Transformants having isoprene-producing ability from methane

Abstract

The present invention relates to a transformant having isoprene-producing ability The transformant of the present invention has superior isoprene-producing ability by introduction, into methanotrophic bacteria, a nucleotide sequence encoding exogenous isoprene synthase, a nucleotide sequence encoding exogenous isopentenyl diphosphate isomerase, and a nucleotide sequence encoding exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, and thus can be effectively used for isoprene biosynthesis.

Description

Transformants capable of producing isoprene from methane

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0008400, filed January 18, 2024, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present invention relates to a transformant having the ability to produce isoprene from methane and its use.

[0005]

[0006] Isoprene is a key component of natural rubber and is also the basis for producing synthetic rubber, which is used in tires, medical supplies, and adhesives. Furthermore, isoprene can be used as a transportation fuel and jet fuel, and its high energy efficiency and low greenhouse gas emissions compared to other biofuels make it an ideal drop-in biofuel.

[0007] Currently, isoprene is obtained through the refining process of petroleum, but the price of isoprene is steadily rising due to the fluctuating oil price and the focus of the petroleum refining process on producing fuel materials that exclude isoprene, while the demand for synthetic rubber is continuously increasing.

[0008] Microbial production of isoprene has the advantages of high purity, eliminating the need for advanced purification processes, being produced from renewable resources and low-cost raw materials, and easily converting it into biofuels and biochemicals. Biologically, isoprene is produced from the precursor dimethylallyl diphosphate (DMAPP) via isoprene synthase. Since the first reports of isoprene production and release in poplar and willow species, research on the mechanisms of isoprene release and related enzymes in plants has continued steadily. However, research on microbial isoprene production and the production of isoprene using microorganisms as hosts has only recently been conducted. However, isoprene production still requires a large amount of carbon, which limits its production efficiency.

[0009] Meanwhile, methane (CH4) is a simple hydrocarbon gas composed of a single carbon atom. While it is relatively inexpensive compared to liquid hydrocarbons like petroleum, converting it into high-value compounds is challenging. Furthermore, it is known to be a greenhouse gas, and efforts are ongoing to reduce methane emissions by converting it into other compounds and apply this to industrial applications.

[0010] To convert methane biologically, one option is to utilize methanotrophs. Methanophores are bacteria that possess the unique ability to utilize methane as a sole carbon or energy source. They can convert methane into methanol using methane monooxygenase (MMO), which can then be converted into various organic compounds through various biosynthetic pathways.

[0011] Under this technical background, the present invention has confirmed that the isoprene production ability of a transformant in which genes for isoprene production enzymes, isopentenyl diphosphate isomerase, and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are introduced into a methanogenic bacterium is remarkably superior, and thus it is expected that the transformant can be efficiently utilized for isoprene production.

[0012]

[0013] The purpose of the present invention is to provide a transformant having isoprene production ability.

[0014] In addition, the present invention aims to provide a composition for producing isoprene comprising the transformant.

[0015] In addition, the present invention aims to provide a method for producing isoprene, which includes a step of culturing the transformant.

[0016]

[0017] In order to achieve the above object, one aspect of the present invention provides a transformant in which a base sequence encoding exogenous isoprene synthase, a base sequence encoding exogenous isopentenyl diphosphate isomerase, and a base sequence encoding exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are introduced into a methanogenic bacterium.

[0018] In addition, to achieve the above purpose, another aspect of the present invention provides a composition for producing isoprene comprising the transformant.

[0019] In addition, in order to achieve the above purpose, another aspect of the present invention provides a method for producing isoprene, including a step of culturing the transformant in the presence of methane.

[0020]

[0021] The transformant of the present invention has excellent isoprene production ability by introducing a base sequence encoding an exogenous isoprene synthase, a base sequence encoding an exogenous isopentenyl diphosphate isomerase, and a base sequence encoding an exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase into a methanogenic bacterium, and thus can be effectively used for isoprene biosynthesis purposes.

[0022] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0023]

[0024] Figure 1 shows a plasmid into which an isoprene production enzyme gene and an idi gene or dxs gene have been introduced.

[0025] Figure 2 shows the isoprene production of transformants into which each of the three plasmids of Figure 1 was introduced.

[0026] Figure 3 shows the isoprene production of transformants expressing the E. coli-derived idi gene or the S. cerevisiae-derived idi gene.

