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  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids
  • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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  • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
  • C12Y114/19001—Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase

Definitions

• This application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates.
• the application further relates to methods of producing unsaturated C&-C24 fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising one or more of these compounds and/or the recombinant microorganisms.
• the present application relates to recombinant microorganisms having a biosynthesis pathway for the production of one or more compounds selected from unsaturated C6-C24 fatty- alcohols, aldehydes, and acetates.
• the recombinant microorganisms described herein may be used for the production of at least one compound, such as an insect pheromone, a fragrance, or a flavoring agent, selected from unsaturated C6-C24 fatty alcohols, aldehydes, and acetates.
• the recombinant microorganism comprises a biosynthesis pathway for the production of an unsaturated C6-C24 fatty aldehyde or fatty alcohol .
• the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty aldehyde or fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses (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 poly-unsaturated C6-C24 fatty acyl-CoA; and (b): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming
• the mono- or poly-unsaturated C6-C24 fatty aldehyde is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty aldehyde is a fragrance or flavoring agent.
• the recombinant microorganism further comprises at least one endogenous or exogenous nucieic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate, (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 C6-C24 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
• the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent.
• 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 the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
• the recombinant microorganism furtlier comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or polyunsaturated C6-C24 fatty acetate.
• 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, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
• the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, CIO, C I 1, C12, or C13 in the fatty acid or its derivatives, such as, for example, fatty' acid CoA esters.
• the fatty-acyl desaturase is a Zl l desaturase.
• the Zl l desaturase, or the nucleic acid sequence that encodes it can be isolated from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichopiusia ni, HeUcoverpa zea, or Thalassiosira pseudonana.
• Zl I-desaturases, or the nucleic acid sequences encoding them can be isolated from Bomhyx mori.
• the Zl l desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF4I6738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. in some embodiments, a nucleic acid sequence encoding a Zl l.
• the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni.
• the Z1 I desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni.
• the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agroiis segetum. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agroiis segetum. In some embodiments, the Z l l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Zl l desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana.
• the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transiiella. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transiiella. In further embodiments, the Zl 1 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z l 1 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea.
• the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, ⁇ 21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481 , AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221. I . In some embodiments, the Zl l desaturase comprises a chimeric polypeptide.
• a complete or partial Zl l desaturase is fused to another polypeptide.
• the N-terminal native leader sequence of a Zl l desaturase is replaced by an oleosin leader sequence from another species.
• the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29.
• the Zl l desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61 , 62, 63, 78, 79 and 80.
• the Zl l desaturase catalyzes the conversion of a fatty acyl- CoA into a mono- or poly-unsaturated product selected from Zl l -13:Acyl ⁇ CoA, El l - 13:Acyl-CoA, (Z,Z)-7,l l-13:Ac l-CoA, Zl l-14:Acyl-CoA, El l-14:Acyl-CoA, (E,E)-9, 11 - 14:Acyl-CoA, (E,Z)-9,l l-14:Acyl-CoA, (Z,E)-9,l l-14:Acyi-CoA, (Z,Z)-9,l l-14:Acyl-CoA, (E,Z)-9,1 l-15:Acyl-CoA, (Z,Z)-9, 1 l-15: Acyl-CoA, Zl-16:Ac
• the fatty-acyl desaturase is a Z9 desaturase.
• the Z9 desaturase, or the nucleic add sequence that encodes it can be isolated from organisms of the species Ostrinia fumacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitelia. Helicoverpa assulta, or Helicoverpa zea.
• the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788.
• a nucleic acid sequence encoding a Z9 desaturase is codon optimized.
• the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis.
• the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis .
• the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitelia.
• the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitelia.
• the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.
• Other Z9 desaturase s of the present disclosure include SEQ ID Nos: 95, 97, 99, 101 , 103, and 105.
• the overexpression of a Z9-18 specific desturase can increase the membrane fluidity to improve the diffustion of fatty alcohols into the supernatant.
• the Z9 desaturase catalyzes the conversion of a fatty acyl- CoA into a monounsaturated or polyunsaturated product selected from Z9-l l :Acyi-CoA, Z9- 12:Acyl-CoA, E9- !
• the recombinant microorganism may express a bifunctional desaturase capable of catalyzing the subsequent desaturation of two double bonds
• the recombinant microorganism may express more than 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.
• the recombinant microorganism may express an exogenous nucleic acid molecule encoding a Zl l desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.
• the recombinant microorganism may express a fatty-acyl conjugase that acts independently or together with a fatty-acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.
• the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 aldehyde or fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyzes the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl- CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty aldehyde or fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated CV C24 fatty acyl-CoA from (a) into the
• the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA.
• the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C6-C24 fatty acyl-CoA, 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 conjugase that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated C6-C24 fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C6-C
• 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 conjugases that catalyze the conversion of a saturated or monounsaturated C6-C24 fatty acyl-CoA to a corresponding polyunsaturated CV C24 fatty acyl-CoA.
• the fatty-acyl conjugase is a conjugase 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.
• the conjugase, or the nucleic acid sequence that encodes it can be isolated from organisms of the species Cydia pomonella, Cydia nigricana, Lobesia boirana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois Iransitella, Manauca sexta, Bombyx rnori, Calendula officinalis, Trichosanthes kirilowii, Punica granaium, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana.
• the conjugase comprises a sequence selected from GenBank Accession No. 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, AF 182521, AF 182520, Q95UJ3.
• the fatty alcohol forming acyl-CoA reductase i.e., fatty alcohol forming fatty-acyl reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Spodoptera littoralis, Hehcoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evcmymellus .
• the reductase comprises a sequence selected from. GenBank Accession Nos. JX679210 and HG423128, and UniProt Accession No. I3PN86.
• a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera littorahs, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus is codon optimized.
• the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum.
• the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from. Agrotis segetum.
• the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littorahs.
• the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera lateralis.
• the reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81.
• the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum.
• the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from. Spodoptera littorahs. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an ammo acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.
• the present disclosure teaches using multiple fatty acyl reductase enzymes. 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 co-factors. For example, the fatty acyl reductase from Euglena gracilis (SEQ ID NO: 75) uses NADH instead of NADPH as reducing equivalent. In some embodiments, this can allow for co-factor balancing using two or more different reductases.
• the fatty acyl reductase is a mutated 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 mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.
• the fatty alcohol forming fatty-acyl reductase catalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoA into a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E) ⁇ 5 ⁇ decenoi, (Z)-7-dodecenol, (E)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (E)-9-dodecenol, (Z) ⁇ 9-tetradecenol, (E)-9-tetradecenol, (Z)-9-hexadecenol, (Z)-l 1-tetradecenol, (Z)-7- hexadeceno
• the recombinant microorganism may 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 -C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated CVC24 fatty alcohol.
• the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ⁇ Cis fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyi 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; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ⁇ Cis fatty acyl-CoA after one or
• the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodopiera litura, Sesamia inferens, Manduca sexta, Ostrinia niibilalis, Helicoverpa zea, Chonsioneura rosaceana, Drosophila melanogasier, Spodopiera littoralis, Lamproma capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase.
• the fatty acyl desaturase has 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 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.
• the acyl-CoA oxidase is selected from Table 5a .
• the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera tittoralis, Euglena gracilis, Yponomeuta evonyrnellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase.
• 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%, 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 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.
• the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii .
• the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ⁇ Ci8 fatty acyl-CoA .
• the acyltransferase is selected from the group consisting of glycerol-3 -phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT).
• the acyltransferase is selected from Table 5b.
• the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C 16, of >C14, of >C 12 or of >C10 acylglycerol substrates.
• the acylglycerol lipase is selected from Table 5c.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ⁇ Cis fatty alcohol.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase: (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.
• one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase: (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or
• one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y lipofytica PO 1 (YALI0E32835g), Y. lipolytica POX2 (YALIOFl 0857g), Y. lipolytica POX3 (YALI0D24750gj, Y.
• lipolytica POX4 (YALiOE27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g); S. cerevisiae POX1 (Y GL205W); Candida POX2 (Ca019, 1655, CaO 19.9224, CTRG_02374, Ml 8259), Candida POX4 (Ca019.1652, Ca()19.9221, CTRGJ32377, M12160), and Candida POX5 (CaO 19 5723. CaOI9.13146, CTRG . 02721, M12161).
• a recombinant microorganism capable of producing a mono- or poly -unsaturated ⁇ Cis fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated CG ⁇ C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, dismpt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes.
• the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde and/or fatty acetate. In other embodiments, the one or more acyl-CoA oxidase enzymes being expressed are selected from Table 5a.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y, lipolytica YAL10E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YAL10C 14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBLOl lw, S.
• Candida CTRG_02630 Candida CaO19.250, Candida Ca019.7881, Candida CTRG_02437, Candida Ca019.1881, Candida CaO 19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida Ca019.8645, Candida CTRG 0475Q, Candida Ca019.13439, Candida CTRG 04390, Canada Ca019.6941, Canada CaO19.14203, and Candida CTRG 06209.
• a recombinant microorganism capable of producing a mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid is provided, wherein the recombinant microorganism expresses one or more acyitransferase enzymes, and wherein the recombinant microorganism is manipulated to delete, dismpt, mutate, and/or reduce the activity of one or more endogenous acyitransferase enzymes.
