Production of non-native monounsaturated fatty acids in bacteria
Recombinant proteobacteria, including γ-proteobacteria, comprising a heterologous acyl-ACP desaturase and a heterologous acyl-ACP thioesterase, wherein the native dual 3-hydroxy acyl-ACP dehydratase/isomerase is deleted are provided herein. The recombinant proteobacteria produce non-native monounsaturated free fatty acids or derivatives thereof. Methods of producing non-native monounsaturated free fatty acids or derivatives thereof are also provided, in addition to cell cultures and fatty acid compositions produced by the recombinant proteobacteria. The recombinant proteobacteria may be used to produce insect pheromones or precursors thereof, and fragrances or precursors thereof.
1 . A recombinant proteobacterium, comprising:
a heterologous acyl-ACP desaturase, wherein the heterologous acyl-ACP desaturase has at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 12; and
an acyl-ACP thioesterase, wherein the acyl-ACP thioesterase i) is native to the recombinant proteobacterium or ii) is heterologous to the recombinant proteobacterium and is a plant FatA-type thioesterase, a plant FatB-type thioesterase, or a bacterial acyl-ACP thioesterase;
wherein expression of a native dual 3-hydroxy acyl-ACP dehydratase/isomerase is deleted or attenuated; and
wherein the recombinant proteobacterium produces a non-native monounsaturated free fatty acid or a derivative thereof.
2 . The recombinant proteobacterium of claim 1 , wherein the heterologous acyl-ACP desaturase is a Δ9-tetradecanoyl-acyl-ACP desaturase.
3 . The recombinant proteobacterium of claim 1 , wherein the non-native monounsaturated free fatty acid or derivative thereof is an ω-3, ω-5, ω-6, ω-8, ω-9, ω-11, or ω-12 monounsaturated free fatty acid or derivative thereof.
4 . The recombinant proteobacterium of claim 1 , further comprising
one or more of a ferredoxin, a ferredoxin reductase, or a flavodoxin reductase, wherein:
one or more of the ferredoxin, ferredoxin reductase, or flavodoxin reductase is heterologous to the recombinant proteobacterium; or
the recombinant proteobacterium overexpresses endogenous flavodoxin reductase or ferredoxin reductase.
5 . The recombinant proteobacterium of claim 1 , further comprising a heterologous 3-hydroxy acyl-ACP dehydratase.
6 . The recombinant proteobacterium of claim 1 , further comprising a heterologous dual 3-hydroxy acyl-ACP dehydratase/isomerase.
7 . The recombinant proteobacterium of claim 1 , further comprising one or more additional heterologous polypeptides selected from a ferredoxin, a ferredoxin reductase, a flavodoxin reductase, a 3-hydroxyacyl-ACP dehydratase, a dual 3-hydroxy acyl-ACP dehydratase/isomerase, a fatty acid metabolism regulator protein, a β-ketoacyl-ACP synthase, a carboxylic acid reductase, an alcohol dehydrogenase, a phosphopantetheinyl transferase, an alcohol acetyl-CoA transferase, an ω-hydroxylase, an alcohol oxidase/dehydrogenase, and an aldehyde dehydrogenase.
8 . The recombinant proteobacterium of claim 1 , wherein the recombinant proteobacterium produces a non-native monounsaturated free fatty acid or a derivative thereof that is a fatty alcohol, an ω-hydroxy fatty acid, a fatty alcohol acetate ester, a fatty aldehyde, or an α/ω-dicarboxylic acid.
9 . The recombinant proteobacterium of claim 1 , wherein the non-native monounsaturated free fatty acid is Z9-tetradecenoic acid, Z11-hexadecenoic acid, Z13-octadecenoic acid, or a combination thereof.
