WO2015056813A1 - Method for producing isoprene using bacterium - Google Patents

Abstract

The present invention provides a bacterium which has been modified to overexpress the hepS and hepT genes, a method for producing isoprene by fermentation using the bacterium, and a method for producing an isoprene polymer by polymerization of the isoprene.

Description

DESCRIPTION

METHOD FOR PRODUCING ISOPRENE USING BACTERIUM

Background of the Invention

Field of the Invention

The present invention relates to the microbiological industry, and specifically to a method for producing isoprene by fermentation of a bacterium which has been modified to overexpress the hepS and hepT genes.

Description of the Related Art

Isoprene is the volatile monomer of natural rubber that is insoluble in water and soluble in many organic solvents such as, for example, alcohols. Most animals, plants, and bacteria naturally produce isoprene. Isoprene serves as a building-block for a vast variety of naturally occurring compounds, cooperatively termed the isoprenoids. In bacteria, isoprenoids are involved in numerous pivotal functions such as protein degradation and hormone-based signaling, some are structural components of membranes such as sterols, carotenoids, ubiquinone, and dolichols. Isoprene is an important organic compound used in a wide variety of industrial applications such as, for example, as a starting material or an intermediate in the synthesis of many chemical compounds and polymers including fine chemicals, elastomers, and synthetic hydrocarbons rubbers.

Naturally produced isoprene is commonly used for production of natural rubber. A method for improvement of productivity of rubber by plants, such as Hevea brasiliensis transformed by the geranylgeranyl diphosphate synthase-encoding genes, is known (U.S. patent Nos. 8,450,560 and 7,692,066). To reduce the environmental impact and increase the commercially available quantities of rubber, the renewable methods for producing isoprene are required to meet demands of the synthetic chemistry industry. Isoprene can be obtained by direct petroleum cracking to the C5 cracking fraction (K. Weissermel and H.J. Arpe, Industrial organic chemistry, 4^th ed., Wiley-VCH, 2003), or by oxidative dehydrogenation of isopentane and isoamylenes over oxide catalysts (Lisova N.N. et al., Effect of method of zinc-titanium catalyst preparation on its catalytic properties in oxidative dehydrogenation of isopentane, Nefiepererab. Neftekhim. {Kiev), 1984, 27, 15; Gusman T.Ya. et al., Oxidative dehydrogenation of isoamylenes on bismuth molybdates, Khim. Tekh. Topi. Masel, 1967, 12, 1) and zeolites (Okel'son I.I. and Kuznetsov A.V. Effect of cation nature on zeolite activity in the oxidative dehydrogenation of isopentane, Vopr. Kinetiki i Kataliza, 1973, (1), 68). The C5 isoprene skeleton can also be synthesized from smaller subunits such as, for example, isobutylene and formaldehyde (Sharf V.Z. et al., Production of isoprene from formaldehyde and isobutylene through 3-methylbutanediol- l,3, Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, 1965, 9: 1663- 1665). As bacteria are known to produce isoprene (Kuzma J. et al., Bacteria produce the volatile hydrocarbon isoprene, Curr. Microbiol, 1995, 30(2):97- 103), methods for production of isoprene using recombinant microorganisms are developed that utilize bacteria as producing hosts (see, for example, U.S. patent Nos. 5,849,970, 8,288, 148, and 8,361 ,762; or WO2012149491 A2).

Isoprene synthase of unknown amino acid sequence has been shown to catalyze formation of isoprene from 3,3-dimethylallyl diphosphate (DMAPP) in Bacillus subtilis (B. subtilis) (Sivy T.L. et al., Isoprene synthase activity parallels fluctuations of isoprene release during growth of Bacillus subtilis, Biochem. Biophys. Res. Commun., 2002, 294(l):71-75). Several attempts were undertaken to elucidate the isoprene synthase-encoding gene in B. subtilis. For example, conditional deletion of the yqiD gene [ispA), a homolog of the ispA gene from Escherichia coli (£. coft) encoding farnesyl diphosphate synthase (geranyltranstransferase), did not significantly change the isoprene emission (Julsing M.K. et al., Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis, Appl. Microbiol. Biotechnol. , 2007, 75(6): 1377- 1384). It has been even suggested that such an enzyme for isoprene production in B. subtilis might not exist, and isoprene might be a by-product of a reaction catalyzed by the nudF and yhfR gene products which consume DMAPP (Sivy T.L. et al., Evidence of isoprenoid precursor toxicity in Bacillus subtilis, Biosci. Biotechnol. Biochem., 2011, 75(12):2376-2383; Hess B.M. et al., Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics, PLoS One, 2013, 8(6):e66104). The role of heptaprenyl diphosphate synthase (HepS) was studied in B. subtilis. It was found that the increase of expression of the HepS gene results in a shift from isoprene production to geranyl diphosphate (GPP)/farnesyl diphosphate (FPP) production (Hess B.M. et al., PLoS One, 2013, 8(6):e66104).

Until now, no data has been reported demonstrating the influence of the hepS and hepT genes on isoprene production in a bacterium and the effect from overexpression of the hepS and hepT genes on isoprene production by the bacterium.

Brief Summary of the Invention

An aspect of the present invention is to provide a bacterium which has been modified to overexpress the hepS and hepT genes.

Another aspect of the present invention is to provide a method for producing isoprene using a bacterium as described hereinafter.

These aims were achieved by the unexpected findings that heptaprenyl diphosphate synthase comprising the component I (HepS) encoded by the hepS gene and the component II (HepT) encoded by the hepT gene is responsible, apart from the synthesis of heptaprenyl diphosphate, for isoprene production by a bacterium; and that overexpression of the hepS and hepT genes or increasing of an isoprene- synthesizing activity in a bacterium confers on the bacterium higher productivity of isoprene. These findings have resulted in the following non-limiting aspects of the present invention. An aspect of the present invention is to provide an isoprene- producing bacterium, wherein the bacterium has been modified to overexpress a DNA encoding an enzyme comprising a combination of the following (A) and (B):

(A) a protein selected from the group consisting of the proteins (A- l) to (A- 31:

(A- l) a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17;

(A-2) a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17, but which includes substitution, deletion, insertion, and/or addition of one or several amino acid residues and has activity of heptaprenyl diphosphate synthase with a protein of (B); and

(A-3) a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17, but which has an identity of amino acid sequence of not less than 50% (preferably, not less than 55%, not less than 60%, not less than 65%, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 95%, or not less than 98%) with respect to the entire amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17, and having activity of heptaprenyl diphosphate synthase with a protein of (B); and

(B) a protein selected from the group consisting of the proteins (B- l) to (B-3):

(B-l) a protein having the amino acid sequence of SEQ ID NO: 4,

SEQ ID NO: 15 or SEQ ID NO: 19;

(B-2) a protein having the amino acid sequence of SEQ ID NO: 4,

SEQ ID NO: 15 or SEQ ID NO: 19, but which includes substitution, deletion, insertion, and/ or addition of one or several amino acid residues and has activity of heptaprenyl diphosphate synthase with a protein of

(A); and (B-3) a protein having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 19, but which has an identity of amino acid sequence of not less than 50% (preferably, not less than 55%, not less than 60%, not less than 65%, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 95%, or not less than 98%) with respect to the entire amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 19, and having activity of heptaprenyl diphosphate synthase with a protein of (A); wherein the enzyme has an isoprene-synthesizing activity.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the enzyme is derived from a bacterium belonging to the genus Bacillus.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the enzyme is derived from a bacterium belonging to the species Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium harbors a DNA comprising a combination of the following (C) and (D):

(C) a DNA selected from the group consisting of the DNAs (C- l) to (C-3):

(C-l) a DNA comprising a hepS gene encoded by the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 12 or SEQ ID NO: 16;

(C-2) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 12 or SEQ ID NO: 16 encoding a protein as defined in the preceding (A); and

(C-3) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 12 or SEQ ID NO: 16 due to degeneracy of genetic code; and

(D) a DNA selected from the group consisting of the DNAs (D- l) to (D-3):

(D-l) a DNA comprising a hepT gene encoded by the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18; (D-2) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18 encoding a protein as defined in the preceding (B); and

(D-3) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18 due to degeneracy of genetic code.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium has been modified to overexpress the hepS and hepT genes.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the DNA is overexpressed by increasing a copy number of the DNA or modifying an expression control sequence of the DNA so that the expression of the DNA is enhanced as compared to a non-modified bacterium.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Bacillus or the family Enterobacteriaceae.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Bacillus, Escherichia or Pantoea.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Bacillus or the family Enterobactenaceae.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium is Bacillus subtilis, Escherichia coli or Pantoea ananatis.

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium has been modified to increase the isoprene-synthesizing activity of the enzyme encoded by the hepS and hepT genes.

An aspect of the present invention is to provide a method for producing isoprene comprising: (i) cultivating the bacterium as described above in a culture medium to produce the isoprene; and

(ii) collecting the isoprene from a medium.

An aspect of the present invention is to provide a method for producing an isoprene polymer comprising:

(i) cultivating the bacterium as described above in a culture medium to produce isoprene;

(ii) collecting the isoprene from a medium; and

(iii) polymerizing the isoprene to produce the isoprene polymer.

An aspect of the present invention is to provide a polymer derived from isoprene produced by the method as described above.

An aspect of the present invention is to provide a rubber composition comprising the polymer as described above.

An aspect of the present invention is to provide a tire manufactured by using the rubber composition as described above.

Industrial Applicability

According to the present invention, production of isoprene by fermentation using a bacterium and production of isoprene polymer by polymerization of the isoprene can be improved.

Brief Description of Drawings

FIG. 1 shows the scheme for construction of the artificial hepS- menH*-hepT operon.

FIG. 2 shows the results of GC-analysis for isoprene after

fermentation of MI-3 medium (A) or MI-3 medium with BS168-C (B) or BS168-4 (C), and a standard of isoprene in methanol (D) (0.1 mg/L).

FIG. 3 shows the chromatogram profiles (0 - 33 min) of samples from fermentation probes of BS168-C (A) and BS 168-4 (B).

FIG. 4 shows the chromatogram profiles (2 - 7 min) of samples from fermentation probes of BS 168-C (A) and BS 168-4 (B). FIG. 5 shows the chromatogram profiles (0 - 33 min) of samples from fermentation probes of BS 168-C (A) and BS 168-4 (B) resolved for the peaks with WM of 68 Da.

FIG. 6 shows data of the MS-analysis of the peak eluted at 3.05 min (A) (BS 168-4) and a standard of isoprene (B).

FIG. 7 shows kinetics (growth (A) and isoprene accumulation (B)) during fermentation in GC-vial of B. subtilis strains BS 168-C, BS 168-4, BS168-9, and BS 168- 10.

FIG. 8 shows the scheme for the downstream region of mevalonate pathway and its proximal region fixed on chromosome.

FIG. 9 shows the scheme for the downstream region of mevalonate pathway and its proximal region controlled by the tac promoter and fixed on chromosome.

FIG. 10 shows the map of pAH 162-Para-mvaES plasmid.

FIG. 11 shows the map of pAH 162-KKDyI-ispS(K) plasmid.

FIG. 12 shows the map of pAH162-Ptac-ispS(M)-mvk(Mma) plasmid. FIG. 13 shows the scheme of genome modifications of P. ananatis SC I 7(0): A) AampC:: «Lphi80-kan- iii?phi80, B) AampH:: iiLphi80-kan- af£Rphi80, C) Acrt::atiLphi80-kan- iii?phi80.

FIG. 14 shows the scheme for AampC::KKDyI-ispS(K) chromosome modification.

FIG. 15 shows the scheme for AampH::Para-mvaES chromosome modification.

FIG. 16 shows the scheme for Acrt::Ptac-ispS(M)-mvk(Mma) chromosome modification.

FIG. 17 shows the map of pAH162-Ptac vector.

FIG. 18 shows the map of pAH 162-Ptac-mvk(M. paludicola) plasmid.

FIG. 19 shows the nucleotide sequence of the hepS gene from B. subtilis (SEQ ID NO: l).

FIG. 20 shows the the amino acid sequence of the HepS protein encoded by the hepS gene from B. subtilis (SEQ ID NO:2). FIG. 21 shows the nucleotide sequence of the hepT gene from B. subtilis (SEQ ID NO: 3).

FIG. 22 shows the the amino acid sequence of the HepT protein encoded by the hepT gene from B. subtilis (SEQ ID NO:4).

FIG. 23 shows the nucleotide sequence of the hepS gene from B. licheniformis (SEQ ID NO : 12) .

FIG. 24 shows the the amino acid sequence of the HepS protein encoded by the hepS gene from B. licheniformis (SEQ ID NO: 13).

FIG. 25 shows the nucleotide sequence of the hepT gene from B. licheniformis (SEQ ID NO : 14) .

FIG. 26 shows the the amino acid sequence of the HepT protein encoded by the hepT gene from B. licheniformis (SEQ ID NO: 15).

FIG. 27 shows the nucleotide sequence of the hepS gene from B. amyloliquefaciens (SEQ ID NO: 16).

FIG. 28 shows the the amino acid sequence of the HepS protein encoded by the hepS gene from B. amyloliquefaciens (SEQ ID NO: 17).

FIG. 29 shows the nucleotide sequence of the hepT gene from B. amyloliquefaciens (SEQ ID NO: 18).

FIG. 30 shows the the amino acid sequence of the HepT protein encoded by the epT gene from B. amyloliquefaciens (SEQ ID NO: 19).

Detailed Description of the Invention

The present invention is described in detail below.

1. Bacterium

The phrase "isoprene-producing bacterium" can mean any bacterium which has an ability to produce, emit, and/or cause accumulation of isoprene in a medium when the bacterium is grown in the culture medium. Specific examples of an isoprene-producing bacterium include, but are not limited to, a bacterium belonging to the genus Bacillus or the family Enterobacteriaceae. As an isoprene- producing bacterium, any bacterium of the genus Bacillus or the family Enterobacteriaceae can be used as long as the bacterium has an ability to produce, emit, and/ or cause accumulation of isoprene in a medium when the bacterium is grown in the culture medium. Specific examples of a bacterium belonging to the family Enterobacteriaceae, such as a bacterium belonging to the genera Escherichia and Pantoea, or a bacterium belonging to the genus Bacillus are given hereinafter.

The phrase "isoprene-producing bacterium" can also mean a bacterium which is able to produce, emit, and/ or cause accumulation of isoprene in a medium in an amount larger than a wild-type or parental strain, such as the strain E. coli MG1655, P. ananatis SC17(0) or B. subtilis 168.

The phrase "isoprene-producing ability" is equivalent to the phrase "ability to produce isoprene" and can mean the ability of the bacterium to produce, emit, and/ or cause accumulation of isoprene in a medium to such a level that the isoprene can be collected from the medium, when the bacterium is grown in the culture medium.

The phrase "isoprene" refers to 2 -methyl- 1 ,3 -butadiene or isopentadiene (CH₂=C(CH₃)-CH=CH2, CAS No.78-79-5). It is a C5 hydrocarbon product which can be obtained by eliminating pyrophosphate from 3,3-dimethylallyl diphosphate (DMAPP). DMAPP can be produced from its isomer isopentenyl diphosphate (IPP, 3-methyl-3- butenyl pyrophosphate). DMAPP and IPP are structural isomers, intramolecular conversion of which is catalyzed by isopentenyl pyrophosphate delta-isomerase (Idi) encoded by the idi gene (ypgA). The phrase "isoprene" can mean the isoprene in a gaseous or liquid form, or a mixture thereof, under the conditions which are used in the method for producing isoprene as described herein. The phrase "isoprene" can mean the isoprene in a free form as a liquid and/ or gas, or in a dissolved form so long as the isoprene is soluble in a culture medium or medium, whereto the isoprene produced by the method is emitted, and/ or wherein the isoprene produced by the method is accumulated, and/ or wherefrom the isoprene produced by the method is collected. The phrase "culture medium" can mean a medium appropriate for growth of the bacterium and containing at least the nutrients, supplements, and water, which are required for fermentation of an isoprene-producing bacterium. The more detailed explanations as to the phrase "culture medium" are given hereinafter.

The phrase "medium" can mean an environment, whereto the isoprene produced by the modified bacterium is emitted, and/or wherein the isoprene produced by the method is accumulated, and/ or wherefrom the isoprene produced by the method is collected. Isoprene produced by the method can be emitted by the bacterium directly into the culture medium as a result of the naturally occurring efflux process, because it is known that, for example, bacteria of the genus Bacillus can synthesize and emit isoprene (Julsing M.K. et al., Appl. Microbiol. Biotechnol., 2007, 75(6): 1377- 1384). Furthermore, as the solubility of isoprene in water- containing media such as, for example, a culture medium may be limited under the fermentation conditions, the isoprene produced by the method can be emitted from the culture medium into an outside medium. Specifically, the isoprene produced by the method may be emitted from the culture medium into a gaseous phase such as, for example, a fermentation gas or off-gas stream, or absorbed by an absorbent material or condensed from the gaseous phase, and so forth, for the purpose of accumulating and collecting isoprene as described hereinafter. Therefore, the phrase "medium" can mean, but is not limited to, a culture medium and/or an outside medium, whereto the isoprene produced by the method is emitted, and/or wherein the isoprene produced by the method is accumulated, and/ or wherefrom the isoprene produced by the method is collected.

