyl-CoA + 1,2-diacyl-sn-glycerol → triacyl-sn-glycerol + coenzyme A.

[0386] DGAT accepts a wide range of acyl-CoAs as acyl donors, including C18:1, C18:2, and C20:1 acyl-CoAs as demonstrated for Arabidopsis DGAT (Jako C et al. (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126(2):861-874). Expression of the Arabidopsis cDNA for DGAT in insect cell culture and yeast, as well as overexpression of the cDNA in wild-type Arabidopsis, demonstrated that DGAT activity is the transfer of an acyl group to the sn-3 position of DAG (Hobbs DH et al. (1999) Cloning of acDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett 452(3):145-149; Zou J et al. (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerolacyltransferase gene. Plant J 19(6):645-653). Overexpression of the Arabidopsis cDNA in wild-type Arabidopsis increased oil deposition in seeds, and this increase correlated with increased DGAT mRNA expression levels. This suggests that DGAT is a regulatory point in the triacylglycerol biosynthesis pathway.

[0387] A gene encoding a bifunctional acyl-CoA: acylglycerol acyltransferase (DGAT) has been identified in Saccharomyces cerevisiae and is a major contributor to triacylglycerol biosynthesis (Sandager L et al. (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277(8):6478-6482). This gene (DGA1) belongs to the DGAT2 family, whose members are characterized as acyl-CoA-dependent acyltransferases (Lardizabal KD et al. (2001) DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity." J Biol Chem 276(42):38862-38869). DGA1p has been shown to be the only acyl-CoA-dependent acyltransferase in the yeast genome that catalyzes the esterification of diacylglycerol (DAG) to triacylglycerol (TAG). This has been shown in a DGA1 deletion mutant (Δdga1) in combination with the deletion of another important diacylglycerol acyltransferase in yeast, LRO1, which esterifies DAG using a phosphatidyl donor (Δlro1). In the Δdga1Δlro1 double mutant, almost all diacylglycerol acyltransferases have been lost, and TAG synthesis is abolished.Plasmids carrying the DGA1 gene can rescue the TAG synthesis defect in mutants, indicating that DGA1 is significantly involved in the TAG biosynthetic pathway in vivo (Sorger D, Daum G (2002). Synthesis of triacylglycerols by the acyl-coenzyme A: diacyl-glycerol acyltransferase Dga1p, lipid particles of the yeast Saccharomyces cerevisiae. J Bacteriol 184(2):519-524; Oelkers P et al. (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277(11):8877-8881). In vitro, DGA1p was observed to have a preference for oleoyl-CoA and palmitoyl-CoA, which is opposite to the phospholipid-dependent acyltransferase LRO1p (Oelkers et al. 2002).

[0388] Furthermore, the function of DGA1p as an acyl-CoA-dependent monoacylglycerol acyltransferase (MGAT) was demonstrated in vivo using a Δdga1 mutant that had lost more than 60% of its MGAT activity. DGA1's in vitro MGAT activity was demonstrated by the oleoyl-CoA-dependent esterification of 2-oleoylglycerol, generating 1,2-dioleoylglycerol (Heier C et al. (2010) Identification of Yju3p as a functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1801(9):1063-1071).

[0389] Further insights into the functional importance and topological orientation of sequence motifs in the primary sequence of DGA1p have been obtained through computer simulation analysis, site-directed mutagenesis of the tag motif, and deletion mutations at the C and N termini. It can be shown that, in addition to the tag motif found in other DGAT2 family members, yeast also has a unique hydrophilic extension that has been shown to significantly regulate enzyme activity. Moreover, the histidine residue 195 in the second of the four identified transmembrane domains has been shown to be essential for enzyme activity. The topological structure of DGA1 reveals that both the C-terminus and the N-terminus face the cytoplasm, and that the C-terminus is more important for DGA1 activity than the N-terminus (Liu Q et al. (2011) Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 286(15):13115-13126).

[0390] Using highly purified cell fractions and immunoblotting, Sorger et al. (2002) and Liu et al. (2011) demonstrated that DGA1 is localized to lipid droplets and microsomal membranes, most likely the endoplasmic reticulum.

[0391] Acinetobacter sp. ADP1 expresses a bifunctional enzyme that exhibits both wax ester synthase (WS) and acyl-coA:diacylglycerol acyltransferase (DGAT) activities (Kalscheuer R and Steinbuchel A (2003) A novel bifunctional wax ester synthase / acyl-CoA:diacylglycerolacyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278(10):8075-8082). This homodimer catalyzes the final step in TAG and WE biosynthesis (Stoveken T et al. (2005) The wax ester synthase / acylcoenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187(4):1369-1376). It mediates the formation of oxoester and thioester bonds and has a broad substrate range, accepting medium-chain fatty alcohols and acyl-CoA esters as well as monoacylglycerol (MAG) (Uthoff S et al. (2005) Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase / acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1. Appl Environ Microbiol 71(2):790-796).

[0392] In some embodiments, the diacylglycerol acyltransferase is TAG1 from Arabidopsis thaliana (Gene ID AT2G19450). In some embodiments, the diacylglycerol acyltransferase is DGA1 from Saccharomyces cerevisiae (YOR245c). In some embodiments, the diacylglycerol acyltransferase is atfA from Acinetobacter sp. ADP1 (MetaCyc Accession ID ACIAD0832). In some embodiments, the diacylglycerol acyltransferase is YALI0E32769g from Yarrowia lipolytica. In some embodiments, the diacylglycerol acyltransferase is CaO19.6941 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CaO19.14203 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CTRG_06209 from Candida tropicalis.

[0393] Phospholipid:diacylglycerol acyltransferase (PDAT) catalyzes the following reaction: phosphatidylcholine + 1,2-diacyl-sn-glycerol → triacyl-sn-glycerol + 1-acyl-sn-glycero-3-phosphocholine.

[0394] Arabidopsis PDAT can use different phospholipids as acyl donors, using acyl groups with carbon chain lengths of 10-22 at any sn-position (Stahl U et al. (2004) Cloning and functional characterization of aphospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant Physiol 135(3):1324-1335). However, the amount of acyl group at the sn-2 position of phosphatidylcholine is three times greater than that at the sn-1 position. The highest activity is achieved with acyl groups having multiple double bonds, epoxy groups, or hydroxyl groups. In the test, the enzyme activity was highest with castor oil acyl groups. 18:0- and 22:1-acyl groups gave the lowest enzyme activity. Among the different phospholipid types, the activity with phosphatidylethanolamine was higher than that with phosphatidate or phosphatidylcholine.

[0395] PDAT activity was detected in the microsomal fraction of castor bean seeds. Radiolabeled ricinoleoyl and vernoloyl groups were efficiently transferred from phosphatidylcholine to DAG to form triacylglycerol (Dahlqvist A et al. (2000) Phospholipid:diacylglycerol acyltransferase:an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97(12):6487-6492).

[0396] In other embodiments, the diacylglycerol acyltransferase is a phospholipid:diacylglycerol acyltransferase (PDAT). In some embodiments, the PDAT is from Arabidopsis thaliana (Gene ID AT5G13640). In some embodiments, the PDAT is from Ricinus communis. In some embodiments, the PDAT is LRO1 (YNR008w) from Saccharomyces cerevisiae. In some embodiments, the PDAT is YALI0E16797g from Yarrowia lipolytica. In some embodiments, the PDAT is CaO19.13439 from Candida albicans. In some embodiments, the PDAT is CTRG_04390 from Candida tropicalis.

[0397] In some embodiments, a recombinant microorganism is provided that is capable of producing saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18In some embodiments, the recombinant microorganism expresses one or more acyltransferases, and the recombinant microorganism is manipulated to delete, destroy, mutate, and / or reduce the activity of one or more endogenous acyltransferases. In some embodiments, the one or more acyltransferases expressed are different from the one or more endogenous acyltransferases deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acyltransferases comprise a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAAT), a glycerophospholipid acyltransferase (GPLAT), and / or a diacylglycerol acyltransferase (DGAT). In some embodiments, the one or more acyltransferases expressed preferably store short-chain fatty acyl-CoA. In other embodiments, the one or more acyltransferases expressed are selected from Table 5b. In some embodiments, the one or more endogenous acyltransferases deleted or downregulated are selected from Yarrowia lipolytica YALI0C00209g, Yarrowia lipolytica YALI0E18964g, Yarrowia lipolytica YALI0F19514g, Yarrowia lipolytica YALI0C14014g, Yarrowia lipolytica YALI0E16797g, Yarrowia lipolytica YALI0E32769g, Yarrowia lipolytica YALI0D07986g, Saccharomyces cerevisiae YBL011w, Saccharomyces cerevisiae YDL052c, Saccharomyces cerevisiae YOR175C, Saccharomyces cerevisiae YPR139C, Saccharomyces cerevisiae YNR008w, Saccharomyces cerevisiae YOR245c, Candida sp. I503-0 In some embodiments, the recombinant microorganism further expresses a pheromone biosynthetic pathway enzyme. In additional embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturases and / or fatty acyl ligases. In yet another embodiment, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol-forming fatty acyl reductases.