• one or more genes of the microbial host encoding GPATs, LPAATs, GPLATs and/or DGATs are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs.
• the one or more acyitransferase enzymes being expressed are different from the one or more endogenous acyitransferase enzymes being deleted or downregulated.
• the one or more acyitransferase enzymes being expressed are selected from Table Sb.
• one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacyiglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YAL10E32035g, Y.
• lipolytica YALiOD17534g Y. lipotytica YALIOFlOOl Og
• Y. lipolytica YALI0C14520g Y. lipolytica YALI0E00528g
• S. cerevisiae YKL140w S. cerevisiae YMR313c
• S. cerevisiae YKR089c S. cerevisiae YOR081C
• S. cerevisiae YKL094W S. cerevisiae YLL012W
• S. cerevisiae YLL012W S.
• Candida CaO 19.2050 Candida Ca019,9598, Candida CTRG__01 138, Candida W5Q__03398, Candida CTRG_00057, Candida CaO 19.5426, Candida Ca019.12881 , Candida CTRG 06185, Candida CaO 19.4864.
• the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g iALK I), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B.138l6g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y.
• endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g iALK I), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica Y
• lipolytica YALI0AI 5488g (ALK7), Y. lipolytica YALI0CI2122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YAU0B207G2g (AL 10), 5 ' . lipolytica YALI0C10054g ⁇ ALK 11 ⁇ and Y. lipolytica YALI0A2()130g iAL I 2 .
• a recombinant microorganism capable of producing a mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated Ce-Cw fatty acid is provided, wherein the recombinant microorganism expresses one or more acyigiycerol lipase and/or sterol ester esterase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyigiycerol lipase and/or sterol ester esterase enzymes.
• the one or more acyigiycerol lipase and/or sterol ester esterase enzymes being expressed are different from the one or more endogenous acyigiycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated.
• the one or more endogenous or exogenous acyigiycerol lipase and/or sterol ester esterase enzymes being expressed prefer to hydroiyze ester bonds of long-chain acylglycerols.
• the one or more acyigiycerol lipase and/or sterol ester esterase enzymes being expressed are selected from Table Sc.
• the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7- 12:Acyl-CoA, E9-14:Acyl-CoA, El l-16:Acyl-CoA, E13-18:Acyl-CoA,Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Zl i-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl- CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Zl l- 14:Acyl-CoA, Z13-16:
• the mono- or poly-unsaturated ⁇ Cis fatty alcohol is selected from the group consisting of E5- 10.01 L Z8- 12:OH, Z9- 12:QH, Z 1 1-14:QH, Z i 1 - 16.01 1. H i i - 14.0! I. E8E10- 12:OH, E7Z9- i .: OH. Z 1 1Z13-16QH, /' ⁇ - ! 4 OH. Z9- 16:OH, and Z 13-18:OH.
• the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyi- CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ⁇ Cis fatty acid into a corresponding Cis fatty aldehyde.
• the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A.
• 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 poly-unsaturated ⁇ Cis fatty alcohol into a corresponding ⁇ Cis fatty aldehyde, in some preferred embodiments, the ⁇ Cis fatty aldehyde is selected from the group consisting of Z9- 1 Aid . / i 1 - 1 6 A id.. Z l 1 / 1 3- 1 6 Aid., and Z 13- 18:Ald.
• the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol, dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ⁇ Cis fatty alcohol mto a corresponding ⁇ Cis fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ⁇ Cis fatty alcohol into a corresponding ⁇ Cis fatty acetate.
• the mono- or polyunsaturated ⁇ Cis fatty aldehyde and ⁇ Cis fatty acetate is selected from the group consisting of E5-10:Ac, Z7- 12:Ac, Z8- 12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12- I4:Ac, El l- 14:Ac, Z l l-14:Ac, Z l i - 1 6 Ac. Z9-16:Ac, Z9-16:Ald, Z l l-16:Ald,Z l lZ 13-16:Ald, and Z 13- 1 8:Ald.
• the disclosure provides a method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ⁇ Cis fatly alcohol. from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into a microorganism the following: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated CG- C2 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C6-C24 fatty acyl-CoA from (a) into a mono- or poly-unsaturated ⁇ CI 8 fatty acyl
• the microorganism is MATA ura3-302::SUC2 ⁇ ⁇ 2 ⁇ 3 ⁇ 4 ⁇ 5 ⁇ Afadh Aadhl Aadh2 Aadh3 Aadh4 AadhS Aad G Aadh7 Afa,ol ::URA3.
• the disclosure provides a method of producing a mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde or fatty acetate from, an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde or fatty acetate.
• the method further comprises a step of recovering die mono- or poly-unsaturated ⁇ Cis fatty alcohol, fatty aldehyde or fatty- acetate.
• the recovery step comprises distillation.
• the recovery step comprises membrane-based separation.
• the mono- or poly-unsaturated ⁇ Cis fatty alcohol is converted into a corresponding ⁇ Cis fatty aldehyde using chemical methods.
• the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, ⁇ - PhI(OAc)2, Swem oxidation and noble rnetal-air.
• the mono- or polyunsaturated ⁇ Cis fatty alcohol is converted into a corresponding ⁇ Cis fatty acetate using chemical methods.
• the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N, N- dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated
• a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N, N- dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated
• the disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the recombinant Yarrowia lipolytica microorganism, comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase 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%, 5
• the recombinant Yarrovjia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated CVC24 fatty alcohol.
• the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YAL10E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YAL10E17787g (ADH2), YALI0A16379g (ADH3), YALI.0E158.18g (ADH4), YALI
• the recombinant Yarrovjia lipofytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YAI,T0E27654g (POX4), YALI0C23859g (POX5), YAI,T0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5),
• cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALKl),YALI0F01320g (ALK2), YAL10E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C 12122g (ALK8),YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C 10054g (ALK11) and YALI0A20130g (Aikl2); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA 1) and YALI0D07986g (DGA2).
• the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from. Z9 ⁇ 14:Acy]-CoA, Zl 1 - 14; Acyl-CoA, El l-14:Acyl-CoA, Z9-16:Acy]-CoA, and Zl l -16:Acyl- CoA.
• the mono- or poly-unsaturated C6-C24 fatty alcohol is selected from the group consisting of ⁇ 9-14 ⁇ , / ! 1 - 14:0: 1. Ei l-14:GH, Z9-16:OH, Zl l -16:OH, Zl 1Z13-16: OH, and Z 13- 18 : OH.
• the recombinant Yarrowia lipofytica 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 poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty aldehyde.
• the alcohol dehydrogenase is selected from Table 3a.
• the C6-C/.4 fatty aldehyde is selected from the group consisting of Z9-14:Ald, Zl 1-14: Aid, El I-14:Ald, Z9-16:Ald, ZI l-16:Ald, Zl lZI3-16:Ald and Z13-18:Ald.
• 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 poly-un saturated C6-C24 fatty alcohol into a corresponding C&-C24 fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
• the mono- or poly-unsaturated CG- C24 fatty aldehyde and C6-C24 fatty acetate is selected from the group consisting of Z9-14:Ac, Zl l-14:Ac, El l-14:Ac, Z9-16:Ac, Zl l-16:Ac, Zl lZ13-16:Ac, Z13-18:Ac, Z9-14:Ald, Zl l- 14:Ald, El M4:Ald, Z9-16:Ald, Zl l-16:Ald, Zl lZ 13-16:Ald and Z 13-18:Ald.
• 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 an Amyelois transitella, Spodoptera littoralis, Agrotis segelum, or Trichophisia ni derived desaturase.
• the disclosure provides a method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of saturated C6-C24 fatty acid, wherein the method comprises introducing into the Yarrowia lipolytica microorganism the following: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase 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%
• the microorganism is MATA ura3-302: :SUC2 ⁇ ⁇ 2 ⁇ 3 ⁇ 4 ⁇ 5 ⁇ Afadh Aadhl Aadh2 Aadh3 Aadh4 Aadh5 Aadh6 Aadh7 Afaol ::URA3.
• the disclosure provides a method of producing a mono- or poly-unsaturated C6-C2 fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C6-C24 fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C6-C24 fatty alcohol, fatty aldehyde or fatty acetate.
• the method further comprises a step of recovering the mono- or poly-unsaturated CVC24 fatty alcohol, fatty aldehyde or fatty acetate.
• the recovery step comprises distillation.
• the recovery step comprises membrane-based separation.
• the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty aldehyde using chemical methods.
• the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPQ-Phi(QAc)2, S ern oxidation and noble metal-air.
• the mono- or poly-unsaturated C6-C24 fatty alcohol is converted into a corresponding C6-C24 fatty acetate using chemical methods.
• the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4- N, N-dimethylan inopyridine (DMAP) or sodium acetate to esterify the mono- or polyunsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
• a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4- N, N-dimethylan inopyridine (DMAP) or sodium acetate to esterify the mono- or polyunsaturated C6-C24 fatty alcohol to the corresponding C6-C24 fatty acetate.