The bacterium that can be used (as a host cell) in the present invention may be a gram-positive bacterium or a gram-negative bacterium. Examples of the gram-positive bacterium may include bacteria belonging to the genus Bacillus, such as, for example, a bacterium belonging to the species Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens. Examples of the gram-negative bacterium may include bacteria belonging to the family Enter obacteriaceae, such as, for example, bacteria belonging to the genus Escherichia or Pantoea, specific examples of which include, but are not limited to, Escherichia coli and Pantoea ananatis.

Specific examples of the bacterium belonging to the genus Bacillus include those classified into the genus Bacillus according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http:/ /www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=U ndef&id= 1386&lvl=3&lin=f&keep= 1 &srchmode= 1 &unlock) . Exemplary bacterial species include B. subtilis subsp. subtilis strain 168 {B. subtilis 168, ATCC 23857) and B. amyloliquefaciens. B. amyloliquefaciens is a heterogenic species. A number of B. amyloliquefaciens strains are known, such as SB, T, P, W, F, N, K, and H (Welker N.E. and Campbell L.L., Unrelatedness of B. amyloliquefaciens and B. subtilis. J. Bacteriol., 1967, 94: 1124- 1130). Recently, Bacillus strains were isolated from plants, and these are usually considered as a distinct ecotype of B. amyloliquefaciens (Reva O.N. et at, Taxonomic characterization and plant colonizing abilities of some bacteria related to B. amyloliquefaciens and B. subtilis. FEMS Microbiol. Ecol, 2004, 48:249-259). The entire nucleotide sequence of one of the strains, B. amyloliquefaciens FZB42, has been reported (Chen X.H. et al., Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium B. amyloliquefaciens FZB42. Nat. Biotechnol, 2007, 25: 1007-1014). In addition, the complete genome sequence of the B. amyloliquefaciens DSM7 strain was recently determined as well (Ruckert C. et al., Genome sequence of B. amyloliquefaciens type strain DSM7(T) reveals differences to plant- associated B. amyloliquefaciens FZB42. J. Biotechnol, 201 1, 155:78-85). The B. subtilis strains such as, for example, B. subtilis 6051 (ATCC 6051), B. subtilis 23059 (ATCC 23059), and B. subtilis 23856 (ATCC 23856) are known to produce and emit isoprene in a high amount (Kuzma J. et al., Curr. Microbiol, 1995, 30(2) :97- 103). These strains are available from, for example, the American Type Culture Collection (ATCC; P.O. Box 1549, Manassas, VA 20108, USA). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to www.atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection. Examples of bacteria belonging to genus Bacillus can also include Bacillus licheniformis, Bacillus pumilis, Bacillus megaterium, Bacillus brevis, Bacillus (now classified as Paenibacillus) polymyxa, Bacillus stearothermophilus, and so forth.

Specific examples of the bacteria belonging to the family Enterobacteriaceae include those classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov/Taxonomy/ Browser/ wwwtax.cgi?id=543).

Examples of bacterial strains of the family Enterobacteriaceae that may be used include strains of the bacteria belonging to the genera Escherichia, Enterobacter, Pantoea, and so forth.

Strains of Escherichia bacterium which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used (Bachmann, B.J., Derivations and genotypes of some mutant derivatives of E. coli K- 12, p. 2460-2488. In F.C. Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2^nd ed. ASM Press, Washington, D.C., 1996). The species E. coli is a particular example. Specific examples of E. coli include E. coli W31 10 (ATCC 27325), E. coli MG1655 (ATCC 47076, ATCC 700926), and so forth, which are derived from the prototype wild-type strain, E. coli K- 12 strain. These strains are available from, for example, the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to www.atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.

Examples of the Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteria include Pantoea ananatis [P. ananatis), and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc. A bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae. When a Pantoea ananatis strain is bred by genetic engineering techniques, P. ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ 13601 strain (FERM BP-7207) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as P. ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above. Derivatives of the above mentioned P. ananatis strains include the P. ananatis SCI 7 strain (FERM BP- 11091) and SC17(0) strain (VKPM B-9246), and the like.

The bacterium of the present invention modified to overexpress the hepS and hepT genes, which is able to produce isoprene, can be used.

The bacterium may inherently have the isoprene-producing ability or may be modified to have an isoprene-producing ability by using a mutation method or DNA recombination techniques. The bacterium can be obtained by overexpressing the hepS and hepT genes in a bacterium, which inherently has the ability to produce isoprene. Alternatively, the bacterium can be obtained by imparting the ability to produce isoprene to a bacterium already having the hepS and hepT genes overexpressed.

The bacterium of the present invention has been modified to overexpress the hepS and hepT genes. It is also possible that the bacterium of the present invention has been modified to increase an isoprene-synthesizing activity of heptaprenyl diphosphate synthase encoded by the hepS and hepT genes. Equivalently, it is also possible that the bacterium of the present invention has been modified to increase an isoprene-synthesizing activity of isoprene synthase encoded by the hepS and hepT genes. Generally, it is also possible that the bacterium of the present invention has been modified to increase an isoprene-synthesizing activity of the HepST protein complex encoded by the hepS and hepT genes.

For the purposes of the present invention, explanations given hereinafter as to HepST protein complex can be applied mutatis mutandis to heptaprenyl diphosphate synthase and isoprene synthase encoded by the hepS and hepT genes.

The phrase "a bacterium modified to overexpress the hepS and hepT genes" can mean that the bacterium has been modified in such a way that in the modified bacterium the total enzymatic activity of the corresponding gene protein products such as HepS and HepT is increased as compared with a non-modified strain, for example, a wild- type or parental strain. As the heptaprenyl diphosphate synthase (HepST), which is a heterodimer of HepS (component I) encoded by hepS and HepT (component II) encoded by hepT, comprises protein products of the hepS and hepT genes, the phrase "a bacterium modified to overexpress the hepS and hepT genes" can also mean that the bacterium has been modified in such a way that in the modified bacterium the total enzymatic activity of the HepST protein complex encoded by the corresponding genes is increased as compared with that level in a non- modified strain. Examples of a non-modified strain serving as a reference for the above comparison can include a wild-type strain of a bacterium belonging to the genus Bacillus such as the strain B. subtilis 168. Another examples of a non-modified strain serving as a reference for the above comparison can include a wild-type strain belonging to the genus Escherichia or Pantoea such as E. coli W31 10 (ATCC 27325), E. coli MG1655 (ATCC 47076, ATCC 700926), P. ananatis AJ 13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207). The total enzymatic activity of the corresponding gene protein products such as HepS and HepT can be increased by increasing enzymatic activity of HepS and HepT proteins, or by increasing enzymatic activity of HepS or HepT so that the total enzymatic activity of the HepST protein complex comprising HepS and HepT is increased as compared with that level in a non-modified strain.

The phrase "a bacterium modified to overexpress the hepS and hepT genes" can mean that the bacterium has been modified in such a way that in the modified bacterium the total expression level of the hepS and hepT genes is higher than that level in a non-modified strain, for example, a wild-type or parental strain. The phrase "a bacterium modified to overexpress the hepS and hepT genes" can also mean that the total expression level of the hepS and hepT genes can be increased by increasing expression level of hepS and hepT genes, or by increasing expression level of hepS or hepT so that the total expression level of the hepS and hepT genes is increased as compared with that level in a non- modified strain. Therefore, the phrase "a bacterium modified to overexpress the hepS and hepT genes" may be equivalent to the phrase "a bacterium modified to overexpress at least one of the hepS and hepT genes". That is, the hepS gene, or the hepT gene, or the hepS and hepT genes may be overexpressed, so long as the total expression level of the genes is higher than that level in a non-modified strain.

The phrase "the hepS and hepT genes are overexpressed" can also mean that the expression level of the hepS gene, or the hepT gene, or the hepS and hepT genes is higher than that level in a non-modified strain. Therefore, the phrase "the hepS and hepT genes are overexpressed" is equivalent to the phrase "expression of the hepS and hepT genes is enhanced". Specifically, the phrase "a bacterium modified to overexpress the hepS and hepT genes" can mean that the bacterium has been modified in such a way that in the modified bacterium the hepS and/ or epT gene(s) are/ is overexpressed as described hereinafter. The phrase "the hepS and hepT genes are overexpressed" can be essentially applied to the phrase "an isoprene-synthesizing activity of the HepST protein complex is increased". That is, the phrase "the hepS and hepT genes are overexpressed" can mean that the total enzymatic activity of the HepS and HepT proteins or a HepST protein complex encoded by the corresponding genes is increased by, for example, introducing and/ or increasing the copy number of the hepS gene, or the hepT gene, or the hepS and hepT genes in bacterial genome, or increasing the activity per molecule (may be referred to as a specific activity) of the HepS protein, or the HepT protein, or the HepST protein complex encoded by hepS and hepT genes, as compared with a non-modified strain. The bacterium can be modified so that the activity of the HepS protein, or the HepT protein, or the HepST protein complex per cell is increased to 1 10% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, of the activity of a non-modified strain.

Activity of HepST protein complex can be determined by evaluating the activity of heptaprenyl diphosphate synthase, which can be used to determine a specific enzymatic activity of the HepST protein complex per mg.

Activity of heptaprenyl diphosphate synthase means activity of catalyzing the following reaction: (2£,6£)-farnesyl diphosphate + 4 isopentenyl diphosphate <→ 4 diphosphate + all-frans-heptaprenyl diphosphate.

In the modified bacterium, the heptaprenyl diphosphate synthase activity of HepST encoded by the hepS and hepT genes per mg crude protein or an amount of cells expressed as an optical density (OD) at a specific wavelength can be determined by measuring the amount of relabeled isopentenyl pyrophosphate incorporated into acid-labile allylic pyrophosphates (Takahashi I. et al., Heptaprenyl pyrophosphate synthetase from Bacillus subtilis, J. Biol. Chem., 1980, 255(10):4539- 4543). The specific heptaprenyl diphosphate synthase activity of HepS or HepT, or their combination such as HepS and HepT, can be determined by measuring activity of the entire HepST protein complex comprising HepS and HepT as described above. The crude protein concentration can be determined by the Bradford protein assay (Bradford M.M., Anal. Biochem., 1976, 72:248-254) using bovine serum albumin as a standard.

The phrase "enzyme" is generally apparent to the person skilled in the art and can mean any kinds of proteins, capable of catalyzing one or more chemical changes in one or more organic and/ or inorganic substances. Specifically, the phrase "enzyme" as used herein can mean, but is not limited to, a heptaprenyl diphosphate synthase (HepST) (EC: 2.5.1.30), isopentenyl pyrophosphate delta-isomerase (Idi) (EC: 5.3.3.2), and isoprene synthase (IspS) (EC: 4.2.3.27).

Activity of heptaprenyl diphosphate synthase is explained above. Activity of isopentenyl pyrophosphate delta-isomerase means activity of catalyzing the following reaction: isopentenyl diphosphate _*→ 3,3-dimethylallyl diphosphate (Reardon J.E. and Abeles R.H. Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate, Biochemistry, 1986, 25(19):5609-5616).

Activity of isoprene synthase means activity of catalyzing the following reaction: 3,3-dimethylallyl diphosphate <→ isoprene + diphosphate (Sivy T.L. et al., Biochem. Biophys. Res. Commun., 2002, 294(l):71-75).

The phrase "enzyme has an isoprene-synthesizing activity" can mean that a protein, such as an enzyme, has the activity of isoprene synthase. Specifically, the phrase "enzyme has an isoprene-synthesizing activity" can mean that the enzyme is capable of synthesizing the isoprene from a substrate such as, for example, 3,3-dimethylallyl diphosphate (DMAPP).

As the isoprene-producing bacterium has been modified to overexpress the hepS and hepT genes or to increase an isoprene- synthesizing activity of the enzyme encoded by these genes, activity of HepST protein complex can be also determined by evaluating the activity of isoprene synthase, which can be used to determine a specific enzymatic activity of the HepST protein complex per mg. In the modified bacterium, the isoprene-synthesizing activity of HepST encoded by the hepS and hepT genes per mg crude protein or an amount of cells expressed as an optical density (OD) at a specific wavelength can be determined by measuring the amount of isoprene produced from DMAPP using gas chromatography (GC) analysis (Sivy T.L. et ai., Biochem. Biophys. Res. Commun., 2002, 294(l):71-75). Exemplary, the isoprene- synthesizing activity can be determined as described in Experiment 4 of Example 2 hereinafter. The specific isoprene synthase activity of HepS or HepT, or their combination such as HepS and HepT, can be determined by measuring activity of the entire HepST protein complex comprising HepS and HepT as described above. The crude protein concentration can be determined by the Bradford protein assay (Bradford M.M., Anal. Biochem., 1976, 72:248-254) using bovine serum albumin as a standard.

The explanations given hereinafter and concerning methods which can be used to enhance expression of the hepS gene can also be applied mutatis mutandis to the hepT gene or a combination of the hepS and hepT genes.

Methods which can be used to enhance expression of the hepS gene include, but are not limited to, increasing the hepS gene copy number in bacterial genome (in the chromosome and/ or in the autonomously replicated plasmid) and /or introducing the hepS gene into a vector that is able to increase the copy number and/ or the expression level of the hepS gene in a bacterium of the genus Bacillus according to genetic engineering methods known to the person skilled in the art. Vectors which can be used include E. coli - B. subtilis shuttle vectors such as pHY300PLK, pMWMXl, pLF22, pKSl, pGK12, pLF14, pLF22, or the like, phage vectors such as 11059, IBF101 , M 13mp9, Mu phage (Japanese patent application, publication No. 2-109985), or the like, plasmid-based expression vectors (Nguyen H.D. et al., Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability, Plasmid, 2005, 54(3):241-248), or the like. Methods which can be used to enhance expression of the hepS and/ or hepT gene in in bacteria belonging to the family Enterobacteriaceae, vector autonomously replicable in Enterobacteriaceae can be used. Specific examples of vector autonomously replicable in bacteria belonging to the family Enterobacteriaceae such as Escherichia coli, Pantoea ananatis include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pATV28, pSTV29 (all of these are available from Takara Bio), pACYC184, pMW218, pMW219 (NIPPON GENE), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (QIAGEN), and broad host-range vector RSF1010.

Enhancing of the hepS gene expression can also be achieved by increasing the expression level of the hepS gene by modification of adjacent regulatory regions of the hepS gene or introducing native and/ or modified foreign regulatory regions. Regulatory regions or sequences can be exemplified by promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements (e.g., regions to which repressors or inducers bind and/ or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA). Such regulatory regions are described, for example, in Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2^nd ed., Cold Spring Harbor Laboratory Press (1989). Modifications of regions controlling gene expression can be combined with increasing the copy number of the hepS gene in bacterial genome.

The exemplary promoters enhancing the hepS gene expression can be the potent promoters. For example, the Pspac, Pgrac, PrplU, PrepAB from plF22, PgsiB, and Ppur promoters, T7 promoter, trp promoter, lac promoter, tac promoter, thr promoter, trc promoter, tet promoter, PR promoter, and PL promoter are known to be potent promoters. Potent promoters providing a high level of gene expression in a bacterium can be used. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of the hepS gene to strengthen the promoter function, thus resulting in the increased transcription level of the hepS gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/ or in the spacer between the SD sequence and the start codon, and/ or a sequence immediately upstream and/ or downstream from the start codon in the ribosome-binding site greatly affects the translation efficiency of mRNA. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol, 1981 , 35:365-403; Hui A. et al., EMBO J., 1984, 3:623-629).

The following method can be employed to introduce a site- specific mutation into chromosome of a isoprene-producing bacterium. Firstly, the delivery plasmid carrying a site-directed mutation is constructed and then transformed into the targeted isoprene-producing bacterium. Secondly, the two-step replacement recombination procedure is performed in the resulting transformants to incorporate a site-specific mutation into the chromosome by gene substitution. To transfer the delivery plasmid into naturally non-transformable isoprene-producing bacterium, an intermediate host isoprene-producing bacterium harboring the delivery plasmid can be obtained by transformation. Then, a bacterial phage propagated on the obtained transformants is used for transduction of the delivery plasmid into the targeted bacterium. This method is suitable for introducing marker-free deletions, insertions, point mutations, and so forth into the chromosomes of isoprene-producing bacterium strains (Zakataeva N.P. et al., Appl. Microbiol. Biotechnol., 2010, 85(4): 1201-1209). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid which is unable to replicate in the host.