[0027] Figure 4 shows a plasmid into which an isoprene production enzyme gene and an idi gene, dxs gene, or ispH gene derived from S. cerevisiae have been introduced.

[0028] Figure 5 shows the isoprene production of transformants into which each of the eight plasmids of Figure 4 was introduced.

[0029] Figure 6 shows the isoprene production amount of a transformant expressing an isoprene production enzyme gene, an idi gene derived from S. cerevisiae, and an ispH gene derived from R. communis.

[0030]

[0031] Hereinafter, the present invention will be described in detail.

[0032]

[0033] 1. Transformants with isoprene production ability

[0034] One aspect of the present invention provides a transformant having isoprene production ability.

[0035] The above transformant may be one in which a base sequence encoding an exogenous isoprene synthase, a base sequence encoding an exogenous isopentenyl diphosphate isomerase, and a base sequence encoding an exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are introduced into a methanogenic bacterium.

[0036] The transformant of the present invention can have isoprene production ability by introducing a base sequence encoding an exogenous isoprene synthase.

[0037] The above isoprene synthase (IspS) has the activity of converting dimethylallyl pyrophosphate (DMAPP), an isomer of isopentenyl pyrophosphate (IPP), into isoprene.

[0038] In the present invention, the exogenous isoprene synthase may be derived from a plant of the genus Populus, for example, from Populus trichocarpa, Populus alba, Populus deltoides, and Populus davidiana, and specifically from Populus trichocarpa, but any enzyme having isoprene synthesis activity may be used without limitation.

[0039] In the present invention, the exogenous isoprene synthase may include the amino acid sequence of SEQ ID NO: 24.

[0040] The amino acid sequences of the present invention may include variants having different sequences by deletion, insertion, substitution, or a combination thereof of amino acid residues, within a range that does not affect the structure, function, activity, etc. of a polypeptide including the same. In addition, the amino acid sequences may include amino acids that have undergone conventional modifications known in the art, and the amino acid modifications may be, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. The amino acid sequences of the present invention include those having substantially the same amino acid sequence as the above or variants thereof. The meaning of having the substantially identical amino acid sequence may include, but is not limited to, an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the amino acid sequence described above.

[0041] Additionally, the amino acid sequences described above may include conservative substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. For example, conservative substitutions include substitutions among amino acid residues having basic side chains, such as lysine, arginine, and histidine. In addition, amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; Substitutions among amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, are also conservative substitutions.

[0042] In the present invention, the exogenous isoprene synthase may be encoded by the base sequence of SEQ ID NO: 25.

[0043] The base sequence of the present invention may include a base sequence substantially identical to the base sequence encoding the amino acid sequence of the present invention. The substantially identical base sequence includes, for example, a case where the same amino acid can be synthesized when transcribed and translated, and may be a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology with the base sequences listed above, but is not limited thereto.

[0044] The base sequence of the present invention may include an optimized base sequence depending on the type of organism to which it is introduced and expressed and the expression system of said organism, such as transcription and translation. This is due to the degeneracy of codons, and thus, various combinations of nucleotide sequences capable of encoding the expressed protein may exist, all of which are included within the scope of the present invention. The modification of the polynucleotide according to the above codon optimization may be determined depending on the type of organism to which the exogenous base sequence of the present invention is to be expressed and applied.

[0045] The above isopentenyl-diphosphate delta isomerase (IDI) has the activity of converting IPP to DMAPP or converting DMAPP to IPP, and thus performs the function of regulating the balance of DMAPP and IPP concentrations.

[0046] In the present invention, the exogenous isopentenyl diphosphate isomerase may be derived from yeast, and the yeast may be a microorganism belonging to the genus Saccharomyces, the genus Schizosaccharomyces, the genus Phaffia, the genus Kluyveromyces, the genus Pichia, the genus Candida, or the genus Yarrowia.

[0047] Specifically, the exogenous isopentenyl diphosphate isomerase may be derived from the genus Saccharomyces, and more specifically, the yeast of the genus Saccharomyces may be Saccharomyces cerevisiae.

[0048] In one embodiment of the present invention, it was confirmed that a methanogenic bacterium expressing isopentenyl diphosphate isomerase derived from Saccharomyces cerevisiae has significantly superior isoprene production ability compared to a methanogenic bacterium expressing isopentenyl diphosphate isomerase derived from Escherichia coli. Therefore, by introducing a base sequence encoding isopentenyl diphosphate isomerase derived from Saccharomyces cerevisiae, isoprene production ability can be significantly improved.