10 . The recombinant proteobacterium of claim 1 , wherein:
(a) the recombinant proteobacterium comprises a carboxylic acid reductase, or comprises a carboxylic acid reductase and a phosphopantetheinyl transferase, wherein the recombinant proteobacterium produces a non-native monounsaturated fatty aldehyde or a non-native monounsaturated fatty alcohol;
(b) the recombinant proteobacterium comprises a carboxylic acid reductase and an alcohol dehydrogenase, or comprises a carboxylic acid reductase, an alcohol dehydrogenase, and a phosphopantetheinyl transferase, wherein the recombinant proteobacterium produces a non-native monounsaturated fatty alcohol;
(c) the recombinant proteobacterium comprises a carboxylic acid reductase and an alcohol acetyl-CoA transferase, or comprises a carboxylic acid, an alcohol acetyl-CoA transferase, and a phosphopantetheinyl transferase, wherein the recombinant proteobacterium produces a non-native monounsaturated fatty alcohol acetate ester (FACE);
(d) the recombinant proteobacterium comprises an ω-hydroxylase, wherein the recombinant proteobacterium produces a non-native monounsaturated ω-hydroxy fatty acid; or
(e) the recombinant proteobacterium comprises an ω-hydroxylase, and further comprises an alcohol oxidase/dehydrogenase and an aldehyde dehydrogenase, wherein the recombinant proteobacterium produces a non-native monounsaturated α/ω-dicarboxylic acid.
11 . The recombinant proteobacterium of claim 8 , wherein:
the non-native monounsaturated fatty aldehyde is Z9-tetradecenal, Z11-hexadecenal, Z13-octadecenal, or a combination thereof;
the non-native monounsaturated fatty alcohol is Z9-tetradecenol, Z11-hexadecenol, Z13-octadecenol, or a combination thereof;
the non-native monounsaturated fatty alcohol acetate ester (FACE) is Z9-tetradecenyl acetate, Z11-hexadecenyl acetate, Z13-octadecenyl acetate, or a combination thereof;
the non-native monounsaturated ω-hydroxy fatty acid is (Z9)-14-hydroxy-tetradecenoic acid, (Z11)-16-hydroxy-hexadecenoic acid, (Z13)-18-hydroxy-octadecenoic acid, or a combination thereof; or
the non-native monounsaturated α/ω-dicarboxylic acid is (Z5)-1,14-tetradecenedioic acid, (Z5)-1,16-hexadecenedioic acid, (Z5)-1,18-octadecenedioic acid, or a combination thereof.
12 . The recombinant proteobacterium of claim 1 , further comprising a carboxylic acid reductase, wherein the recombinant proteobacterium has one or more deletions in endogenous alcohol dehydrogenase genes, endogenous aldehyde reductase genes, or both.
13 . A cell culture, comprising the recombinant proteobacterium of claim 1 and one or more non-native monounsaturated free fatty acids or derivatives thereof.
14 . A method for producing a non-native monounsaturated free fatty acid or a derivative thereof, the method comprising culturing the recombinant proteobacterium of claim 1 .
15 . The method of claim 14 , further comprising isolating the non-native monounsaturated free fatty acid or derivative thereof.
16 . The method of claim 14 , wherein:
the non-native monounsaturated free fatty acid or derivative thereof is an ω-3, ω-5, ω-6, ω-8, ω-9, ω-11, or ω-12 monounsaturated free fatty acid or derivative thereof;
the non-native monounsaturated free fatty acid or derivative thereof is a fatty acid, a fatty alcohol, ω-hydroxy fatty acid, a fatty alcohol acetate ester, a fatty aldehyde, or an α/ω-dicarboxylic acid; and
the non-native monounsaturated free fatty acid or derivative thereof is an insect pheromone, an insect pheromone precursor, a fragrance, or a fragrance precursor.
17 . The method of claim 14 , wherein the recombinant proteobacterium further comprises one or more additional heterologous polypeptides selected from a ferredoxin, a ferredoxin reductase, a flavodoxin reductase, a 3-hydroxyacyl-ACP dehydratase, a dual 3-hydroxy acyl-ACP dehydratase/isomerase, a fatty acid metabolism regulator protein, a β-ketoacyl-ACP synthase, a carboxylic acid reductase, an alcohol dehydrogenase, a phosphopantetheinyl transferase, an alcohol acetyl-CoA transferase, an ω-hydroxylase, an alcohol oxidase/dehydrogenase, and an aldehyde dehydrogenase.