Mutants having capability to produce 1, 4-butanediol and method for preparing 1, 4-butanediol using the same

Patent metadata

Field	Value
Publication number	US-2015353964-A1
Application number	US-201514802681-A
Country	US
Kind code	A1
Filing date	Jul 17, 2015
Priority date	Sep 7, 2007
Publication date	Dec 10, 2015
Grant date	—

Abstract

A mutant capable of producing 1,4-butanediol and a method of preparing 1,4-butanediol using the same are provided. The mutant microorganism is prepared by introducing and amplifying genes encoding enzymes converting succinate into 4-hydroxybutyrate and 4-hydroxybutyrate into 1,4-butanediol in a microorganism capable of producing succinate. The method includes culturing the mutant in a medium containing carbohydrate and obtaining 1,4-butanediol from the culture. Thus, 1,4-butanediol, which is essential in chemical industry, can be prepared in a biological process.

First claim

1 - 32 . (canceled)

33 . An isolated mutant microorganism exhibiting high production of 1,4-butanediol, which is prepared by introducing or amplifying genes encoding enzymes converting succinate into 4-hydroxybutyrate, and 4-hydroxybutyrate into 1,4-butanediol, in a Lumen bacteria capable of producing succinate.

34 . The isolated mutant microorganism according to claim 33 , wherein the Lumen bacteria have inactive genes encoding lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pfl), and produce succinate in high concentration without substantial production of other organic acids in an anaerobic condition.

35 . The isolated mutant microorganism according to claim 33 , wherein the Lumen bacteria have inactive genes encoding lactate dehydrogenase (ldhA), pyruvate-formate lyase (pfl), phosphotransacetylase (pta) and acetate kinase (ackA), and produce succinate in high concentration without substantial production of other organic acids in an anaerobic condition.

36 . The isolated mutant microorganism according to claim 33 , wherein the Lumen bacteria have inactive genes encoding lactate dehydrogenase (ldhA), pyruvate-formate lyase (pfl) and phosphopyruvate carboxylase (ppc), and produce succinate in high concentration without substantial production of other organic acids in an anaerobic condition.

37 . The isolated mutant microorganism according to claim 33 , wherein the Lumen bacteria are selected from the group consisting of Mannheimia species, Actinobacillus species and Anaerobiospirillum species.

38 . The isolated mutant microorganism according to claim 37 , wherein the Lumen bacteria are Mannheimia species.

39 . The isolated mutant microorganism according to claim 38 , wherein the Lumen bacteria are selected from the group consisting of Mannheimia succiniciproducens MBEL55E (KCTC 0769BP), and Mannheimia species LPK (KCTC 10558BP), LPK4 and LPK7 (KCTC 10626BP).

40 . The isolated mutant microorganism according to claim 33 , wherein the gene encoding the enzyme converting succinate into 4-hydroxybutyrate is derived from Clostridium kluyveri.

41 . The isolated mutant microorganism according to claim 33 , wherein the gene encoding the enzyme converting succinate into 4-hydroxybutyrate is selected from the group consisting of genes encoding succinyl-CoA transferase (Cat1), succinate semialdehyde dehydrogenase (SucD), 4-hydroxybutyrate dehydrogenase (4hbD) and 4-hydroxybutyrate dehydrogenase (GHB).

42 . The isolated mutant microorganism according to claim 41 , wherein the gene encoding Cat1 has a base sequence of SEQ ID NO: 1, the gene encoding SucD has a base sequence of SEQ ID NO: 2, the gene encoding 4hbD has a base sequence of SEQ ID NO: 3, and the gene encoding GHB has a base sequence of SEQ ID NO: 4.

43 . The isolated mutant microorganism according to claim 41 , wherein the mutant comprises a gene encoding Cat1; a gene encoding SucD; and a gene encoding 4hbD or a gene encoding GHB.

44 . The isolated mutant microorganism according to claim 33 , wherein the gene encoding the enzyme converting 4-hydroxybutyrate into 1,4-butanediol is derived from Clostridium acetobutylicum.

45 . The isolated mutant microorganism according to claim 33 , wherein the gene encoding the enzyme converting 4-hydrxoybutyrate into 1,4-butanediol is a gene encoding 4-hydroxybutyrate-CoA transferase and a gene encoding alcohol dehydrogenase reducing 4-hydroxybutyrate-CoA; or a gene encoding phosphotransbutyrylase, a gene encoding butyryl kinase and a gene encoding alcohol dehydrogenase reducing 4-hydroxybutyrate-CoA.

46 . The isolated mutant microorganism according to claim 45 , wherein the gene encoding 4-hydroxybutyrate-CoA transferase has a base sequence of SEQ ID NO: 5.

47 . The isolated mutant microorganism according to claim 45 , wherein the gene encoding phosphotransbutyrylase and the gene encoding butyryl kinase have base sequences of by SEQ ID NOs: 6 and 7, respectively.

48 . The isolated mutant microorganism according to claim 45 , wherein the alcohol dehydrogenase is butyl-CoA dehydrogenase derived from Clostridium acetobutylicum.

49 . The isolated mutant microorganism according to claim 48 , wherein the gene encoding butyl-CoA dehydrogenase has a base sequence of SEQ ID NO: 8 or 9.

50 . The isolated mutant microorganism according to claim 33 , wherein the mutunt has an inactive gene associated with conversion of succinate semialdehyde into succinate.

51 . The isolated mutant microorganism according to claim 50 , wherein the gene associated with conversion of succinate semialdehyde into succinate is a gene encoding succinic semialdehyde dehydrogenase (GabD).

52 . The isolated mutant microorganism according to claim 51 , wherein the gene encoding GabD has a base sequence of SEQ ID NO: 10.

53 . The isolated mutant microorganism according to claim 33 , wherein a gene encoding C4-dicarboxylate transport protein (DctA) associated with transport of succinate is further introduced or amplified in the mutant.

54 . The isolated mutant microorganism according to claim 53 , wherein the gene encoding DctA has a base sequence of SEQ ID NO: 11.

55 . A isolated mutant microorganism exhibiting high production of 1,4-butanediol, which is prepared by introducing or amplifying a gene encoding Cat1; a gene encoding SucD; a gene encoding 4hbD or GHB; a gene encoding 4-hydroxybutyrate-CoA transferase, or a gene encoding Ptb and a gene encoding Buk; and a gene encoding butyl-CoA dehydrogenase, in a microorganism capable of producing succinate.

56 . The isolated mutant microorganism according to claim 55 , wherein a gene encoding GabD is inactivated in the mutant.

57 . The isolated mutant microorganism according to claim 55 , wherein a gene encoding DctA associated with transport of succinate is introduced or amplified in the mutant.

58 . A method of preparing 1,4-butanediol, comprising:
culturing the mutant microorganism according to claim 33 in a medium containing a carbon source; and obtaining 1,4-butanediol from the medium.

Assignees

• Lg Chemical Ltd

Inventors

• Park Si Jae
• Lee Sang Hyun
• Lee Sang Yup
• Lee Eun Jeong

Classifications

• C12Y101/01061

  4-Hydroxybutyrate dehydrogenase (1.1.1.61) · CPC title

• C12N9/13

  transferring sulfur containing groups (2.8) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N9/001

  acting on the CH-CH group of donors (1.3) · CPC title