• DMAP 4- N, N-dimethylan inopyridine
• the present application provides an additional biosynthetic pathway for the production of an unsaturated C6-C24 fatty alcohol utilizing a saturated C6-C2.4 fatty acyl-ACP intermediate derived from a C6-C24 fatty acid.
• the application relates to a recombinant microorganism capable of producing an unsaturated C6-C24 fatty alcohol from an endogenous or exogenous source of C6-C2.4 fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl- ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acy l-ACP; (c) one or more endogenous or exogenous nucleic acid molecules encoding a
• the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent.
• 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 con version of the mono- or poly-unsaturated Ce- C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty- aldehyde.
• the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty acetate.
• acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 carbon atoms.
• the acyl-ACP synthetase, or the nucleic acid that encodes it can be isolated from organisms of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrow ia lipolytica.
• the fatty-acyl-ACP desaturase is a soluble desaturase.
• the fatty-acyl-ACP desaturase, or the nucleic acid that encodes it can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.
• the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty-acyi desaturase that catalyzes the conversion of a saturated Ce-CiA fatty acyl-ACP to a corresponding mono- or polyunsaturated C6-C24 fatty acyl-ACP.
• fatty acid elongation enzymes i.e. , a fatty acid synthase complex
• a fatty acid synthase complex can be utilized to extend the chain length of a mono- or poly-unsaturated C6-C24 fatty acyl- ACP by two additional carbons at the alpha carbon.
• the two additional carbons are derived from endogenous malonyl-CoA.
• the one or more nucleic acid molecules encoding a fatty acid synthase complex are endogenous nucleic acid molecules, i. e. , the nucleic acid molecule(s) is/are native to the recombinant microorganism.
• the one or more nucleic acid molecules encoding a fatty acid synthase complex are exogenous nucleic acid molecules.
• the fatty aldehyde forming acyl-ACP reductase i. e. , fatty aldehyde forming fatty-acyi reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species can be isolated from organisms of the species Pelargonium horiorum, Asclepias syriaca, and Uncaria tomentosa.
• 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 conversion of the mono- or poly -unsaturated C6-C24 fatty aldehyde from (d) into a corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
• the dehydrogenase is encoded by an endogenous nucleic acid molecule.
• the dehydrogenase is encoded by an exogenous nucleic acid molecule.
• the endogenous or exogenous nucleic acid molecule encoding a dehydrogenase is isolated from organisms of the species Saccharomyces cerevisiae, Escherichia coti, Yarrowia lipoiytica, or Candida tropicalis.
• the present application provides an additional biosynthetic pathway for the production of an unsaturated Ce-Cw fatty alcohol utilizing a saturated C6-C24 fatty acyl-ACP intermediate derived from a CVC24 fatty acid.
• the application relates to a recombinant microorganism capable of producing an unsaturated C&-C24 fatty alcohol from an endogenous or exogenous source of C6-C24 fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated CVC24 fatty acyl-ACP to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-ACP; (c) at least one exogenous fatty acyl-ACP thioesterase that cataly
• the mono- or poly-unsaturated C6-C24 fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent.
• 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 the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
• the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (e) into a corresponding mono- or polyunsaturated C6-C24 fatty acetate
• a fatty acyl-ACP thioesterase can be utilized to convert a mono- or poly-unsaturated C6-C24 fatty acyl-ACP into a corresponding mono- or poly-unsaturated C6-C24 fatty acid.
• soluble fatty acyl-ACP thioesterases can be used to release free fatty acids for reactivation to a CoA thioester.
• Fatty acyl-ACP thioesterases that can be included within the embodiment include, but are not limited to, including Q41635, Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AA077182, CAH09236, ACL08376, and homologs thereof may be used.
• the mono- or poly-unsaturated C6-C24 fatty acyl-CoA may serve as a substrate for an elongase, which can be utilized to extend the chain length of a mono- or poly- unsaturated C6-C24 fatty acyl-CoA by two additional carbons at the alpha carbon.
• the two additional carbons are derived from endogenous malonyl-CoA.
• the recombinant microorganism according to die 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 a mono- or poly-unsaturated C6-C24 fatty alcohol into a corresponding mono- or polyunsaturated C6-C24 fatty aldehyde.
• the alcohol oxidase, or the nucleic acid sequence that encodes it can be isolated from organisms of the species Candida boidinii, Komagataella pastoris, Tanacetum v lgare, Simmondsia chinensis.
• the alcohol oxidase comprises a sequence selected from GenBank Accession Nos. Q00922, F2QY27, Q6QIR6, Q8LDP0, and L7VFV2.
• the recombinant microorganism according to the first or second aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of a C6-C24 fatty alcohol into a corresponding C6-C24 fatty acetate.
• the acetyl transferase, or the nucleic acid sequence that encodes it can be isolated from organisms of the species Saccharomyces cerevisi e, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum, Euonymus alatus.
• the acetyl transferase 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. L XP_505595.1, and XP_505513.1.
• the fatty alcohol may be converted into a fatty acetate using chemical methods, e.g., via chemical catalysis utilizing a chemical agent such as acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride.
• a chemical agent such as acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride.
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express one or more nucleic acids encoding protein or polypeptide which, when expressed, is toxic to an insect.
• Exemplary toxicant producing genes suitable for the present disclosure can be obtained from entomopathogenic organism., such as Bacillus thiiringiensis, Pseudomonas aeruginosa, Serratia marcescens, and members of the genus Streptomyces .
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding a.
• Bacillus thuringiensis (' " Bi " ) toxin in additional or alternative embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding other toxic proteins such as spider venom ,
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express an RNAi molecule which, when expressed, produces an oligonucleotide that is toxic to an insect.
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may further be engineered to express a metabolic pathway which, when expressed, produces a small molecule that is toxic to an insect.
• toxic small molecules include azadirachtin, spinosad, avermectin, pyrethnns, and various terpenoids.
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may be a eukaryotic microorganism, such as a yeast, a filamentous fungi, or an algae, or alternatively, a prokaryotic microorganism, such as a bacterium.
• suitable host cells can include cells of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, and Streptomyces .
• a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Coryn
• the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate is a yeast.
• suitable yeasts include yeasts of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, or Myxozyma,
• the yeast is an oleaginous yeast.
• Exemplary oleaginous yeasts suitable for use in the present disclosure include members of the genera Yarrowia, Candida, Rliodotorula , Rhodosporidium, Cr ptococcus , Trichosporon, and Lipomyces, including, but not limited to the species of Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloid.es, Lipomyces starkey, L. lipoferiis, C. revêti, C. pulcherrirna, C. utilis, Rhodotorula minula, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutims, and R, graminis.
• endogenous enzymes can convert critical substrates and/or intermediates upstream of or within the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate biosynthesis pathway into unwanted by-products. Accordingly, in some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate biosynthesis pathway.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acid into a ⁇ -hydroxyfatty acid.
• the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from XP 504406, XP 504857, XP_504311, XP_500855, XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211, BAA02212, BAA02213, BAA02214, AA073952, AA073953, AA073954, AA073955, AA073956, AA073958, AA073959, AAO73960, AA073961, AA073957, XP_002546278, or homologs thereof.
• the recombinant bacterial microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from BAM49649, AAB80867, ⁇ ⁇ 7462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CABS 1047, AAC82967, WP_011027348, or homologs thereof.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acyl-CoA into ⁇ , ⁇ -enoyl-CoA.
• the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361 , AAA34363, CAA29901 , BAA04761, AAA34891, or homologs thereof.
• the recombinant bacterial microorganism is engineered to delete, disnipt, mutate, and/or reduce the activity of one or more enzyme selected from.
• 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 enzyme involved in peroxisome assembly and/or peroxisome enzyme import.
• the recombinant yeast microorganism is engineered to delete, disnipt, mutate, and/or reduce the activity of one or more enzyme selected from XP 505754, XP 501986, XP 501311, XP J504845, XP 503326, XP_504029, XP_ 002549868, XP_ 002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW1 1472, EIW09743, EIW08286, or homologs thereof.
• the recombinant microorganism is manipulated to delete, disnipt, mutate, and/or reduce the activity of one or more endogenous reductase or desaturase enzymes that interferes with the unsaturated -C24 fatty alcohol, aldehyde, or acetate, i.e., catalyzes the conversion of a pathway substrate or product into an unwanted by-product.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous alcohol oxidase or alcohol dehydrogenase enzymes that catalyzes the unwanted conversion of the desired product, e.g. , unsaturated C&-C24 fatty alcohol into a corresponding unsaturated C6-C24 fatty aldehyde.
• one or more endogenous alcohol oxidase or alcohol dehydrogenase enzymes that catalyzes the unwanted conversion of the desired product, e.g. , unsaturated C&-C24 fatty alcohol into a corresponding unsaturated C6-C24 fatty aldehyde.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for one or more unsaturated fatty acyl-CoA intermediates.
• the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases.
• 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 homolog thereof.
• the one or more endogenous enzymes comprise one or more glycerolphospholipid acyltransferases.
• the recombinant yeast microorganism is engineered to delete, disrapt, mutate, and/or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALT0EI 6797g and CTG_04390, or homolog thereof.
• the one or more endogenous enzymes comprise one or more acyi-CoA/steroi acyltransferases.
