Vectors and strains for producing myrcene and method of producing myrcene using the same

Patent metadata

Field	Value
Publication number	US-9719095-B2
Application number	US-201514950425-A
Country	US
Kind code	B2
Filing date	Nov 24, 2015
Priority date	Jul 8, 2015
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

Disclosed herein are an expression vector capable of expressing myrcene, an Escherichia coli strain transformed with the vector and having improved capability of producing myrcene and a method for producing myrcene and a method for recycling glycerol using the same. In an aspect, the transformed Escherichia coli strain of the present disclosure can produce myrcene with high purity on a large scale using glycerol or glucose as a carbon source. Also, the Escherichia coli strain of the present disclosure is economical and environment-friendly because it can produce high value-added myrcene using waste glycerol as a carbon source. In addition, the strongly volatile myrcene can be produced and isolated at the same time.

First claim

What is claimed is:

1. A transformed Escherichia coli strain transformed with a first vector and a second vector, the first vector comprising, in sequence:
a chloramphenicol resistance gene as a selection marker;
a p15A replication origin;
a lacUV5 promoter;
a first domain comprising a series of genes encoding a series of enzymes that contribute to the production of mevalonate from acetyl-CoA; and
a second domain comprising a series of genes encoding a series of enzymes that contribute to the production of dimethylallyl pyrophosphate (DMAPP) from mevalonate; and
the second vector comprising, in sequence:
an ampicillin resistance gene as a selection marker;
a ColE1 replication origin;
a trc promoter; and
a gene encoding an enzyme capable of producing myrcene from geranyl pyrophosphate (GPP).

2. The transformed Escherichia coli strain according to claim 1 ,
wherein the first domain of the first vector comprises, in sequence:
a gene encoding acetyl-CoA thiolase (ACAT);
a gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA synthase (HMGS); and
a gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR), and

wherein the second domain comprises, in sequence,
a gene encoding mevalonate kinase (MK);
a gene encoding phosphomevalonate kinase (PMK);
a gene encoding mevalonate diphosphate decarboxylase (PMD); and
a gene encoding isopentenyl diphosphate isomerase (IDI).

3. The transformed Escherichia coli strain according to claim 2 ,
wherein the gene encoding acetyl-CoA thiolase (ACAT) comprises a sequence of SEQ ID NO: 1,
the gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA synthase (HMGS) comprises a sequence of SEQ ID NO: 2,
the gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) comprises a sequence of SEQ ID NO: 3,
the gene encoding mevalonate kinase (MK) comprises a sequence of SEQ ID NO: 4,
the gene encoding phosphomevalonate kinase (PMK) comprises a sequence of SEQ ID NO: 5,
the gene encoding mevalonate diphosphate decarboxylase (PMD) comprises a sequence of SEQ ID NO: 6, and
the gene encoding isopentenyl diphosphate isomerase (IDI) comprises a sequence of SEQ ID NO: 7.

4. The transformed Escherichia coli strain according to claim 1 , wherein the enzyme capable of producing myrcene from geranyl pyrophosphate (GPP) is myrcene synthase (MS).

5. The transformed Escherichia coli strain according to claim 4 , wherein the gene encoding myrcene synthase (MS) comprises a sequence of SEQ ID NO: 9.

6. The transformed Escherichia coli strain according to claim 1 ,
wherein the first vector further comprises one or more selected from the group consisting of: a trc promoter, and a gene encoding an enzyme capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP),
wherein the trc promoter is located between the first domain and the second domain, and
wherein the gene encoding the enzyme capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) is located downstream of the second domain.

7. The transformed Escherichia coli strain according to claim 6 , wherein the enzyme capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) is geranyl pyrophosphate synthase (GPPS).

8. The transformed Escherichia coli strain according to claim 7 , wherein the gene encoding geranyl pyrophosphate synthase (GPPS) comprises a sequence of SEQ ID NO: 8.

9. The transformed Escherichia coli strain according to claim 1 , wherein the second vector further comprises, between the trc promoter and the gene encoding the enzyme capable of producing myrcene from geranyl pyrophosphate (GPP), a gene encoding an enzyme capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP).

10. The transformed Escherichia coli strain according to claim 9 , wherein the enzyme capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) is geranyl pyrophosphate synthase (GPPS).

11. The transformed Escherichia coli strain according to claim 10 , wherein the gene encoding geranyl pyrophosphate synthase (GPPS) comprises a sequence of SEQ ID NO: 8.

12. The transformed Escherichia coli strain according to claim 1 , wherein the first vector comprises a sequence of any of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

13. The transformed Escherichia coli strain according to claim 1 , wherein the second vector further comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

14. The transformed Escherichia coli strain according to claim 1 , wherein the Escherichia coli strain is Escherichia coli DH1 transformed with the first vector and the second vector.

15. The transformed Escherichia coli strain according to claim 14 , wherein the Escherichia coli strain produces at least 45 mg/L of myrcene in 70 hours under a condition of 37° C. and 1% (w/v) glycerol.

16. The transformed Escherichia coli strain according to claim 14 , wherein, the Escherichia coli strain produces at least 3 times an amount of myrcene when glycerol is used as a carbon source as compared to when glucose is used as a carbon source.

17. The transformed Escherichia coli strain according to claim 14 , wherein the Escherichia coli strain is an Escherichia coli strain of accession number KCTC12850BP or KCTC12851BP.

Assignees

• Korea Inst Sci & Tech

Inventors

• Woo Han Min
• Kim Eun Mi
• Um Youngsoon
• Gong Gyeongtaek
• Lee Sun Mi
• Kim Yunje

Classifications

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12Y205/01

  transferring alkyl or aryl groups, other than methyl groups (2.5.1) · CPC title

• C12Y207/01036

  Mevalonate kinase (2.7.1.36) · CPC title

• C12N15/72

  Expression systems using regulatory sequences derived from the lac-operon · CPC title