Biofuel and chemical production by recombinant microorganisms via fermentation of proteinaceous biomass

Patent metadata

Field	Value
Publication number	US-9284566-B2
Application number	US-201113883531-A
Country	US
Kind code	B2
Filing date	Nov 3, 2011
Priority date	Nov 3, 2010
Publication date	Mar 15, 2016
Grant date	Mar 15, 2016

Abstract

Provided herein are metabolically modified microorganisms characterized by having an increased keto-acid flux when compared with the wild-type organism and comprising at least one polynucleotide encoding an enzyme that when expressed results in the production of a greater quantity of a chemical product when compared with the wild-type organism. The recombinant microorganisms are useful for producing a large number of chemical compositions from various nitrogen containing biomass compositions and other carbon sources. More specifically, provided herein are methods of producing alcohols, acetaldehyde, acetate, isobutyraldehyde, isobutyric acid, n-butyraldehyde, n-butyric acid, 2-methyl-1-butyraldehyde, 2-methyl-1-butyric acid, 3-methyl-1-butyraldehyde, 3-methyl-1-butyric acid, ammonia, ammonium, amino acids, 2,3-butanediol, 1,4-butanediol, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine, isobutene, itaconate, acetoin, acetone, isobutene, 1,5-diaminopentane, L-lactic acid, D-lactic acid, shikimic acid, mevalonate, polyhydroxybutyrate (PHB), isoprenoids, fatty acids, homoalanine, 4-aminobutyric acid (GABA), succinic acid, malic acid, citric acid, adipic acid, p-hydroxy-cinnamic acid, tetrahydrofuran, 3-methyl-tetrahydrofuran, gamma-butyrolactone, pyrrolidinone, n-methylpyrrolidone, aspartic acid, lysine, cadeverine, 2-ketoadipic acid, and/or S-adenosyl-methionine (SAM) from a suitable nitrogen rich biomass.

First claim

What is claimed is:

1. A recombinant microorganism comprising a deletion, reduced expression, or reduced function of a gene involved in synthesis or uptake of a quorum sensing-related autoinducer molecule and/or of a gene involved in ammonia reuptake, and further comprising at least one heterologous polynucleotide encoding a dehydrogenase, a transaminase, or a deaminase that deaminates an amino acid substrate to produce an increased keto-acid flux when compared with the wild-type organism;
wherein the dehydrogenase is selected from the group consisting of glutamate dehydrogenase (E.C. 1.4.1.2 and E.C. 1.4.1.4), glutamic dehydrogenase (E.C. 1.4.1.3), valine dehydrogenase (E.C. 1.4.1.8), leucine dehydrogenase (E.C. 1.4.1.9), and phenylalanine dehydrogenase (E.C. 1.4.1.20);
wherein the deaminase is selected from the group consisting of aspartate ammonia lyase (4.3.1.1), L-serine ammonia lyase (E.C. 4.3.1.17), D-serine ammonia lyase (4.3.1.18), threonine ammonia lyase (E.C. 4.3.1.19), tyrosine ammonia lyase (E.C. 4.3.1.23), phenylalanine ammonia lyase (E.C. 4.3.1.24), and phenylalanine/tyrosine ammonia lyase (E.C. 4.3.1.25);
wherein the transaminase is an L-α-transaminase (E.C. 2.6.I.X, where X is any number);
wherein the deletion, reduced expression, or reduced function of a gene involved in synthesis or uptake of a quorum sensing related autoinducer molecule is from the deletion, or reduced expression or function of the genes luxS or lsrA; and
wherein the deletion, reduced expression, or reduced function of a gene involved in ammonia reuptake is from the deletion, or reduced expression or function of the genes gdhA or glnA.

2. The recombinant microorganism according to claim 1 , wherein the microorganism further metabolizes the keto-acid into a chemical product.

3. The recombinant microorganism according to claim 1 , wherein the leucine dehydrogenase is LeuDH.

4. The recombinant microorganism according to claim 3 , wherein the leucine dehydrogenase LeuDH is from Thermoactinomyces intermedins.

5. The recombinant microorganism according to claim 1 , wherein the deaminase is the serine deaminase SdaB.

6. The recombinant microorganism according to claim 5 , wherein the serine deaminase SdaB is from Escherichia coli, Rosebacter atrosepticum, Corynebacterium diphtheriae, Salmonella enerica, Yersinia enterocolitica , or Burkholderia pseudomallei.

7. The recombinant microorganism according to claim 1 , wherein the L-α-transaminase is selected from the group consisting of L-aspartate transaminase (E.C. 2.6.1.1), L-alanine transaminase (E.C. 2.6.1.12 and E.C. 2.6.1.47), L-asparagine transaminase (E.C. 2.6.1.14), and glycine transaminase (E.C. 2.6.1.35).