The copy number, presence or absence of the gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), or mass spectrometry analysis of the protein samples, and the like.

Methods for manipulation with recombinant molecules of DNA and molecular cloning such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well- known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2^nd ed., Cold Spring Harbor Laboratory Press (1989) or Green M.R. and Sambrook J.R., "Molecular Cloning: A Laboratory Manual", 4^th ed., Cold Spring Harbor Laboratory Press (2012); Glick B.R., Pasternak J.J. and Patten C.L., "Molecular Biotechnology: principles and applications of recombinant DNA", 4^th ed., Washington, DC, ASM Press (2009).

The hepS gene (synonyms: gerCl, gerCA, hepA) encodes the heptaprenyl diphosphate synthase component I, HepS (Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P31 1 12). The hepS gene (GenBank accession No. NC_000964.3; nucleotide positions: 2383615 to 2384370, complement; Gene ID: 938998) is located between the mtrB and ubiE genes on the same strand on the chromosome of B. subtilis subsp. subtilis strain 168. The nucleotide sequence of the hepS gene from B. subtilis and the amino acid sequence of the HepS protein encoded by the hepS gene from B. subtilis are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The nucleotide sequence of the hepS gene from B. licheniformis and the amino acid sequence of the HepS protein encoded by the hepS gene from B. licheniformis are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The nucleotide sequence of the hepS gene from B. amyloliquefaciens and the amino acid sequence of the HepS protein encoded by the hepS gene from B. amyloliquefaciens are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

The hepT gene (synonyms: gerC3, gerCC, hepB) encodes the heptaprenyl diphosphate synthase component II, HepT (Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P31 1 14). The hepT gene (GenBank accession No. NC_000964.3; nucleotide positions: 2381919 to 2382965, complement; Gene ID: 939002) is located between the ubiE and ndk genes on the same strand on the chromosome of B. subtilis subsp. subtilis strain 168. The nucleotide sequence of the hepT gene from B. subtilis and the amino acid sequence of the HepT protein encoded by the hepT gene from B. subtilis are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The nucleotide sequence of the hepT gene from B. licheniformis and the amino acid sequence of the HepT protein encoded by the hepT gene from B. licheniformis are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The nucleotide sequence of the hepT gene from B. amyloliquefaciens and the amino acid sequence of the HepT protein encoded by the hepT gene from B. amyloliquefaciens are shown in SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

The homology of nucleotide sequences of the hepS and hepT genes amino acid sequences of heptaprenyl diphosphate synthases HepS and HepT encoded by these genes derived from bacteria belonging to the genus Bacillus is shown in Table 1, which homology was determined as the percent of identity using the computer program BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/). Table 1.

Since there may be some differences in DNA sequences between the bacterial species or strains, the hepS and hepT genes are not limited to the genes shown in SEQ ID NOs: 1 and 3, SEQ ID NOs: 12 and 14, and SEQ ID NOs: 16 and 18, respectively, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1 and 3, SEQ ID NOs: 12 and 14, and SEQ ID NOs: 16 and 18, and which encode variants of the HepS and HepT proteins, respectively.

The phrase "a variant protein" can mean a protein which has one or several changes in the sequence compared with SEQ ID NO: 2 or 4, SEQ ID NO: 13 or 15, or SEQ ID NO: 17 or 19, whether they are substitutions, deletions, insertions, and/ or additions of one or several amino acid residues, but still maintains an activity or function similar to that of the HepS or HepT protein, respectively, or the three-dimensional structure of the HepS or HepT protein is not significantly changed relative to the wild-type or non-modified protein. The number of changes in the variant protein depends on the position or the type of amino acid residues in the three-dimensional structure of the protein. It can be, but is not strictly, limited to, 1 to 60, in another example 1 to 30, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2 or 4, SEQ ID NO: 13 or 15, or SEQ ID NO: 17 or 19. This is because some amino acids have high homology to one another so that the activity or function is not affected by such a change, or the three-dimensional structure of HepS or HepT protein is not significantly changed relative to the wild-type or non-modified protein. Therefore, the protein variants encoded by the hepS and hepT genes may have a homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 50%, not less than 55%, not less than 60%, not less than 65%, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 95%, or not less than 98% with respect to the entire amino acid sequence shown in SEQ ID NO: 2 or 4, SEQ ID NO: 13 or 15, or SEQ ID NO: 17 or 19, respectively, as long as the activity or function of the HepS and HepT proteins is maintained, or the three-dimensional structure of the HepS and HepT is not significantly changed relative to the wild-type or non- modified proteins.

The exemplary substitution, deletion, insertion, and/ or addition of one or several amino acid residues can be a conservative mutation(s). The representative conservative mutation is a conservative substitution. The conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, lie and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gin, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gin and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution Asn, Glu, Lys, His, Asp or Arg for Gin, substitution Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for lie, substitution of He, Met, Val or Phe for Leu, substitution Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He or Leu for Val.

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/ are compensated by one or more secondary mutations in the different position(s) of amino acids sequence so that the activity or function of the variant protein is maintained and similar to that of the HepS or HepT protein, or the three- dimensional structure of HepS and HepT is not significantly changed relative to the wild-type or non-modified protein.

To evaluate the degree of protein or DNA homology, several calculation methods can be used, such as BLAST search, FASTA search and ClustalW method. The BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin S. and Altschul S.F. (Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes, Proc. Natl. Acad. Sci. USA, 1990, 87:2264-2268; Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877). The computer program BLAST calculates three parameters: score, identity and similarity. The FASTA search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods Enzymol, 1990, 183:63-98). The ClustalW method is described by Thompson J.D. et al. (CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice, Nucleic Acids Res., 1994, 22:4673-4680).

Moreover, the hepS and hepT genes can be variant nucleotide sequences. The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes "a variant protein" using any synonymous amino acid codons according to the standard genetic code table (see, e.g., Lewin B., "Genes VIIF, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Therefore, the hepS and hepT genes can be variant nucleotide sequences due to degeneracy of genetic code.

The phrase "a variant nucleotide sequence" can also mean, but is not limited to, a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1 or 3, SEQ ID NO: 12 or 14, or SEQ ID NO: 16 or 18, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it encodes active or functional protein. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology, defined as the parameter "identity" when using the computer program BLAST, of not less than 65%, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% is formed, and a nonspecific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of 1 ^XSSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate), or in another example, O. l xSSC, 0.1% SDS at 60°C or 65°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham Hybond™-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. Alternatively, the stringent conditions may include hybridization in 6*SCC at about 45°C followed by one or two or more washings in 0.2xSCC and 0.1% SDS at 50 to 65°C. As the probe, a part of the sequences complementary to the sequences shown in SEQ ID NO: 1 or 3, SEQ ID NO: 12 or 14, or SEQ ID NO: 16 or 18 may also be used. Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequences shown in SEQ ID NO: 1 or 3, SEQ ID NO: 12 or 14, or SEQ ID NO: 16 or 18, and a DNA-fragment containing the nucleotide sequence as template. The length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp. For example, when a DNA-fragment having a length of about 300 bp is used as the probe, the washing conditions after hybridization can be exemplified by 2xSSC, 0.1% SDS at 50°C, 60°C or 65°C.

As the genes encoding the HepS and HepT proteins of the species B. subtilis, B. licheniformis and B. amyloliquefaciens have already been elucidated (see above), the variant nucleotide sequences encoding variant proteins of HepS and HepT proteins can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., The polymerase chain reaction, Trends Genet, 1989, 5: 185- 189) utilizing primers prepared based on the nucleotide sequences of the hepS or hepT gene; or the site-directed mutagenesis method by treating DNA containing the wild-type hepS or hepT gene in vitro, for example, with hydroxy lamine, or a method for treating a bacterium, for example, a bacterium harboring the wild-type hepS and hepT genes with ultraviolet (UV) irradiation or a mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the such treatment; or chemically synthesized as full- length gene structures. Genes encoding the HepS and HepT proteins or its variant proteins of other bacteria can be obtained in a similar manner.

Furthermore, as amino acid sequences of the HepS and HepT proteins, or variant proteins thereof, and nucleotide sequences of the hepS and hepT genes, or variant nucleotide sequences thereof, are elucidated, the phrase "derived from" as to the HepS and HepT proteins, such as in the phrase "enzyme is derived from a bacterium belonging to the genus Bacillus" or "enzyme is derived from a bacterium belonging to the species Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens", can refer to the amino acid sequence shown in SEQ ID NO: 2 or 4, SEQ ID NO: 13 or 15, or SEQ ID NO: 17 or 19, respectively, or variant proteins thereof as explained hereinabove. Yet furthermore, the phrase "derived from" as to the hepS and hepT genes, such as in the phrase "gene is derived from a bacterium belonging to the genus Bacillus" or "enzyme is derived from a bacterium belonging to the species Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens", can refer to the nucleotide sequence shown in SEQ ID NO: 1 or 3, SEQ ID NO: 12 or 14, or SEQ ID NO: 16 or 18, respectively, or variant nucleotide sequences thereof as explained hereinabove. For example, the phrase "enzyme is derived from a bacterium belonging to the genus Bacillus" can refer to the amino acid sequence shown in SEQ ID NO: 2 or 4, SEQ ID NO: 13 or 15, or SEQ ID NO: 17 or 19, encoded by the nucleotide sequence shown in SEQ ID NO: 1 or 3, SEQ ID NO: 12 or 14, or SEQ ID NO: 16 or 18, respectively. Specifically, the phrase "enzyme in derived from a bacterium belonging to the genus Bacillus" can mean the amino acid sequence of the enzyme encoded by the nucleotide sequence, which is a part of the native or wild-type genomic DNA of the bacterium belonging to the genus Bacillus.

The phrase "a wild-type protein" can mean a native protein naturally produced by a wild-type or parent bacterial strain of the genus Bacillus, for example, by the wild-type B. subtilis 168 strain. A wild-type protein can be encoded by the "wild-type gene", or the "non-modified gene" naturally occurring in genome of a wild-type bacterium.

The bacterium as described herein can be obtained by introducing the aforementioned DNAs into a bacterium inherently having an ability to produce an isoprene. Alternatively, the bacterium as described herein can be obtained by imparting the ability to produce an isoprene to a bacterium already harboring the aforementioned DNAs.

The bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence, without departing from the scope of the present invention.

2. Method

2-1. Method for producing isoprene

The method for producing isoprene includes the steps of cultivating the bacterium in a culture medium to allow the isoprene to be produced, emitted, and accumulated in the medium, and collecting the isoprene from the medium.

The cultivation of an isoprene-producing bacterium in the culture medium and collecting isoprene from the medium may be performed in a manner similar to conventional fermentation, wherein isoprene is produced using a bacterium. The culture medium for production of the isoprene can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, inorganic ions, and other organic and inorganic components as required. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soy bean hydrolyzates; ammonia gas; aqueous ammonia; and the like can be used. The sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like. Vitamins such as vitamin Bl , required substances, for example, organic nutrients such as nucleic acids and amino acids, or yeast extract or tryptone, and the like may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, iron ions, manganese ions, and the like may be added, if necessary. The culture medium can also be supplemented with selective agents, such as antibiotics, to select for the cells maintaining certain genetic constructs such as plasmids, vectors, and the like.

Cultivation can be performed under aerobic conditions for 16 to 72 hours, the culture temperature during cultivation is controlled within 28 to 45°C, or within 32 to 37°C, and the pH is adjusted between 5 and 8, or between 5.5 and 6.5. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.

The produced isoprene can be recovered from the medium in a gaseous or liquid form by any conventional techniques, which are routinely used to isolate gas or separate gas from liquid. Such techniques include, but are not limited to, gas stripping, membrane separation, absorption, fractionation, adsorption/ desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent, and so forth. Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner using, for example, adsorption to a solid phase, partition into a liquid phase, or direct condensation. Membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene can also be used. If necessary, the isoprene is compressed and condensed. Isoprene can be recovered using the extractive distillation with a lower alkyl alcohol such as methanol, ethanol, propanol, or a mixture thereof.

The recovery of isoprene may be performed in one step or multiple steps. For example, the removal of isoprene vapor from the fermentation off-gas stream and the conversion of isoprene to a liquid phase can be performed simultaneously. Specifically, isoprene is directly condensed from the off- gas stream to form a liquid. Alternatively, the isoprene vapor can be removed from the fermentation off-gas stream and then converted into a liquid phase. It is also possible to adsorb isoprene from the off-gas stream to a solid phase and then desorb from the solid phase or extract with a solvent.

Furthermore, the isoprene produced by the method of the present invention can be purified from one or more components (such as solid, liquid, and gaseous admixtures) using conventional techniques such as filtration, distillation from a solution in a liquid extractant, and chromatography .

A portion of the fermentation gas or off-gas stream, remaining after the removal of isoprene, can be recuperated by introducing into a fermentation vessel used for the production of isoprene.

2-2. Method for producing an isoprene polymer

The method for producing an isoprene polymer includes steps of producing isoprene, and a step of polymerizing the isoprene to produce the isoprene polymer. The steps of producing isoprene can be performed in the same manner as in the method for producing isoprene as described above. The polymerization of the isoprene can be performed by any methods known in the art including chemical synthesis methods such as addition polymerization.

2-3. Method for producing a rubber composition

The rubber composition of the present invention comprises a polymer derived from isoprene produced by the method for producing isoprene according to the present invention. The polymer derived from isoprene may be a homopolymer (i.e., isoprene polymer) or a heteropolymer comprising isoprene and one or more monomer units other than the isoprene (e.g., a copolymer such as a block copolymer). Preferably, the polymer derived from isoprene is a homopolymer (i.e., isoprene polymer) produced by the method for producing isoprene polymer according to the present invention. The rubber composition of the present invention may further comprise one or more polymers other than the above polymer, one or more rubber components, and/ or other components. The rubber composition of the present invention can be manufactured using the polymer derived from isoprene. For example, the rubber composition of the present invention can be prepared by mixing the polymer derived from isoprene with one or more polymers other than the above polymer, one or more rubber components, and/ or other components such as a reinforcing filler, a crosslinking agent, a vulcanization accelerator and an antioxidant.

2-4. Method for producing a tire

The tire of the present invention is manufactured by using the rubber composition of the present invention. The rubber composition of the present invention may be applied to any portion of the tire without limitation, which may be selected as appropriate depending on the application thereof. For example, the rubber composition of the present invention may be used in a tread, a base tread, a sidewall, a side reinforcing rubber and a bead filler of a tire. The tire can be manufactured by a conventional method. For example, a carcass layer, a belt layer, a tread layer, which are composed of unvulcanized rubber, and other members used for the production of usual tires are successively laminated on a tire molding drum, then the drum is withdrawn to obtain a green tire. Thereafter, the green tire is heated and vulcanized in accordance with an ordinary method, to thereby obtain a desired tire (e.g., a pneumatic tire).

Examples

The present invention will be more specifically explained below with reference to the following non-limiting Examples.

Example 1. Cloning of the hepS-menH-hepT operon from B. subtilis into a plasmid vector The hepS and hepT genes were overexpressed in B. subtilis 168 by cloning the native hepS-menH-hepT operon and the artificial hepS- menH*-hepT operon having inactivated the menH gene into a plasmid vector pMWALl.

A DNA-fragment I was amplified by PCR using primers PI (SEQ ID

NO: 5) and P2 (SEQ ID NO: 6), and chromosome of B. subtilis 168 (ATCC 23857) as the template, which was isolated and purified using GenElute Bacterial Genomic DNA Kit (Sigma, USA, catalog No. NA21 10). The PCR- mixture I of a total volume of 50 pL contained 5 pL lOxPfu-Buffer supplemented with MgSO4 (Fermentas, Lithuania), 5 pL dNTPs solution (2 mM each, Fermentas, Lithuania), 1 pL DMSO (Sigma, USA, catalog No. D8418), primers PI and P2 (20 pmol each), 50 ng chromosomal DNA, and 1 pL Pfu DNA-polymerase (Fermentas, Lithuania, catalog No. EP0561). PCR-protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/45°C 30 sec/72°C 6 min, 5 cycles; 94°C 30 sec/55°C 30 sec/72°C 6 min, 25 cycles; 72°C 10 min, 1 cycle.