• 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 homolog thereof.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that oxidizes fatty aldehyde intermediates.
• the one or more endogenous enzymes comprise one or more fatty aldehyde dehydrogenases.
• 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 homolog thereof.
• the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that consumes fatty acetate products.
• the one or more endogenous enzymes comprise one or more sterol esterases.
• 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 homolog thereof.
• the one or more endogenous enzymes comprise one or more triacyiglycerol lipases.
• the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more triacyiglycerol lipases selected from the group consisting of YALI0D17534g, YALlOFlOOlOg, CTRG 00057 and CTRG 06185, or homolog thereof.
• the one or more endogenous enzymes comprise one or more monoacylglycerol lipases.
• 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 YALI0C 14520g, CTRG__03360 and CTRG_05049, or homolog thereof.
• the one or more endogenous enzymes comprise one or more extracellular lipases.
• 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, YALI0B0936Ig, CTRG . 05930, CTRG 04188, CTRG 02799, CTRG_03052 and CTRG 03885, or homolog thereof.
• one or more of the exogenous unsaturated C6-C24 fatty alcohol, aldehyde, or acetate pathway genes encodes an enzyme that is localized to a yeast compartment selected from the group consisting of the cytosol, the mitochondria, or the endoplasmic reticulum.
• one or more of the exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum.
• at least two exogenous pathway genes encode an enzyme that is localized to the endoplasmic reticulum.
• all exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum.
• the present application provides methods of producing an unsaturated C6-C24 fatty alcohol, aldehyde, or acetate using a recombinant microorganism as described herein.
• the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate is produced and optionally, recovering the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate.
• the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate may be isolated from the fermentation medium using various methods known in the art including, but not limited to, distillation, membrane -based separation gas stripping, solvent extraction, and expanded bed adsorption.
• the recombinant microorganism e.g. , a yeast
• the yeast may be recovered and produced in dry particulate form.
• the yeast may be dried to produce powdered yeast.
• the process for producing powdered yeast comprises spray drying a liquid yeast composition in air, optionally followed by further drying.
• the recombinant microorganism composition will comprise the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate when dried.
• preferred recombinant microorganisms of the disclosure will have the ability to utilize alkanes and fatty acids as carbon sources.
• carbon sources may 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, as well as the aforementioned alkanes and fatty acids.
• the recombinant microorganism will convert the carbon source to the unsaturated C6-C24 fatty alcohol, aldehyde, or acetate under aerobic conditions.
• the present application provides methods of producing one or more unsaturated C6-C24 fatty alcohols, aldehydes, or acetates using a recombinant microorganism as described herein.
• the product is an insect pheromone.
• exogenous and endogenous enzymes can be expressed in a recombinant host microorganism to produce a desired insect pheromone.
• Exemplary insect pheromones in the form of fatty alcohols, fatty aldehydes, or fatty acetates capable of being generated using the recombinant microorganisms and methods described herein include, but are not limited to, (Z)-l l -hexadecenal, (Z)-l l-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)- i l , 13 ⁇ hexadecadienal, (9Z, 1 l E)-hexadecadienal, (E,E)-8, 10-dodecadien- l-ol, (7E,9Z)- dodecadienyl acetate, (Z)-3-nonen-l-oi, (Z)-5-decen-l-ol, (Z)-5-decenyi acetate, (E)-5-decen- l-ol, (
• compositions comprising one of more of the insect pheromone-producing recombinant microorganisms described herein can be provided.
• the composition may further comprise one or more insect pheromones produced by the recombinant microorganism.
• Figure 1 illustrates the conversion of a saturated fatty acyl-CoA to an unsaturated fatty alcohol.
• Figure 2 illustrates the conversion of a saturated fatty acid to a mono- or polyunsaturated fatty aldehyde, alcohol, or acetate.
• Figure 3 illustrates an additional pathway for the conversion of a saturated fatty acid to a mono- or poiy-unsaturated fatty aldehyde, alcohol, or acetate
• Figure 4 illustrates a pathway for the conversion of a saturated fatty acid to various trienes, dienes, epoxides, and odd-numbered pheromones.
• Figure 5 shows Zl l-hexadecenol production from W303A and BY4742 ⁇ 1.
• Figure 6A- Figure 6B shows sample chromatograms of biotransformation product of Zl 1 -hexadecenoic acid using S. cerevisiae expressing either an empty vector (Figure A), or Helicoverpa arrnigera alcohol-forming reductase (Figure 6B). Black lines: no substrate added. Purple line: Zl 1-hexadecenoic acid was added as substrate.
• Figure 7A- Figure 7B shows a comparison of GC-MS fragmentation pattern of Zl l- hexadecenol authentic compound ( Figure 7A), and Zl l-hexadecenol biologically derived ( Figure 7B).
• Figure 8 shows biomass at the time of harvesting for product analysis of W303A (wild type) and BY4742 ⁇ 1 (beta-oxidation deletion mutant).
• Strain expressing empty vector (EV), S. littoralis reductase (FAR-SL), H. arrnigera reductase (FAR-HA), A. segetum reductase (FAR-AS). Error bars represent standard deviation derived from. N 2 biologically independent samples.
• Figure 10 shows a pOLEl cassette comprising an extended OLE! promoter sequence (light yellow), OLE1 promoter (orange), OLE1 leader sequence (dark grey), a synthon such as an insect desaturase sequence (light grey), and the VSP13 terminator sequence (blue).
• Figure llA- Figure HE shows validation of the pOLEl cassette, and complementation assay.
• Figure HA YPD + palmitoleic acid
• Figure 11B YPD - palmitoieic acid
• Figure 11C CM-Ura glucose + palmitoleic acid
• Figure 11D CM-Ura glucose - palmitoleic acid
• Figure HE Map of strains in Figure 11 A- Figure 11D.
• Dasher GFP synthon.
• Figure 12A shows complementation of AOLEl growth without UFA on YPD.
• Figure 12B shows complementation of AOLEl growth without UFA on CM-Ura glucose.
• Figure OA shows the full fatty acid spectrum of a AOLEl strain expressing: S. cerevisiae OLE! desaturase (blue), chimeric T. ni desaturase (red).
• Figure 13B shows a focused fatty acid spectrum within 5.5-min - 8-min retention time of S. cerevisiae AOLEl strain expressing S. cerevisiae OLE1 desaturase (red) and chimeric / ' ni desaturase (blue).
• Figure 14A- Figure 14B shows a comparison of GC-MS fragmentation pattern of (Z)- 11-hexadecenoic acid from an authentic compound ( Figure 14A) and biologically derived ( Figure 14B).
• Figure 15 shows C16 fatty alcohol production from AOLEl expressing various fatty- alcohol pathway variants in culture supplemented with palmitic and palmitoleic acid. Error bars represent 5% uncertainty of metabolite quantification accuracy.
• Figure 16 shows representative chromatograms of biotransformation product C16 fatty acids using S. cerevisiae expressing fatty alcohol pathways TN_desat - HA_reduc when fed with palmitic acid (black) and when fed with palmitic and palmitoleic acids (orange). Profile of a negative control strain (harboring an empty vector) fed with palmitic acid (purple).
• Figure 17 shows that (Z)- l l-hexadecenoic acid was detected in the cell pellets of S. cerevisiae expressing fatty alcohol pathways TN desat-SL reduc (blue), SC_desat-HA_reduc (red), TN desat-HA reduc (green), SC desat-SL reduc (pink).
• Figure 18 shows C16 fatty alcohol production from AOLEl expressing various fatty alcohol pathway variants in culture supplemented with palmitic acid only. Error bars represent 5% uncertainty of metabolite quantification accuracy.
• Figure 19A- Figure 19C shows detection of (Z)- l l-hexadecenol.
• Figure 19A Fragmentation partem of an authentic standard. The m/z 297.3 was used in follow up experiments to selectively detect the alcohol. To also detect the internal standard, the masses 208 and 387.3 were included too.
• Figure 19B In addition to the detection of the specific mass fragment, the retention time was used as second stage confirmation. The retention time is 6.22.
• Figure 19C Comparison of the two different regioisomers 9Z ⁇ and 1 IZ-hexadecenol when detected in SIM mode (297.3) with the same method.
• Figure 20 shows pXICL expression cassette architecture. Hie C. albicans OLE1 leader-/!, segetum desaturase fusion is also shown.
• Figure 21A- Figure 21D shows mCherry control integration.
• Figure 21A Negative (water-only) control tra sformation plate.
• Figure 21B pPVO.137 mCherry transformation plate.
• Figure 21C Patch plates from negative control clones.
• Figure 21 D Patch plates from pPVO 137 clones.
• Figure 22 shows integration efficiency as a function of total observed colonies.
• a control plate with no DNA added to the transformation was obsen/ed to have 350 colonies (indicated by orange line).
• the fraction of clones confirmed to be positive integrants is positively correlated with total colony count. A sharp increase is observed above 6,000 total colonies. The data suggests that the presence of positive integrants increases the observed background growth. For some transformations the efficiency was high enough that the background population was small relative to the positive integrant population.