[0398] In some embodiments, saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C18A method for producing fatty alcohols, fatty aldehydes, and / or fatty acetates, wherein the method comprises introducing at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase into a recombinant microorganism or expressing at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase in a recombinant microorganism, or introducing a deletion, insertion, or loss-of-function mutation in one or more genes encoding an acyltransferase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase introduced or expressed is different from the one or more genes encoding an acyltransferase that are deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase introduced or expressed, or the one or more genes encoding an acyltransferase that are deleted or downregulated comprises a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAAT), a glycerophospholipid acyltransferase (GPLAT), and / or a diacylglycerol acyltransferase (DGAT). In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase that preferably stores short-chain fatty acyl-CoA. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase selected from Table 5b. In some embodiments, the one or more endogenous acyltransferases deleted or downregulated are selected from Yarrowia lipolytica YALI0C00209g, Yarrowia lipolytica YALI0E18964g, Yarrowia lipolytica YALI0F19514g, Yarrowia lipolytica YALI0C14014g, Yarrowia lipolytica YALI0E16797g, Yarrowia lipolytica YALI0E32769g, Yarrowia lipolytica YALI0D07986g, Saccharomyces cerevisiae YBL011w, Saccharomyces cerevisiae YDL052c, Saccharomyces cerevisiae YOR175C, Saccharomyces cerevisiae YPR139C, Saccharomyces cerevisiae YNR008w, Saccharomyces cerevisiae YOR245c, Candida sp. 503_0 2577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203 and Candida CTRG_06209. In some embodiments, the method further comprises introducing at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase into the recombinant microorganism or expressing at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase in the recombinant microorganism.In additional embodiments, the method further comprises introducing into the recombinant microorganism or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol.

[0399] Table 5b. Exemplary acyltransferases

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409] Acylglycerol lipase and sterol esterase

[0410] In some embodiments, recombinant microorganisms are provided for producing short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates. In certain embodiments, the short-chain fatty alcohols, fatty aldehydes, and / or fatty acetates have a carbon chain length shorter than or equal to C18. In some preferred embodiments of the methods for producing short-chain pheromones, a selected enzyme that preferably hydrolyzes the ester bonds of long-chain acylglycerols is co-expressed with one or more fatty acyl desaturases. Such suitable enzymes are exemplified by heterologous or engineered acylglycerol lipases. Examples of acylglycerol lipases suitable for this purpose are listed in Table 5c.

[0411] In some preferred embodiments of the methods for producing fatty alcohols, fatty aldehydes, and / or fatty acetates, one or more genes encoding acylglycerol lipases (monoacyl-, diacyl-, or triacylglycerol lipases) and sterol ester esterases of the microbial host are deleted or downregulated and replaced with one or more acylglycerol lipases that prefer long-chain acylglycerol substrates. Such deletion or down-regulation targets include but are not limited to Yarrowia lipolytica YALI0E32035g, Yarrowia lipolytica YALI0D17534g, Yarrowia lipolytica YALI0F10010g, Yarrowia lipolytica YALI0C14520g, Yarrowia lipolytica YALI0E00528g, Saccharomyces cerevisiae YKL140w, Saccharomyces cerevisiae YMR313c, Saccharomyces cerevisiae YKR089c, Saccharomyces cerevisiae YOR081c, Saccharomyces cerevisiae YKL094W, Saccharomyces cerevisiae YLL012W, Saccharomyces cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683 and Candida CTRG_04630.

[0412] Carboxylester hydrolases (EC 3.1.1) are a large class of enzymes that catalyze the hydrolysis or synthesis of ester bonds. They have been described in all domains of life (prokaryotic and eukaryotic). Most of them belong to -hydrolase superfamily, and has a conserved "catalytic triad" formed by His, an acidic amino acid and a Ser residue located in a highly conserved GXSXG sequence. During the hydrolysis period, the catalytic Ser will initiate a nucleophilic attack on the substrate with the help of the other two residues in the triad (which are very close in space). It is speculated that they promote the hydrolysis of esters through a mechanism similar to that of chymotrypsin-like serine proteases. Another characteristic feature is the presence of an amino acid region (the sequence of this region is not as conserved as the catalytic triad), an oxyanion hole, which serves to stabilize the transition state generated during catalysis. In addition, these enzymes generally do not require cofactors. Acylglycerol lipases and sterol esterases belong to the carboxylic ester hydrolase family.

[0413] Acylglycerol lipase catalyzes a chemical reaction that breaks down monoglycerides of long-chain fatty acids using a water molecule. The systematic name for this enzyme class is glyceride acylhydrolase. Other common names include monoacylglycerol lipase, monoacylglycerol lipase, monoglyceride lipase, monoglyceride hydrolase, fatty acyl monoester lipase, monoacylglycerol hydrolase, monoglyceride lipase, and monoglyceridase. This enzyme is involved in glycerolipid metabolism.

[0414] Sterol esterase catalyzes the chemical reaction:

[0415]

[0416] Thus, the two substrates of this enzyme are sterol esters and H2O, while its two products are sterols and fatty acids.

[0417] The systematic name of this enzyme class is sterol ester acyl hydrolase. Other commonly used names include cholesterol esterase, cholesterol ester synthase, triterpenoid esterase, cholesterol-based esterase, cholesterol-based ester hydrolase, sterol ester hydrolase, cholesterol ester hydrolase, cholesterol esterase and acyl cholesterol lipase. This enzyme participates in the biosynthesis of bile acids. Sterol esterase is widely distributed in nature and has been identified from mammalian tissues (such as pancreas, intestinal mucosa, liver, placenta, aorta and brain), filamentous fungi, yeast and bacteria.

[0418] In terms of substrate specificity, many sterol esterases are able to catalyze the hydrolysis or synthesis of a rather wide range of other substrates containing ester bonds, such as acylglycerols, aryl esters, and in some cases alcohol esters, cinnamyl esters, xanthogenates or synthetic polymers.

[0419] In some embodiments, a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18A recombinant microorganism of a fatty alcohol, fatty aldehyde and / or fatty acetate, wherein the recombinant microorganism expresses one or more acylglycerol lipases and / or sterol ester esterases, and wherein the recombinant microorganism is manipulated to delete, destroy, mutate one or more endogenous acylglycerol lipases and / or sterol ester esterases and / or reduce the activity of one or more endogenous acylglycerol lipases and / or sterol ester esterases. In some embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are different from the one or more endogenous acylglycerol lipases and / or sterol ester esterases deleted or lowered. In some embodiments, the one or more endogenous or exogenous acylglycerol lipases and / or sterol ester esterases expressed preferably hydrolyze the ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are selected from Table 5c. In some embodiments, the one or more endogenous acylglycerol lipases and / or sterol ester esterases deleted or downregulated are selected from Yarrowia lipolytica YALI0E32035g, Yarrowia lipolytica YALI0D17534g, Yarrowia lipolytica YALI0F10010g, Yarrowia lipolytica YALI0C14520g, Yarrowia lipolytica YALI0E00528g, Saccharomyces cerevisiae YKL140w, Saccharomyces cerevisiae YMR313c, Saccharomyces cerevisiae YKR089c, Saccharomyces cerevisiae YOR081c, Saccharomyces cerevisiae YKL094W, Saccharomyces cerevisiae YLL012W, Saccharomyces cerevisiae YLR020C, Candida sp. CaO19.2050, Candida sp. CaO19.9598, Candida sp. In some embodiments, the recombinant microorganism further expresses a pheromone biosynthetic pathway enzyme. In additional embodiments, the pheromone biosynthetic enzymes comprise one or more fatty acyl desaturases and / or fatty acyl ligases. In yet another embodiment, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol-forming fatty acyl reductases.

