KAS-III free FA synthesis
The present disclosure describes a genetically engineered a KASIII-independent fatty acid biosynthetic pathway that makes use of the promiscuous nature of the rest of the FAS enzymes (3-ketoacyl-ACP synthetase, 3-ketoacyl-ACP reductase, 3-hydroxyacyl ACP dehydrase, enoyl-ACP reductase) to bypass the KASIII step by providing a Co-A precursor of two or higher than two carbons (such as the four carbon butyryl-CoA) as the starting molecule. Since many CoA-based starter molecules can be supplied for the fatty acid synthesis, much more diversified products can be obtained with various carbon-chain lengths. As such, this disclosure will serve as a powerful and efficient platform to produce low to medium chain length products carrying many different functional groups.
1. A genetically engineered microbe comprising:
a) a β-ketoacyl-acyl carrier protein synthase III (KASIII) independent fatty acid synthesis (FAS) pathway that makes a product from a primer excluding acetyl coA or propionyl-coA using FAS enzymes (except for KASIII);
b) said microbe having an overexpressed acyl ACP thioesterase (TE);
c) said microbe also having one or more expression vectors overexpressing enzymes selected from the group consisting of 3-ketoacyl-ACP synthetase, 3-ketoacyl-ACP reductase, 3-hydroxyacyl ACP dehydrase, enoyl-ACP reductase, and Co-A transferase;
d) said microbe comprising reduced KASIII activity (KASIII − ) or null KASIII activity (ΔKASIII); and
e) said microbe having one or more expression vectors overexpressing beta-ketothiolase, acetoacetyl-CoA reductase, trans-enoyl-CoA reductase, and either 3-hydroxyacyl-[ACP] dehydratase or crotonase.
2. A method of making a product, comprising: a) growing the microbe of claim 1 in a medium allowing cell growth; b) elongating a starter molecule or primer molecule having 2 or >2 carbons using the FAS enzymes (except for KASIII) to make a product; and, c) isolating said product.
3. A method of making a product, comprising: a) growing the microbe of claim 1 in a medium allowing cell growth; b) elongating a starter molecule or primer molecule having >2 carbons using the FAS enzymes (except for KASIII) to make a product; and, c) isolating said product.
4. The method of claim 3 , wherein said product is selected from the group consisting of C6-C16 hydroxy fatty acids, C6-C16 amino fatty acids, C6-C16 halogenated fatty acids, C6-C16 branched fatty acids, C6-C16 unsaturated fatty acids, C6-C16 ω-hydroxy fatty acids, C6-16 α,ω-dicarboxylic acids, C6-16 α,ω-diol fatty acids or derivatives thereof.
5. The method of claim 3 , wherein said product is C6-C16 ω-hydroxy fatty acids or derivatives thereof.
6. The method of claim 5 , wherein bifunctional α,ω-dicarboxylic acids are obtained by the oxidation of said ω-hydroxy fatty acids.
7. The method of claim 5 , wherein bifunctional α,ω-diols are obtained by the reduction of said ω-hydroxy fatty acids.
8. A genetically engineered microbe having a β-ketoacyl-acyl carrier protein synthase III (KASIII) independent fatty acid synthesis (FAS) pathway that makes a product from a starter molecule of >2 carbons or >3 carbons, said starter molecule excluding acetyl-CoA or propionyl-coA, using FAS enzymes (except for KASIII), said microbe having reduced KASIII activity, an overexpressed acyl ACP thioesterase (TE), and reduced native acyl-carrier protein (ACP) dependent fatty acid biosynthesis, malonyl-CoA-ACP transacylase, and acetyl-CoA carboxylase.
9. A method of making a product; a) growing the microbe of claim 8 in a medium; b) elongating a coA-activated starter molecule having >2 carbons using the FAS enzymes to make a product; and, c) isolating said product.
10. The method of claim 9 , further comprising adding said starter molecule to said medium.
11. The microbe of claim 8 , having one or more overexpressed enzymes selected from the group consisting of 3-ketoacyl-ACP synthetase, 3-ketoacyl-ACP reductase, 3-hydroxyacyl ACP dehydrase, enoyl-ACP reductase, thioesterase or Co-A transferase.
12. The microbe of claim 11 , wherein said starter molecule is produced in vivo by a native pathway or by a genetically engineered pathway.
13. A method of making a product; a) growing the microbe of claim 11 in a medium; b) elongating a coA-activated starter molecule having >2 carbons using the FAS enzymes to make a product; and, c) isolating said product.
14. The microbe of claim 8 , wherein said starter molecule is produced in vivo by a native pathway or by a genetically engineered pathway.
15. A method of making a product; a) growing the microbe of claim 14 in a medium; b) elongating a coA-activated starter molecule having >2 carbons using the FAS enzymes to make a product; and, c) isolating said product.
16. A recombinant bacteria comprising KASIII − (β-ketoacyl-acyl carrier protein synthase III − ), TE + (thioesterase + ), PhaA + (beta-ketothiolase + ), PhaB + (acetoacetyl-CoA reductase + ), TER + (trans-enoyl-CoA reductase + ), and either FabZ + (3-hydroxyacyl-[acyl-carrier-protein] dehydratase + ) or crt + (crotonase + ).
17. The bacteria of claim 16 , further comprising PrpE + (propionyl-CoA synthase).