• Figure 23 shows a chromatogram overlay of Candida tropicalis SPV053 strains. Compared to the mCherry (red) control experiment a clear peak at 6.22 min is observable for the A. transitella (blue) and H. zea (green) desaturase. Therefore, the formation of Z-l l- hexadecenoic acid is only observable in strains expressing an active Zl 1 -desaturase.
• Figure 24A- Figure 24E shows confirmation of the 11 Z-regioisomer.
• Figure 24A The specific peak with an ion fragment of 245 m/z was only observed in C. tropicalis SPV053 expressing either the Zl l-desaturase from transitella or H. zea.
• Figure 24B The fragmentation patterns of the authentic standard.
• Figure 24D The fragmentation patterns of the newly formed compound in samples with expressed desaturase from H. zea match those of the standard.
• Figure 24E The fragmentation patterns of the newly formed compound in samples with expressed desaturase from A, transitella match those of the standard.
• Figure 24C Tire fragmentation patterns of the mCherry control significantly differ from those of Figure 24B, Figure 24D and Figure 24E.
• Figure 25A- Figure 25B shows a GC-FID cliromatogram of different C. tropicalis SPV053 strains incubated with methyl tretradecanoate.
• Figure 25A Overall spectrum. The occurrence of the ZH -C16: ! peak is observable for the strains expressing the Zl l- desaturases f om A. transitella and H. zea.
• Figure 25B Zoom of the C14 to C18 area. A new peak is visible at 4.8 min, which could correspond to Zl l-C14: l . Another peak near Z9- C18: 1 is also visible, which could correspond to Zl 1-C18: i .
• Figure 27 shows only codon optimized H. zea desaturase variants produce detectable Zl l-hexadecenoic acid in SPV300 screen. Labels indicate parent strain and piasmid of desaturase expression cassette.
• pPV101 nrGFP control
• pPV198 H zea Zl l desaturase with native codon usage
• pPV199 H. zea Zl 1 desaturase with Homo sapiens codon optimization
• pPV200 H. zea Zl l desaturase with Homo sapiens codon optimization and swapped Y. lipolytica OLE1 leader sequence
• ⁇ 201 A. transitella Zl l desaturase with native codon usage.
• Figure 28 shows final cell densities for desaturase screen in SPV 140 and SPV300 backgrounds. SPV300 strains with integrated desaturase cassettes grew to higher cell densities.
• Figure 29 shows individual isolate Z l l-hexadecenoic acid titers for SPV140 and SPV300 strains expressing H. zea lA 1 desaturase with H. sapiens codon optimization.
• Figure 30 shows a cliromatogram overlay of extracted metabolites for Z11 -160H producing strain (SPV0490) versus control strain (SPV0488) of Candida viswanathii (tropicalis).
• Figure 31 illustrates pathways that cars be deleted or disrupted to reduce or eliminate competition with the biosynthesis pathway for the production of a mono- or poly-unsaturated C6-C24 fatty alcohol, aldehyde, or acetate.
• Figure 32A- Figure 32B shows Z9-160H and Zl M60H titers in YPD ( Figure 32A) and Semi-Defined C:N ⁇ 80 ( Figure 32B) media for pEXP clones.
• Ten isolates expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the EXP promoter from, two independent competent cell preparations (Comp. Cell Preparation 1, Comp. Cell Preparation 2) were compared to a parental negative control (SPV300) and a desaturase only negative control (SPV459 Hz desat only). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N 2).
• H. zea desaturase under the TEF promoter and H. armigera reductase under the TAL promoter were compared to a parental negative control (SPV300) and positive Bdr pathway controls using the EXP promoter to drive H. armigera FAR expression (SPV575, SPV578).
• the 16-carbon lipid profiles of 5 select clones expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the EXP promoter are compared to a parental negative control (SPV300) and positive Bdr pathway controls using the EXP promoter to drive H. armigera FAR expression (SPV575, SPV578).
• Figure 40 shows examples of acyl-CoA intermediates generated through selective ⁇ - oxidation controlled by acyl-CoA oxidase activity.
• Figure 41 shows Zl l- 14 Acid (methyl myristate fed - 14ME) and Zl l -16Acid (methyl palmitate fed - 16ME) titers of characterized Al l desaturases.
• SPV300 desaturase library integration parent.
• SPV298 prototrophic parent of SPV300, negative control.
• SPV459 SPV300 with current best desaturase (Helicoverpa zea, SEQ ID NO: 54), positive control.
• the desaturase in DST006 is genetically equivalent to the H. zea desaturase expressed in SPV459 and served as an internal library control.
• Figure 42 shows C14 and C 18 product profiles of SPV298 (negative control, parent strain) and SPV459 (SPV298 lineage with H. zea desaturase, SEQ ID NO: 54) fed on either methyl palmitate (16ME) or methyl myristate ( 14ME).
• Figure 43 shows bioinformatic analysis of potential serine, threonine and tyrosine phosphorylation sites of the H. amigera FAR enzyme (SEQ ID NO: 41). The horizontal line resembles the threshold for potential phosphorylation.
• Figure 44 shows bioinformatic analysis of potential serine and threonine phosphorylation sites of the Helicoverpa amigera derived FAR enzyme upon expression in yeast.
• the used server (world wide web address: ebs.dm.dk/serac6s/NetPhosYeast/: Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphoiylation sitesl . J. Mol. Biol. 294, 1351-1362 (1999)) predicts phosphoryiated amino acids specifically in yeast.
• the horizontal line resembles the threshold for possible phosphoiylation sites.
• Figure 45 shows analysis of the Z9/Z11-160H titers of HaFAR mutant library upon expression in Y. lipolytica SPV603. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy of the parental strain.
• Figure 46 shows analysis of the Z9/Z1 l-16Acid titers of HaFAR mutant library upon expression in Y. lipolytica SPV603. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain.
• Figure 47 shows analysis of the fatty alcohol titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 72h after addition of 10 g/L methyl palmitate. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain. The analysis is based on technical quadruplicates.
• Figure 48 shows analysis of the fatty acid titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 72h after addition of 10 g/L methyl palmitate. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain. The analysis is based on technical quadruplicates.
• Figure 49 shows analysis of the fatty alcohol titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 20h upon addition of 10 g/L methyl palmitate. The analysis is based on technical quadruplicates.
• Figure 50 shows analysis of the fatty alcohol titers of selected strains in a time course experiment in shake flasks.
• a copy of the enzyme HaFAR or HaS195A was introduced into the strains SPV1053 (Adgal AURA, ALeu, leu2: :pTEF-HZ_Z 1 l_desat_Hs-tXPR2_loxP) and SPV1054 (Adga2 AURA, ALeu, leu2: :pTEF-HZ_Zl l_desat_Hs-tXPR2_loxP). Cultivation was performed as biological triplicates in shake flasks.
• FIG. 51 shows analysis of the fatty acid titers of selected strains in a time course experiment in shake flasks.
• a copy of the enzyme HaFAR or HaS1 5A was introduced into the strains SPV1053 (Adgal AURA, ALeu, Ieu2::pTEF-HZ_Zl l_desat_Hs-tXPR2_loxP) and SPV1054 (Adga2 AURA, ALeu, 1eu2: :pTEF-HZ_Zl l_desat_Hs-tXPR2_loxP).
• Cultivation was performed as biological triplicates in shake flasks. Strains were cultivated in shake flasks over a period of 72h upon addition of 10 g/L methyl palmitate.
• Figure 52 shows analysis of the fatty alcohol titers of new? strains in a FAR library screening in 24 well plates.
• a copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Adga2 AURA, ALeu, leu2::pTEF-HZ_Zl l_desat_Hs- tXPR2_loxP).
• Cultivation was performed as biological quadruplicates in 24 well plates. Strains were cultivated over a period of 96h upon addition of 10 g/L methyl palmitate.
• Figure 53 shows analysis of the fatty acid titers of new strains in a FAR library screening in 24 well plates.
• a single copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Adga2 AURA, ALeu, leu2: :pTEF-HZ_Z 1 l_desat_Hs- tXPR2__loxP), Cultivation was performed as biological quadruplicates in 24 well plates. Strains were cultivated over a period of 96h upon addition of 10 g/L methyl palmitate.
• Figure 54 shows a biosynthetic pathway capable of using tetradecyl-ACP (14:ACP) inputs to produce a blend of E- and Z- tetradecenyl acetate (El l ⁇ 14:OAc and Zl l-14:OAC) pheromones in a recombinant microorganism of the present disclosure.
• a sequence listing for SEQ ID NO: 1 - SEQ ID NO: 105 is part of this application and is incorporated by reference herein. Hie sequence listing is provided at the end of this ed in computer readable format.
• compositions comprising, “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.
• '"or refers to an inclusive “or” and not to an exclusive “or.”
• microbial As used herein, the terms "microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic ceils or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species 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 a chemical.
• the recombinant microorganisms are prokaryotic microorganism .
• the prokaryotic microorganisms are bacteria.
• Bacteria or “eubacteria”, refers to a domain of prokaryotic organisms.
• Bacteria include at least eleven distinct groups as follows: (1 ) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group ⁇ Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g.. Purple photosynthetic +non- photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g.
• oxygenic phototrophs oxygenic phototrophs
• Spirochetes 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.
• Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
• the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacier, Agrobacierium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia. Rickettsia, Treponema, and Fusobacterium.