[0420] In some embodiments, a saturated C6-C 24 Fatty acyl-CoA produces mono- or polyunsaturated ≤C18A method for producing fatty alcohols, fatty aldehydes and / or fatty acetates, wherein the method comprises introducing at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase into a recombinant microorganism or expressing at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase in a recombinant microorganism, or introducing a deletion, insertion or loss-of-function mutation of one or more genes encoding an acylglycerol lipase or sterol ester esterase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase introduced or expressed is different from the one or more genes encoding an acylglycerol lipase or sterol ester esterase that are deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase introduced or expressed preferably hydrolyzes ester bonds of long-chain acylglycerols. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase introduced or expressed is selected from Table 5c. In some embodiments, the one or more genes deleted or downregulated encode an acylglycerol lipase or sterol ester esterase selected from the group consisting of: Yarrowia lipolytica YALI0E32035g, Yarrowia lipolytica YALI0D17534g, Yarrowia lipolytica YALI0F10010g, Yarrowia lipolytica YALI0C14520g, Yarrowia lipolytica YALI0E00528g, Saccharomyces cerevisiae YKL140w, Saccharomyces cerevisiae YMR313c, Saccharomyces cerevisiae YKR089c, Saccharomyces cerevisiae YOR081c, Saccharomyces cerevisiae YKL094W, Saccharomyces cerevisiae YLL012W, Saccharomyces cerevisiae YLR020C, Candida sp. CaO19.2050, Candida sp. CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630. In some embodiments, the method further comprises introducing at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase into the recombinant microorganism, or expressing at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and / or a fatty acyl ligase in the recombinant microorganism.In additional embodiments, the method further comprises introducing into the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol.

[0421] Table 5c. Exemplary acylglycerol lipases

[0422]

[0423]

[0424] Expression of toxic proteins or peptides

[0425] This disclosure describes toxic proteins, peptides, or small molecules that can be encoded by recombinant microorganisms. In some embodiments, the toxic proteins, peptides, or small molecules are biosynthetically produced along with insect pheromones.

[0426] In some embodiments, the recombinant microorganism expresses one or more nucleic acid molecules encoding proteins or polypeptides toxic to insects. In some embodiments, the toxic protein or polypeptide is from an insect pathogenic organism. In some embodiments, the insect pathogenic organism is selected from Bacillus thuringiensis, Pseudomonas aeruginosa, and Serratia marcescens. In a specific embodiment, the nucleic acid molecule encodes a Bacillus thuringiensis toxin.

[0427] In some embodiments, the recombinant microorganism is engineered to express a metabolic pathway that, when expressed, produces a small molecule that is toxic to insects.

[0428] In an exemplary embodiment, insect pheromones produced by the recombinant microorganisms described herein can be used to attract pests, and then the pests can be eradicated using toxic substances such as toxic proteins, peptides, or small molecules that have been co-produced by the recombinant microorganisms described herein.

[0429] Pheromone biosynthesis using recombinant microorganisms

[0430] As discussed above, in a first aspect, the present disclosure relates to a recombinant microorganism capable of producing a saturated C6-C 24 Fatty acyl-CoA produces mono- or polyunsaturated C6-C 24 Fatty alcohol. An illustrative embodiment of the first aspect is Figure 1. The blue line represents the biochemical pathway for producing saturated acyl-CoA, which serves as the substrate for the conversion of unsaturated fatty acyl-CoA. The conversion of substrate to unsaturated fatty acyl-CoA can be carried out by endogenous or exogenous enzymes in the host. The green line indicates the conversion catalyzed by the exogenous nucleic acid molecule encoding the enzyme. Therefore, in some embodiments, the conversion of saturated fatty acyl-CoA to mono- or polyunsaturated fatty acyl-CoA is catalyzed by at least one desaturase encoded by an exogenous nucleic acid molecule. In other embodiments, the conversion of mono- or polyunsaturated fatty acyl-CoA to mono- or polyunsaturated fatty alcohol is catalyzed by at least one reductase encoded by an exogenous nucleic acid molecule. The gray dotted line indicates the downstream steps for synthesizing pheromones, spices, flavorings, and polymer intermediates, such as using alcohol oxidase or oxidants to produce mono- or polyunsaturated fatty aldehydes, and using acetyltransferases or chemicals (such as acetyl chloride) to produce mono- or polyunsaturated fatty acetates. The red cross indicates the host's natural pathway that is missing or downregulated, which increases the flux towards the engineered pathway.

[0431] Thus, in one embodiment, the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, the fatty acyl reductase catalyzing the conversion of a mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, endogenous enzymes in the host microorganism can be used to produce saturated C6-C 24 In other embodiments, one or more exogenous enzymes in the host microorganism can be used to produce saturated C6-C 24 Fatty acyl-CoA.

[0432] As described above, fatty acyl desaturases catalyze the desaturation of the hydrocarbon chain of, for example, a saturated fatty acyl-CoA molecule to produce a corresponding unsaturated fatty acyl-CoA molecule. In some embodiments, an exogenous fatty acyl desaturase can be selected and expressed in a recombinant microorganism to catalyze the formation of at least one double bond in a fatty acyl-CoA molecule having 6 to 24 carbon atoms in the hydrocarbon chain. Thus, in some embodiments, the fatty acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate having a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0433] The exogenous fatty acyl desaturase described herein can be selected to catalyze the desaturation of a desired position on the hydrocarbon chain. Thus, in some embodiments, the fatty acyl desaturase is capable of generating a double bond at the C5, C6, C7, C8, C9, C10, C11, C12, or C13 position of a fatty acid or derivative thereof, such as, for example, a fatty acid CoA ester.

[0434] One or more fatty acyl-CoA desaturases can be expressed in a host to catalyze the desaturation of multiple positions on the hydrocarbon chain. In some embodiments, the fatty acyl-CoA desaturase is heterologous to the host microorganism. Thus, various embodiments provide recombinant microorganisms comprising at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the desaturation of C6-C8 hydrocarbon chains. 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty acyl-CoA.

[0435] In an exemplary embodiment, the fatty acyl desaturase is a Z11 desaturase. The Z11 fatty acyl desaturase catalyzes the formation of a double bond between the 11th and 12th carbon atoms relative to the carbonyl group in a substrate. In various embodiments described herein, the Z11 desaturase or the nucleic acid sequence encoding it can be isolated from an organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosirapseudonana. Additional Z11 desaturases or nucleic acid sequences encoding them can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutellaxylostella, Bombyx mori, or Diaphania nitidalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase is codon-optimized and is from an organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs: 9, 18, 24, and 26 from Trichoplusia ni.In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO:49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs:10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO:53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs:11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs:12, 17, and 30 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 52 from the navel orange borer. In further embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 19, 25, 27, and 31 from the corn moth (Helicoverpa zea). In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 54 from the corn moth (Helicoverpa zea). In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 39 from the rice borer. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in GenBank Accession Nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1, and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, all or part of the Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase is replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 28, and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 78, 79, and 80.

[0436] In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA to a mono- or di-unsaturated product selected from the group consisting of: Z11-13: acyl-CoA, E11-13: acyl-CoA, (Z,Z)-7,11-13: acyl-CoA, Z11-14: acyl-CoA, E11-14: acyl-CoA, (E,E)-9,11-14: acyl-CoA, (E,Z)-9,11-14: acyl-CoA, (Z,E)-9,11-14: acyl- CoA, (Z,Z)-9,11-14: acyl-CoA, (E,Z)-9,11-15: acyl-CoA, (Z,Z)-9,11-15: acyl-CoA, Z11-16: acyl-CoA, E11-16: acyl-CoA, (E,Z)-6,11-16: acyl-CoA, (E,Z)-7,11-16: acyl-CoA, (E,Z)-8,11-16: acyl-CoA, (E,E)-9,11-16: acyl-CoA, (E ,Z)-9,11-16: acyl-CoA, (Z,E)-9,11-16: acyl-CoA, (Z,Z)-9,11-16: acyl-CoA, (E,E)-11,13-16: acyl-CoA, (E,Z)-11,13-16: acyl-CoA, (Z,E)-11,13-16: acyl-CoA, (Z,Z)-11,13-16: acyl-CoA, (Z,E)-11,14-16: acyl-CoA, (E,E,Z)-4, 6,11-16: acyl-CoA, (Z,Z,E)-7,11,13-16: acyl-CoA, (E,E,Z,Z)-4,6,11,13-16: acyl-CoA, Z11-17: acyl-CoA, (Z,Z)-8,11-17: acyl-CoA, Z11-18: acyl-CoA, E11-18: acyl-CoA, (Z,Z)-11,13-18: acyl-CoA, (E,E)-11,14-18: acyl-CoA or a combination thereof.