[0049] In the present invention, the exogenous isopentenyl diphosphate isomerase may include the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and specifically, may include the amino acid sequence of SEQ ID NO: 21.

[0050] In the present invention, the exogenous isopentenyl diphosphate isomerase may be encoded by the base sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and specifically may be encoded by the base sequence of SEQ ID NO: 23.

[0051] The above 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH) has the activity of converting 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) into IPP or DMAPP.

[0052] In the present invention, the 4-hydroxy-3-methylbut-2-enyl diphosphate reductase may be derived from a plant of the genus Populus, for example, from Populus trichocarpa, Populus alba, Populus deltoides, Populus davidiana, and specifically from Populus trichocarpa.

[0053] Additionally, the above 4-hydroxy-3-methylbut-2-enyl diphosphate reductase may be derived from Ricinus communis.

[0054] In one embodiment of the present invention, it was confirmed that when 4-hydroxy-3-methylbut-2-enyl diphosphate reductase was further expressed in addition to isoprene synthase and isopentenyl diphosphate isomerase in a methanogenic bacterium, isoprene productivity was significantly improved. In particular, when 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Ricinus communis was expressed, isoprene productivity was higher than when 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from poplar Trichokappa was expressed. In addition, it was confirmed that when 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Ricinus communis was further expressed, the growth level of the transformant was higher than when only isoprene synthase and isopentenyl diphosphate isomerase were introduced, so a transformant further including a base sequence encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Ricinus communis can be usefully utilized for isoprene biosynthesis.

[0055] In the present invention, the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase may include the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, and specifically, may include the amino acid sequence of SEQ ID NO: 27.

[0056] In the present invention, the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase may be encoded by the base sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and specifically, may be encoded by the base sequence of SEQ ID NO: 29.

[0057] In the present invention, the base sequence encoding the exogenous isoprene synthase, the base sequence encoding the exogenous isopentenyl diphosphate isomerase, and the base sequence encoding the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are P o It may be operably linked to a promoter.

[0058] The above “operably linked” means a state in which a nucleic acid expression control sequence and a sequence encoding a desired protein are functionally linked to each other so as to perform a general function. The above ‘expression control sequence’ means a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. For example, it includes a promoter for performing transcription, an arbitrary operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence controlling the termination of transcription and translation, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer.

[0059] The above P o The promoter can react with phenol to bind multimerized DmpR, thereby activating transcription of a target polypeptide operably linked thereto.

[0060] In the present invention, the transformant may further have a base sequence encoding an exogenous 1-deoxy-D-xylulose 5-phosphate synthase introduced therein.

[0061] The above 1-deoxy-D-xylulose-5-phosphate synthase (dxs) has the activity of synthesizing 1-deoxy-D-xylulose-5-phosphate using pyruvate and glyceraldehyde-3-phosphate as substrates.

[0062] In the present invention, the base sequence encoding the exogenous 1-deoxy-D-xylulose 5-phosphate synthase may be introduced downstream of the base sequence encoding the exogenous isopentenyl diphosphate isomerase.

[0063] In one embodiment of the present invention, pAWPS-P o -The isoprene production capacity of the methanotroph containing the PtIspS-idi-dxs plasmid was measured to be 2.24 mg / L, but pAWPS-P with the order of the idi and dxs genes changed o - In the case of methanotrophs introduced with the PtIspS-dxs-idi plasmid, it was confirmed that the isoprene production capacity was significantly low at 0.09 mg / L. Therefore, it is desirable for the base sequence encoding 1-deoxy-D-xylulose 5-phosphate synthase to be introduced downstream of the base sequence encoding isopentenyl diphosphate isomerase in terms of improving isoprene production capacity.

[0064] In the present invention, the exogenous 1-deoxy-D-xylulose 5-phosphate synthase may include the amino acid sequence of SEQ ID NO: 30.

[0065] In the present invention, the exogenous 1-deoxy-D-xylulose 5-phosphate synthase may be encoded by the base sequence of SEQ ID NO: 31.

[0066] The above "transformant" may be a methanogenic bacterium comprising exogenous isoprene synthase, exogenous isopentenyl diphosphate isomerase and exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, a base sequence encoding them, or an expression vector comprising the base sequence.