• Gram positive bacteria include cocci, nonsporulating rods, and sporulating rods.
• the genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynehacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces .
• recombinant microorganism and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene.
• alteration it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration.
• alter can mean “inhibit,” but the use of the word “alter” is not limited to this definition.
• recombinant microorganism and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
• expression of a protein results from transcription and translation of the open reading frame sequence.
• the level of expression of a desired product in a host ceil may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence.
• mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et ai, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
• Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al, 1989, supra,
• polynucleotide is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA.
• DNA single stranded or double stranded
• RNA ribonucleic acid
• nucleotide refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids.
• nucleoside refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids.
• nucleotide analog or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
• the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.”
• the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise 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 under which the gene is expressed.
• the transcribed region of the gene may include untranslated regions, including introns, 5' ⁇ untranslated region (UTR), and 3'-UTR, as well as the coding sequence.
• enzyme refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.
• non -naturally occurring when used in reference to a microorganism organism or enzyme activity of the disclosure, is intended to mean that the microorganism organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species.
• Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microorganism's genetic material.
• modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species.
• Additional modifications include, for example, non-coding regulator ⁇ ' regions in which the modifications alter expression of a gene or operon.
• Exemplary non-naturally occurring microorganism or enzyme activity includes the hydroxylation activity described above.
• exogenous refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
• endogenous or “native” as used herein with reference to various molecules refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
• an endogenous or exogenous source of saturated C6-C24 fatty acid refers to a source of saturated C6-C24 fatty acid originating from within the microorganism (endogenous), such as when a saturated C6-C24 fatty acid is produced or synthesized inside the microorganism, or originating from outside the microorganism (exogenous), such as when a saturated C6-C24 fatty acid is provided to the microorganism during the course of culturing or cultivating the microorganism in media in flasks or other containers.
• heterologous refers to various molecules, e.g. , polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign ("exogenous") to (i. e. , not naturally- found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g.
• molecule(s) is "endogenous to") a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g. , greater than naturally found) amount in the cell.
• homologous sequences refers to related sequences (nucleic or amino acid) that are functionally related to the referenced sequence.
• a functional relationship may be indicated in any 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.
• Use of the term homolog in this disclosure refers to instances in which both (a) and (b) are indicated.
• the degree of sequence identity may vary, but in one embodiment, 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 when using standard sequence alignment programs known in the art (e.g., Clustal Omega alignment using default parameters).
• Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel ei al., eds., 1987) Supplement 30, section 7.718, Table 7.71.
• Some alignment programs are Mac Vector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania).
• Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector ⁇ (Invitrogen, Carlsbad, CA).
• fatty acid refers to a compound of structure R-COOH, wherein R is a C& to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon and the carboxvl group is at position 1 , In a particular embodiment, R is a Ce to C24 saturated or unsaturated linear hydrocarbon and the carboxvl group is at position 1.
• fatty alcohol refers to an aliphatic alcohol having the formula R-OH, wherein R is a Ce to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon. In a particular embodiment, R is a Ce to C24 saturated or unsaturated linear hydrocarbon.
• fatty acyl-CoA refers to a compound having the structure R-(CO)-S-Ri, wherein R; is Coenzyme A
• fatty acyl-ACP refers to a compound having the structure R-(CO)-S-Ri , wherein Ri is a acyl carrier protein ACP.
• short chain' 1 or ' “ short-chain” refers to fatty alcohols, fatty aldehydes, and/or fatty acetates, including pheromones, fragrances, flavors, and polymer intermediates with carbon chain length shorter than or equal to CI 8.
• the present disclosure addresses the need for novel technologies for the cost-efficient production of valuable products from Sow-cost feedstocks. Specifically, the present inventors have addressed this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C6-C24 fatty alcohols, aldehydes, and acetates including synthetic insect pheromones, fragrances, flavors, and polymer intermediates from low-cost feedstocks. Thus, aspects of the disclosure are based on the inventors' discovery that recombinant microorganisms can be engineered in order to produce valuable products from low-cost feedstocks, which circumvents conventional synthetic methodologies to produce valuable products.
• recombinant microorganisms can be engineered to synthesize mono- or poly-unsaturated C6-C24 fatty alcohols.
• Mono- or poly-unsaturated C6-C24 fatty alcohols synthesized as described herein can be further converted into the corresponding aldehydes or acetates.
• various embodiments of the present disclosure can be used to synthesize a variety of insect pheromones selected from fatty alcohols, aldehydes, and acetates.
• embodiments described herein can also be used for the synthesis of fragrances, flavors, and polymer intermediates.
• Engineering of the microbial hosts entail the expression of a non-native pheromone biosynthetic pathway which is comprised of but not limited to one or multiple fatty acyl desaturases, and fatty alcohol-forming or fatty aldehyde-forming reductases.
• Fatty acids produced by desaturation reactions can be stored intracellularly as triacyiglycerides or reduced enzyrnaticaily by reductases to form fatty alcohols or aldehydes.
• Triacyiglycerides containing unsaturated fatty acids can be extracted, esterified, and chemically reduced to produce unsaturated fatty alcohols.
• Fatty alcohols produced via the described pathways can be further converted into fatty aldehyde pheromones, and fatty acetate pheromones via subsequent chemical oxidation, and esterification methods, respectively. Methods of chemical oxidation and esterification are known in the arts. Fatty alcohols produced via the described pheromone biosynthetic pathway can also be further converted into fatty aldehyde pheromones, and fatt ' acetate pheromones using enzymatic conversion such as alcohol dehydrogenases, and acetyltransferase, respectively.
• fatty acyl-CoA or fatty acyl- ACP formed as intermediates in the pheromone biosyntlietic pathway can be released as free fatty acids by native or heterologously derived thioesterases, to become substrates for synthesis of pheromones using metathesis.
• embodiments of the disclosure provide for the synthesis of one or more insect pheromones using a recombinant microorganism.
• a pheromone is a volatile chemical compound that is secreted by a particular insect for the function of chemical communication within the species. That is, a pheromone is secreted or excreted chemical factor that triggers a social response in members of the same species.
• alarm pheromones there are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.
• Non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include linear alcohols, aldehydes, and acetates listed in Table 1.
• the pheromones synthesized as taught in this disclosure include at least one pheromone listed in Table 2a to modulate the behavior of an insect listed in Table 2a
• non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include alcohols, aldehydes, and acetates listed in Table 2a.
• the microorganisms described herein are not limited to the synthesis of C6-C20 pheromones listed in Table 1 and Table 2a. Rather, the disclosed microorganisms can also be utilized in the synthesis of various C6-C24 mono- or poly-unsaturated fatty alcohols, aldehydes, and acetates, including fragrances, flavors, and polymer intermediates.
• Table 2 a Exemplary pheromones that can be synthesized according to methods described in the present disclosure.
• each double bond is represented by a numeral corresponding to that of the carbon from which it begins, with each carbon numbered from that attached to the functional group.
• the carbon to which the functional group is attached is designated -1-.
• Pheromones may have, but are not limited to, hydrocarbon chain lengths numbering 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca- ), or 18 (octadeca-) carbons long,
• the presence of a double bond has another effect. It precludes rotation of the molecule by fixing it in one of two possible configurations, each representing geometric isomers that are different molecules. These are designated either E (from the German word Eni restroom, opposite) or Z (Zusammen, together), when the carbon chains are connected on the opposite (trans) or same (cis) side, respectively, of the double bond.
• Aldehyde -CH O Formvi- -a.i
• Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations or variations known to one skilled in the art.
• (11Z)- hexadecen-l-al can also be written as Z-l l-hexadecen-l-al, Z-l l- hexadecenal, or Z-x- y:Aki, wherein x represents the position of the double bond and y represents the number of carbons in the hydrocarbon skeleton.
• the fatty acyl-CoA precursors of (1 lZ)-hexadecen-l-al can be identified as (HZ)-hexadecenyl-CoA or Z-1 l-16:Acyl-CoA.
• the present disclosure relates to the synthesis of mono- or poly-unsaturated Ce-C 2 4 fatty alcohols, aldehydes, and acetates using a recombinant microorganism comprised of one or more heterologous enzymes, which catalyze substrate to product conversions for one or more steps in the synthesis process.
• the present disclosure describes enzymes that desaturaie fatty acyl substrates to corresponding unsaturated fatty acyl substrates.
• a desaturase is used to catalyze the conversion of a fatty acyl- CoA or acyl-ACP to a corresponding unsaturated fatty acyl-CoA or acyl-ACP.
• a desaturase is an enzyme thai catalyzes the formation of a carbon-carbon double bond in a saturated fatty- acid or fatty acid derivative, 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 a corresponding unsaturated fatty acid/acyl.
• Desaturases are classified with respect to the ability of the enzyme to selectively catalyze double bond formation at a subterminal carbon relative to the methyl end of the fatty acid/acyl or a subterminal carbon relative to the carbonyl end of the fatty acid/acyl.
• Omega ( ⁇ ) desaturases catalyze the formation of a carbon-carbon double bond at a fixed subterminal carbon relative to the methyl end of a fatty acid/acyl.
