# Patent

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

## 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.

## Background
FIELD OF THE INVENTION
Disclosed herein is an expression vector capable of expressing myrcene. Also disclosed herein are a 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.
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to Korean Patent Application No. 10-2015-0097250, filed on Jul. 8, 2015, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference.
DESCRIPTION ABOUT NATIONAL SUPPORT RESEARCH AND DEVELOPMENT
This study is made by the support of Cooperative Study Business (Creative Allied Project, CAP) of Korea Ministry of Science, ICT and Future Planning under the supervision of Korea Institute of Science and Technology, and the subject name thereof is Development of technology for producing next generation fuel/material by integrative use of woody biomass (Subject Identification No. CAP-11-1).
BACKGROUND OF THE INVENTION
Monoterpenes are naturally occurring substances which are very important in cosmetic or pharmaceutical industry. Among them, myrcene is a substance that can be used as a starting material for various substances. In particular, it can be widely used as a precursor for menthol or other alcohol substances (e.g., linalool, geraniol). Until now, various myrcene-synthesizing genes have been found in different plants and have been characterized. Nevertheless, the currently available method of producing myrcene is limited only to extraction from plants or pyrolysis of β-pinene.
REFERENCES OF THE RELATED ART
Non-Patent Documents


    (Non-patent document 1) Sarria S et al., (2014) Microbial Synthesis of Pinene”, ACS Synthetic Biology 3, pp. 466-475.


SUMMARY
In an aspect, the present disclosure is directed to providing an expression vector capable of expressing myrcene by transforming Escherichia coli. 
In another aspect, the present disclosure is directed to providing an Escherichia coli strain having improved capability of producing myrcene.
In another aspect, the present disclosure is directed to production of myrcene on a large scale using a transformed Escherichia coli strain.
In another aspect, the present disclosure is directed to production and isolation/extraction of strongly volatile myrcene simultaneously.
In another aspect, the present disclosure is directed to production of high value-added myrcene using waste glycerol.
In an aspect, the present disclosure relates to a first vector containing, in sequence, a chloramphenicol resistance gene as a selection marker; a p15A replication origin as a replication origin; a lacUV5 promoter; a first domain containing a gene encoding an enzyme which produces mevalonate from acetyl-CoA; and a second domain containing a gene encoding an enzyme which produces dimethylallyl pyrophosphate (DMAPP) from mevalonate.
In another aspect, the present disclosure relates to a second vector containing, in sequence, an ampicillin resistance gene as a selection marker; a ColE1 replication origin as a replication origin; a trc promoter; and a gene encoding an enzyme which is capable of producing myrcene from geranyl pyrophosphate (GPP).
In another aspect, the present disclosure relates to an Escherichia coli strain transformed with the first vector and the second vector.
In another aspect, the present disclosure relates to an Escherichia coli strain producing 45 mg/L or more of myrcene in 70 hours under a condition of 37° C. and 1% (w/v) glycerol.
In another aspect, the present disclosure relates to a method for producing myrcene, including a step of cultuirng an Escherichia coli strain.
In another aspect, the present disclosure relates to a method for recycling glycerol, including a step of cultuirng an Escherichia coli strain.
In another aspect, the present disclosure relates to a kit for producing myrcene.
In an aspect, an Escherichia coli strain transformed with the vector of the present disclosure can produce myrcene with high purity on a large scale using glycerol or glucose as a carbon source. The Escherichia coli strain is economical because it can produce high value-added myrcene using waste glycerol as a carbon source. Also, it is environment-friendly because the microorganism can remove waste glycerol.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show depository receipts of transformed Escherichia coli strains of the present disclosure.
FIG. 2 shows a myrcene production pathway of a transformed Escherichia coli strain of the present disclosure.
FIG. 3 shows the structure of a first plasmid vector (plasmid 1) and a second plasmid vector (plasmid 2) of the present disclosure.
FIG. 4 shows six transformed strains of the present disclosure.
FIG. 5A and FIG. 5B show a result of measuring myrcene production by a transformed Escherichia coli strain of the present disclosure by gas chromatography-mass spectrophotometry (A: standard myrcene, B: myrcene produced by transformed Escherichia coli).
FIG. 6A, FIG. 6B and FIG. 6C show a result of measuring myrcene production by transformed Escherichia coli strains of the present disclosure when incubated in a medium supplemented with 1% glucose (first bar: 24 hours later, second bar: 48 hours later, third bar: 72 hours later, A: LB medium, B: EZ-rich medium, C: M9-MOPS medium).
FIG. 7A and FIG. 7B show a result of measuring myrcene production by transformed Escherichia coli strains of the present disclosure when incubated in a medium supplemented with 1% glycerol (first bar: 24 hours later, second bar: 48 hours later, third bar: 72 hours later, A: EZ-rich medium, B: M9-MOPS medium).
FIG. 8 shows recovery of myrcene with (w) or without (w/o) dodecane overlay.
FIG. 9 shows the sequence of an atoB gene (SEQ ID NO: 1).
FIG. 10 shows the sequence of an HMGS gene (SEQ ID NO: 2).
FIG. 11 shows the sequence of an HMGR gene (SEQ ID NO: 3).
FIG. 12 shows the sequence of an MK gene (SEQ ID NO: 4).
FIG. 13 shows the sequence of a PMK gene (SEQ ID NO: 5).
FIG. 14 shows the sequence of a PMD gene (SEQ ID NO: 6).
FIG. 15 shows the sequence of an IDI gene (SEQ ID NO: 7).
FIG. 16 shows the sequence of a tGPPS gene (SEQ ID NO: 8).
FIG. 17 shows the sequence of a tMS-Qi gene (SEQ ID NO: 9).
FIG. 18A-FIG. 18E show the sequence of a pBbA5c-MevT(co)-MBI(co) plasmid vector (SEQ ID NO: 10), in sequence.
FIG. 19A-FIG. 19E show the sequence of a pBbA5c-MevT(co)-MBIG(co) plasmid vector (SEQ ID NO: 11), in sequence.
FIG. 20A-FIG. 20E show the sequence of a pBbA5c-MevT(co)-T1-MBI(co) plasmid vector (SEQ ID NO: 12), in sequence.
FIG. 21A-FIG. 21E show the sequence of a pBbA5c-MevT(co)-T1-MBIG(co) plasmid vector (SEQ ID NO: 13), in sequence.
FIG. 22A-FIG. 22B show the sequence of a pBbE1a-tMS(co.Qi) plasmid vector (SEQ ID NO: 14), in sequence.
FIGS. 23A-FIG. 23C show the sequence of a pBbE1a-tGPPS2(co)-tMS(co.Qi) plasmid vector (SEQ ID NO: 15), in sequence.