Resulting DNA-fragment I (2,750 bp) was purified using QIAquick GelExtraction Kit (Qiagen, Germany, catalog No. 28704), digested by BamHl and EcoRl (Fermentas, Lithuania) to obtain a BamHI-EcoRI DNA- fragment (2,742 bp) of SEQ ID NO: 26, and ligated with pM WAL 1 / BamHl- EcoRl vector. Thus the plasmid pMWALl-hepS-menH-hepT was constructed (Table 2). Construction of the pMWALl vector (SEQ ID NO: 9) is described in Auxiliary example 1. The obtained ligation mixture was introduced into TGI strain (Stratagene, La Jolla, CA, USA, catalog No. 200123) using standard calcium-dependent transformation. Colonies resistant to ampicillin (Ap^R-colonies) were selected. A plasmid DNA from a dozen of Ap^R-colonies was isolated and its structure was verified by the restriction and sequence analyses.

The plasmid harboring hep S- menH- hepT operon placed under the control of the Prep promoter was constructed using the following procedure. The 2,538 bp DNA-fragment II was amplified by PCR using primers P2 (SEQ ID NO: 6) and P4 (SEQ ID NO: 8) and the PCR-protocol as described above for obtaining the DNA-fragment I, digested by BamHl and EcoRl to obtain a BamHI-EcoRI DNA-fragment (2,530 bp) of SEQ ID NO: 27, and cloned into pMWALl -Prep/ BamHI-EcoRI vector. Thus the plasmid pMWALl-Prep-hepS-menH-hepT plasmid was constructed (Table 2). Construction of the pMWALl-Prep vector is described in Auxiliary example 2. The obtained ligation mixture was introduced into TGI strain using standard calcium-dependent transformation. Colonies resistant to ampicillin (Ap^R-colonies) were selected. A plasmid DNA from a dozen of Ap^R-colonies was isolated and its structure was verified by the restriction and sequence analyses.

An artificial operon having inactivated the menH gene was constructed using the following procedure (FIG. 1). Firstly, a DNA- fragment III was amplified by PCR using the primer P3 (SEQ ID NO: 7), which contains two parts homologous to the N- and C-end-located segments of the structural part of menH, and the primer P4 (SEQ ID NO: 8). The structure of P3 allows the in-frame shortening of the menH gene and maintaining the intergenic translational coupling of hepS and hepT in the operon. The PCR-mixture II of a total volume of 50 iL contained 5iL lOxPfu-Buffer supplemented with MgSO4, 5 dNTPs solution (2 mM each), 1 pL DMSO, primers P3 and P4 (20 pmol each), 25 ng pMWALl- hepS-menH-hepT plasmid DNA, and 1 iL Pfu DNA-polymerase. PCR- protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/50°C 30 sec/72°C 3 min, 25 cycles; 72°C 5 min, 1 cycle.

Secondly, the unidirect PCR was performed using the minus chain (- chain) of the DNA-fragment III for amplification to obtain the DNA- fragment IV. The PCR-mixture III of a total volume of 50 contained 5 yL lOxPfu-Buffer supplemented with MgSO₄, 5 xh dNTPs solution (2 mM each), 1 L DMSO, 10 iL of the PCR-mixture II subjected to PCR as described above, 100 ng pMWALl-hepS-menH-hepT plasmid DNA, and 1xL Pfu DNA-polymerase. PCR-protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/50°C 30 sec/72°C 3 min, 25 cycles; 72°C 7 min, 1 cycle. Thirdly, the target DNA-fragment IV was amplified by PCR using primers PI (SEQ ID NO: 5) and P4 (SEQ ID NO: 8). The PCR-mixture IV of a total volume of 100 μί, contained 10 iL lOxPfu-Buffer supplemented with MgSO₄, 10 ]iL dNTPs solution (2 mM each), 2 pL DMSO, primers PI and P4 (100 pmol each), 10 \xL of the PCR-mixture III subjected to PCR as described above, and 1 L Pfu DNA-poiymerase. PCR-protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/50°C 30 sec/72°C 3 min, 25 cycles; 72°C 5 min, 1 cycle.

The obtained DNA-fragment IV (2,348 bp) hepS-menH*-hepT was purified using QIAquick GelExtraction Kit, digested by BamHl and EcoRl to obtain a BamHI-EcoRI DNA-fragment (2,340 bp) of SEQ ID NO: 28, and ligated with pMWALl / BamHl- EcoRl vector. Thus the plasmid pMWALl-hepS-menH*-hepT was constructed (Table 2). The obtained ligation mixture was introduced into TGI strain using standard calcium- dependent transformation. Colonies resistant to ampicillin (Ap^R-colonies) were selected. A plasmid DNA from a dozen of Ap^R-colonies was isolated and its structure was verified by the restriction and sequence analyses.

able 2.

Example 2. Production of isoprene by B. subtilis having overexpressed the hepS and hepT genes from B. subtilis

Experiment 1.

The control vector pMWAL- 1 and plasmids pMWALl-hepS-menH- hepT, pMWALl-Prep-hepS-menH-hepT and pMWALl-hepS-menH*-hepT (Example 1) were each introduced into B. subtilis 168 using electroporation (Eppendorf AG, Germany; Protocol No. 4308 915.504 - 08/2003). Thus the B. subtilis strains BS 168-C, BS 168-4, BS 168-9 and BS 168- 10 were obtained which contained the plasmids pMWAL- 1 , pMWALl-hepS-menH-hepT, pMWALl-Prep-hepS-menH-hepT and pMWALl-hepS-menH*-hepT, respectively (Table 2). The strains were each grown at 37°C in LB-medium (also referred to as lysogenic broth or Luria- Bertani medium as described in Sambrook, J. and Russell, D.W. «Molecular Cloning: A Laboratory Manual», 3^rd ed., Cold Spring Harbor Laboratory Press (2001)) supplemented with chloramphenicol (Cm, 5 g/mL) to OD595 of about 2. Glycerol was added to the obtained cells cultures in amount of 20% (v/v). Thus obtained cells were aliquoted ( 150jiL in 1.5 mL Eppendorf vials) and stored at -70°C.

An aliquot of cells (25 μί) was inoculated into 1 mL of cultivation medium MI-3 (tryptone 10 g/L, yeast extract 20 g/L) supplemented with Cm (5 μg/mL). Cells were cultivated in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/PTFE molded septa (Macherey-Nagel, catalog No. 70234.9) at 37°C with shaking (250 rpm) for 16 hours. Cultivation of each strain was repeated six times. The fermentation gas was subjected to GC-analysis using the following procedure as described hereinafter. A calibration curve (linear regression, R² = 0.9983, n = 7, range of isoprene concentration from 0 to 500 μg/L; Auxiliary example 3) was used for evaluation of an amount of isoprene in fermentation probes.

GC isoprene analysis:

Gas chromatography (GC) system: GC-2014 (Shimadzu, Japan) equipped with HTA HT200H Headspace GC Autosampler. GC-Column: Rxi- lms 30 m, 0.53 mm ID (Internal Diameter), 1.5 μιη (Restek, catalog No. 13370)

Temperature of column: 37°C for 15 min

Temperature of split: 250°C

Temperature of FID (Flame Ionization Detector): 250°C

Split ratio: 5

Pressure: 64.7 kPa

Total flow: 105.1 mL/min

Column flow: 17.02 mL/min

Sampler volume: 0.4 mL

Incubation time: 13 min at 37°C

The chromatogram profiles of an isoprene standard in methanol (D), and a fermentation gas from the culture medium MI-3 (A), BS168-C (B) and BS 168-4 (C) are shown on FIG. 2.

As one can see from Table 3, the modified B. subtilis strains

BS 168-4, BS 168-9 and BS168- 10 having overexpressed the hepS and hepT genes accumulated a higher amount of isoprene than the control BS168-C strain. The ANOVA, Tukey-Kramer Multiple Comparisons Test (GraphPad Instat, USA) showed the statistically significant difference between the control BS168-C and tested strains BS168-4, BS 168-9 or BS 168- 10 (for each strain: sample size = 6; normality test is passed, P < 0.001), and did not show the statistically significant difference between the tested strains BS 168-4, BS 168-9, and BS 168- 10 (P > 0.05). Table 3.

Strain OD595 Isoprene, g/L

BS168-C (control) 3.7 ± 0.1 43.9 ± 3.4

BS168-4 2.7 ± 0.3 60.2 ± 4.9

BS 168-9 3.4 ± 0.1 58.1 ± 2.7

BS168- 10 3.3 ± 0.2 58.7 ± 2.8 Experiment 2.

A production of isoprene was also determined using GC/MS- analysis. The fermentation probes of BS 168-4 and BS 168-9 were obtained as described in Experiment 1, but the MI-3 medium (tryptone 10 g/L, yeast extract 20 g/L), used for fermentation, was supplemented with Cm in amount of 10 g/mL. Cells were cultivated in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/PTFE molded septa (Macherey- Nagel, catalog No. 70234.9) at 37°C with shaking (250 rpm) for 24 hours. Cultivation of each strain was repeated ten times.

Fermentation gases from two GC-vials of BS 168-4 were analyzed as described in Experiment 1. As one can see from Table 4, the modified B. subtilis strain BS 168-4 having overexpressed the hepS and hepT genes and analyzed using gas chromatography (GC) as described in Experiment 1 accumulated a higher amount of isoprene than the control BS 168-C strain.

Fermentation gases from eight GC-vials of BS 168-4 were analyzed using GC/MS. The solid adsorption and thermal desorption system was used for GC/MS-analysis. A sample tube was filled with an adsorption material (Carbopack Z 60/80, CAMSCO, USA). Then, a head space gas of eight GC-vials was aspirated by a vacuum pump, and organic molecules were adsorbed to the adsorption material and analyzed for isoprene using GC/MS.

GC/MS isoprene analysis:

Thermal desorption gas chromatograph-mass spectrometer (GC/MS) system: gas chromatograph GC6890N (Agilent Technologies, USA), mass spectrometer JMS-KP (JEOL Ltd., Japan), and thermal desorption autosampler Unity 2-Ultra (Markes, England)

Adsorbent: Carbopack Z 60/80 (CAMSCO, USA)

GC-column: DB-lms 60 m, 0.25 mm ID, 0.25 m (Agilent Technologies, USA, catalog No. 122-0162)

Conditions for thermal desorption: 8 min at 300°C T (column): 40°C for 7 min, then 120°C with 10°C/min, then 270°C with 20°C/min, and then 270°C for 10 min.

The results of GC/MS-analysis are shown on FIGs. 3-6. FIG. 3 shows the chromatogram profiles obtained for samples from fermentation probes of BS 168-C (A), BS 168-4 (B), and BS 168-9 (C) in a range of 0 - 33 min. FIG. 4 shows the chromatogram profiles obtained for the same samples within the range from 2 to 7 min. FIG. 5 shows the chromatogram profiles obtained for samples from fermentation probes of BS168-C (A), BS 168-4 (B), and BS 168-9 (C) and resolved for the peaks with a molecular weight (MW) of 68 Da. As one can see from the FIG. 5, the major peak having MW of 68 Da is eluted at 3.05 min (peak No. 3 on FIG. 4). FIG. 6 shows data of the MS-analysis of the peak from a fermentation probe of BS168-C (or BS 168-4, or BS168-9) eluted at 3.05 min (A), and its comparison with a reference spectrum for a standard of isoprene (B). As one can see from the FIG. 6, the peak having MW of 68 Da and eluted at 3.05 min corresponds to the isoprene.

Table 4.

Experiment 3.

The kinetics of isoprene synthesis in BS 168-C, BS 168-4, BS168-9, and BS 168-10 was investigated. For this purpose, 25 ]iL of frozen cells stock (see Experiment 1) was inoculated into 5 mL of MI-3 broth (tryptone 10 g/L, yeast extract 20 g/L) supplemented with chloramphenicol (10 μg/mL) and cultivated in 50-mL test tubes at 37°C for overnight (16 hours). Then, 20 iL of overnight- grown cell culture was inoculated into 2 mL of MI-3 broth supplemented with chloramphenicol (10 g/mL) and cultivated in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/PTFE molded septa (Macherey-Nagel, catalog No. 70234.9) at 37°C with shaking (250 rpm). Ten identical vials were used for each strain. One vial was frozen at -20°C at each hour during the 10-hours fermentation for isoprene measurement. The last (tenth) vial was frozen after cultivation for overnight (16 hours).

Isoprene analysis was performed as described in Experiment 1. For this purpose, each collected GC-vial was thawed immediately before measurement. The OD595 of cell culture was measured in each GC-vial immediately after isoprene measurement.

FIG. 7 shows the growth curves (A) and isoprene accumulation curves (B) for BS168-C, BS168-4, BS168-9, and BS168-10 strains. As one can see from the FIG. 7(A), the modified B. subtilis strains BS 168-4, BS 168-9 and BS168-10 having overexpressed the hepS and hepT genes accumulated a higher amount of isoprene than the control BS168-C strain.

Experiment 4.

An isoprene-synthesizing activity was tested in crude cell lysates of BS 168-C and BS 168-9 strains.

Both strains were cultivated for 6 hours as described in Experiment 3. Four identical GC-vials were used for each strain. One GC- vial was frozen at -20°C after the cultivation. Biomass from other three GC-vials was harvested by centrifugation at 4°C for 10 minutes (16.1 rcf, relative centrifugal force), washed by 4 mL of buffer A (25 mM Tris-HCl pH 8.5, 10 mM β-mercaptoethanol, 1 mM MgC ), re-suspended in 1 mL of buffer A supplemented with 10 mg/mL lysozyme (Sigma-Aldrich, catalog No. L7651). The obtained suspension of cells was incubated at 37°C for 45 min. Then, the cell debris was removed by centrifugation for 15 min at 4C° (16.1 rcf) and thus obtained the protein preparation was used for measuring activity of isoprene synthase as described hereinafter.

The test reaction mixture contained: 0.5 mL of protein preparation and 10 L of γ,γ-dimethylallyl pyrophosphate (DMAPP) triammonium salt (Sigma-Aldrich, D4287; 1 mg/mL solution in methanol and 10 mM aqueous NH₄OH as 7 : 3, v/v). The control reaction mixture was of the same composition but without DMAPP. Both test and control reaction mixtures were each incubated at 37°C for overnight (16 hours) in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/ PTFE molded septa (Macherey-Nagel, catalog No. 70234.9). Synthesized isoprene was measured as described in Experiment 1. An activity of isoprene synthase was expressed as an amount of synthesized isoprene (in pmol) per a weight of total protein in crude lysate (in mg) per the time of incubation of a reaction mixture (in hours), that is as pmol/(mg*hour).

The results are shown in Table 5. As one can see from Table 5, the crude cell lysates obtained from the modified B. subtilis BS 168-9 strain having overexpressed the kepS and hepT genes showed the higher activity of isoprene synthase than from the control BS 168-C strain. Table 5.

Example 3. Cloning of the hep S- menH- hepT oper on from B. licheniformis into a plasmid vector

The hepS and hepT genes of the hep S- menH- hepT operon from B. licheniformis were cloned using the procedure described in Example 1.

Specifically, a DNA-fragment V was amplified by PCR using primers P7 (SEQ ID NO: 20) and P8 (SEQ ID NO: 21), and chromosome of B. licheniformis (ATCC 14580) as the template, which was isolated and purified using GenElute Bacterial Genomic DNA Kit (Sigma, USA, catalog No. NA21 10). The PCR-mixture V of a total volume of 50 contained 5iL lOxPfu-Buffer supplemented with MgSO4 (Fermentas, Lithuania), 5 L dNTPs solution (2 mM each, Fermentas, Lithuania), 1 ]iL DMSO (Sigma, USA, catalog No. D8418), primers P7 and P8 (20 pmol each), 50 ng chromosomal DNA, and 1 L Pfu DNA-polymerase (Fermentas, Lithuania, catalog No. EP0561). PCR-protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/55°C 30 sec/72°C 6 min, 40 cycles; 72°C 10 min, 1 cycle.

Resulting DNA- fragment V (2,768 bp) was purified using QIAquick GelExtraction Kit, digested by BamHl and EcoRl to obtain a BamHI-EcoRI DNA-fragment (2,760 bp) of SEQ ID NO: 29, and ligated with pMWALl / BamHl- EcoRl vector. Thus the plasmid pMWALl -hepS-menH- hepT-BL was constructed (Table 2).

The plasmid harboring hepS-menH-hepT operon from B. licheniformis placed under the control of the Prep promoter was constructed using the following procedure. The 2,556 bp DNA-fragment VI was amplified by PCR using primers P9 (SEQ ID NO: 22) and P8 and the PCR-protocol as described above for obtaining the DNA-fragment V, digested by BamHl and EcoRl to obtain a BamHI-EcoRI DNA-fragment (2,548 bp) of SEQ ID NO: 30, and cloned into pMWALl -Prep/ BamHl - Eco vector. Thus the plasmid pMWALl -Prep-hepS-menH-hepT-BL was constructed (Table 2).