[0067] The above transformant includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an exogenous gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein or product.

[0068] The above "methanotroph" refers to a bacterium that can metabolize methane as a carbon source and energy source, and any strain with this property can be used. Methanophores can be classified into two types, Type I (γ-proteobacteria) and Type II (α-proteobacteria), depending on which pathway they use in the process of producing organic compounds from methane. The methanotroph of the present invention may perform carbon assimilation using the RuMP (ribulose monophosphate) pathway or may perform carbon assimilation using the serine pathway, and for example, may be a methanotroph that can produce organic acids from methane using the RuMP pathway. The above-mentioned methanogenic bacteria may be strains classified as Methylococcus capsulatus, Methylosinus trichosporium, or Methylomonassp., and specifically may be Methylococcus capsulatusBath strains. However, any strain capable of producing isoprene by introducing the base sequence of the present invention may be used without limitation.

[0069] In the present invention, the term "transformation" refers to a phenomenon in which a vector containing a polynucleotide encoding a target polypeptide is introduced into a cell or microorganism, thereby artificially causing a genetic change by introducing an external polynucleotide into the cell or microorganism, so that the polypeptide encoded by the polynucleotide can be expressed in the introduced cell or microorganism. The transformed polynucleotide may be located within the chromosome of the microorganism or located outside the chromosome, as long as it can be expressed in the microorganism. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence necessary for expression in the microorganism, but is not limited thereto.

[0070] In the present invention, any transformation method can be used, and can be easily performed according to a conventional method in the art. Common transformation methods include CaCl2 precipitation method, Hanahan method which increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in CaCl2 method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation method, etc. The transformation method of the present invention is not limited to the above examples, and transformation or transfection method conventionally used in the art can be used without limitation.

[0071] In addition to the base sequence encoding the isoprene synthase, the transformant of the present invention may additionally have base sequences encoding other enzymes acting in the isoprene biosynthetic pathway introduced. The isoprene biosynthetic pathway is the mevalonic acid pathway (also called the MVA pathway) or the non-mevalonic acid pathway (also called the MEP pathway). The mevalonic acid pathway uses acetyl CoA as a starting material, and enzymes acting in the mevalonic acid pathway include, in order from upstream, acetyl CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonic acid kinase, 5-phosphomevalonic acid kinase, diphosphomevalonic acid decarboxylase, and isopentenyl diphosphate isomerase. Meanwhile, the non-mevalonate pathway starts from glyceraldehyde 3-phosphate and pyruvate, and enzymes acting in the non-mevalonate pathway include, in order from upstream, DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, and HMB-PP reductase.

[0072] The transformant of the present invention is a methanotroph, wherein a base sequence encoding an exogenous isoprene synthase and a base sequence encoding an exogenous isopentenyl diphosphate isomerase are introduced into the methanotroph, and can produce acetyl-CoA and pyruvate from methane using the RuMP pathway. The produced acetyl-CoA is converted into IPP and DMAPP through the actions of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, 5-phosphomevalonate kinase, diphosphomevalonate decarboxylase, and isopentenyl diphosphate isomerase through the mevalonate pathway, and DMAPP can be converted into isoprene by isoprene synthase. In addition, the produced pyruvate is converted into IPP and DMAPP through the non-mevalonate pathway, through the actions of DOXP synthase, DOXP reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, HMB-PP synthase, and HMB-PP reductase, and DMAPP can be converted into isoprene by isoprene synthase.

[0073] That is, the transformant of the present invention has the effect of significantly increasing isoprene production capacity compared to existing methanogenic bacteria by introducing exogenous base sequences of other species encoding enzymes that function in the mevalonic acid pathway and / or non-mevalonic acid pathway in addition to enzymes inherent in methanogenic bacteria.

[0074]

[0075] 2. Composition and production method for isoprene production

[0076] Another aspect of the present invention provides a composition for producing isoprene comprising the transformant.

[0077] Since the overlapping description is the same as that described above in '1. Transformant having isoprene production ability', the description is omitted.

[0078] The above composition can be usefully used for isoprene production purposes by including the transformant of the present invention having excellent isoprene production ability.

[0079] The above composition is P introduced into the transformant. o To induce expression of the promoter, phenol may be further included.

