Synthesis of short chain fatty acids from bacteria
The present invention relates to an engineered bacteria for producing short chain fatty acid with the overexpression of a long chain (>C12) acyl-ACP thioesterases (long-TE) and a short chain (≦C12) acyl-ACP thioesterases (short-TE).
1. A genetically engineered E. coli comprising an overexpressed gene encoding i) a long chain (>C12) acyl-ACP thioesterases (long-TE) and ii) an overexpressed gene encoding a short chain (≦C12) acyl-ACP thioesterases (short TE), wherein said bacteria makes more short chain free fatty acids than a control strain lacking i).
2. The bacteria of claim 1 , further comprising a reduction in activity of at least one enzyme in the TCA cycle and at least one enzyme of glycolysis.
3. The bacteria of claim 2 , wherein said at least one protein from the tricarboxylic acid cycle is succinyl-CoA synthetase.
4. The bacteria of claim 2 , wherein said at least one protein from glycolysis is selected from glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phophoglycerate mutate, enolase, pyruvate kinase, and glucose phosphotransferase.
5. The bacteria of claim 2 , wherein said at least one enzyme from glycolysis is glucokinase or glucose phosphotransferase.
6. The bacteria of claim 2 , further comprising at least one further modification selected from the group consisting of i) overexpressed malonyl coenzyme A-acyl carrier protein transacylase, ii) overexpressed transhydrogenase, iii) moderately overexpressed acetyl-CoA carboxylase, iv) overexpressed NAD kinase and v) reduced activity of endogenous fatty acyl-CoA synthetase.
7. The bacteria of claim 1 , further comprising ΔfadD and ΔsucC.
8. The bacteria of claim 1 , further comprising ΔfadD and ΔpfkA.
9. The bacteria of claim 1 , having a genotype further comprising:
ΔfadD, ΔsucC
ΔfadD, ΔfumAC and optional ΔsucC
ΔfadD, ΔgapA and optional ΔsucC
ΔfadD, ΔptsG and optional ΔsucC
ΔfadD, ΔpfkA and optional ΔsucC
ΔfadD, Δglk and optional ΔsucC
TE + and fabD +
TE + and udhA +
TE + and pntAB +
ΔsucC
ΔfumAC and optional ΔsucC
ΔgapA and optional ΔsucC
ΔptsG and optional ΔsucC
ΔpfkA and optional ΔsucC
Δglk and optional ΔsucC
NAD-kinase +
acc + and/or fabD + and/or udhA + and/or pntAB + and/or NAD-kinase + combined with any genotypes in this table.
10. A genetically engineered bacteria comprising i) an overexpressed long chain (>C12) acyl-ACP thioesterases (long-TE) and an ii) overexpressed short chain (≦C12) acyl-ACP thioesterases (short-TE), wherein said bacteria makes more short chain free fatty acids than a control strain lacking i).
11. The bacteria of claim 10 , wherein said bacteria makes 50% more short chain free fatty acids than said control strain.
12. A genetically engineered bacteria comprising I an overexpressed long chain (>C12) acyl-ACP thioesterases (long-TE) and ii) an overexpressed short chain (≦C12) acyl-ACP thioesterases (short-TE), and iii) wherein at least one gene from the tricarboxylic acid cycle or glycolysis, or both, is inactivated, wherein said bacteria makes more short chain fatty acids than a control strain lacking i).
13. A method of making short chain fatty acids, comprising:
a) inoculating culture broth in a container having walls with a bacteria of claim 1 ;
b) growing said bacteria until said bacteria secrete fatty acids into said culture broth; and
c) collecting said fatty acids.
14. The method of claim 13 , wherein said culture broth is acidified.
15. The method of claim 13 , further comprising supplementing said culture broth with 0.1-1% acetic acid.
16. The method of claim 13 , wherein collecting said fatty acids comprises collecting a solid fraction of said fatty acids by filtration of said culture broth.
17. The method of claim 13 , wherein collecting said fatty acids comprises extracting solids from the walls of said container with a hydrophobic solvent.
18. The method of claim 13 , wherein collecting said fatty acids comprises rinsing said walls with an alkali solution.
19. The method of claim 13 , wherein collecting said fatty acids comprising skimming said fatty acids from a top of said culture broth.
20. A method of making short chain fatty acids, comprising:
a) inoculating culture broth in a container having walls with a bacteria of claim 12 ;
b) growing said bacteria until said bacteria secrete fatty acids into said culture broth; and
c) collecting said fatty acids.