DETAILED DESCRIPTION
Hereinafter, the present disclosure is described in detail.
In an aspect, the present disclosure relates to a transformed Escherichia coli strain transformed with a first vector and a second vector, the first vector containing, in sequence, a chloramphenicol resistance gene as a selection marker; a p15A replication origin as a replication origin; a lacUV5 promoter; a first domain containing a gene encoding an enzyme which produces mevalonate from acetyl-CoA; and a second domain containing a gene encoding an enzyme which produces dimethylallyl pyrophosphate (DMAPP) from mevalonate, and the second vector containing, in sequence, an ampicillin resistance gene as a selection marker; a ColE1 replication origin as a replication origin; a trc promoter; and a gene encoding an enzyme which is capable of producing myrcene from geranyl pyrophosphate (GPP).
In the present disclosure, a pathway of synthesizing mevalonate from acetyl-CoA is denoted by the acronym MevT, and a pathway of synthesizing isopentenyl diphosphate (IPP) from mevalonate is denoted by the acronym MBI.
In the present disclosure, the first vector is also called a first plasmid or a first plasmid vector, and the second vector is also called a second plasmid or a second plasmid vector.
In this aspect, the first vector may further contain one or more selected from a trc promoter; and a gene encoding an enzyme which is capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP). The trc promoter may be located between the first domain and the second domain, and the gene encoding an enzyme which is capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) may be located downstream of the second domain.
In the transformed Escherichia coli strain according to an aspect of the present disclosure, the first domain of the first vector may contain, 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). In this aspect, the second domain of the first vector may contain, 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).
In this aspect, the enzyme which is capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) may be geranyl pyrophosphate synthase (GPPS).
In the transformed Escherichia coli strain according to an aspect of the present disclosure, the gene encoding acetyl-CoA thiolase (hereinafter, atoB gene) may contain a sequence of SEQ ID NO: 1, the gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA synthase (hereinafter, HMGS gene) may contain a sequence of SEQ ID NO: 2, the gene encoding 3-hydroxyl-3-methyl-glutaryl-CoA reductase (hereinafter, HMGR gene) may contain a sequence of SEQ ID NO: 3, the gene encoding mevalonate kinase (hereinafter, MK gene) may contain a sequence of SEQ ID NO: 4, the gene encoding phosphomevalonate kinase (hereinafter, PMK gene) may contain a sequence of SEQ ID NO: 5, the gene encoding mevalonate diphosphate decarboxylase (hereinafter, PMD gene) may contain a sequence of SEQ ID NO: 6, and the gene encoding isopentenyl diphosphate isomerase (hereinafter, IDI gene) may contain a sequence of SEQ ID NO: 7.
And, the gene encoding geranyl pyrophosphate synthase (hereinafter, GPPS or tGPPS gene) may contain a SEQ ID NO: 8.
In the transformed Escherichia coli strain according to an aspect of the present disclosure, the first vector may contain a sequence of any of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, OR SEQ ID NO: 13.
In this aspect, the enzyme which is capable of producing myrcene from geranyl pyrophosphate (GPP) may be myrcene synthase (MS).
And, in the transformed Escherichia coli strain according to an aspect of the present disclosure, the second vector may further contain, between the trc promoter and the gene encoding an enzyme which is capable of producing myrcene from geranyl pyrophosphate (GPP), an enzyme which is capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP).
The enzyme which is capable of producing geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) may be geranyl pyrophosphate synthase (GPPS).
In this aspect, the gene encoding myrcene synthase (MS) (hereinafter, MS or tMS gene) may contain a sequence of SEQ ID NO: 9, and the gene encoding geranyl pyrophosphate synthase (GPPS) (hereinafter, GPPS or tGPPS gene) may contain a sequence of SEQ ID NO: 8.
In the transformed Escherichia coli strain according to an aspect of the present disclosure, the second vector may contain a sequence of SEQ ID NO: 14 or 15. In the present disclosure, pM1 denotes a first vector containing a chloramphenicol resistance gene, a p15A replication origin, an atoB gene, an HMGS gene and an HMGR gene, and further containing an MK gene, a PMK gene, a PMD gene and an IDI gene, and is also expressed as pBbA5c-MevT(co)-MBI(co). And, in the present disclosure, pM2 denotes a first vector (pM1) further containing a tGPPS gene, and is also expressed as pBbA5c-MevT(co)-MBIG(co). And, in the present disclosure, pM3 denotes a first vector (pM1) further containing a promoter, e.g., a Trc promoter (PTrc). The promoter may be located between the HMGR gene and the MK gene. pM3 is also expressed as pBbA5c-MevT(co)-T1-MBI(co). In the present disclosure, pM4 denotes a first vector (pM3) further containing a tGPPS gene, and is also expressed as pBbA5c-MevT(co)-T1-MBIG(co). And, in the present disclosure, pM(Qi) denotes a second vector containing a trc promoter and an MS gene, and is also expressed as pBbE1a-tMS(co.Qi). And, in the present disclosure, pGM(Qi) denotes a second vector (pM(Qi)) further containing a GPPS gene. The GPPS gene may be located between the tMS gene and the promoter. pGM(Qi) is also expressed as pBbE1a-tGPPS2(co)-tMS(co.Qi).
pM1 may contain a sequence of SEQ ID NO: 10, pM2 may contain a sequence of SEQ ID NO: 11, and pM3 may contain a sequence of SEQ ID NO: 13. pM(Qi) may contain a sequence of SEQ ID NO: 14, and pGM(Qi) may contain a sequence of SEQ ID NO: 15.
The vectors according to an aspect of the present disclosure may be those described in Table 1.