[0437] In another exemplary embodiment, the fatty acyl desaturase is a Z9 desaturase. The Z9 fatty acyl desaturase catalyzes the formation of a double bond between the 9th and 10th carbon atoms relative to the carbonyl group in a substrate. In various embodiments described herein, the Z9 desaturase or the nucleic acid sequence encoding it can be isolated from an organism of the species Ostriniafurnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In an exemplary embodiment, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon-optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.

[0438] In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA to a monounsaturated or polyunsaturated product selected from the group consisting of: Z9-11: acyl-CoA, Z9-12: acyl-CoA, E9-12: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, (Z,E)-7,9-12: acyl-CoA, (Z,Z)-7,9-12: acyl-CoA, Z9-13: acyl-CoA, E9-13: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, (Z,Z)-7,9-12: acyl-CoA, Z9-14: acyl-CoA, E9-14: acyl-CoA, (E,E)-7,9-12: acyl-CoA, (E,Z)-7,9-12: acyl-CoA, Z)-5,9-13: acyl-CoA, (Z,E)-5,9-13: acyl-CoA, (Z,Z)-5,9-13: acyl-CoA, Z9-14: acyl-CoA, E9-14: acyl-CoA, (E,Z)-4,9-14: acyl-CoA, (E,E)-9,11-14: acyl-CoA, (E,Z)-9,11-14: acyl-CoA, (Z,E)-9,11-14: acyl-CoA, (Z,Z)-9,11-14: acyl-CoA, (E,E)-9,1 2-14: acyl-CoA, (Z,E)-9,12-14: acyl-CoA, (Z,Z)-9,12-14: acyl-CoA, Z9-15: acyl-CoA, E9-15: acyl-CoA, (Z,Z)-6,9-15: acyl-CoA, Z9-16: acyl-CoA, E9-16: acyl-CoA, (E,E)-9,11-16: acyl-CoA, (E,Z)-9,11-16: acyl-CoA, (Z,E)-9,11-16: acyl-CoA, (Z,Z)- 9,11-16: acyl-CoA, Z9-17: acyl-CoA, E9-18: acyl-CoA, Z9-18: acyl-CoA, (E,E)-5,9-18: acyl-CoA, (E,E)-9,12-18: acyl-CoA, (Z,Z)-9,12-18: acyl-CoA, (Z,Z,Z)-3,6,9-18: acyl-CoA, (E,E,E)-9,12,15-18: acyl-CoA, (Z,Z,Z)-9,12,15-18: acyl-CoA or a combination thereof.

[0439] Saturated C6-C 24 The desaturation of fatty acyl-CoA can proceed through multiple reactions to produce polyunsaturated C6-C 24 In some embodiments, the recombinant microorganism can express a bifunctional desaturase capable of catalyzing the formation of at least two double bonds. In some embodiments, the recombinant microorganism can express more than one exogenous nucleic acid molecule encoding more than one fatty acyl desaturase that catalyzes the desaturation of C6-C 24Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA. For example, the recombinant microorganism can express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase. Thus, the resulting polyunsaturated fatty acyl-CoA has a double bond between the 9th and 10th carbon atoms and another double bond between the 11th and 12th carbon atoms.

[0440] In some embodiments, the recombinant microorganism can express a fatty acyl conjugating enzyme that functions independently or in conjunction with a fatty acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.

[0441] In one embodiment, the present disclosure provides a recombinant microorganism capable of producing saturated or monounsaturated C6-C 24 Fatty acyl-CoA produces polyunsaturated C6-C 24 Fatty alcohols, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugating enzyme that catalyzes the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, the fatty acyl reductase that catalyzes the conversion of a polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty alcohol.

[0442] In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty acyl conjugating enzymes that catalyze the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA.

[0443] In another embodiment, the present disclosure provides a recombinant microorganism capable of producing a saturated or monounsaturated C6-C 24 Fatty acyl-CoA produces polyunsaturated C6-C 24 Fatty alcohols, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugating enzyme, the fatty acyl desaturase and the fatty acyl conjugating enzyme catalyzing the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C24 Fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty alcohol, the fatty acyl reductase that catalyzes the conversion of a polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty alcohol.

[0444] In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty acyl conjugating enzymes that catalyze the conversion of saturated or monounsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding polyunsaturated C6-C 24 Fatty acyl-CoA.

[0445] In yet another embodiment, the fatty acyl conjugating enzyme is one that is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0446] In certain embodiments, the conjugating enzyme or the nucleic acid sequence encoding it can be isolated from the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowii, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana). In an exemplary embodiment, the conjugating enzyme comprises the following sequence, which is selected from GenBank Accession Nos. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

[0447] As described above, fatty acyl reductases catalyze the reduction of carbonyl groups on, for example, unsaturated fatty acyl-CoA molecules to produce corresponding unsaturated fatty acid molecules. In some embodiments, the fatty acyl-CoA reductase that forms fatty alcohols is heterologous to the microorganism. Thus, various embodiments provide recombinant microorganisms comprising at least one exogenous nucleic acid molecule encoding a fatty acyl-CoA reductase that forms fatty alcohols, which catalyzes the reduction of carbonyl groups on unsaturated fatty acyl-CoA molecules to produce corresponding unsaturated fatty acid molecules.

[0448] In some embodiments, the fatty acyl reductase is from an organism of the species Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus, or Helicoverpa amigera. In some embodiments, the nucleic acid sequence encoding the fatty acyl reductase is codon-optimized. In some embodiments, the fatty acyl reductase comprises the sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises the sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In other embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 32, 40, 72, 74, 76, and 81. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 55 from Agrotissima lutea. In other embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises the amino acid sequence set forth in SEQ ID NO: 77 from Prunella vulgaris.

[0449] In some embodiments, producing an unsaturated fatty alcohol in a recombinant microorganism comprises expressing one or more mutant FARs. In certain embodiments, a Helicoverpa amigera fatty acyl-CoA reductase (HaFAR) variant is provided that exhibits a net increase in fatty alcohol production compared to a wild-type Helicoverpa amigera fatty acyl-CoA reductase encoded by the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the increased enzymatic activity is a net increase in the amount of fatty alcohol produced relative to the amount of fatty alcohol produced by the wild-type enzymatic activity of the HaFAR encoded by the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the wild-type HaFAR comprises the nucleotide sequence set forth in SEQ ID NO:90. In some embodiments, the variant of the wild-type HaFAR encoded by the amino acid sequence set forth in SEQ ID NO:41 comprises point mutations at the following positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises amino acid F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, the variant of the wild-type HaFAR encoded by the amino acid sequence set forth in SEQ ID NO:41 comprises a combination of point mutations selected from mutations at the following amino acid positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises amino acid F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutant fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.

[0450] In an exemplary embodiment, the fatty acyl reductase catalyzes the conversion of a mono- or polyunsaturated fatty acyl-CoA to a fatty alcohol product selected from the group consisting of: (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (Z)-9-tetradecenol, (Z)-10 ... )-9-hexadecenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or a combination thereof.

[0451] In some embodiments, the recombinant microorganisms described herein can include multiple fatty acyl reductases. Thus, in such embodiments, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty acyl reductases that catalyze the conversion of mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty alcohol.

[0452] In another embodiment, the present disclosure provides a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 The recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the oxidation of a mono- or polyunsaturated C6-C6 fatty acyl-CoA fatty acid from (a) after one or more consecutive cycles of acyl-CoA oxidase activity. 24 Fatty acyl-CoA is converted to mono- or polyunsaturated ≤C 18 Fatty acyl-CoA, where a given cycle produces a mono- or polyunsaturated C6-C 24 Fatty acyl-CoA substrates have two carbon-truncated mono- or polyunsaturated C4-C 22a fatty acyl-CoA intermediate; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of the mono- or polyunsaturated ≤C18 fatty acyl-CoA from (b) to the corresponding mono- or polyunsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from the group consisting of fatty acyl desaturases derived from Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusiani, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana. In some embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession Nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1, and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol-forming fatty acyl reductase is selected from the group consisting of fatty alcohol-forming fatty acyl reductases derived from Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus, and Helicoverpa armigera.In additional embodiments, the fatty alcohol-forming fatty acyl reductase has 95% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77, and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Candida viswinii.