Example 4. Cloning of the hepS-menH-hepT operon from B.

amyloliquefaciens into a plasmid vector

The hepS and hepT genes of the HepS-menH-hepT operon from B. amyloliquefaciens were cloned using the procedure described in Example 1.

Specifically, a DNA-fragment VII was amplified by PCR using primers P10 (SEQ ID NO: 23) and PI 1 (SEQ ID NO: 24), and chromosome of B. amyloliquefaciens strain K (JCM 20197, Japan Collection of Microorganisms, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan) as the template, which was isolated and purified using GenElute Bacterial Genomic DNA Kit (Sigma, USA, catalog No. NA2110). The PCR-mixture VI of a total volume of 50 \iL contained 5 L lOxPfu-Buffer supplemented with MgSO₄ (Fermentas, Lithuania), 5 μL dNTPs solution (2 mM each, Fermentas, Lithuania), 1 μL DMSO (Sigma, USA, catalog No. D8418), primers PIO and Pl l (20 pmol each), 50 ng chromosomal DNA, and 1 L Pfu DNA-polymerase (Fermentas, Lithuania, catalog No. EP0561). PCR-protocol was as follows: 94°C 5 min, 1 cycle; 94°C 30 sec/55°C 30 sec/72°C 6 min, 40 cycles; 72°C 10 min, 1 cycle.

Resulting DNA-fragment VII (2,763 bp) was purified using QIAquick

GelExtraction Kit, digested by Kprd and EcoKi (Fermentas, Lithuania) to obtain a KpnI-EcoRI DNA-fragment (2,755 bp) of SEQ ID NO: 31, and ligated with pMWALl /Kpnl-EcoRl vector. Thus the plasmid pMWALl - hepS-menH-hepT-BA was constructed (Table 2).

The plasmid harboring hepS-menH-hepT operon from B. amyloliquefaciens placed under the control of the Prep promoter was constructed using the following procedure. The 2,551 bp DNA-fragment VIII was amplified by PCR using primers P12 (SEQ ID NO: 25) and Pl l and the PCR-protocol as described above for obtaining the DNA-fragment VII, digested by Kprd and EcoRl to obtain a KpnI-EcoRI DNA-fragment (2,543 bp) of SEQ ID NO: 32, and cloned into pMWALl -Prep/ Kpnl- EcoRl vector. Thus the plasmid pMWALl -Prep-hepS-menH-hepT-BA was constructed (Table 2). Example 5. Production of isoprene by B. subtilis having overexpressed the hepS and hepT genes from B. subtilis, B. licheniformis and B.

amyloliquefaciens

The control vector pMWAL-1 and plasmids pMWALl -hepS-menH- hepT, pMWALl -Prep-hepS-menH-hepT, pMWALl -hepS-menH-hepT-BL, pMWALl -Prep-hepS-menH-hepT-BL, pMWALl -hepS-menH-hepT-BA, and pMWALl -Prep-hepS-menH-hepT-BA were each introduced into B. subtilis 168 using electroporation (Eppendorf AG, Germany; Protocol No. 4308 915.504 - 08/2003). Thus the B. subtilis strains BS 168-C, BS168-4, BS168-9, BS168- 12, BS168- 13, BS168- 14, and BS168-15 were obtained respectively (Table 2).

The strains were each grown at 37°C in LB-medium supplemented with chloramphenicol (Cm, 5 g/mL) for overnight (16- 18 hours). Glycerol was added to the obtained cell cultures in amount of 8% (v/v). Thus obtained solutions of cells were divided into aliquots (150 μΐ,- each in 1.5 mL Eppendorf vials) and stored at -70°C.

An aliquot (25 μί,) of each solution of cells was inoculated into 5 mL of MI-3 broth (tryptone 10 g/L, yeast extract 20 g/L) supplemented with chloramphenicol (10 g/mL) and cultivated in 50-mL test tubes at 37°C for overnight (16 hours). Then, 20 iL of the overnight- grown cell cultures was each inoculated into 2 mL of MI-3 broth supplemented with chloramphenicol (10 μg/mL) and cultivated in 20-mL GC-vials crimped by caps with 3 -mm grey Butyl/ PTFE molded septa (Macherey-Nagel, catalog No.70234.9) at 37°C with shaking (250 rpm) for 10 hours. Seven identical vials were used for each strain. Two vials were used for culture optical density (OD595) measurements, and five vials were subjected to GC-analysis (Experiment 2 of Example 2).

The obtained data for OD595 and accumulation of isoprene by B. subtilis strains are shown in Table 6. As one can see from Table 6, the modified B. subtilis strains BS 168-4, BS 168-9, BS168-12, BS168-13, BS168-14, and BS168-15 having overexpressed the epSand hepT genes accumulated a higher amount of isoprene than the control BS168-C strain.

The ANOVA, Tukey-Kramer Multiple Comparisons Test (GraphPad Instat, USA) showed the statistically significant difference between the control BS168-C and tested strains BS 168-4, BS 168-9, BS168-12, BS168-13, BS168-14 or BS168-15 (for each strain: sample size = 5; normality test is passed, P < 0.001 for BS168-4, BS168-9, BS168-12, BS168-14, BS168-15 and P < 0.05 for BS168-13), and did not show the statistically significant difference between the tested strains BS 168-4, BS168-9, BS168-12, BS168-13, BS168-14, and BS168-15 (P > 0.05). Table 6.

Example 6. Production of isoprene by E. coli having overexpressed the hepS and hepT genes from B. subtilis

The plasmid pMWALl-Prep-hepS-menH*-hepT were constructed as described in Example 1. The DNA-fragment IV (2,348 bp, Example 1) hepS-menH*-hepT was purified using QIAquick GelExtraction Kit, digested by BamHl and EcoRl, and ligated with pMWALl-Prep/BamHI- EcoRI vector (Auxiliary example 2). Thus the plasmid pMWALl-Prep- hepS-menH*-hepT was obtained (Table 2) .

Experiment 1.

The control vector pMWAL- 1 and plasmids pMWALl-hepS-menH- hepT and pMWALl-Prep-hepS-menH*-hepT are each introduced into isoprene-producing E. coli strain EI20 [E. coli MG1655 Tn7: :Ptac-KKDyI ldhA:: Para-mvaES] using electroporation. A method for construction of the E. coli strain EI20 is described in Auxiliary example 4. For electroporation, 10 mL of 2YT-broth (Sambrook, J. and Russell, D.W. «Molecular Cloning: A Laboratory Manual*, 3^rd ed., Cold Spring Harbor Laboratory Press (2001)) is inoculated with biomass of each strain which is grown on and picked up from 2YT-agar plate. Cells are cultivated at 37°C for about 2-3 hours to OD600 of about 1. Then, cells from 1.5 mL of grown cell cultures are harvested by centrifugation in 1.5 mL Eppendorf vials, washed three times with 1.5 mL of cold 10% (v/v) glycerol solution, and re-suspended in 100 ]iL of cold 10% (v/v) glycerol solution supplemented with 10-20 ng of plasmid DNA. Obtained mixtures are each transferred into 0.2 -cm Gene Pulser/MicroPulser Electroporation Cuvette (Bio-Rad) and subjected to electroporation using MicroPulser™ Electroporator (Bio-Rad) and the prescribed protocol Ec-2. The cells are transferred into 1 mL of 2YT-broth supplemented with 0.1% (w/v) glucose and cultivated at 37°C for about 2 hours. Then 10 L of obtained cell cultures is diluted in 100 pL of 2YT-broth and plated onto 2YT-agar plates supplemented with chloramphenicol (final concentration 100 pg/mL) and ampicillin (final concentration 100 pg/mL). Agar-plates are cultivated at 37°C for about 24 hours, and colonies resistant to ampicillin, and chloramphenicol are selected and re-plated on the same agar- medium. Thus the E. coli strains EI20/pMWALl (EI20-C), EI20/pMWALl-hepS- menH-hepT (EI20- 1) and EI20/pMWALl-Prep-hepS-menH*-hepT (EI20-2) are obtained. Experiment 2.

5 mL of Terrific Broth (Sambrook, J. and Russell, D.W. «Molecular

Cloning: A Laboratory Manual», 3rd ed., Cold Spring Harbor Laboratory

Press (2001)) supplemented with chloramphenicol (100 pg/mL), and ampicillin (100 pg/mL) (referred to as TB-CA medium) is inoculated with biomass of E. coli strains EI20-C, EI20- 1 and EI20-2 picked up from freshly prepared agar-containing plates and cultivated for 4 hours at

37°C to ODeoo of about 5-6.

An aliquot (20 pL) of each obtained cell culture is inoculated into 2 mL of TB-CA medium or TB-CA medium supplemented with 100 μΜ or 1 mM L-arabinose. Cells are cultivated in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/ PTFE molded septa (Macherey-Nagel, catalog No.

70234.9) at 37°C with shaking (250 rpm) for 16 hours. Cultivation of each strain is repeated four times. The fermentation gas is subjected to

GC-analysis (Experiment 2 of Example 2).

Based on data for OD600 and accumulation of isoprene by E. coli strains, the modified E. coli strains EI20-1 and EI20-2 having overexpressed the hepS and hepT genes accumulate a higher amount of isoprene than the control EI20-C strain.

Example 7. Production of isoprene by P. ananatis having overexpressed the hepS and hepT genes from B. subtilis

The control vector pMWAL- 1 (Auxiliary example i) and the plasmid pMWALl-Prep-hepS-menH*-hepT (Example 6) were each introduced into P. ananatis strain ISP3-mvk(Mpd) [P. ananatis SC 17(0) AampC::atiLphi80-KKDyI-ispS(K)-atti?phi80 AampH::attLphi80-Para- mvaES- iii?phi80 Acrt::pAH 162-Ptac-mvk(M. paludicola)] using electroporation as described in Experiment 1 of Example 6, except that the cultivation medium did not contain antibiotics. A method for construction of the P. ananatis strain ISP3-mvk(Mpd) is described in Auxiliary example 5. Cultivation temperature was 34°C, and cells were selected on plates containing 2YT-agar supplemented with chloramphenicol (10 pg/mL). Thus the P. ananatis strains ISP3- mvk(Mpd)/pMWALl (ISP3-C) and ISP3-mvk(Mpd)/pMWALl-Prep-hepS- menH*-hepT (ISP3-T) were obtained.

5 mL of 2YT Broth (Sambrook, J. and Russell, D.W. «Molecular Cloning: A Laboratory Manual», 3rd ed., Cold Spring Harbor Laboratory Press (2001)) supplemented with chloramphenicol (20 pg/mL) was inoculated with biomass of P. ananatis strains ISP3-C and ISP3-T picked up from freshly prepared agar-containing plates and cultivated for 4 hours at 34°C.

An aliquot (20 pL) of each obtained cell culture was each inoculated into 2 mL of Terrific Broth supplemented with chloramphenicol (20 pg/mL) and 1 mM L-arabinose. Cells were cultivated in 20-mL GC-vials crimped by caps with 3-mm grey Butyl/ PTFE molded septa (Macherey-Nagel, catalog No. 70234.9) at 34°C with shaking (250 rpm) for 24 hours. Cultivation of each strain was repeated four times. The fermentation gas was subjected to GC-analysis (Experiment 2 of Example 2). The data for OD600 and accumulation of isoprene by P. ananatis strains are shown in Table 7. As one can see from Table 7, the modified P. ananatis strain ISP3-T having overexpressed the hepS and hepT genes accumulated a higher amount of isoprene than the control ISP3-C strain.

Table 7.

Auxiliary example 1. Construction of the pMWALl vector

The pMWALl vector was obtained from the collection of N.P. Zakataeva and S.V. Gronsky (Closed Joint-Stock Company «Ajinomoto- Genetika Research Institute» (ZAO AGRI), Russian Federation, 117545 Moscow, 1^st Dorozhny pr., 1 , bldg. 1).

The low-copy shuttle vector pMWALl was constructed by modifying the pALl plasmid. The pALl plasmid is a derivative of theta-replicating B. subtilis plasmid pBS72 (Titok M.A. et al., Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector, Plasmid, 2003, 49(l):53-62; Titok M.A. et al., Molecular and genetic analysis of rep region of the theta-type plasmid pBS72 from Bacillus subtilis, Doklady Natsional'noi Akademii Nauk Belarusi (Russian), 2003, 47(4):67-70) described in (Lagodich A.V. et al., Construction of vector system for molecular cloning in Bacillus subtilis and Escherichia coli, Mol. Biol. (Mosk., Russian), 2005, 39(2):345-348) and obtained by cloning the pBS72 replicon into the E. coli ColEl -replicon vector pMTL21C (Chambers S.P. et al., The pMTL cloning vectors, I. Improved polylinker regions to facilitate the generation of sonicated DNA for nucleotide sequencing, Gene, 1988, 68: 139-149).

To construct pMWALl (SEQ ID NO: 9), the Nhel-Smul fragment of pALl was blunted using Klenow fragment (Fermentas, Lithuania) and ligated with blunted (Klenow fragment, Fermentas) the Pvull-EcoKL fragment of the pMW1 18-S vector. The pMW1 18-S is a derivative of pMW1 18 (Nippon Gene Co., Ltd.) and has deleted one of two the Pvull sites. The pMW1 18-S vector was constructed by digesting pMW1 18 with Ndel-Ηί^ηάΊΙΙ (Fermentas), blunting using Klenow fragment, and self- ligating using a ligase (Fermentas).

Auxiliary example 2. Construction of the pMWALl -Prep vector

The pMWALl-Prep vector was obtained from the collection of N.P. Zakataeva and S.V. Gronsky (Closed Joint-Stock Company «Ajinomoto- Genetika Research Institute* (ZAO AGRI), Russian Federation, 1 17545 Moscow, 1^st Dorozhny pr., 1 , bldg. 1).

The pMWALl-Prep expression vector was constructed as follows. A PCR-fragment containing the repAB promoter (Prep) was amplified by PCR using primers P5 (SEQ ID NO: 10) and P6 (SEQ ID NO: 1 1) and plasmid DNA of pLF22 (Tarakanov B.V. et al., Expression vector pLF22 for the lactic acid bacteria, Mikrobiologiia (Russian), 2004, 73(2):21 1-217) as the template. The obtained PCR-fragment was digested with Pael and Xbal and cloned into Pael - Xbal sites of pMWALl (SEQ ID NO: 9). Thus the pMWALl -Prep vector was obtained.

Auxiliary example 3. Preparation of standards for isoprene

7 iL of cold isoprene (4°C) (Sigma- Aldrich, catalog No. 464953) was mixed with 963 μΐ.· of cold methanol (-20°C) (Sigma- Aldrich, catalog No. 322415) and kept for about 1 hour at 4°C to obtain the 4.8 mg/mL standard of isoprene in methanol. The standard was diluted 1000-times with cold methanol, and then diluted further with 2.5% (v/v) methanol in water to final concentrations of isoprene 10, 20, 50, 100, 200, and 500 μg/L. 1 mL of each standard solutions was transferred to 20-mL GC-vials crimped by caps with 3-mm grey Butyl/ PTFE molded septa (Macherey- Nagel, catalog No. 70234.9) and used for GC-analysis (Experiment 1 of Example 2, GC isoprene analysis). Auxiliary example 4. Construction of the E. coli strain EI20

A method for construction of the E. coli strain EI20, which is also referred to as E. coli MG1655 Tn7::Ptac-KKDyI ldhA::Para-mvaES, is described below.

4.1. Construction of arabinose-inducible expression vector

An arabinose-inducible expression vector for the mevalonate pathway upstream genes mvaE and mvaS was constructed by the following procedure. A DNA-fragment containing Para consisting of araC and araBAD promoter sequences derived from E. coli was obtained by PCR using the plasmid pKD46 as the template and primers P13 (SEQ ID NO: 33) and P14 (SEQ ID NO: 34). The plasmid pKD46 (Datsenko K.A. and Wanner B.L., One-step inactivation of chromosomal genes in Escherichia coli K- 12 using PCR-products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) includes a 2, 154 nucleotides DNA-fragment of phage λ (nucleotide positions from 31088 to 33241 , GenBank accession number J02459), and contains genes of the λ-Red homologous recombination system (γ, β exo genes) under the control of the arabinose- inducible ParaB promoter. The pKD46 plasmid is necessary for integration of a PCR-product into chromosome of the E. coli MG1655 strain. The E. coli MG1655 strain containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA (Accession No. CGSC7669).