TABLE 1





Plasmid vectors
Characteristics







pBbA5c-MevT(co)-MBI(co)
Contains p15A, CmR, PlacUV5 and



mevalonate pathway gene


pBbA5c-MevT(co)-MBIG(co)
Contains p15A, CmR, PlacUV5,



mevalonate pathway gene and tGPPS



gene


pBbA5c-MevT(co)-T1-MBI(co)
Contains p15A, CmR, PlacUV5, Ptrc



and mevalonate pathway gene


pBbA5c-MevT(co)-T1-MBIG(co)
Contains p15A, CmR, PlacUV5, Ptrc,



mevalonate pathway gene and tGPPS



gene


pBbE1a-tMS(co.Qi)
Contains ColE1, AmpR, Ptrc and



myrcene synthase (MS) from



Q. ilex L.


pBbE1a-tGPPS2(co)-tMS(co.Qi)
Contains ColE1, AmpR, Ptrc, GPPS2



gene from A. grandis and myrcene



synthase (MS) from Q. ilex L.









In Table 1, CmR denotes a chloramphenicol resistance gene and AmpR denotes an ampicillin resistance gene.
Also, the first vector and the second vector may be Escherichia coli expression vectors.
The transformed Escherichia coli strain according to an aspect of the present disclosure may be Escherichia coli DH1 transformed with the first vector and the second vector. Specifically, it may contain two vectors, i.e., the first vector containing a gene which encodes an enzyme capable of producing dimethylallyl pyrophosphate (DMAPP) or geranyl pyrophosphate (GPP) from a carbon source and the second vector containing a gene which encodes an enzyme capable of producing myrcene from dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) or from geranyl pyrophosphate (GPP).
In the present disclosure, the ‘first’ vector and the ‘second’ vector do not mean that the parent strain should be transformed with the vectors in that order.
In this aspect, the strain may be one described in Table 2.










TABLE 2







Strains
Characteristics (introduced plasmid vectors)









Ec-pM1/pGM(Qi)
DH1, pBbA5c-MevT(co)-MBI(co) and




pBbE1a-tGPPS2(co)-tMS(co.Qi)



Ec-pM2/pM(Qi)
DH1, pBbA5c-MevT(co)-MBIG(co) and




pBbE1a-tMS(co.Qi)



Ec-pM2/pGM(Qi)
DH1, pBbA5c-MevT(co)-MBIG(co) and




pBbE1a-tGPPS2(co)-tMS(co.Qi)



Ec-pM3/pGM(Qi)
DH1, pBbA5c-MevT(co)-T1-MBI(co) and




pBbE1a-tGPPS2(co)-tMS(co.Qi)



Ec-pM4/pM(Qi)
DH1, pBbA5c-MevT(co)-T1-MBIG(co) and




pBbE1a-tMS(co.Qi)



Ec-pM4/pGM(Qi)
DH1, pBbA5c-MevT(co)-T1-MBIG(co) and




pBbE1a-tGPPS2(co)-tMS(co.Qi)