8. The recombinant microorganism according to claim 7 , wherein the L-aspartate transaminase is AvtA.

9. The recombinant microorganism according to claim 8 , AvtA is from Escherichia coli, Neisseria meningitidis, Pantoea ananatis, Amycolatopsis mediterranei, Mannheimia succinicproducens, Salmonella enterica , or Yersinia pestis.

10. The recombinant microorganism according claim 1 , wherein the microorganism is further characterized by, a reduced autoinducer 2 reuptake activity, a reduced glutamate dehydrogenase activity, a reduced glutamine synthase activity, a reduced glutamate synthase activity, and/or a reduced global regulator activity when compared with the wild-type organism.

11. The recombinant microorganism according to claim 10 , wherein the reduced global regulator activity is from the deletion, or reduced expression or function of the genes CRP, LRP, Fis, and/or IHF.

12. The recombinant microorganism according to claim 2 , wherein the chemical product is selected from the group consisting of an alcohol, an acetaldehyde, acetate, isobutyraldehyde, isobutyric acid, n-butyraldehyde, n-butyric acid, 2-methyl-1-butyraldehyde, 2-methyl-1-butyric acid, 3-methyl-l-butyraldehyde, 3-methyl-1-butyric acid, ammonia, ammonium, glutamic acid, threonine, methionine, isoleucine, valine, leucine, tryptophan, tyrosine, phenylalanine, 2,3-butanediol, 1,4-butanediol, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine, isobutene, itaconate, acetoin, acetone, isobutene, 1,5-diaminopentane, L-lactic acid, D-lactic acid, shikimic acid, mevalonate, polyhydroxybutyrate (PHB), isoprenoids, fatty acids, homoalanine, 4-aminobutyric acid (GABA), succinic acid, malic acid, citric acid, adipic acid, p-hydroxy-cinnamic acid, tetrahydrofuran, 3-methyl-tetrahydrofuran, gamma-butyrolactone, pyrrolidinone, n-methylpyrrolidone, aspartic acid, lysine, cadeverine, 2-ketoadipic acid, and S-adenosyl-methionine (SAM).

13. The recombinant microorganism according to claim 12 , wherein the alcohol is selected from the group consisting of ethanol, 1-proponal, n-butanol, isobutanol, 2-methyl-l-butanol and 3-methyl-butanol.

14. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from pyruvate.

15. The recombinant microorganism of claim 14 , wherein the chemical product is selected from the group consisting of acetate, acetaldehyde, isobutyraldehyde, n-butyraldehyde, 2,3-butanediol, L-lactic acid, D-lactic acid, an aromatic, polyhydroxybutyrate (PHB), mevalonate, an isoprenoid, ethanol, isobutanol, n-butanol, and a fatty acid.

16. The recombinant microorganism of claim 15 , wherein the aromatic is selected from the group consisting of tryptophan, tyrosine, phenylalanine, and shikimic acid.

17. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from 2-ketoglutarate.

18. The recombinant microorganism according to claim 17 , wherein the chemical product is selected from the group consisting of GABA (4-aminobutyric acid), glutamic acid, succinate and malic acid.

19. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from oxaloacetate.

20. The recombinant microorganism according to claim 19 , wherein the chemical product is selected from the group consisting of aspartic acid, lysine, cadeverine, 2-ketoadipic acid, threonine, methionine and S-adenosyl-methionine (SAM).

21. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from 2-ketobutyrate.

22. The recombinant microorganism of claim 21 , wherein the chemical product is 2-methyl-l-butyraldehyde, isoleucine, homoalanine, and 2-methyl-l-butanol.

23. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from 2-ketoisovalerate.

24. The recombinant microorganism of claim 23 , wherein the chemical product is selected from the group consisting of isobutyraldehyde, 3-methyl-l-butyraldehyde, isobutanol, 3-methyl-l-butanol, and valine.

25. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from 2-ketoisocaproate.

26. The recombinant microorganism of claim 25 , wherein the chemical product is selected from the group consisting of 3-methyl-l-butyraldehyde, leucine, and 1-butanol.

27. The recombinant microorganism according to claim 2 , wherein the chemical product is produced from 2-keto-3-methylvalerate.

28. The recombinant microorganism of claim 27 , wherein the chemical product is selected from the group consisting of 2-methyl-l-butyraldehyde, isoleucine, and 2-methyl-1-butanol.

29. The recombinant microorganism according to claim 1 , wherein the

Assignees

• Liao James C
• Cho Kwang Myung
• Yan Yajun
• Huo Yixin
• Univ California

Inventors

• Liao James C
• Cho Kwang Myung
• Yan Yajun
• Huo Yixin

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12P7/16

  Butanols · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title