• an ⁇ 3 desaturase catalyzes the formation of a double bond between the third and fourth carbon relative the methyl end of a fatty acid/acyl.
• Delta ( ⁇ ) 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.
• a ⁇ 9 desaturase catalyzes the formation of a double bond between the C9 and Cio carbons with respect to the carboxyl end of the fatty acid or the carbonyl group of a fatty acyl CoA.
• a desaturase can be described with reference to the location in which the desaturase catalyzes the formation of a double bond and the resultant geometric configuration (i.e., E/Z) of the unsaturated hydrocarbon.
• a Z9 desaturase refers to a ⁇ desaturase that catalyzes the formation of a double bond between the (3 ⁇ 4 and Cio carbons with respect to the carbonyl end of a fatty acid/acyl, thereby orienting two hydrocarbons on opposing sides of the carbon-carbon double bonds in the cis or Z configuration .
• a Zl l desaturase refers to a ⁇ desaturase that catalyzes the formation of a double bond between the C1 1 and C12 carbons with respect to the carbonyl end of a fatty acid/acyl.
• Desaturases have a conserved structural motif. This sequence motif of transmembrane desaturases is characterized by [HX3-4HX7-41 (3 non-His)HX2-3(l nonHis)HHX61- 189(40 non-His)HX2-3(l non-His)HH], The sequence motif of soluble desaturases is characterized by two occurrences of [D/EEXXHJ.
• the desaturase is a fatty acyl-CoA desaturase that catalyzes the formation of a double bond in a fatty acyl-CoA.
• the fatty acyl- CoA desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
• the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
• the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal CoA on the unsaturated fatty acyl-CoA.
• a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-CoA.
• the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl -CoA such that the resultant unsaturated fatty acyl-CoA has a cis or trans (i.e., Z or E) geometric configuration.
• the desaturase is a fatty acyl-ACP desaturase that catalyzes the formation of a double bond in a fatty acyl-ACP.
• the fatty acyl-ACP desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
• the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
• the fatty acyl-ACP desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal carbonyl on the unsaturated fatty acyl-ACP.
• a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-ACP.
• the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-ACP has a cis or trans (i.e., Z or E) geometric configuration.
• the fatty acyl desaturase is a Zl l desaturase.
• a nucleic acid sequence encoding a Zl l desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia vehitiana, Choristoneura rosaceana, Lampronia capitella, Trichopliisia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized.
• the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichopliisia ni.
• the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Tnchoplusia ni. In other embodiments, the Z l l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Zl 1 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana.
• the Zl l desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea.
• the Zl 1 desaturase comprises an ammo acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Zl l desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, ⁇ 21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1.
• the Zl l desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Zl l desaturase is fused to another polypeptide. In certain embodiments, the N-tenninal native leader sequence of a Zl l desaturase is replaced by an oieosin leader sequence from another species. In certain embodiments, the Zl l desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Zl l desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.
• the fatty acyl desaturase is a Z9 desaturase.
• a nucleic acid sequence encoding a Z9 desaturase is codon optimized.
• the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis.
• the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis
• the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella.
• the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an ammo acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.
• the present disclosure teaches a recombinant microorganism comprising a Zl l or Z9 desaturase 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 with any one of SEQ ID Nos.
• the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a Zl l or Z9 desaturase, wherein said 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 with any one of SEQ ID Nos.
• the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyi desaturase 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%, 5
• Fatty Acyl Reductase [0196] The present disclosure describes enzymes that reduce fatty acyl substrates to corresponding fatty alcohols or aldehydes.
• a fatty alcohol forming fatty acyl -reductase is used to catalyze the conversion of a fatty acyl-CoA to a corresponding fatty alcohol .
• a fatty aldehyde forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl- ACP to a corresponding fatty aldehyde.
• a fatty acyl reductase is an enzyme that catalyzes the reduction of a fatty acyl-CoA to a corresponding fatty alcohol or the reduction of a fatty acyl- ACP to a corresponding fatty aldehyde.
• a fatty acyl -Co A and fatty' acyl -A CP has a structure of R-(CO)-S-Ri, wherein R is a Ce to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon, and Ri represents CoA or ACP. In a particular embodiment, R is a Ce to C24 saturated or unsaturated linear hydrocarbon.
• CoA is a non-protein acyl carrier group involved in the synthesis and oxidation of fatty acids.
• ACP is an acyl carrier protein, i.e. , a polypeptide or protein subunit, of fatty acid synthase used in the synthesis of fatty acids.
• the disclosure provides for a fatty alcohol forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl -CoA to the corresponding fatty alcohol .
• R-(CO)-S-CoA is converted to R-CH2OH and CoA-SH when two molecules of NAD(P)H are oxidized to NAD(P) + .
• a recombinant microorganism described herein can include a heterologous fatty' alcohol forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-CoA to the corresponding fatty alcohol.
• a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase which catalyzes the conversion of a mono- or polyunsaturated C6-C24 fatty acyl-CoA into the corresponding mono- or poly-unsaturated C6-C2.4 fatty alcohol.
• the disclosure provides for a fatty aldehyde forming fatty acyl- reductase which catalyzes the reduction of a fatty acyl-ACP to the corresponding fatty- aldehyde.
• R-(CO)-S-ACP is converted to R-(CO)-H and ACP-SH when one molecule of NAD(P)H is oxidized to NAD(P) ' .
• a recombinant microorganism described herein can include a heterologous fatty aldehyde forming fatty acyl- reductase, which catalyzes the reduction a fatty acyl-ACP to the corresponding fatty aldehyde.
• a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acy] reductase which catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-ACP into the corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
• the respective alcohol-forming fatty acyl reductase (FAR) enzymes 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, L. I. Insect Endocrinology. (Academic Press)).
• site specific dephosphorylation of heterologouslv expressed FAR enzymes in yeast such as Y. lipoiytica can lead to inactivation, and results in low fatty alcohol titers.
• a bioinformatic approach can be used to predict phosphorylated residues within FAR.
• Alanine substitution of serine and threonine residues has been shown to abolish phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snfl -Dependent Regulation of Accl . mBio 5 (2014)).
• the impact of alanine substitutions to prevent phosphorylation of serine residues and its impact on fatly alcohol titers can be tested.
• improvement of FAR activity can also be achieved by other amino acid substitutions.
• methods are provided to identify beneficial mutations of FAR based on selection and alteration of phosphorylation-sensitive residues upon its expression in a host microorganism.
• the host microorganism is yeast selected from the group consisting of Yarrowia, Candida, Saccharom ces, Pichia, Hansemda, and Kluyveromyces .
• a nucleic acid sequence encoding a fatty-acy! reductase from organisms of the species Agrotis segetum, Spodoptera exigua, Spodoptera liitoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera is codon optimized.
• the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum.
• the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littor lis.
• the fatty acyl reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an 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 SEQ ID NOs: 41 and 57 from Helicoverpa armigera.
• the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an ammo acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an ammo acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.
• the production of unsaturated fatty alcohols in a recombinant microorganism comprises the expression of one or more mutant FARs.
• HaFAR Helicoverpa amigera fatty acyl-CoA reductase
• HaFAR Helicoverpa amigera fatty acyl-CoA reductase
• the increased enzymatic activity is a net activity increase in amount of fatty alcohol produced relative to the amount of fatty alcohol produced by a wild type enzymatic activity of HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41.
• a wild type HaFAR comprises a nucleotide sequence set forth in SEQ ID NO: 90.
• a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: S60X, S 195X, S298X, S378X, S3 ! - ) ⁇ .
• a variant of a wild type HaFA encoded by an 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 an amino acid selected from F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W and R.
• the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42- 8. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.
• the present disclosure teaches a recombinant microorganism comprising a fatty acyi 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 with any one of SEQ ID Nos. selected from the group consisting of, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 73, 75, 77, and 82.
• the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a fatty acyi reductase, wherein said 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 with any one of SEQ ID Nos, selected from the group consisting of 1, 2, 3, 32, 37, 40, 72, 74, 76, 81,
• the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyi reductase 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 a fatty acyi reductase selected from the group consisting of SEQ ID NOs: 41 -48, 57, 73, 75 and 77 that catalyzes the conversion of the group consisting of SEQ ID NO
• the present disclosure describes enzymes that ligate a fatty acid to the corresponding fatty acyl-ACP.
• an acyl-ACP synthetase is used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP.
• An acyl-ACP synthetase is an enzyme capable of ligating a fatty acid to ACP to produce a fatty acid acyl-ACP.
• an acyl -ACP synthetase can be used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP.
• the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.
• a recombinant microorganism described herein can include a heterologous acyl-ACP synthetase, which catalyzes the conversion of a fatty acid to a corresponding fatty acyl-ACP.
• a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule which encodes an acyl-ACP synthetase that catalyzes the conversion of a saturated C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP.
• the present disclosure describes enzymes that catalyze the elongation of a carbon chain in fatty acid.
• a fatty acid synthase complex is used to catalyze initiation and elongation of a carbon chain in a fatty acid.
• a "fatty acid synthase complex” refers to a group of enzymes that catalyzes the initiation and elongation of a carbon chain on a fatty acid.
• the ACP along with the enzymes in the fatty acid synthase (FAS) pathway control the length, degree of saturation, and branching of the fatty acids produced.