The transformed Escherichia coli strain according to an aspect of the present disclosure may produce 45 mg/L or more of myrcene in 70 hours under a condition of 37° C. and 1% (w/v) glycerol. It may produce 5 mg/L or more, 10 mg/L or more, 15 mg/L or more, 20 mg/L or more, 25 mg/L or more, 30 mg/L or more, 35 mg/L or more, 40 mg/L or more, 45 mg/L or more, 50 mg/L or more, 55 mg/L or more, 60 mg/L or more, 65 mg/L or more or 70 mg/L or more of myrcene, although not being limited thereto.
The transformed Escherichia coli strain according to an aspect of the present disclosure may use glucose or glycerol as a carbon source, although not being limited thereto.
In this aspect, when glycerol is used as a carbon source, the strain may produce 3 times or more, 1.5 times or more, 1.7 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.3 times or more, 2.5 times or more, 2.7 times or more, 2.9 times or more or 3.1 times or more of myrcene as compared to when glucose is used as a carbon source, although not being limited thereto. In this aspect, the amount of myrcene produced may vary depending on the components of a medium in which the Escherichia coli strain is cultured.
Also, the Escherichia coli strain may be a strain of an accession number KCTC12850BP or KCTC12851BP.
In another aspect, the present disclosure relates to a method for producing myrcene, including a step of culturing a transformed Escherichia coli strain.
In this aspect, the method for producing myrcene may further include a step of supplying a carbon source to the culture medium. The carbon source may be glucose or glycerol, although not being limited thereto.
Also, the method for producing myrcene according to an aspect of the present disclosure may further include a step of adding 10-30% (w/v) of dodecane based on the volume of the culture medium. The addition amount of dodecane may be 5-35%, 10-30%, 10-25%, 10-20%, 10-15%, 15-30%, 15-25% or 15-20%, although not being limited thereto. The dodecane may be added simultaneously with the culture medium or the carbon source, or may be added after the culture medium and/or the carbon source has been supplied. The dodecane may be located naturally above the culture medium without being mixed with the culture medium. Specifically, it may be added on top of the culture medium. When the dodecane is added, evaporation and loss of the strongly volatile myrcene into the atmosphere may be prevented.
In another aspect, the present disclosure relates to a method for recycling glycerol, including a step of culturing the Escherichia coli strain. In this aspect, the method may further include a step of supplying glycerol, specifically waste glycerol, to the Escherichia coli strain. The waste glycerol may be a byproduct from biodiesel production.
Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples.
Example 1 Establishment of Strategy for Producing Myrcene
A myrcene metabolic pathway as shown in FIG. 1 was designed to prepare an Escherichia coli strain having superior capability of producing myrcene.
Also, literature search was conducted to select a gene encoding a myrcene synthase (MS) gene. Candidate genes were selected based on the literature search and synthesized after codon optimization for expression in Escherichia coli. The selected gene was myrcene synthase derived from pine tree (Quercur liex L.) (Fischbach R et al., Eur. J. Biochem., 2001).
Example 2 Preparation of Plasmid Vector and Strain
Two types of plasmids were constructed for production of myrcene. To a first plasmid, a gene necessary for producing IPP and DMAPP from acetyl-CoA was introduced. The first plasmid was cloned using a pBbA5c-RFP vector (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). pBbA5c-RFP was treated with restriction enzymes EcoRI and BamHI and pBbA5c and RFP fragments were isolated by agarose gel electrophoresis. Only the purified pBbA5c vector was used for cloning. A DMAPP producing gene synthesized in the same manner was treated with the same restriction enzymes and then purified. The restriction enzyme-treated pBbA5c vector and DMAPP producing gene were transformed into E. coli by treating with ligase and then cloning was conducted. All the genes introduced into the plasmids were prepared by GenScriptR. Additionally, a strong pTrc promoter was introduced upstream of a mevalonate kinase (MK) gene. A total of four plasmids pM1, pM2, pM3 and pM4 were constructed.