[0080] The composition may comprise, as a carbon source, at least one C1 compound selected from the group consisting of methane, methanol, methylamine, formic acid, formaldehyde and formamide.

[0081] Meanwhile, the composition may not contain any other carbon source other than methane (CH4). The methane may be dissolved and included in the composition, or may be provided in the form of methane gas being supplied to the composition. The other carbon source other than methane may be, for example, methanol, glucose, starch, or sugar, but is not limited thereto. The non-inclusion of any other carbon source other than methane may mean that the concentration of the other carbon source in the composition is 0, or that the carbon source is included in the composition at a level where the transformant cannot continuously grow using the carbon source other than methane. However, although the initial composition does not contain any other carbon source other than methane, as the transformant in the composition grows and metabolizes using methane as a carbon source, isoprene produced by the transformant using methane may be included in the composition.

[0082] In addition to the carbon source, the above composition may further include various nitrogen sources, trace element components, amino acids, vitamins, minerals, and appropriate precursors.

[0083] In addition, the composition may further include an additive. Any additive may be used as long as it does not inhibit the growth, metabolism, etc. activities of the transformant included in the composition of the present invention. For example, the additive may be a protective agent, a buffer, or a carrier. The protective agent may be any agent that functions to protect the transformant and prevent damage or death during the process of storing, storing, or distributing the composition of the present invention without affecting the growth, metabolism, etc. activities of the transformant. When the composition of the present invention is stored, stored, distributed, or used in a lyophilized state, the protective agent may be a cryoprotectant. The cryoprotectant may be at least one selected from the group consisting of skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol, chicory, potassium phosphate, and honey, but is not limited thereto, and any agent that can prevent the transformant from being damaged or killed during the freeze-drying process of the composition may be included. When the cryoprotectant is included in the composition of the present invention, the composition can be used in the form of a lyophilized product, for example, a powder, through a freeze-drying process. The freeze-dried composition has advantageous advantages in formulation, packaging, storage, etc., and has the effect of preserving the transformant included therein for a long period of time.

[0084]

[0085] Another aspect of the present invention provides a method for producing isoprene, comprising the step of culturing the transformant in the presence of methane.

[0086] The above methane (CH4) may be supplied by a mass flow controller (MFC). When methane is supplied by a mass flow controller, the supply speed of methane can be controlled, and when methane is supplied together with other gases such as oxygen and nitrogen, the ratio of them can be controlled. The methane may be supplied so as to be included in a ratio of 10% to 60% of the air supplied during cultivation, for example, so as to be included in a ratio of 15% to 55%, or so as to be included in a ratio of 20% to 50%. In this case, oxygen may be included in the supplied air in a ratio of 2% to 5%, 2.5% to 4.5%, or 3% to 4%.

[0087] The above culturing step is P introduced into the transformant. o To induce the expression of the promoter, it can be performed in the presence of phenol.

[0088] The above method for producing isoprene may further include a step of recovering isoprene from the culture after the culturing step.

[0089] The above method for producing isoprene may be one that produces isoprene at a concentration of 100 mg / L or more, specifically 500 mg / L or more, 1000 mg / L or more, 1500 mg / L or more, 2000 mg / L or more, 2500 mg / L or more, or 3000 mg / L or more, when the transformant is cultured for 10 hours or more, specifically 15 hours or more, 20 hours or more, 30 hours or more, 40 hours or more, 48 hours or more, 50 hours or more, or 60 hours or more.

[0090] Cultivation of the transformant may be performed at 20°C to 50°C, preferably at 25°C to 45°C, more preferably at 30°C to 40°C. If cultivation of the transformant is performed at a temperature range of less than 20°C or more than 50°C, a sufficient amount of intermediate product may not be produced, which may result in a problem of insufficient production or insufficient production of the final product, isoprene.

[0091] In addition, the culture of the transformant can be performed at pH 5 to pH 8, preferably at pH 6 to pH 7, and more preferably at pH 6.3 to pH 6.7, but is not limited thereto. The culture pH conditions of the transformant as described above can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium of the transformant. If the culture pH conditions of the transformant are outside the above range, it is difficult for the transformant to grow, so there is a problem that isoprene expression is not easy, and isoprene synthesis is not performed efficiently.

[0092]

[0093] Hereinafter, the present invention will be described in detail by examples.