The nucleotide sequence of the mvaE gene from Enterococcus faecalis (E. faecalis) and the amino acid sequence encoded by this gene are shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively. To efficiently express the mvaE gene, the mvaE gene in which the codon usage in E. coli had been optimized was designed and used, and this was designated as EFmvaE (SEQ ID NO: 37). The EFmvaE gene was chemically synthesized, cloned into pUC57 (GenScript), and the resulting plasmid was designated as pUC57-EFmvaE. A DNA-fragment containing the EFmvaE gene was obtained by PCR using the plasmid pUC57- EFmvaE as the template and primers PI 5 (SEQ ID NO: 38) and P16 (SEQ ID NO: 39).

The nucleotide sequence of the mvaS gene from E. faecalis and the amino acid sequence encoded by this gene are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. To efficiently express the mvaS gene, the mvaS gene in which the codon usage in E. coli had been optimized was designed and used, and this was designated as EFmvaS (SEQ ID NO: 42). The EFmvaS gene was chemically synthesized, cloned into pUC57 (GenScript), and the resulting plasmid was designated as pUC57-EFmvaS. A DNA-fragment containing the EFmvaS gene was obtained by PCR using the plasmid pUC57-EFmvaS as the template and primers P17 (SEQ ID NO: 43) and P18 (SEQ ID NO: 44). A DNA-fragment containing a Ttrp sequence was obtained by PCR using the plasmid pSTV28-Ptac-Ttrp (SEQ ID NO: 47) as the template (U.S. patent application publication No. 2014/01 13344 Al) and primers P19 (SEQ ID NO: 45) and P20 (SEQ ID NO: 46).

Prime Star polymerase (Takara Bio Inc.) was used for PCR to obtain the above mentioned four DNA-fragments. Reaction solutions were prepared according to the composition attached to the kit, and DNA was amplified through 30 cycles of the reactions at 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 1 min per 1 kb. PCR with the purified PCR- product containing Para and the PCR-product containing the EFmvaE gene as the template was carried out using primers PI 3 and PI 6. PCR with the purified PCR-product containing the EFmvaS gene and the PCR- product containing Ttrp as the template was carried out using primers PI 7 and P20. As a result, a PCR-product containing Para and the EFmvaE gene and a PCR-product containing the EFmvaS gene and Ttrp were obtained. A plasmid pMW219 (Nippon Gene Co., Ltd.) was digested with Smal according to a standard method and ligated to the PCR- product containing Para and the EFmvaE gene and the PCR-product containing the EFmvaS gene and Ttrp using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid was designated as pMW-Para-mvaES- Ttrp.

4.2. Construction of E. coli MG1655 Tn7::Ptac-KKDyI

4.2.1. Cloning a downstream region of the mevalonate pathway

A downstream region of the mevalonate pathway was obtained from Saccharomyces cerevisiae (S. cerevisiae) S288c (WO2009076676 A2, Saccharomyces Genome Database http:/ /www.yeastgenome.org/, Cherry J.M. et al., Saccharomyces Genome Database: the genomics resource of budding yeast, Nucleic Acids Res., 2012, 40 (Database issue): D700- D705). An ERG12 gene encoding mevalonate kinase (EC: 2.7.1.36, NCBI accession number NP_013935), an ERG8 gene encoding phosphomevalonate kinase (EC: 2.7.4.2, NCBI accession number NPJ313947), an ERG19 gene encoding diphosphomevalonate decarboxylase MVD 1 (EC: 4.1.1.33, NCBI accession number NP_014441), and an IDI1 gene encoding isopentenyl-diphosphate delta-isomerase (EC: 5.3.3.2, NCBI accession number NP_015208) were amplified by PCR using genomic DNA of S. cerevisiae S288c (ATCC 204508D) as the template and the primers shown in Table 8. The nucleotide sequences of the amplified genes and the amino acid sequences of the enzymes encoded by these genes are available on Saccharomyces Genome Database (http:/ /www.yeastgenome.org/).

Prime Star Max Premix (Takara Bio Inc.) was used for a PCR enzyme, and the reaction was performed at 98°C for 2 minutes and 30 cycles at 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 1 minute per 1 kb. Cloning and construction of an expression vector were performed by inserting the obtained DNA-fragment into the pSTV28-Ptac- Ttrp vector (SEQ ID NO: 47) treated with the restriction enzyme Smal by an In-fusion cloning method (Clontech). The pSTV28-Ptac-Ttrp vector is a derivative of the commercially available pSTV28 vector (Takara Bio Inc.), and it contains the Ptac-Ttrp DNA-fragment containing the tac promoter (synonym: Ptac) region (de Boer H.A. et al., The tac promoter: a functional hybrid derived from the trp and lac promoters, Proa Natl. Acad. Sci. USA, 1983, 80(l):21-25) and the trp terminator (synonym: Ttrp) region of a tryptophan operon derived from E. coli (Wu A.M. and Piatt T., Transcription termination: nucleotide sequence at 3' end of tryptophan operon in Escherichia coli, Proc. Natl. Acad. Sci. USA, 1978, 75(11):5442- 5446) and having a Kpnl site at 5 '-terminus and a BamHl site at 3'- terminus (U.S. patent application publication No. 2014/01 13344 Al).

E. coli DH5a (Takara Bio Inc.) was transformed with the obtained expression vector, clones having desired sequence length of each gene were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed.

Table 8.

4.2.2. Construction of an artificial operon downstream of mevalonate pathway

A sequence, in which the ERG12 gene encoding mevalonate kinase and the ERG8 gene encoding phosphomevalonate kinase were arranged in straight, was constructed by the In-fusion cloning method (Clontech). The ERG 12 and ERG8 genes were amplified by PCR using genomic DNA from S. cerevisiae as the template and the primers shown in Table 9. KOD plus (Toyobo) was used for the PCR enzyme, and the reaction was performed at 94°C for 2 minutes and 30 cycles at 94°C for 15 seconds, 45°C for 30 seconds and 68°C for 1 minute per 1 kb. The cloning and the construction of an expression vector were performed by inserting the obtained DNA-fragment into pUCl 18 vector (Takara Bio Inc.) treated with the restriction enzyme Srncd by the In-fusion cloning method. E. coli JM109 (Takara Bio Inc.) was transformed with the obtained expression vector, clones having an objective sequence length of each gene were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed. The produced plasmid was designated as pUC- mvk-pmk (WO2013179722 Al, US20141 13344 Al). The nucleotide sequence of pUC-mvk-pmk is represented by SEQ ID NO: 56.

Table 9.

The sequence, in which the ERG19 gene encoding diphosphomevalonate decarboxylase and the IDI1 gene encoding isopentenyl-diphosphate delta-isomerase were arranged in straight, was constructed by the In-fusion cloning method (Clontech). The ERG19 and IDI1 genes were amplified by PCR with genomic DNA of S. cerevisiae S288c (ATCC 204508D) as the template and the primers shown in Table 10. KOD plus (Toyobo) was used for the PCR enzyme, and the reaction was performed at 94°C for 2 minutes and 30 cycles at 94°C for 15 seconds, 45°C for 30 seconds and 68°C for 1 minute per 1 kb, and then at 68°C for 10 minutes. The cloning and the construction of an expression vector were performed by inserting the obtained DNA- fragment into pTWV228 vector (Takara Bio Inc.) treated with the restriction enzyme Smal by the In-fusion cloning method. E. coli DH5 (Takara Bio Inc.) was transformed with the obtained expression vector, clones having an objective sequence length of each gene were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed. The produced plasmid was designated as pTWV-dmd-yidi (WO2013179722 Al , US20141 13344 Al). The nucleotide sequence of pTWV-dmd-yidi is represented by SEQ ID NO: 61.

Table 10.

The sequence, in which the ERG 12 gene encoding the mevalonate kinase, the ERG8 gene encoding the diphosphomevalonate kinase, the ERG19 gene encoding the diphosphomevalonate decarboxylase and the IDI1 gene encoding the isopentenyl-diphosphate delta-isomerase were arranged in straight, was constructed by the In-fusion cloning method (Clontech). An expression vector in which these four genes were arranged in straight was constructed by amplifying the ERG12 and ERG8 genes by PCR using pUC-mvk-pmk as the template and the primers shown in Table 1 1 and amplifying the ERG19 and IDI1 genes by PCR using pTWV- dmd-yidi as the template and the primers shown in Table 1 1 , followed by cloning the amplified products into pTrcHis2B vector (Invitrogen Corp.) by the In-fusion cloning method. Prime Star HS DNA polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was performed at 98°C for 2 minutes and 30 cycles at 98°C for 10 seconds, 52°C for 5 seconds and 72°C for 1 minute per 1 kb, and then at 72°C for 10 minutes. The obtained DNA-fragment was inserted into pTrcHis2B vector treated with the restriction enzymes iVcoI and Pstl to construct the expression vector. E. coli JM109 (Takara Bio Inc.) was transformed with the obtained expression vector, clones having an objective sequence length were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed. The constructed expression vector was designated as pTrc-KKDyl( ). The nucleotide sequence of pTrc-KKDyl(P) is represented by SEQ ID NO: 66.

Table 1 1.

4.2.3. Fixation of downstream region of mevalonate pathway on

chromosome

The sequence, in which the ERG12, ERG8, ERG19 and IDI1 genes were arranged in straight, was expressed on a chromosome. A glucose isomerase promoter was used for the expression of the genes, and a transcription termination region of aspA gene of E. coli was used for the termination of the transcription (WO2010031062 Al, Takagi J.S. et al., Cloning and nucleotide sequence of the aspartase gene of Eschenchia coli W, Nucleic Acids Res., 1985, 13(6):2063-2074). A translocation site of Tn7 transposon was used as a chromosomal site to be fixed (Lichtenstein C. and Brenner S., Site-specific properties of Tn7 transposition into the E. coli chromosome, Mol. Gen. Genet, 1981, 183(2):380-387). The cat gene was used as an antibiotic-resistance marker after the fixation on the chromosome. A Tn7 downstream region in the chromosome region to be fixed was amplified by PCR using genomic DNA of E. coli MG1655 (Hayashi K. et al., Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110, Mol. Syst. Biol, 2006;2:2006.0007) as the template and the primers shown in Table 12. Prime Star HS DNA polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was carried out at 98°C for 2 minutes and 30 cycles at 98°C for 10 seconds, 52°C for 5 seconds and 72°C for 1 minute per 1 kb, and then at 72°C for 10 minutes. A cat gene region containing a phage λ attachment site was amplified by PCR using pMWl 18- attl_^Cm-attR plasmid (WO2010027022 Al) as the template and the primers shown in Table 12. Prime Star HS DNA polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was carried out at 95°C for 3 minutes followed by 2 cycles at 95°C for 1 minute, 34°C for 30 seconds and 72°C for 40 seconds, 2 cycles at 95°C for 30 seconds, 50°C for 30 seconds and 72°C for 40 seconds, and then at 72°C for 5 minutes. A sequence downstream of the mevalonate pathway to which a promoter and a transcription termination region had been added (hereinafter abbreviated as KKDyl) was amplified using pTrc-KKDyl(P) as the template (Auxiliary example 4.2.2) and the primers shown in Table 12. Prime Star HS DNA polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was carried out at 98°C for 2 minutes and 30 cycles at 98°C for 10 seconds, 52°C for 5 seconds and 72°C for 1 minute per 1 kb, and then at 72°C for 10 minutes.

Table 12.

A vector was constructed using the obtained PCR-products and the plasmid pMW219 (Nippon Gene Co., Ltd.) treated with the restriction enzyme Smal by the In-fusion cloning method (Clontech). E. coli JM109 (Takara Bio Inc.) was transformed with the obtained expression vector, clones having an objective sequence length were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed. The resulting plasmid was designated as pMW219-KKDyI- TaspA. The nucleotide sequence of pMW219 -KKDyl -TaspA is represented by SEQ ID NO: 71.

A Tn7 upstream region in the chromosome region to be fixed was amplified by PCR using the genomic DNA of E. coli MG1655 (Hayashi K. et al., Highly accurate genome sequences of Escherichia coli K- 12 strains MG1655 and W3110, Mol. Syst. Biol, 2006;2:2006.0007) as the template and the primers shown in Table 13. Prime Star HS DNA polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was carried out at 98°C for 2 minutes and 30 cycles at 98°C for 10 seconds, 52°C for 5 seconds and 72°C for 1 minute per 1 kb, and then at 72°C for 10 minutes. A vector was constructed using the obtained PCR-product and pMW219-KKDyI-TaspA (SEQ ID NO: 71) treated with the restriction enzyme SaR by the In-fusion cloning method (Clontech). E. coli JM 109 (Takara Bio Inc.) was transformed with the obtained expression vector, clones having an objective sequence length were selected, a plasmid was extracted according to standard methods, and its sequence was confirmed. The resulting plasmid was designated as pMW-Tn7-Pgi- KKDyI-TaspA-Tn7. The sequence of the constructed plasmid is represented by SEQ ID NO: 78.

Table 13.

A chromosome having a region including the chloramphenicol- resistance gene {cat), the glucose isomerase promoter, the operon downstream of the mevalonate pathway, and the aspA gene transcription termination region was fixed using λ-Red method (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Set USA, 2000, 97(12):6640-6645). A fragment for chromosome fixation was prepared by extracting the plasmid pMW-Tn7-Pgi-KKDyI-TaspA-Tn7 and then treating it with the restriction enzymes Pvul and Sail followed by purifying it. E. coli MG1655 containing a plasmid pKD46 having a temperature-sensitive replication capacity was used for the electroporation (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). After the electroporation, a colony that acquired the resistance to chloramphenicol was obtained. The genomic DNA was extracted, and the objective region fixed on the chromosome was confirmed by PCR using the primers shown in Table 14. Furthermore, the sequence of the objective region was confirmed by sequence analysis of the relevant DNA-fragment. The nucleotide sequence encoding enzymes from the downstream region of the mevalonate pathway and its proximal region fixed on the chromosome is represented by SEQ ID NO: 79, and its structure is shown on FIG. 8. The resulting mutant strain was designated as E. coli MG1655 cat-Pgi-KKDyl.

Table 14.

The chloramphenicol-resistance marker in MG1655 cat-Pgi-KKDyl was removed using the following procedure. The cells of MG1655 cat-Pgi- KKDyl were made competent, and the plasmid pMW-int/xis was introduced therein. pMW-int/xis is a plasmid containing a gene encoding integrase (Int) of the phage λ and a gene encoding excisionase (Xis) of the phage λ and having the temperature-sensitive replication capacity (WO2007037460 Al). It is known that the antibiotic-resistance gene located in a region in between the attL and attR attachment sites of the phage λ can be excised from the chromosome by introducing pMW- int/xis. As a result, the host devoid of the resistance to antibiotic is obtained. Thus, a chloramphenicol-sensitive strain was obtained from the resulting colony, and it was then cultured on the LB-medium at 42°C for 6 hours. The cultured microbial cells were applied onto the plate with LB- medium to allow colonies to grow. A colony that had lost the resistance to ampicillin was selected. The thus obtained mutant strain was designated as E. coli MG 1655 Pgi-KKDyl .

4.2.4. Substitution of promoter downstream of mevalonate pathway on chromosome The promoter of the operon downstream of the mevalonate pathway on the chromosome was substituted using the λ-Red method (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Set USA, 2000, 97(12):6640- 6645). A genomic fragment having iiL-Tet- attR- Ptac (SEQ ID NO: 84) was used as the template for PCR. The fragment aiiL-Tet- attR- Ptac contains the tac promoter, the attL and attR attachment sites of phage λ, and the tetracycline-resistance marker gene. A DNA- fragment was prepared using the primers shown in Table 15. LA-Taq polymerase (Takara Bio Inc.) was used for the PCR enzyme, and the reaction was carried out at 92°C for 1 minute and for 40 cycles at 92°C for 10 seconds, 50°C for 20 seconds and 72°C for 1 minute per 1 kb, and then at 72°C for 7 minutes. The obtained PCR-product was purified. The E. coli MG1655 Pgi-KKDyl containing the plasmid pKD46 (hereinafter referred to as E. coli MG1655 Pgi-KKDyl /pKD46) having the temperature-sensitive replication capacity was used for the electroporation. The plasmid pKD46 is required for incorporating the PCR-product into E. coli MG1655 Pgi- KKDyl.

Competent cells for the electroporation were prepared as follows. E. coli MG1655 Pgi-KKDyl/ pKD46 was cultured in the LB-medium supplemented with ampicillin (100 mg/L) at 30°C for overnight and then 100-times diluted with 5 mL of LB-medium containing ampicillin (100 mg/L) and L-arabinose (1 mM). The resulting cells in diluted suspension were grown until ODeoo of about 0.6 at 30°C with aeration (120 rpm) and washed three times with ice-cold 10% (v/v) glycerol solution. The electroporation was performed using 50 μL of the competent cells and about 100 ng of the PCR-product in 1 mL of SOC-medium (Sambrook, J. et al., «Molecular Cloning: A Laboratory Manual_*, 2^nd ed., Cold Spring Harbor Laboratory Press (1989)). The cells after the electroporation were cultured at 37°C for 1 hour and transferred onto a plate with LB-agar- medium at 37°C to select a chloramphenicol-resistant transformant. To remove the pKD46 plasmid, the transformant was subcultured at 37°C in LB-agar-medium containing tetracycline (20 mg/L). The ampicillin- resistance was examined in the obtained colonies, and the ampicillin- resistant strain not having pKD46 was obtained. A mutant containing the tac promoter substitution, that was selected using the tetracycline- resistant gene, was obtained. The obtained mutant was designated as E. coli MG 1655 tet-Ptac-KKDyl .

