Biological Synthesis of 6-Aminocaproic Acid and Transgenic Microorganism Therefor

Patent metadata

Field	Value
Publication number	US-2016257976-A1
Application number	US-201414889543-A
Country	US
Kind code	A1
Filing date	May 2, 2014
Priority date	May 6, 2013
Publication date	Sep 8, 2016
Grant date	—

Abstract

The present invention relates to a method for preparing a recombinant microorganism simultaneously comprising genes encoding enzymes used in the biosynthesis pathway of 6-aminocaproic acid, which is a precursor of caprolactam, biosynthesizing 6-aminocaproic acid from the microorganism, and producing the same so as to synthesize caprolactam.

First claim

1 . A method for preparing 6-aminocaproic acid comprising:
preparing an expression vector comprising HpaI (4-hydroxy-2-oxoheptane-1,7-dioate aldolase)-HpaH (2-oxohept-3-ene-1,7-dioate dehydratase) gene, nemA (N-ethylmaleimide reductase) gene, and KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) gene and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) gene (step 1); and transforming the expression vector of step 1 into a microorganism (step 2).

2 . The method of claim 1 , wherein the HpaI-HpaH gene comprises a polynucleotide represented by SEQ ID NO: 3 encoding aldolase-dehydratase.

3 . The method of claim 1 , wherein the nemA gene comprises a polynucleotide represented by SEQ ID NO: 4 encoding reductase.

4 . The method of claim 1 , wherein the KIVD (alpha-ketoisovalerate decarboxylase) gene comprises a polynucleotide represented by SEQ ID NO: 5 encoding decarboxylase.

5 . The method of claim 1 , wherein the PdAT (beta-alanine-pyruvate transaminase) gene comprises a polynucleotide represented by SEQ ID NO: 6 encoding transaminase.

6 . The method of claim 1 , wherein BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) gene comprises a polynucleotide represented by SEQ ID NO: 7 encoding transaminase.

7 . The method of claim 1 , wherein the expression vector of step 1 further comprises nucleic acid (polynucleotide) sequences encoding GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, 6H15, STREP, CBP, CBD, or S-tag affinity tag.

8 . The method of claim 1 , wherein the expression vector of step 1 further comprises nucleic acid sequences encoding kex2p in yeasts, purine in mammals, Factor Xa, enterokinase, subtilisin, tobacco etch virus protease, thrombin, or ubiquitin hydrolase.

9 . The method of claim 1 , wherein the microorganism of step 2 is a bacterium, yeast, or fungus.

10 . The method of claim 1 , wherein the method further comprises producing and secreting 6-aminocaproic acid by culturing the transformed microorganism of step 2 in a fed-batch fermentation.

11 . The method of claim 10 , wherein the method further comprises purifying protein secreted.

12 . An expression vector for biosynthesis of 6-aminocaproic acid comprising HpaI-HpaH gene, nemA gene, KIVD (alpha-ketoisovalerate decarboxylase) gene; and at least one of PdAT (beta-alanine-pyruvate transaminase) gene and BcAT (adenosylmethionine-8-amino-7-oxononanoate aminotransferase) gene.

13 . The expression vector of claim 12 , wherein the expression vector is pACYCWG shown in FIG. 2 .

14 . A transformant which is transformed with the expression vector of claim 12 .

15 . The transformant of claim 14 , wherein the transformant is a bacterium, yeast, or fungus.

16 . The transformant of claim 14 , wherein the transformant transforms pyruvate and/or succinic semialdehyde (SSA) into 6-aminocaproic acid.

17 . A method for producing caprolactam further comprises transforming 6-aminocaproic acid produced by the method according to claim 1 into caprolactam.

Assignees

• Korea Res Inst Bioscience & Biotechnology

Inventors

• Lee Hong-Weon
• Ahn Jung Oh
• Jung Joon Ki
• Ko Hee-Ju
• Park Sun-Joo
• Kim Chun Sug
• Lee Hyeok Won
• Lee Eun Gyo
• Lee Joo Hwan

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title