• the steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families.
• one or more of these genes can be attenuated, expressed or over-expressed. In exemplary embodiments, one or more of these genes is over-expressed.
• Type I FAS I
• Type II FAS
• Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acetyl-CoA and malonyl-CoA.
• the steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families.
• fab fatty acid biosynthesis
• acc acetyl-CoA carboxylase
• acetyl-CoA is carboxylated by acetyl-CoA carboxylase (Acc, a multi- subunit enzyme encoded by four separate genes, accABCD), to form malonyl-CoA.
• acetyi-CoA is carboxylated by the yeast equivalents of the acetyi-CoA carboxylase, encoded by ACC 1 and ACC2.
• the malonate group is transferred to ACP by malonyl- CoA:ACP transacylase (FabD) to form malony]-ACP.
• a malonyl-palmityl tranferase domain adds malonyi from malonyl-CoA to the ACP domain of the FAS complex.
• a condensation reaction then occurs, where malonyl-ACP merges with acyl-CoA, resulting in ⁇ -ketoacyl-ACP.
• the hydrocarbon substrate is elongated by 2 carbons.
• the ⁇ -keto group is reduced to the fully saturated carbon chain by the sequential action of a keto-reductase (KR), dehydratase (DH), and enol reductase (ER).
• KR keto-reductase
• DH dehydratase
• ER enol reductase
• the elongated fatty acid chain is carried between these active sites while attached covalently to the phosphopantetheine prosthetic group of ACP.
• the ⁇ -ketoacyl-ACP is reduced by NADPH to form ⁇ -hydroxyacyl-ACP.
• this step is catalyzed by ⁇ - ketoacyl-ACP reductase (FabG).
• the equivalent yeast reaction is catalyzed by the ketoreductase (KR) domain of FAS.
• ⁇ -hydroxyacyl-ACP is then dehydrated to form trans-2- enoyl-ACP, which is catalyzed by either ⁇ -hydroxyacyl-ACP dehydratase/isomerase (FabA) or ⁇ -hydroxyacyl-ACP dehydratase (FabZ) in bacteria or the dehydratase (DH) domain of FAS in yeast.
• FabA ⁇ -hydroxyacyl-ACP dehydratase/isomerase
• FabZ ⁇ -hydroxyacyl-ACP dehydratase
• DH dehydratase domain of FAS in yeast.
• NADPH-dependent trans-2 -enoyl-ACP reductase I, II, or 111 Fabl, FabK, and FabL, respectively
• ER enol reductase
• a fatty acid synthase complex can be used to catalyze elongation of a fatty acyl-ACP to a corresponding fatty acyl-ACP with a two carbon elongation relative to the substrate.
• the present disclosure describes enzymes that catalyze the conversion of a fatty aldehyde to a fatty alcohol .
• an alcohol dehydrogenase (ADH, Table 3 and Table 3a) is used to catalyze the conversion of a fatty aldehyde to a fatty alcohol.
• ADHs were characterized from Geobaculus thermodenitrificans NG80-2, an organism that degrades C15 to C36 alkanes using the LadA hydroxylase. Activity was detected from methanol to 1 - triacontanol (Go) for both ADHs, with 1 -octanol being the preferred substrate for ADH2 and ethanol for ADH 1 (Liu a al. 2009).
• Drosophila simulans (Fruit fly) Adh, GD23968 Q24641
• Cupriavidus necator strain ATCC 17699 / H16 /'
• Mycobacterium tuberculosis (strain CDC 1551 /
• Staphylococcus aureus (strain MW2) adh, MW0568 Q8NXU1
• Mycobacterium tuberculosis (strain ATCC 25618 /
• Staphylococcus aureus (strain N3 I 5) adh, SA0562 Q7A742
• Staphylococcus aureus (strain bovine RF122 /
• Staphylococcus aureus (strain COL) adh Staphylococcus aureus adh
• Staphylococcus aureus (strain MRSA252) adh, SAR0613 Q6GJ63
• Staphylococcus aureus (strain MSSA476) adh, SAS0573 Q6GBM4 adh,
• Staphylococcus aureus (strain Mu50 / ATCC
• Staphylococcus epidermidis (strain ATCC 12228) adh, SK 0375 Q8CQ56
• Staphylococcus epidermidis strain ATCC 35984 /
• ADH1 Q9Z2M2 (Geomys bursarius attwateri)
• Kluyveromyces marxianus (Yeast) (Candida kefyr) ADHl Q07288
• Trifolium repens (Creeping white clover) ADHl PI 3603
• Saccharomyces cerevisiae strain ATCC 204508 / ADHl , ADC 1,
• Arabidopsis thaliana (Mouse-ear cress) Atlg77120, P06525
• Schizosaccharomyces pombe (strain 972 / ATCC adhl, adh,
• Adhl Adh-1 .
• Neurospora crassa (strain ATCC 24698 / 74-OR23-
• Candida albicans (Yeast) ADHl, CAD P43067
• Kluyveromyces laciis strain ATCC 8585 / CBS
• Pongo abelii (Sumatran orangutan) (Pongo
• Macaca rnuiatta (Rhesus macaque) ADHl A, ADHl P28469 Organism Gene Name Accession No.
• Ceratitis rosa (Natal frait fly) (Pterandras rosa) ADH2 Q70UP6
• Kluvveromyces marxianus (Yeast) (Candida kefyr) ADH2 Q9P4C2
• Saccharomyces cerevisiae strain ATCC 204508 /
• Candida albicans (strain SC5314 / ATCC MYA- Ca41 C10.04,
• Oryza sativa subsp. japonica (Rice) Osl lg0210500, Q0ITW7
• Kluvveromvces lactis (strain ATCC 8585 / CBS)
• Kluvveromvces lactis (strain ATCC 8585 / CBS)
• Saccharomyces cerevisiae strain ATCC 204508 /
• Kluyveromyces lactis (strain ATCC 8585 / CBS)
• Saccharomyces cerevisiae (strain YJM789) ADH4, ZRG5,
• Saccharomyces cerevisiae strain ATCC 204508 /
• Saccharomyces cerevisiae strain ATCC 204508 /
• Pongo abelii (Sumatran orangutan) (Pongo ADH6 Q5R7Z8 Organism Gene Name Accession No. pygmaeus abelii)
• Mycobacterium tuberculosis (strain CDC 1551 /
• Rhizobium meliloti strain 1021 (Ensifer meliloti) adhA, RA0704,
• Mycobacterium tuberculosis (strain ATCC 25618 /
• Mycobacterium bovis (strain ATCC BAA-935 /
• Mycobacterium tuberculosis (strain CDC 1551 /
• Mycobacterium tuberculosis (strain ATCC 25618 / adhB, Rv0761c,
• Mycobacterium tuberculosis (strain CDC 1551 /
• Mycobacterium tuberculosis (strain ATCC 25618 /
• Clostridium acetobutylicum (strain ATCC 824 /
• Rhodobacter sphaeroides strain ATCC 17023 /
• Emericella nidulans (strain FGSC A4 / ATCC
• Emericella nidulans (strain FGSC A/4 / ATCC
• Emericella nidulans (strain FGSC A4 / ATCC
• Bacillus subtilis strain 168
• gbsB Bacillus subtilis (strain 168)
• gbsB Bacillus subtilis (strain 168)
• gbsB Bacillus subtilis (strain 168)
• BSU31050 Bacillus subtilis (strain 168)
• Mycobacterium tuberculosis (strain ATCC 25618 /
• Catharantlius roseus (Madagascar periwinkle)
• the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADHl), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A 15147g (ADH6), YALI0E07766g (ADH7).
• endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADHl), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0
• the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by YALI0F09603g (FADH), YALI0D25630g (ADHl ), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A 15147g (ADH6), and YALI0E07766g (ADH7).
• the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database IDs Q6C297 (FADH), Q6C7T0 (ADHl), F2Z678 (ADH2), Q6CGT5 (A D! 13).
• Alcohol Oxidase [0222] The present disclosure describes enzymes that oxidize fatty alcohols to fatty- aldehydes.
• an alcohol oxidase is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde.
• Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product.
• AOX enzymes utilize flavin adenine dmucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium, Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.
• FAD flavin adenine dmucleotide
• AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some members of these four groups are better suited than others as candidates for evaluation.
• Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 4) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005).
• Aliphatic alcohol oxidases from methyl otrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols.
• hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003).
• alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to Cl l when used biphasic organic reaction system (Murray and Duff 1990).
• Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.
• Long chain alcohol oxidases include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013), Banthorpe et al. reported a long chain alcohol oxidase purified from die leaves of Tanacelum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-l-ol and octan-i-ol (Banthorpe 1976) (Cardem.il 1978).
• Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992).
• Significant activity is observed for primary alcohols of length Cs to C;e with reported KM in the 10-50 ⁇ range (Eirich et al. 2004).
• Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida hoidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990).
• Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991).
• the long chain fatty alcohol oxidase (LjFAOl) from Lotus japonicas has been heterologous! ⁇ ' expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).
• Alcohol oxidase enzymes capable of oxidizing short chain alcohols (EC 1.1.3.13)
• Komagataella pastoris (strain ATCC 76273 /

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