Table 15.

The tetracycline-resistance marker gene was deleted using the following procedure. The cells of E. coli MG1655 tet-Ptac-KKDyl were made competent, and the plasmid pMW-int/xis was introduced therein. pMW-int/xis is a plasmid containing a gene encoding integrase (Int) of the phage λ and a gene encoding excisionase (Xis) of the phage λ and having the temperature-sensitive replication capacity (WO2007037460 A 1). It is known that the antibiotic-resistance gene located in a region in between the attL and attR attachment sites of the phage λ can be excised from the chromosome by introducing pMW-int/xis. As a result, the host devoid of the resistance to antibiotic is obtained. Thus, a tetracycline- sensitive strain was obtained from the resulting colony, and it was then cultured in the LB-medium at 42°C for 6 hours. The cultured microbial cells were applied onto the plate with LB-medium to allow colonies to grow. A colony that had lost the resistance to ampicillin was selected. The thus obtained mutant strain was designated as E. coli MG1655 Ptac- KKDyl. The nucleotide sequence encoding enzymes from the downstream region of the mevalonate pathway and its proximal region controlled by the tac promoter fixed on the chromosome is represented by SEQ ID NO: 85, and its structure is shown on FIG. 9. 4.3. Construction of plasmid for expressing isoprene synthase derived from Pueraria montana var. lobata (Kudzu) and mevalonate kinase derived from M. mazei

A nucleotide sequence and an amino acid sequence of the isoprene synthase derived from Pueraria montana var. lobata (Kudzu) were already known (NCBI accession number AY316691 for nucleotide sequence and AAQ84170 for amino acid sequence, P. montana var. lobata isoprene synthase, IspS). The nucleotide sequence of the ispS gene derived from Pueraria montana (P. montana) and the amino acid sequence of the IspS protein encoded by this gene are represented by SEQ ID NO: 88 and SEQ ID NO: 89, respectively. The ispS gene was optimized for codon usage frequency in E. coli in order to efficiently express the ispS gene in E. coli, and further designed to cut off the chloroplast localization signal. The obtained gene was designated as ispSK. A nucleotide sequence of ispSK is represented by SEQ ID NO: 90. The ispSK gene was chemically synthesized and cloned into pUC57 (GenScript), and the resulting plasmid was designated as pUC57-IspSK.

A nucleotide sequence and an amino acid sequence of the mevalonate kinase derived from Methanosarcina mazei Gol were already known (NCBI accession number of nucleotide sequence NC_003901.1, locus tag MM_1762, gene ID 1480104, nucleotides from 2101873 to 2102778; NCBI accession number of amino acid sequence NP_633786.1). The nucleotide sequence of the mvk gene derived from Methanosarcina mazei {M. mazei) and the amino acid sequence of the MVK protein encoded by this gene are represented by SEQ ID NO: 91 and SEQ ID NO: 92, respectively. The mvk gene was optimized for codon usage frequency in E. coli in order to efficiently express the mvk gene in E. coli. The designed gene was designated as Mmamvk. A nucleotide sequence of Mmamvk is represented by SEQ ID NO: 93. The Mmamvk gene was chemically synthesized and cloned into pUC57 (GenScript), and the resulting plasmid was designated as pUC57-Mmamvk. A plasmid for expressing the ispSK and Mmamvk genes in E. coli was constructed by the following procedure. PCR was performed using pUC57-IspSK as the template, primers P53 (SEQ ID NO: 94) and P54 (SEQ ID NO: 95) and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to a composition attached to a kit, and DNA was amplified through 40 cycles of reactions at 98°C for 10 seconds, 54°C for 20 seconds and 68°C for 120 seconds. As a result, a PCR- product containing the ispSK gene was obtained. Likewise, pSTV28-Ptac- Ttrp (SEQ ID NO: 47) was amplified by PCR using primers P55 (SEQ ID NO: 96) and P56 (SEQ ID NO: 97) and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to the composition attached to the kit, and DNA was amplified through 40 cycles of reactions at 98°C for 10 seconds, 54°C for 20 seconds and 68°C for 210 seconds. As a result, a PCR-product containing the pSTV28-Ptac-Ttrp construct was obtained. The purified PCR-product containing the ispSK gene was ligated to the PCR-product containing the pSTV28-Ptac-Ttrp construct using In-Fusion HD Cloning Kit (Clontech). The obtained plasmid for expressing the ispSK gene was designated as pSTV28-Ptac-IspSK.

Then, PCR was performed using pUC57-Mmamvk as the template, primers P57 (SEQ ID NO: 98) and P58 (SEQ ID NO: 99) and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to the composition attached to the kit, and DNA was amplified through 30 cycles of the reactions at 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 1 minute per 1 kb. As a result, a PCR-product containing the Mmamvk gene was obtained. Likewise, pSTV28-Ptac-IspSK (see above) was amplified by PCR using primers P59 (SEQ ID NO: 100) and P60 (SEQ ID NO: 101) and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to the composition attached to the kit, and DNA was amplified through 30 cycles of the reactions at 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 1 minute per 1 kb. As a result, a PCR-product containing pSTV28-Ptac-IspSK construct was obtained. The purified PCR-product containing the Mmamvk gene was ligated to the PCR-product containing the pSTV28-Ptac-IspSK construct using In- Fusion HD Cloning Kit (Clontech). The obtained plasmid for expressing the ispSK and Mmamvk genes was designated as pSTV28-Ptac-IspSK- Mmamvk.

4.4. Fixation of arabinose-inducible mvaES operon on chromosome

The sequence of Para-mvaES which is obtained from pMW-Para- mvaES-Ttrp (Auxiliary example 4.1) is expressed on a chromosome. The IdhA gene site is used as a chromosomal site to be fixed (Bunch PK et al., The IdhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 1997, 143(1): 187- 195). Insertion of Para- mvaES DNA fragment to IdhA gene site can be achieved by the use of Dual In/ Out strategy (Minaeva NI et al., Dual-In/ Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure. BMC Biotechnol. 2008 Aug 12;8:63. doi: 10.1 186/ 1472-6750-8-63.). As a result, the E. coli MG1655 Tn7::Ptac- KKDyl IdhA:: Para-mvaES is obtained and designated as E. coi EI20. 4.5. Construction of strain in which arabinose-inducible expression vector for mvaES is introduced into E. coli MG1655 Ptac-KKDyl strain

The cells of E. coli MG1655 Ptac-KKDyl strain (Auxiliary example 4.2.4) were made competent, and the plasmids pMW-Para-mvaES-Ttrp (Auxiliary example 4.1) and pSTV28-Ptac-IspSK-Mmamvk (Auxiliary example 4.3) were introduced by the electroporation method. The cells were applied onto a plate with LB-medium containing chloramphenicol (60 mg/L) and kanamycin (50 mg/L) and cultured at 37°C for 18 hours. Then, the transformant exhibiting resistance to chloramphenicol and kanamycin was obtained from the plate. Thus, the E. coli MG1655 Tn7::Ptac-KKDyI / pMW-Para-mvaES + pSTV28-Ptac-cplO-ispS(Kudzu)- mvk( mazei strain was obtained and designated as E. coli EI 10. Auxiliary example 5. Construction of the P. ananatis ISP3-mvk(Mpd) strain

A method for construction of the P. ananatis strain ISP3-mvk(Mpd), which is also referred to as P. ananatis SCI 7(0) AampC:: iiLphi80- KKDyI-ispS(K)-artRphi80 AampH::a«Lphi80-Para-mvaES-aiii?phi80 Acrt::pAH 162-Ptac-mvk( . paludicola), is described below.

5.1. Construction of pTrc-KKDyl-ispS(K)

An expression vector comprising a sequence in which the genes encoding mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate dexarboxylase and isopentenyl-diphosphate delta- isomerase were aligned linearly was constructed by an In-fusion cloning method (Clontech). A sequence of the genes encoding mevalonate kinase and phosphomevalonate kinase were amplified by PCR using pUC-mvk- pmk (SEQ ID NO: 56; WO2013179722 Al, US2014113344 Al) as the template and primers shown in Table 16. A sequence of the genes encoding diphosphomevalonate dexarboxylase and isopentenyl- diphosphate delta-isomerase were amplified by PCR using pTWV-dmd- yidi (SEQ ID NO: 61 ; WO2013179722 Al , US2014113344 Al) as the template primers shown in Table 16. The obtained PCR-products were cloned into pTrcHis2B vector (Invitrogen Corp.) by the In-fusion cloning method to construct an expression plasmid in which the four genes were aligned linearly. Prime Star HS DNA polymerase (Takara Bio Inc.) was used as a PCR enzyme. The reaction was performed at 98°C for 2 minutes, 30 cycles at 98°C for 10 seconds, 52°C for 5 seconds and 72°C for 1 minute per 1 kb, and at 72°C for 10 minutes. The PCR-fragments were inserted into pTrcHis2B vector digested with the restriction enzymes Ncol and Pstl by the In-fusion cloning method to construct the expression vector. E. coli JM 109 (Takara Bio Inc.) was transformed with the expression vector, a clone having an objective sequence length was selected, the plasmid was extracted according to a standard method, and its sequence was confirmed. The constructed expression vector was designated as pTrc-KKDyl(a). A nucleotide sequence of pTrc-KKDyl(a) is shown in SEQ ID NO: 103.

Table 16.

Then, the plasmid pTrc-KKDyl-IspS(K) in which ispS(K) gene is joined to pTrc-KKDyl(a) was constructed using the following procedure. The pTrc-KKDyl(a) was digested with the restriction enzyme Pstl (Takara Bio Inc.) to obtain pTrc-KKDyI(a)/Psfl. PCR was performed using pUC57- IspSK (Auxiliary example 4.3) as the template, primers shown in Table 17 and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to the composition attached to the kit, and the reaction at 98°C for 10 seconds, 54°C for 20 seconds and 68°C for 120 seconds was carried out for 30 cycles. As a result, a PCR-product containing the ispSK gene was obtained. The purified PCR-product containing the ispSK gene was ligated to pTrc-KKDyI(oc)/Psfl using In- Fusion HD Cloning Kit (Clontech). The resulting plasmid was designated as pTrc-KKDyl-ispS(K) (SEQ ID NO: 106). Table 17.

5.2. Construction of the integrative conditionally replicated plasmid carrying genes of upper and lower mevalonate pathways

To construct the integrative plasmid carrying genes of upper and lower mevalonate pathways, the pAH162-AaiiL-TcR-Aai£R integrative vector (Minaeva N.I. et al., BMC Biotechnol, 2008; 8:63) was used. The Kpnl-SaU fragment of pMW-Para-mvaES-Ttrp (Auxiliary example 4.1) was cloned into SpM-SaR recognition sites of pAH 162-A iiL-TcR-A iii?. As a result, the pAH 162-Para-mvaES plasmid carrying mvaES operon from E. faecalis under control of the E. coli Para promoter and repressor gene araC was constructed (FIG. 10).

The Eel 13611- SaR fragment of the pTrc-KKDyl-ispS(K) plasmid

(Auxiliary example 5.1) including coding parts of the mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase and isopentenyl-diphosphate delta-isomerase isomerase gepMWALlnes from S. cerevisiae and ispS gene from Kudzu was sub-cloned into Sphl-SaR sites _. of pAH 162-AaiiL-TcR-Aai£R. The resulting plasmid was designated pAH 162-KKDyI-ispS(K) (FIG. 1 1).

Then, the plasmid pSTV28-Ptac-IspS(M)-Mmamvk for expressing the ispS gene from Mucuna bracteata (M. bracteata) (Mucuna) (designated as the ispSM gene (SEQ ID NO: 131), U.S. patent application publication No. 2014/01 13344 Al), which encodes the isoprene synthase IspSM (SEQ ID NO: 132), and the mvk gene from M. mazei in E. coli was obtained. This plasmid was constructed from pSTV28-Ptac-ispSK- Mmamvk (Auxiliary example 4.3) by the substitution of the optimized ispSK gene originally derived from P. montana var. lobata (Kudzu) for the ispSM gene from M. bracteata (Mucuna). The BgRl-EcoRl fragment of the pSTV28-Ptac-ispS(M)-Mmamvk (SEQ ID NO: 133) containing the ispSM and mvk genes under control of Ptac was sub-cloned into BamHl-Ecll36ll recognition sites of the integrative vector pAH 162-A iiL-TcR-Aaiii?. Thus the plasmid pAH 162-Ptac-ispS(M)-mvk(Mma) was obtained (FIG. 12).

5.3. Construction of P. ananatis SC I 7(0) derivatives carrying attB site of phi80 phage in different points of genome The derivatives of P. ananatis SC I 7(0) carrying the attB site of phi80 phage substituting the ampC gene, ampH gene or art operon were constructed. The complete genome sequence of P. ananatis AJ 13355 was annotated, and it is available under the NCBI accession number PRJDA162073 or GeneBank accession numbers AP012032.1 and AP012033.1. The P. ananatis SC 17(0) strain (U.S. patent number 8,206,954 B2) is a strain constructed as a strain resistant to the λ-Red gene product for performing gene disruption in P. ananatis. The SC 17(0) strain was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1^st Dorozhny proezd, 1) on September 21, 2005 with an accession number of VKPM B-9246.

To obtain the specified above strains, λ-Red-dependent integration of the PCR-amplified DNA-fragments carrying iiLphi80-kan-aiii?phi80 flanked by 40 bp regions homologous to the target sites in genomic DNA of P. ananatis was performed according to the reported procedure (Katashkina J.I. et al., Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis, BMC Mol. Biol, 2009, 10:34). After electroporation, cells were plated on L-agar containing kanamycin (50 mg/L). DNA-fragments used for substitution of ampC, ampH genes and art operon for aiiLphi80-kan- iii?phi80 were amplified using primer pairs P64 and P65, P66 and P67, P68 and P69 (Table 18). The pMWattphi plasmid (Minaeva N.I. et al., BMC Biotechnol, 2008; 8:63) was used as the template in PCRs. The obtained integrants were named as P. ananatis strains SC17(0)AampC::attLphi80-kan-aiii¾3hi80, SC 17 (O)AampH : : iiLphi80-kan- iti?phi80 and SC 17(0)Acrt: : ariLphi80- kan-aiii?phi80. Primer pairs P70 and P71 , P72 and P73, P74 and P75 (Table 19) were used for verification by PCR of the SC 17(0)AampC: : a«Lphi80-kan- iii?phi80, SC 17(0)AampH: : arfLphi80- kan-aiii?phi80 and SC 17(0)Acrt::aiiLphi80-kan- iii?phi80 strains, respectively. Schemes of the obtained AampC::aiiLphi80-kan- iii?phi80, AampH::aiiLphi80-kan- iii?phi80 and Acrt::afiLphi80-kan-aiii?phi80 genome modifications are shown on FIG. 13A, FIG. 13B and FIG. 13C, respectively.

Table 18.

The kanamycin-resistance marker gene was deleted from the constructed strains using the pAH129-cat helper plasmid and the procedure described in Andreeva I.G. et al. (FEMS Microbiol. Lett, 201 1, 318(l):55-60). Primer pairs P70 and P71, P72 and P73, P74 and P75 were used for verification by PCR of the obtained SC17(0)AampC::attBphi80, SC17(0)AampH::attBphi80 and SC17(0)Acrt::attBphi80 strains, respectively.