[0094] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0095]

[0096] [Example 1]

[0097] Production of modified methanogenic bacteria with idi or dxs genes

[0098] To produce a modified methanogenic bacterium with enhanced isoprene production, first, phenol-induced P oA plasmid was constructed that additionally contained a promoter, an isoprene-producing enzyme gene (SEQ ID NO: 25) derived from P. trichocarpa, and an idi gene (SEQ ID NO: 22) derived from E. coli or a dxs gene (SEQ ID NO: 31) derived from M. capsulatus Bath to control the IPP / DMAPP ratio (Fig. 1).

[0099] Specifically, the plasmid pAWPS-P o -To build PtIspS-idi, the DNA template pAWPS-P o -pAWPS-P based on PtIspS-MVA o -PtIspS and idiDNA fragments were amplified using KOD One DNA polymerase (Toyobo, Tokyo, Japan). The obtained fragments were assembled using Gibson Assembly (NEB, Ipswich, MA, USA).

[0100] Plasmid pAWPS-P o -PtIspS-idi-dxs and pAWPS-P o -For PtIspS-dxs-idi, the dxs DNA fragment amplified by PCR was cloned into pAWPS-P o -PtIspS-idi fragments were assembled. The primers used are as shown in Table 1 below.

[0101] Plasmid target DNA primer sequence pAWPS-P o -PtIspS-idipAWPS-P o -PtIspSV-Fattgtcgggaagatgcg (SEQ ID NO: 1)PtIspS-idi-Rtacgttacctcctcaggatcttagcgttcgaacggca (SEQ ID NO: 2)idiPtIspS-idi-Fgatcctgaggaggtaacgtatg (SEQ ID NO: 3)idi-Rtcacgcatcttcccgacaatttatttaagctgggtaaatgcaga (SEQ ID NO: 4)pAWPS-P o -PtIspS-idi-dxspAWPS-P o-PtIspS-idiV-Fattgtcgggaagatgcg (SEQ ID NO: 1)idi-VRttatttaagctgggtaaatgcaga (SEQ ID NO: 5)dxsidi-dxs-IFcatttacccagcttaaataagtgacccaacagcaagaac (SEQ ID NO: 6)dxs-Rtcacgcatcttcccgacaattcagtgtttgtccttggc (SEQ ID NO: 7)

[0102]

[0103] The three types of plasmids prepared above were transformed into E. coliCC118(λpir), and then transformed into wild-type M. capsulatusBath or mmoX gene knockout Bath△mmoX strain (M. capsulatusBath with mmoX knockout mutant) through tri-parental mating-based conjugation.

[0104]

[0105] [Example 2]

[0106] Measurement of isoprene production capacity of modified methanogenic bacteria

[0107] An experiment was conducted to measure the isoprene production ability of three strains of modified methanotrophs produced in Example 1. Specifically, frozen cell stocks were inoculated into 3 mL NMS medium containing 25 μg / mL kanamycin and 10 μM CuCl2, and cultured in 15 mL sealed glass vials. At this time, 50% methane / 50% air was used based on headspace, and cultured for 18 to 24 hours at 37°C and 200 rpm. These were used as seeds, and 10% of the cells were inoculated into a new 3 mL medium (containing 25 μg / mL kanamycin and 10 μM CuCl2) and cultured. At the time of inoculation, the initial cell concentration OD 600 The concentration was adjusted to 0.06, and 5 μM phenol was added for gene expression, and the cells were further cultured at 37°C and 200 rpm for 48 hours.

[0108] A 25 μL headspace sample from a sealed vial was analyzed using a gas chromatography-mass spectrometer (GC-MS, 5977A MSD, Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS column (30 m Х 0.25 mm Х 0.25 μm; Agilent Technologies). The column flow rate was set to 1 mL / min, and the injector and oven temperatures were maintained at 260°C and 50°C for 2 min, respectively. Isoprene detection was performed at 67 m / z using selective ion monitoring.

[0109] As a result of the measurement, there was no significant difference in growth among the three types of modified methanogenic bacteria, but pAWPS-P o -The isoprene production ability of the methanogenic bacteria containing the PtIspS-idi-dxs plasmid was the highest at 2.24 mg / L, and pAWPS-P containing only the PtIspS and idi genes without the dxs gene o The isoprene production capacity of the methanogenic bacteria expressing the -PtIspS-idi plasmid was confirmed to be slightly low at 2.09 mg / L (Fig. 2). However, pAWPS-P, in which the order of the idi and dxs genes was changed, o - In the case of methanotrophs introduced with the PtIspS-dxs-idi plasmid, isoprene production was measured to be significantly low at 0.09 mg / L. This implies that maintaining the expression level of the idi gene is very important to increase isoprene production in methanotrophs.