[0453] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase, which preferably stores ≤ C 18 Fatty acyl-CoA. In some embodiments, the acyltransferase is selected from glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerophospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferase (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.

[0454] In some embodiments, co-expression of a wax esterase allows for the storage of fatty alcohols and fatty acids in a 1:1 ratio. Combined with TAG storage, this can result in a desired TAG to fatty alcohol ratio that can then be used in different product streams. Examples of wax ester synthases: Homo sapiens AWAT2 (XM_011530876.2), Mouse (AAT68766.1), Euglena gracilis WS (ADI60058.1), Euglena gracilis WSD2 (BAV82975.1), Euglena gracilis WSD5 (BAV82978.1).

[0455] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, >C14, >C12, or >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.

[0456] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes that catalyze the production of mono- or polyunsaturated C 18In another embodiment, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases; (ii) one or more acyltransferases; (iii) one or more acylglycerol lipases and / or sterol ester esterases; (iv) one or more (fatty) alcohol dehydrogenases; (v) one or more (fatty) alcohol oxidases; and (vi) one or more cytochrome P450 monooxygenases.

[0457] In some preferred embodiments, the microbial host comprises one or more genes encoding acyl-CoA oxidases that are deleted or down-regulated to eliminate or reduce the desired truncation of fatty acyl-CoAs beyond the desired chain length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acyl-CoA oxidases selected from the group consisting of Yarrowia lipolytica POX1 (YALI0E32835g), Yarrowia lipolytica POX2 (YALI0F10857g), Yarrowia lipolytica POX3 (YALI0D24750g), Yarrowia lipolytica POX4 (YALI0E27654g), Yarrowia lipolytica POX5 (YALI0C23859g), Yarrowia lipolytica POX6 (YALI0E27664g), Yarrowia lipolytica POX7 (YALI0E276664g), Yarrowia lipolytica POX8 (YALI0E276665g), Yarrowia lipolytica POX9 (YALI0E276666), Yarrowia lipolytica POX10 (YALI0E276666), Yarrowia lipolytica POX11 (YALI0E276666), Yarrowia lipolytica POX12 (YALI0E276666), Yarrowia lipolytica POX13 (YALI0E276666), Yarrowia lipolytica POX14 (YALI0E276666), Yarrowia lipolytica POX15 (YALI0E276666), Yarrowia lipolytica POX16 (YALI0E276666), Yarrowia lipolytica POX17 Yarrowia lipolytica POX6 (YALI0E06567g); Saccharomyces cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160) and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

[0458] In some embodiments, a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 The invention relates to a recombinant microorganism that produces fatty alcohols, fatty aldehydes and / or fatty acetates, wherein the recombinant microorganism expresses one or more acyl-CoA oxidases, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate and / or reduce the activity of one or more endogenous acyl-CoA oxidases. In some embodiments, the one or more acyl-CoA oxidases expressed are different from the one or more endogenous acyl-CoA oxidases that are deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidases expressed regulate the production of mono- or polyunsaturated C6-C 24Chain length of fatty alcohol, fatty aldehyde and / or fatty acetate. In other embodiments, the one or more acyl-CoA oxidases expressed are selected from Table 5a.

[0459] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acyltransferases selected from the group consisting of Yarrowia lipolytica YALI0C00209g, Yarrowia lipolytica YALI0E18964g, Yarrowia lipolytica YALI0F19514g, Yarrowia lipolytica YALI0C14014g, Yarrowia lipolytica YALI0E16797g, Yarrowia lipolytica YALI0E32769g, and Yarrowia lipolytica YALI0D07986g, Saccharomyces cerevisiae YBL011w, Saccharomyces cerevisiae YDL052c, Saccharomyces cerevisiae YOR175C, Saccharomyces cerevisiae YPR139C, Saccharomyces cerevisiae YNR008w, and Saccharomyces cerevisiae YOR175C. 245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203 and Candida CTRG_06209.

[0460] In some embodiments, a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 A recombinant microorganism of a fatty alcohol, fatty aldehyde and / or fatty acetate, wherein the recombinant microorganism expresses one or more acyltransferases, and wherein the recombinant microorganism is manipulated to delete, destroy, mutate one or more endogenous acyltransferases and / or reduce the activity of one or more endogenous acyltransferases. In some preferred embodiments, one or more genes encoding GPAT, LPAAT, GPLAT and / or DGAT of the microbial host are deleted or downregulated and replaced by one or more GPAT, LPAAT, GPLAT or DGAT that preferably stores short-chain fatty acyl-CoA. In some embodiments, the one or more acyltransferases expressed are different from the one or more endogenous acyltransferases deleted or downregulated. In other embodiments, the one or more acyltransferases expressed are selected from Table 5b.

[0461] In some preferred embodiments, one or more genes encoding acylglycerol lipases (monoacyl-, diacyl-, or triacylglycerol lipases) and sterol ester esterases of the microbial host are deleted or downregulated and replaced with one or more acylglycerol lipases that prefer long-chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous acylglycerol lipases and / or sterol ester esterases selected from the group consisting of Yarrowia lipolytica YALI0E32035g, Yarrowia lipolytica YALI0D17534g, Yarrowia lipolytica YALI0F10010g, Yarrowia lipolytica YALI0C14520g, and Yarrowia lipolytica YALI0E00528g, Saccharomyces cerevisiae YKL140w, Saccharomyces cerevisiae YMR313c, Saccharomyces cerevisiae YKR089c, Saccharomyces cerevisiae YOR081c, Saccharomyces cerevisiae YKL094W, Saccharomyces cerevisiae YLL012W, and Saccharomyces cerevisiae YLR020C, and Candida sp. CaO19.205 0, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.

[0462] In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Yarrowia lipolytica YALI0E25982g (ALK1), Yarrowia lipolytica YALI0F01320g (ALK2), Yarrowia lipolytica YALI0E23474g (ALK3), Yarrowia lipolytica YALI0B13816g (ALK4), Yarrowia lipolytica YALI0B13816g (ALK5), Yarrowia lipolytica YALI0B23982g (ALK6), Yarrowia lipolytica YALI0B33982g (ALK7), Yarrowia lipolytica YALI0B43982g (ALK8), Yarrowia lipolytica YALI0B53982g (ALK9), Yarrowia lipolytica YALI0B63982g (ALK10), Yarrowia lipolytica YALI0B73982g (ALK11), Yarrowia lipolytica YALI0B83982g (ALK12), Yarrowia lipolytica YALI0B93982g (ALK13), Yarrowia lipolytica YALI0B133982g (ALK14), Yarrowia lipolytica YALI0B1 3838g (ALK5), Yarrowia lipolytica YALI0B01848g (ALK6), Yarrowia lipolytica YALI0A15488g (ALK7), Yarrowia lipolytica YALI0C12122g (ALK8), Yarrowia lipolytica YA LIOB06248g (ALK9), Yarrowia lipolytica YALI0B20702g (ALK10), Yarrowia lipolytica YALI0C10054g (ALK11) Yarrowia lipolytica YALI0A20130g (ALK12).

[0463] In some embodiments, a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 A recombinant microorganism of a fatty alcohol, fatty aldehyde and / or fatty acetate, wherein the recombinant microorganism expresses one or more acylglycerol lipases and / or sterol ester esterases, and wherein the recombinant microorganism is manipulated to delete, destroy, mutate one or more endogenous acylglycerol lipases and / or sterol ester esterases and / or reduce the activity of one or more endogenous acylglycerol lipases and / or sterol ester esterases. In some embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are different from the one or more endogenous acylglycerol lipases and / or sterol ester esterases deleted or lowered. In some embodiments, the one or more endogenous or exogenous acylglycerol lipases and / or sterol ester esterases expressed preferably hydrolyze the ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipases and / or sterol ester esterases expressed are selected from Table 5c.