5.4. Construction of P. ananatis ISP3-S

5.4.1. Construction of P. ananatis SC17(0)AampC::KKDyI-ispS(K)

(AG9579)

The pAH 162-KKDyI-ispS(K) plasmid (Auxiliary example 5.2) was integrated to the SC17(0)AampC::attBphi80 strain (Auxiliary example 5.3) using the helper plasmid pAH123-cat and the procedure described in Andreeva I.G. et al. (FEMS Microbiol Lett, 2011, 318(l):55-60). Primer pairs P70 and P76, P71 and P77 (Table 19) were used for verification by PCR of the obtained integrant. The vector part of pAH 162-KKDyI-ispS(K) was removed from the resulting strain SC17(0)AampC::pAH 162-KKDyI- ispS(K) using the pMW-int/xis-cat helper plasmid carrying int and xis genes of phage λ (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34). Thus, the P. ananatis SC17(0)AampC::KKDyI-ispS(K) strain (hereinafter referred to as AG9579) was obtained. Primers P70 and P78 (Table 19) were used for verification by PCR of the tetracycline-sensitive derivative. Scheme of the AampC::KKDyI-ispS(K) chromosome modification is shown on FIG. 14. 5.4.2. Construction of P. ananatis SC17(0)AampC::KKDyI-ispS(K)

AampH : : Para-mvaES

Genomic DNA was isolated from the P. ananatis SC 17(0)AampH::aiiLphi80-kan-aiii?phi80 strain (Auxiliary example 5.3) using GeneElute Bacterial Genomic DNA Kit (Sigma) and electroporated to the P. ananatis SC17(0)AampC::KKDyI-ispS(K) strain (Auxiliary example 5.4.1) according to the method of chromosome electroporation (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34). The transfer of the AampH::aiiLphi80-kan-aiii?phi80 mutation was confirmed by PCR using primers P73 and P74 (Table 19).

The kanamycin-resistance marker gene was deleted from the obtained strain using the phi80 Int/Xis-dependent procedure (Andreeva IG et al., FEMS Microbiol. Lett, 2011 , 318(l):55-60). The AampC::KKDyI- ispS(K) modification in the obtained kanamycin- sensitive (Km^s) recombinant was verified by PCR using primers P70 and P78 (Table 19), and the P. ananatis SC17(0)AampC::KKDyI-ispS(K)AampH::attBphi80 strain was selected.

The pAH 162-Para-mvaES plasmid (Auxiliary example 5.2) was integrated to SC 17(0)AampC::KKDyI-ispS(K)AampH::attBphi80 using the pAH 123-cat helper plasmid (Andreeva I.G. et al., FEMS Microbiol. Lett, 2011 , 318(l):55-60). Primer pairs P72 and P76, P73 and 77 (Table 19) were used for verification by PCR of the obtained integrant. The vector part of pAH 162 -Para- mvaES was deleted from the integrant using the phage λ Int/Xis-dependent technique (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34). Deletion of the vector part from chromosome was confirmed by PCR using primers P72 and P79 (Table 19). As a result, the marker-less P. ananatis SC17(0)AampC::KKDyI-ispS(K)AampH::Para- mvaES strain was obtained. Scheme of the AampH::Para-mvaES chromosome modification is shown on FIG. 15.

5.4.3. Construction of P. ananatis SC 17(0)AampC::aiiLphi80-KKDyI- ispS(K)-aifi?phi80 AampH::affLphi80-Para-mvaES-afii?phi80

Acrt:: riLphi80-Ptac-ispS(M)-mvk(Mma)- iii?phi80

The pAH 162-Ptac-ispS(M)-mvk(Mma) plasmid (Auxiliary example 5.2) was integrated to genome of P. ananatis SC 17(0)Acrt::attBphi80 (Andreeva I.G. et al., FEMS Microbiol. Lett, 201 1 , 318(l):55-60). Integration of the plasmid was confirmed by PCR using primer pairs P74 and P76, and P75 and P77 (Table 19). The chromosome modification SC 17(0)Acrt::pAH 162-Ptac-ispS(M)-mvk(Mma) as constructed above was transferred to the P. ananatis SC17(0)AampC::KKDyI- ispS(K)AampH::Para-mvaES strain (Auxiliary example 5.4.2) using the method of electroporation with genomic DNA (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34). The vector part of pAH 162-Ptac-ispS(M)- mvk(Mma) was deleted from the obtained integrant using the phage λ Int/Xis-dependent technique (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34). Deletion of the vector part from chromosome was confirmed by PCR using primers P74 and P80 (Table . 19). Scheme of the Acrt::Ptac- ispS(M)-mvk(Mma) chromosome modification is shown on FIG. 16.

Re-examination of all integrative expression cassettes introduced by PCR to the obtained P. ananatis strain revealed an unexpected rearrangement at the 5'-portion of the KKDy I oper on containing the genes encoding mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate dexarboxylase and the yldl gene from S. cerevisiae. To restore the cassette, the strain was electroporated with genomic DNA isolated from the P. ananatis SC17(0)AampC::pAH 162-KKDyI-ispS(K) strain (Auxiliary example 5.4.1) using GeneElute Bacterial Genomic DNA Kit (Sigma) as described (Katashkina J.I. et al., BMC Mol Biol, 2009, 10:34). Thus the P. ananatis strain was obtained which contained the genes required for the synthesis of isoprene. The vector part of pAH162- KKDyl-ispS(K) was deleted using the phage λ Int/Xis-dependent technique (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34), and the marker-less P. ananatis ISP3-S strain (P. ananatis SC17(0)AampC::a«Lphi80-KKDyI-ispS(K)- iii?phi80 AampH::attLphi80- Para-mvaES-aiii?phi80 Acrt: : aiiLphi80-Ptac-ispS(M)-mvk(Mma)- arii?phi80) was obtained.

5.5. Insertion of tac promoter

The tac promoter was inserted into P. ananatis SC17(0)AampC::KKDyI-ispS(K) (AG9579) using the λ-Red method which is also referred to as «Red-driven integration» or «Red-mediated integration_* (Datsenko K.A. and Wanner B.L., One-step inactivation of chromosomal genes in Escherichia coli K- 12 using PCR-products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). P. ananatis SCI 7(0) is known to be used as a recipient strain suitable for Red-dependent integration into a chromosome of P. ananatis (U.S. patent No. 7,919,284 B2). The helper plasmid RSF-Red-TER that expresses λ gam, bet and exo genes (also known as «λ-Red genes») was used for the Red-dependent integration. The RSF-Red-TER plasmid also contains the levansucrase- encoding gene (sacB) which allows recovering the plasmid from cells in a medium containing sucrose.

The RSF-Red-TER plasmid was introduced into AG9579 by electroporation using a standard method. The obtained strain was designated as AG9579 /RSF-Red-TER. Genomic DNA was extracted from P. ananatis SCI 7(0) strain Ptac-lacZ (Russian patent application No. 2006134574, PCT application publication No. 2008090770 Al, U.S. patent application publication No. 2010062496 Al) and used as the template in PCR. In P. ananatis SCI 7(0) strain Ptac-lacZ, the A iiL-Km^R- λαίί-R-Ptac fragment having tac promoter located downstream to XattL- Km^R-XattR is incorporated upstream to the lacZ gene (WO201 187139 Al). PCR was performed using primers P81 and P82 (Table 20) and Prime Star polymerase (Takara Bio Inc.). A reaction solution was prepared according to the composition attached to the kit, and the reaction was performed at 98°C for 10 seconds and 30 cycles at 54°C for 20 seconds and 68°C for 120 seconds. As a result, a PCR-fragment containing a kanamycin- resistance marker gene and the tac promoter was obtained. The obtained PCR-fragment was purified and then introduced into AG9579/RSF-Red- TER by electroporation (Katashkina J.I. et al., BMC Mol. Biol, 2009, 10:34).

The P. ananatis AG9579/ RSF-Red-TER strain having the introduced PCR-fragment was selected on the L-medium (10 g/L bactotrypsin, 5 g/L yeast extract, 5 g/L NaCl, 15 g/L agar, pH 7.0) containing kanamycin (40 mg/L). About 20 colonies were obtained as transformants. By PCR using primers P83 and P84 (Table 20), it was confirmed that the sequence derived from the obtained above PCR- fragment was inserted upstream to the KKDyl operon. Then, the helper plasmid RSF-Red-TER was deleted. The strain was inoculated to the L- medium containing 5 g/L sucrose and 1 mM isopropyl β-D- l- thiogalactopyranoside (IPTG) to form a single colony. After obtaining the single colony, it was replicated in the L-medium containing chloramphenicol (25 mg/L) and kanamycin (40 mg/L) and the L-medium containing kanamycin (40 mg/L). The chloramphenicol- sensitive (Cm^s) colony was selected. Thus the P. ananatis strain SC17(0) Ptac-KKDyl- ispS(K)(Km^R) was obtained.

Table 20.

5.6. Construction of an integrative plasmid carrying the the mevalonate kinase mvk (M. paludicola) gene

A DNA-fragment containing tac promoter was amplified by PCR using primers P85 and P86 (Table 20) and genomic DNA from P. ananatis strain SC17(0) Ptac-KKDyI-ispS(K)(Km^R) (Auxiliary example 5.5) as the template. The obtained PCR-producr was cloned into the Hind l-Sphl recognition sites of the pAH162-AafiL-TcR-Aa£ii? integrative vector (Minaeva N.I. et al., BMC Biotechnol, 2008; 8:63). The cloned promoter fragment was sequenced. Map of the obtained integrative expression vector pAH162-Ptac is shown on FIG. 17.

A chemically synthesized DNA-fragment (SEQ ID NO: 130) containing the mvk gene from Methanocella paludicola (M. paludicola) strain SANAE (complete genome sequence, GeneBank accession number APO 11532) linked to the canonical Shine-Dalgarno (SD) sequence was cloned into Pstl-Kpnl recognition sites of pAH 162-Ptac (see above). Thus the integrative plasmid pAH 162-Ptac-mvk(M. paludicola) carrying the mvk gene from M. paludicola was obtained. A map of this plasmid is shown on FIG. 18.

5.7. Construction of the P. ananatis ISP3-mvk(Mpd) strain

The pAH162-Ptac-mvk(M. paludicola) plasmid (Auxiliary example 5.6) was integrated into genome of P. ananantis SC 17(0)Acrt::attBphi80 (Auxiliary example 5.3) using pAH 123-cat helper plasmid (Andreeva I.G. et al., FEMS Microbiol. Lett, 2011, 318(l):55-60) to obtain the P. ananatis SC 17(0)Acrt::pAH 162-Ptac-mvk(M. paludicola) strain. The constructed Acrt::pAH162-Ptac-mvk(M. paludicola) chromosome modification was transferred to the P. ananatis ISP3-S strain (Auxiliary example 5.4) by electroporation of genomic DNA isolated from P. ananatis SC17(0)Acrt::pAH 162-Ptac-mvk(M. paludicola). Thus, the P. ananatis SC 17(0) AampC: : aiiLphi80-KKDyI-ispS(K)-aiii?phi80 AampH: : aiiLphi80- Para-mvaES-aiiJ¾)hi80 Acrt::pAH162-Ptac-mvk(M. paludicola) strain was obtained and designated as P. ananatis ISP3-mvk(Mpd).

Example 8. Production of polyisoprene

Isoprene is collected with a trap cooled with liquid nitrogen by passing the fermentation exhaust. Collected of isoprene is mixed with 35g of hexane (Sigma- Aldrich, catalog No. ) and lOg of silica gel (Sigma- Aldrich, catalog No. 236772) and lOg of alumina (Sigma- Aldrich, catalog No. 267740) under a nitrogen atmosphere in 100 mL glass vessel that is sufficiently dried. Resulting mixture is left at room temperature for 5 hours. Then supernatant liquid is skimmed and is added into 50ml glass vessel that is sufficiently dried.

Meanwhile, in a glove box under a nitrogen atmosphere, 40.0 μπιοΐ of Tris[bis(trimethylsilyl)amido]gadrinium, 150.0 μπιοΐ of tributylaluminium, 40.0 μπιοΐ of Bis[2-(diphenylphosphino)phenyl]amine, 40.0 mu.mol of triphenylcarbonium tetrakis(pentafluorophenyl) borate (Ph3CBC6F5)4) are provided in a glass container, which was dissolved into 5 mL of toluene (Sigma-Aldrich, catalog No. 24551 1), to thereby obtain a catalyst solution. After that, the catalyst solution is taken out from the glove box and added to the monomer solution, which is then subjected to polymerization at 50°C for 120 minutes.

After the polymerization, 1 mL of an isopropanol solution containing, by 5 mass %, 2,2'-methylene-bis(4-ethyl-6-t-butylphenol) (NS-5), is added to stop the reaction. Then, a large amount of methanol is further added to isolate the copolymer, and the copolymer is vacuum dried at 70°C to obtain a polymer.

Example 9. Production of rubber compound

The rubber compositions formulated as shown in Table 21 are prepared, which are vulcanized at 145°C for 35 minutes.

Table 21

*1 N-(l ,3-dimethyrbutyr)-N' -p-phenylenediamine

*2 N-cyclohexyl-2-benzothiazolesulfenamide

While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to the person skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated by reference as a part of this application.

Claims

1. An isoprene-producing bacterium, wherein said bacterium has been modified to overexpress a DNA encoding an enzyme comprising a combination of the following (A) and (B) :

(A) a protein selected from the group consisting of the proteins (A- l) to (A- 3):

(A-l) a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17;

(A-2) a protein having the amino acid sequence of SEQ ID NO: 2,

SEQ ID NO: 13 or SEQ ID NO: 17, but which includes substitution, deletion, insertion, and/ or addition of one or several amino acid residues and has activity of heptaprenyl diphosphate synthase with a protein of

(B) ; and

(A-3) a protein having the amino acid sequence of SEQ ID NO: 2,

SEQ ID NO: 13 or SEQ ID NO: 17, but which has an identity of amino acid sequence of not less than 50% with respect to the entire amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 17, and having activity of heptaprenyl diphosphate synthase with a protein of (B); and

(B) a protein selected from the group consisting of the proteins (B- l) to (B-3):

(B-l) a protein having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 19;

(B-2) a protein having the amino acid sequence of SEQ ID NO: 4,

SEQ ID NO: 15 or SEQ ID NO: 19, but which includes substitution, deletion, insertion, and/ or addition of one or several amino acid residues and has activity of heptaprenyl diphosphate synthase with a protein of (A); and

(B-3) a protein having the amino acid sequence of SEQ ID NO: 4,

SEQ ID NO: 15 or SEQ ID NO: 19, but which has an identity of amino acid sequence of not less than 50% with respect to the entire amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 15 or SEQ ID NO: 19, and having activity of heptaprenyl diphosphate synthase with a protein of (A); wherein said enzyme has an isoprene-synthesizing activity.

2. The bacterium according to claim 1 , wherein said enzyme is derived from a bacterium belonging to the genus Bacillus.

3. The bacterium according to claim 2, wherein said enzyme is derived from a bacterium belonging to the species Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens.

4. The bacterium according to any one of claims 1 to 3, wherein said bacterium harbors a DNA comprising a combination of the following (C) and (D):

(C) a DNA selected from the group consisting of the DNAs (C- l) to (C-3):

(C- l) a DNA comprising a hepS gene encoded by the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 12 or SEQ ID NO: 16;

(C-2) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 12 or SEQ ID NO: 16 encoding a protein as defined in the preceding (A); and

(C-3) a DNA comprising a variant nucleotide sequence of SEQ ID

NO: 1 , SEQ ID NO: 12 or SEQ ID NO: 16 due to degeneracy of genetic code; and

(D) a DNA selected from the group consisting of the DNAs (D- l) to (D-3):

(D- l) a DNA comprising a hepT gene encoded by the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18;

(D-2) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18 encoding a protein as defined in the preceding (B); and (D-3) a DNA comprising a variant nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 14 or SEQ ID NO: 18 due to degeneracy of genetic code.

5. The bacterium according to any one of claims 1 to 4, wherein said bacterium has been modified to overexpress the hepS and hepT genes.

6. The bacterium according to any one of claims 1 to 5, wherein said DNA is overexpressed by increasing a copy number of the DNA or modifying an expression control sequence of the DNA so that the expression of said DNA is enhanced as compared to a non-modified bacterium.

7. The bacterium according to any one of claims 1 to 6, wherein said bacterium belongs to the genus Bacillus or the family Enterobactenaceae.

8. The bacterium according to claim 7, wherein said bacterium belongs to the genus Bacillus, Escherichia or Pantoea.

9. The bacterium according to claim 8, wherein said bacterium is Bacillus subtilis, Escherichia coli or Pantoea ananatis.

10. The bacterium according to any one of claims 1 to 9, wherein said bacterium has been modified to increase the isoprene-synthesizing activity of the enzyme encoded by the hepS and hepT genes.

11. A method for producing isoprene comprising:

(i) cultivating the bacterium according to any one of claims 1 to 10 in a culture medium to produce the isoprene; and

(ii) collecting the isoprene from a medium.

12. A method for producing an isoprene polymer comprising: (i) cultivating the bacterium according to any one of claims 1 to 10 in a culture medium to produce isoprene;

(ii) collecting the isoprene from a medium; and

(iii) polymerizing the isoprene to produce the isoprene polymer.

13. A polymer derived from isoprene produced by the method according to claim 11.

14. A rubber composition comprising the polymer according to claim 13.

15. A tire manufactured by using the rubber composition according to claim 14.