[0110]

[0111] [Example 3]

[0112] Optimization of isoprene production by modified methanogenic bacteria

[0113] To further improve the isoprene production ability of methanogenic bacteria, plasmid pAWPS-P was constructed by replacing the E. coli-derived idi gene (SEQ ID NO: 22) in the plasmid constructed in Example 1 with the Saccharomyces cerevisiae-derived idi gene (SEQ ID NO: 23). o -PtIspS-Scidi and pAWPS-P o -PtIspS-Scidi-dxs was created.

[0114] Specifically, the DNA fragment pAWPS-Po-PtIspS was amplified and then assembled with the Scidi-1 DNA fragment amplified from the genomic DNA of Saccharomyces cerevisiae to form pAWPS-P o -PtIspS-Scidi plasmid was constructed.

[0115] Plasmid pAWPS-P o -PtIspS-Scidi-dxs was assembled by amplifying the DNA fragment pAWPS-Po-PtIspS-dxs and then assembling it with the Scidi-2 DNA fragment amplified from the genomic DNA of cerevisiae. The primers used are as shown in Table 2 below.

[0116] Plasmid target DNA primer sequence pAWPS-P o -PtIspS-ScidipAWPS-P o -PtIspSV-Fattgtcgggaagatgcg (SEQ ID NO: 1)PtIspS-idi-Rtacgttacctcctcaggatcttagcgttcgaacggca (SEQ ID NO: 2)Scidi-1ScIDI1-IF1agatcctgaggaggtaacgtatgactgccgacaacaatag (SEQ ID NO: 8)ScIDI1-IR3tcacgcatcttcccgacaatttatagcattctatgaatttgcctgtc (SEQ ID NO: 9)pAWPS-P o -PtIspS-Scidi-dxspAWPS-P o-PtIspS-dxsRBS-PmoB1-VFgtgacccaacagcaagaac (SEQ ID NO: 10) PtIspS-idi-Rtacgttacctcctcaggatcttagcgttcgaacggca (SEQ ID NO: 2) Scidi-2ScIDI1-IF1agatcctgaggaggtaacgtatgactgccgacaacaatag (SEQ ID NO: 8) ScIDI1-IR4agttcttgctgttgggtcacttatagcattctatgaatttgcctgtc (SEQ ID NO: 11)

[0117]

[0118] Afterwards, this was introduced into methanotrophs to produce two new strains of methanotrophs expressing the plasmid, and isoprene production ability was measured using the same method as in Example 2.

[0119] As a result, it was confirmed that the isoprene production ability of the methanogenic bacteria into which the idi gene derived from S. cerevisiae was introduced was significantly superior to that of the methanogenic bacteria into which the idi gene derived from E. coli was introduced (Fig. 3).

[0120] Next, to derive a combination of genes that could optimize isoprene production in methanotrophs, eight plasmids were constructed with different ispH gene origins and different presences of the idi gene, dxs gene, and ispH gene (Fig. 4). The primers used are shown in Table 3 below.