A second plasmid was prepared as two types, one in which a myrcene synthase gene and a GPP synthase gene were introduced and the other in which only a myrcene synthase gene was introduced (Table 1). The second plasmid was cloned using a pBbE1a-RFP vector (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011 b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). The second plasmid was constructed in the same manner as the first plasmid. The constructed plasmids are shown in Table 2 and FIG. 3.
The constructed first plasmid and second plasmid were introduced into an Escherichia coli DH1 strain through transformation. The parent strain Escherichia coli DH1 was acquired from the Coli Genetic Stock Center (CGSC). A total of six myrcene-producing strains were prepared (Table 2).
Example 3 Production of Myrcene Using Transformed Escherichia coli Strain
The transformed Escherichia coli strain was pre-cultured in a Luria-Bertani (LB) medium for 24 hours and then cultured again in three media: 1. LB, 2. EZ-rich (Teknova, Hollister, Calif.), 3. M9-Mops (M9 salt, 75 mM MOPS, 2 mM MgSO4, 0.01 mM CaCl2, 1 mg/L thiamine HCl, 2.78 mg/L FeSO4, micronutrients: 3 nM ammonium molybdate, 0.4 M boric acid, 30 nM cobalt chloride, 23 nM cupric sulfate, 80 nM manganese chloride, 10 nM zinc sulfate). 1% glucose or 1% glycerol was supplied as a carbon source. After inoculating the strain to the three media and incubating for 4 hours in a shaking incubator at 37° C. at 200 rpm, enzymatic expression was induced by adding 100 μM IPTG (isopropyl β-D-1-thiogalactopyranoside) within an OD600 value range of 0.8-1. Then, myrcene was produced by covering the culture medium with 20% (w/v) of dodecane based on the volume of the culture medium. Myrcene production was measured 24 hours, 48 hours and 72 hours later. The amount of myrcene produced for each medium was measured for the cases when 1% glucose was used as a carbon source (FIG. 5) and when 1% glycerol was used (FIG. 6).
For quantitative analysis of the produced myrcene, comparative analysis was conducted with respect to standard myrcene by gas chromatography-mass spectrometry (GC-MS, Agilent 6890N series GC/TOF-MS (LECO)) (FIG. 4): injector temperature: 250° C., flow rate: 1.2 mL/min, split ratio=2:1, oven temperature: 60° C. for initial 5 minutes, raised at 4° C./min (to 240° C.), He gas used, HP-Ultra2 column: 25 m, 0.2 mm diameter, film thickness: 0.11 μm. All the reagents used in the experiment were acquired from Sigma-Aldrich.
Example 4 Confirmation of Dodecane Overlay Effect
The dodecane overlay method was used to increase recovery of the strongly volatile myrcene. Because dodecane is separated on top of the medium without being mixed with the medium, the myrcene which is evaporated as soon as it is produced can be extracted from the above dodecane layer. The dodecane overlay effect was compared using the strain (Ec-pM2/pM(Qi) and condition (1% glycerol, EZ-rich) that showed the highest productivity. As seen from FIG. 7, when dodecane overlay was not used, all the produced myrcene was evaporated and nothing remained.
Biological material was deposited under the terms of the Budapest Treaty, as described below.
Accession Numbers
Depositor: Korea Research Institute of Bioscience & Biotechnology
Accession number: KCTC12850BP
Date of accession: 20150623
Depositor: Korea Research Institute of Bioscience & Biotechnology
Accession number: KCTC12851BP
Date of accession: 20150623
The biological material was accepted by the depository, all restrictions on the availability to the public of the deposited material will be removed, and the viability of the deposits will be maintained for the duration of the patent term or for a period of twenty years in accordance with 37 CFR 1.805-1.807.

## Claims

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.