[0464] In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from the group consisting of E5-10: acyl-CoA, E7-12: acyl-CoA, E9-14: acyl-CoA, E11-16: acyl-CoA, E13-18: acyl-CoA, Z7-12: acyl-CoA, Z9-14: acyl-CoA, Z11-16: acyl-CoA, Z13-18: acyl-CoA, Z8-12: acyl-CoA, Z 10-14: acyl-CoA, Z12-16: acyl-CoA, Z14-18: acyl-CoA, Z7-10: acyl-CoA, Z9-12: acyl-CoA, Z11-14: acyl-CoA, Z13-16: acyl-CoA, Z15-18: acyl-CoA, E7-10: acyl-CoA, E9-12: acyl-CoA, E11-14: acyl-CoA, E13-16: acyl-CoA, E15-18: acyl-CoA, E5Z7-12: acyl- CoA, E7Z9-12: acyl-CoA, E9Z11-14: acyl-CoA, E11Z13-16: acyl-CoA, E13Z15-18: acyl-CoA, E6E8-10: acyl-CoA, E8E10-12: acyl-CoA, E10E12-14: acyl-CoA, E12E14-16: acyl-CoA, Z5E8-10: acyl-CoA, Z7E10-12: acyl-CoA, Z9E12-14: acyl-CoA, Z11E14- 16: acyl-CoA, Z13E16-18: acyl-CoA, Z3-10: acyl-CoA, Z5-12: acyl-CoA, Z7-14: acyl-CoA, Z9-16: acyl-CoA, Z11-18: acyl-CoA, Z3Z5-10: acyl-CoA, Z5Z7-12: acyl-CoA, Z7Z9-14: acyl-CoA, Z9Z11-16: acyl-CoA, Z11Z13-16: acyl-CoA and Z13Z15-18: acyl-CoA. In another embodiment, the mono- or polyunsaturated ≤C 18 The fatty alcohol is selected from E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH and Z13-18:OH.

[0465] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde-forming fatty acyl-CoA reductase capable of catalyzing the conversion of mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 Fatty aldehyde. In some preferred embodiments, the aldehyde-forming fatty acyl-CoA reductase is selected from Acinetobacter calcoaceticus A0A1C4HN78, Acinetobacter calcoaceticus N9DA85, Acinetobacter calcoaceticus R8XW24, Acinetobacter calcoaceticus A0A1A0GGM5, ​​Acinetobacter calcoaceticus A0A117N158 and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 In some preferred embodiments, the ≤C 18 The fatty aldehyde is selected from Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0466] In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 Fatty acetate. In some embodiments, the acetyltransferase is selected from Table 5d. In some preferred embodiments, the ≤C18 fatty acetate is selected from E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.

[0467] In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from the group consisting of alcohol oxidase, alcohol dehydrogenase, and aldehyde-forming fatty acyl-CoA reductase, wherein the alcohol oxidase, alcohol dehydrogenase, and aldehyde-forming fatty acyl-CoA reductase are capable of catalyzing the conversion of mono- or polyunsaturated ≤C 18 Fatty alcohols are converted to the corresponding ≤C 18 Fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of mono- or polyunsaturated ≤C 18Fatty alcohols are converted to the corresponding ≤C 18 In some preferred embodiments, the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C 18 The fatty acetate is selected from E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0468] In another embodiment, the present disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 Fatty alcohols, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 (a) a fatty acyl-CoA-containing fatty acyl group having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105; and (b) at least one nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase having 95% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75, and 77, and catalyzing the conversion of a mono- or polyunsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty alcohol.

[0469] In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes that catalyze the production of mono- or polyunsaturated C6-C 24In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), ... ), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenases selected from YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0 E07766g (ADH7); (iii) (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzymes selected from YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6 ), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferases selected from YALI0E32791g (DGA1) and YALI0D07986g (DGA2).In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06 567g (POX6); (ii) one or more (fatty) alcohol dehydrogenases selected from YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzymes selected from YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), ... LI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferases selected from YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

[0470] In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from the group consisting of Z9-14: acyl-CoA, Z11-14: acyl-CoA, E11-14: acyl-CoA, Z9-16: acyl-CoA, and Z11-16: acyl-CoA. In other embodiments, the mono- or poly-unsaturated C6-C 24 The fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH and Z13-18:OH.

[0471] In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C6-C 24 The fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

[0472] In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In some embodiments, the acetyltransferase is selected from Table 5d. In some embodiments, the C6-C 24 The fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac and Z13-18:Ac.

[0473] In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 Fatty aldehydes; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty aldehydes and C6-C 24 The fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

[0474] In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66, and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from the group consisting of Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturases.

[0475] In some embodiments, the present disclosure provides an engineered saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 A method for producing fatty alcohols from a Yarrowia lipolytica microorganism, wherein the method comprises introducing into the Yarrowia lipolytica microorganism: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 and (b) at least one nucleic acid molecule encoding a fatty alcohol-forming fatty acyl reductase that catalyzes the conversion of a mono- or polyunsaturated C6-C8 fatty acyl-CoA to a fatty acyl-CoA and has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105; and 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 The microorganism comprises a fatty alcohol and has 95% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75, and 77. In some embodiments, the microorganism is MATAura3-302::SUC2Δpox1Δpox2Δpox3Δpox4Δpox5Δpox6ΔfadhΔadh1Δadh2Δadh3Δadh4Δadh5Δadh6Δadh7Δfao1::URA3.

[0476] In some embodiments, the present disclosure provides a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated C6-C 24 The method comprises: culturing the recombinant microorganism described herein in a culture medium containing a raw material, the raw material providing a sufficient amount of mono- or polyunsaturated C6-C 24In some embodiments, the method further comprises recovering the mono- or polyunsaturated C6-C 24 In another embodiment, the recovery step comprises a step of recovering fatty alcohols, fatty aldehydes or fatty acetates. In another embodiment, the recovery step comprises a distillation. In yet another embodiment, the recovery step comprises a membrane-based separation.

[0477] In some embodiments, chemical methods are used to convert the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In other embodiments, the chemical method is selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)2, Swern oxidation and noble metal-air. In some embodiments, the mono- or polyunsaturated C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In another embodiment, the chemical method utilizes a chemical reagent to convert the mono- or polyunsaturated C6-C8 fatty acid ester into a fatty acid ester in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate. 24 Esterification of fatty alcohols to the corresponding C6-C 24 Fatty acetate, the chemical reagent is selected from acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propionyl chloride and propionic anhydride.

[0478] As discussed above, in a second aspect, the present application relates to a recombinant microorganism capable of converting C6-C 24 Fatty acids produce unsaturated C6-C 24 Fatty alcohol. An illustrative embodiment of the second aspect is Figure 2In the embodiment of the present invention, the host of the invention is shown in Figure 2. The blue line represents the biochemical pathway endogenous to the host, for example, n-alkanes, fatty alcohols or fatty aldehydes are converted into the approach of fatty acids, or fatty acids are converted into the approach of fatty acyl-CoA, acetyl-CoA or dicarboxylic acids. The conversion of substrate to unsaturated fatty acids can be carried out by the endogenous or exogenous enzymes in the host. The yellow line indicates the conversion catalyzed by the exogenous nucleic acid molecules of the encoding enzyme. Therefore, in some embodiments, saturated fatty acids can be catalyzed by at least one saturated fatty acyl-ACP synthase to the conversion of saturated fatty acyl-ACP, wherein the fatty acyl-ACP synthase is encoded by exogenous nucleic acid molecules. In other embodiments, saturated fatty acyl-ACP can be catalyzed by at least one fatty acyl-ACP desaturase to the conversion of single or polyunsaturated fatty acyl-ACP, wherein the fatty acyl-ACP desaturase is encoded by exogenous nucleic acid molecules. In still other embodiments, using fatty acid synthase complex and carbon source (for example malonyl-ACP), single or polyunsaturated fatty acyl-ACP can be relatively extended by at least 2 carbons. In one such embodiment, the conversion of a mono- or poly-unsaturated fatty acyl-ACP to a corresponding mono- or poly-unsaturated fatty acyl-ACP extended by two carbon atoms can be catalyzed by at least one fatty acid synthase complex, wherein the fatty acid synthase complex is encoded by one or more exogenous nucleic acid molecules. In yet another embodiment, the conversion of the extended mono- or poly-unsaturated fatty acyl-ACP to a mono- or poly-unsaturated fatty aldehyde can be catalyzed by a fatty acyl reductase that forms a fatty aldehyde, wherein the fatty acyl reductase that forms a fatty aldehyde is encoded by an exogenous nucleic acid molecule. In some embodiments, a mono- or poly-unsaturated fatty aldehyde can be converted to a corresponding mono- or poly-unsaturated fatty alcohol, wherein the conversion of the substrate to the product is catalyzed by a dehydrogenase, wherein the dehydrogenase is encoded by an endogenous or exogenous nucleic acid molecule. Dashed lines indicate downstream steps of the present disclosure, such as utilizing an acetyltransferase or translocation, or subsequent chemical transformation to produce a functionalized pheromone. Red crosses indicate missing or downregulated host native pathways, which increase flux toward the engineered pathway.

[0479] In one embodiment, the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the synthesis of C6-C 24 Fatty acids are converted to the corresponding saturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding a fatty acyl-ACP desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or polyunsaturated C6-C 24The fatty acyl-ACP is converted to the corresponding mono- or polyunsaturated C6-C ... 24 Fatty acyl-ACP; (d): at least one exogenous nucleic acid molecule encoding a fatty acyl reductase that forms a fatty aldehyde, the fatty acyl reductase that catalyzes the conversion of a mono- or polyunsaturated C6-C 24 Fatty acyl-ACP is converted into the corresponding mono- or polyunsaturated C6-C 24 and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of a mono- or polyunsaturated C6-C 24 Fatty aldehyde C6-C 24 Converted into the corresponding mono- or polyunsaturated C6-C 24 In some embodiments, endogenous enzymes in the host microorganism can be used to produce C6-C 24 In other embodiments, saturated C6-C8 fatty acids can be produced by one or more exogenous enzymes in the host microorganism. 24 fatty acid.

[0480] In some embodiments, the recombinant microorganisms disclosed herein include an acyl-ACP synthetase to catalyze the conversion of C6-C 24 Fatty acids are converted to the corresponding saturated C6-C 24 Fatty acyl-ACP. In some embodiments, the acyl-ACP synthetase is a synthetase capable of utilizing as a substrate a fatty acid having a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In exemplary embodiments, the recombinant microorganism can include a heterologous acyl-ACP synthetase from an organism of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.

[0481] In some embodiments, the recombinant microorganism comprises a fatty acyl-ACP desaturase. In some embodiments, the fatty acyl-ACP desaturase is a soluble desaturase. In other embodiments, the fatty acyl-ACP desaturase is from an organism of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.

[0482] In some embodiments, the recombinant microorganism comprises a fatty acid synthase complex. In some embodiments, the one or more nucleic acid molecules encoding the fatty acid synthase complex are endogenous nucleic acid molecules. In other embodiments, the one or more nucleic acid molecules encoding the fatty acid synthase complex are exogenous nucleic acid molecules.

[0483] In some embodiments, the recombinant microorganisms disclosed herein include a fatty acyl reductase that forms a fatty aldehyde, which catalyzes the conversion of a C6-C 24 Fatty acyl-ACP is converted to the corresponding C6-C 24 Fatty aldehydes. In exemplary embodiments, the fatty acyl reductase that forms fatty aldehydes is from an organism of the species Pelargonium hortorum, Asclepiassyriaca, and Uncaria tomentosa. In some embodiments, the recombinant microorganism includes a dehydrogenase to convert an unsaturated fatty aldehyde into a corresponding unsaturated fatty alcohol. In some embodiments, the nucleic acid molecule encoding the dehydrogenase is endogenous to the recombinant microorganism. In other embodiments, the nucleic acid molecule encoding the dehydrogenase is exogenous to the recombinant microorganism. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding the dehydrogenase is isolated from an organism of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.

[0484] As discussed above, in a third aspect, the present application relates to a recombinant microorganism capable of converting C6-C 24 Fatty acids produce unsaturated C6-C 24 Fatty alcohol. An illustrative embodiment of the second aspect is Figure 3In the figure, the blue line represents the biochemical pathway endogenous to the host, such as the pathway for converting n-alkanes, fatty alcohols or fatty aldehydes into fatty acids, or the pathway for converting fatty acids into fatty acyl-CoA, acetyl-CoA or dicarboxylic acids. The conversion of substrates to unsaturated fatty acids can be carried out by endogenous or exogenous enzymes in the host. The yellow line indicates the conversion catalyzed by the exogenous nucleic acid molecule encoding the enzyme. Therefore, in some embodiments, the conversion of saturated fatty acids to saturated fatty acyl-ACP can be catalyzed by at least one saturated fatty acyl-ACP synthase, wherein the fatty acyl-ACP synthase is encoded by an exogenous nucleic acid molecule. Non-natural saturated fatty acyl-ACP thioesters establish substrates suitable for desaturation and are different from CoA-thioesters for β-oxidation or fatty acid elongation. In other embodiments, the conversion of saturated fatty acyl-ACP to single or polyunsaturated fatty acyl-ACP can be catalyzed by at least one fatty acyl-ACP desaturase, wherein the fatty acyl-ACP desaturase is encoded by an exogenous nucleic acid molecule. In some embodiments, the mono- or polyunsaturated fatty acyl-ACP can be converted into the corresponding mono- or polyunsaturated fatty acid by fatty acyl-ACP thioesterase. In a specific embodiment, soluble fatty acyl-ACP thioesterase can be used for releasing free fatty acids to reactivate to CoA thioester. Fatty acyl-ACP thioesterases can be used, including Q41635, Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AAO77182, CAH09236, ACL08376 and homologues thereof. In another embodiment, mono- or polyunsaturated fatty acyl-ACP can be converted into the corresponding mono- or polyunsaturated fatty acid by fatty acyl-ACP thioesterase. In yet other embodiments, the conversion of the extended mono- or polyunsaturated fatty acyl-CoA to a mono- or polyunsaturated fatty alcohol can be catalyzed by a fatty alcohol-forming fatty acyl reductase, wherein the fatty alcohol-forming fatty acyl reductase is encoded by an exogenous nucleic acid molecule. Dashed lines indicate downstream steps of the present disclosure, such as the use of acetyltransferases or translocations, or subsequent chemical conversions to produce functionalized pheromones. Red crosses indicate missing or downregulated host native pathways, which increase flux toward the engineered pathway.

[0485] The fatty alcohols produced as taught herein can be further converted to produce downstream products, such as insect pheromones, fragrances, flavorings, and polymer intermediates, which utilize the aldehyde or acetate functional groups. Thus, in some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of C6-C 24 Fatty alcohols are converted to the corresponding C6-C24 In other embodiments, the recombinant microorganism may further comprise at least one endogenous or exogenous nucleic acid molecule encoding an acetyltransferase capable of catalyzing the conversion of C6-C 24 Fatty alcohols are converted to the corresponding C6-C 24 In certain embodiments, the acetyltransferase or the nucleic acid sequence encoding it can be isolated from organisms of the species Candida glabrata, Saccharomyces cerevisiae, Monarch butterfly, Heliothis virescens, Bombyx mori, Agrotissima cutworm, Agrotissima fulva, Euonymus japonicus, Homo sapiens, Kluyveromyces thermotolerans, and Yarrowia lipolytica. In exemplary embodiments, the acetyltransferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1.

[0486] Recombinant microorganisms

[0487] The present disclosure provides microorganisms that can be engineered to express various exogenous enzymes.

[0488] In various embodiments as herein described, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microorganism is a yeast. In an exemplary embodiment, the yeast is a member of the genus selected from the group consisting of: Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.

[0489] The present inventors have found that oleaginous yeasts such as Candida and Yarrowia are 24Fatty alcohol substrates and products have unexpectedly high tolerance.Therefore, in such an exemplary embodiment, the recombinant microorganism of the present invention is an oleaginous yeast.In other embodiments, the oleaginous yeast is a member of the genus selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In even further embodiments, the oleaginous yeast is a member of a species selected from the group consisting of Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

[0490] In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In an exemplary embodiment, the prokaryotic microorganism is a member of the genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

[0491] In some embodiments, the recombinant microorganism is used to produce the mono- or polyunsaturated C6-C 24 Fatty alcohol, fatty aldehyde or fatty acetate.

[0492] Thus, in another aspect, the present invention provides methods for producing mono- or polyunsaturated C6-C 24 In one embodiment, the method comprises culturing the recombinant microorganism in a medium containing a raw material providing a carbon source until a mono- or polyunsaturated C6-C 24 In some embodiments, the method comprises culturing a recombinant microorganism as described herein in a culture medium containing a feedstock sufficient to produce a mono- or polyunsaturated fatty acid. C18 In another embodiment, the carbon source of the mono- or polyunsaturated C 18 Fatty alcohol, fatty aldehyde or fatty acetate. Recovery can be carried out by methods known in the art, such as distillation, membrane-based separation stripping, solvent extraction and expanded bed adsorption.

[0493] In some embodiments, the feedstock comprises a carbon source. In various embodiments described herein, the carbon source can be selected from sugar, glycerol, alcohol, organic acid, alkane, fatty acid, lignocellulose, protein, carbon dioxide, and carbon monoxide. In another embodiment, the sugar is selected from the group consisting of glucose, fructose, and sucrose.

[0494] Engineering can produce mono- or polyunsaturated C6-C 24 Microbial method for producing fatty alcohols, fatty aldehydes and / or fatty acetates

[0495] In one aspect, the present disclosure provides a saturated C6-C 24 Fatty acids produce mono- or polyunsaturated ≤C 18 A method for producing fatty alcohols in a microorganism, wherein the method comprises introducing into the microorganism: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the oxidation of a mono- or polyunsaturated C6-C6 fatty acyl-CoA fatty acid from (a) after one or more consecutive cycles of acyl-CoA oxidase activity. 24 Fatty acyl-CoA is converted to mono- or polyunsaturated ≤C 18Fatty acyl-CoA, where a given cycle produces a mono- or polyunsaturated C6-C 24 Fatty acyl-CoA substrates have two carbon-truncated mono- or polyunsaturated C4-C 22 Fatty acyl-CoA intermediate; and (c) at least one exogenous nucleic acid molecule encoding a fatty acyl reductase to form a fatty alcohol, the fatty acyl reductase to form a fatty alcohol catalyzing the mono- or polyunsaturated ≤C 18 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated ≤C 18 In some embodiments, the microorganism is MATA ura3-302::SUC2Δpox1Δpox2Δpox3Δpox4Δpox5Δpox6ΔfadhΔadh1Δadh2Δadh3Δadh4Δadh5Δadh6Δadh7Δfao1::URA3.

[0496] In some embodiments, the fatty acyl desaturase is selected from the group consisting of fatty acyl desaturases derived from Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana. In some preferred embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession Nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In further embodiments, the acyl-CoA oxidase is selected from Table 5a. In yet another embodiment, the fatty alcohol-forming fatty acyl reductase is selected from the group consisting of fatty alcohol-forming fatty acyl reductases derived from Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus, and Helicoverpa armigera. In yet another embodiment, the fatty alcohol-forming fatty acyl reductase has 90% sequence identity to a fatty alcohol-forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77, and 82.In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, and Candida viswinii.

[0497] In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from the group consisting of E5-10: acyl-CoA, E7-12: acyl-CoA, E9-14: acyl-CoA, E11-16: acyl-CoA, E13-18: acyl-CoA, Z7-12: acyl-CoA, Z9-14: acyl-CoA, Z11-16: acyl-CoA, Z13-18: acyl-CoA, Z8-12: acyl-CoA, Z 10-14: acyl-CoA, Z12-16: acyl-CoA, Z14-18: acyl-CoA, Z7-10: acyl-CoA, Z9-12: acyl-CoA, Z11-14: acyl-CoA, Z13-16: acyl-CoA, Z15-18: acyl-CoA, E7-10: acyl-CoA, E9-12: acyl-CoA, E11-14: acyl-CoA, E13-16: acyl-CoA, E15-18: acyl-CoA, E5Z7-12: acyl- CoA, E7Z9-12: acyl-CoA, E9Z11-14: acyl-CoA, E11Z13-16: acyl-CoA, E13Z15-18: acyl-CoA, E6E8-10: acyl-CoA, E8E10-12: acyl-CoA, E10E12-14: acyl-CoA, E12E14-16: acyl-CoA, Z5E8-10: acyl-CoA, Z7E10-12: acyl-CoA, Z9E12-14: acyl-CoA, Z11E14- 16: acyl-CoA, Z13E16-18: acyl-CoA, Z3-10: acyl-CoA, Z5-12: acyl-CoA, Z7-14: acyl-CoA, Z9-16: acyl-CoA, Z11-18: acyl-CoA, Z3Z5-10: acyl-CoA, Z5Z7-12: acyl-CoA, Z7Z9-14: acyl-CoA, Z9Z11-16: acyl-CoA, Z11Z13-16: acyl-CoA and Z13Z15-18: acyl-CoA. In another embodiment, the mono- or polyunsaturated ≤C 18 The fatty alcohol is selected from E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH and Z13-18:OH.

[0498] In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nuc...

Claims

1. A recombinant Yarrowia lipolytica ( Yarrowia lipolytica ) microorganism, wherein the recombinant Yarrowia lipolytica microorganism comprises: a) a heterologous nucleic acid molecule encoding and expressing a fatty alcohol-forming fatty acyl reductase, the fatty alcohol-forming fatty acyl reductase being derived from Euglena gracilis ( Euglena gracilis ) and having at least 99% sequence identity to SEQ ID NO: 75, wherein the fatty alcohol-forming fatty acyl reductase catalyzes the conversion of mono- or poly-unsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty alcohol.

2. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the fatty alcohol-forming fatty acyl reductase comprises the amino acid sequence of SEQ ID NO:

75.

3. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises at least one nucleic acid molecule encoding a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105, which catalyzes a saturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty acyl-CoA.

4. The recombinant Yarrowia lipolytica microorganism according to claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation and / or reduction of the fao1 gene.

5. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: a) one or more enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA; b) one or more fatty alcohol dehydrogenases; c) one or more fatty alcohol oxidases; d) one or more enzymes that catalyze the conversion of fatty acids into ω-hydroxy fatty acids; and e) one or more diacylglycerol acyltransferases.

6. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: (i) one or more acyl-CoA oxidases selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), and YALI0E06567g (POX6); (ii) one or more fatty alcohol dehydrogenases selected from YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) fatty alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzymes selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11), and YALI0A20130g (ALK12); and (v) one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

7. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of each of the following enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA: CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, and CAG79214.

8. The recombinant Yarrowia lipolytica microorganism of claim 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of the FAO1 enzyme and each of the following enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA: CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, and CAG79214.

9. The recombinant Yarrowia lipolytica microorganism according to claim 1, wherein the mono- or polyunsaturated C6-C 24 The fatty alcohol is selected from Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH and Z13-18:OH.

10. A recombinant Yarrowia lipolytica ( Yarrowia lipolytica ) microorganism, wherein the recombinant Yarrowia lipolytica microorganism comprises: a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 95% sequence identity to SEQ ID NO: 54, which catalyzes the saturation of C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 fatty acyl-CoA; and b) at least one nucleic acid molecule encoding a fatty alcohol-forming acyl reductase from Euglena gracilis having at least 99% sequence identity to SEQ ID NO: 75, which catalyzes the conversion of a mono- or poly-unsaturated C6-C 24 Fatty acyl-CoA is converted to the corresponding mono- or polyunsaturated C6-C 24 Fatty alcohol.

11. The recombinant Yarrowia lipolytica microorganism of claim 10, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: a) one or more fatty alcohol dehydrogenases; b) one or more enzymes that catalyze the conversion of fatty acids into ω-hydroxy fatty acids; and c) one or more diacylglycerol acyltransferases.

12. The recombinant Yarrowia lipolytica microorganism of claim 10, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of one or more endogenous enzymes selected from the group consisting of: a) one or more fatty alcohol dehydrogenases; b) one or more cytochrome P450 enzymes selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11), and YALI0A20130g (ALK12); and c) one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

13. The recombinant Yarrowia lipolytica microorganism of claim 10, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of each of the following enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA: CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, and CAG79214.

14. The recombinant Yarrowia lipolytica microorganism of claim 10, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and / or reduction in activity of the FAO1 enzyme and each of the following enzymes that catalyze the conversion of fatty acyl-CoA to α,β-enoyl-CoA: CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, and CAG79214.

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