[0121] Plasmid target DNA primer sequence pAWPS-Po-PtIspS-PtIspHpAWPS-Po-PtIspSV-Fattgtcgggaagatgcg(SEQ ID NO: 1)V-Rttagcgttcgaacggca(SEQ ID NO: 12)PtIspH-1IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc(SEQ ID NO: 13)PtIspH-IR1tcacgcatcttcccgacaatttaggccacctgcagg(SEQ ID NO: 14)pAWPS-Po-PtIspS-RcIspHpAWPS-Po-PtIspSV-Fattgtcgggaagatgcg(SEQ ID NO: 1)V-Rttagcgttcgaacggca(SEQ ID NO: 12) RcIspH-1IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc (SEQ ID NO: 13) RcIspH-IR1tcacgcatcttcccgacaatttaggccagctgcagg (SEQ ID NO: 15) pAWPS-Po-PtIspS-PtIspH-ScIDIpAWPS-Po-PtIspS-ScIDIPtIspS-idi-Fgatcctgaggaggtaacgtatg (SEQ ID NO: 3) V-Rttagcgttcgaacggca (SEQ ID NO: 12) PtIspH-2IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc (SEQ ID NO: 13) PtIspH-IR2tacgttacctcctcaggatcttaggccacctgcagg (SEQ ID NO: 16)pAWPS-Po-PtIspS-RcIspH-ScIDIpAWPS-Po-PtIspS-ScIDIPtIspS-idi-Fgatcctgaggaggtaacgtatg(SEQ ID NO: 3)V-Rttagcgttcgaacggca(SEQ ID NO: 12)RcIspH-2IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc(SEQ ID NO: 13)RcIspH-IR2tacgttacctcctcaggatcttaggccagctgcagg(SEQ ID NO: 17)pAWPS-Po-PtIspS-PtIspH-dxspAWPS-Po-PtIspS-dxsRBS-PmoB1-VFgtgacccaacagcaagaac(SEQ ID NO:10)V-Rttagcgttcgaacggca(SEQ ID NO: 12)PtIspH-3IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc(SEQ ID NO: 13)PtIspH-IR3agttcttgctgttgggtcacttaggccacctgcagg(SEQ ID NO: 18)pAWPS-Po-PtIspS-RcIspH-dxspAWPS-Po-PtIspS-dxsRBS-PmoB1-VFgtgacccaacagcaagaac(SEQ ID NO: 10)V-Rttagcgttcgaacggca(SEQ ID NO: 12)RcIspH-3IspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc(SEQ ID NO: 13) RcIspH-IR3agttcttgctgttgggtcacttaggccagctgcagg (SEQ ID NO: 19) pAWPS-Po-PtIspS-PtIspH-ScIDI-dxspAWPS-Po-PtIspS-ScIDIPtIspS-idi-Fgatcctgaggaggtaacgtatg (SEQ ID NO: 3) V-Rttagcgttcgaacggca (SEQ ID NO: 12) PtIspH-dxsIspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc (SEQ ID NO: 13) PtIspH-IR2tacgttacctcctcaggatcttaggccacctgcagg (SEQ ID NO: 16)pAWPS-Po-PtIspS-RcIspH-ScIDI-dxspAWPS-Po-PtIspS-ScIDIPtIspS-idi-Fgatcctgaggaggtaacgtatg(SEQ ID NO: 3)V-Rttagcgttcgaacggca(SEQ ID NO: 12)RcIspH-dxsIspH-IFttctgccgttcgaacgctaagtggcatcaatccaacacc(SEQ ID NO: 13)RcIspH-IR2tacgttacctcctcaggatcttaggccagctgcagg(SEQ ID NO: 17)

[0122]

[0123] Afterwards, this was introduced into methanotrophs to produce 8 new methanotrophs expressing the plasmid, and the isoprene production ability was measured using the same method as in Example 2.

[0124] As a result, it was confirmed that the isoprene productivity of the methanotroph introduced with the S. cerevisiae-derived idi gene (SEQ ID NO: 23) and the R. communis-derived ispH gene (SEQ ID NO: 29) was the highest at 3.56 mg / L (Fig. 5). In addition, it was shown that the isoprene productivity was further improved when the R. communis-derived ispH gene (SEQ ID NO: 29) was introduced compared to the P. trichocarpa-derived ispH gene (SEQ ID NO: 28). Meanwhile, the methanotroph introduced with only the S. cerevisiae-derived idi gene (SEQ ID NO: 23) without the ispH gene also showed excellent isoprene productivity, but the OD 600 The value was lower than that of the methanotroph introduced with the R. communis-derived isopH gene (SEQ ID NO: 29). Therefore, the methanotroph introduced with the S. cerevisiae-derived idi gene (SEQ ID NO: 23) and the R. communis-derived isopH gene (SEQ ID NO: 29) not only has the highest isoprene production ability, but also maintains normal cell growth, so it is expected to be most useful for isoprene biosynthesis.

[0125] Next, the plasmid pAWPS-P, which was shown to have the best isoprene production ability in the above experiment, o -Plasmid pAWPS-P, which further introduced the R. communis-derived ispH gene (SEQ ID NO: 29) into PtIspS-Scidi o -After producing a methanogenic bacterium expressing PtIspS-RcispH-Scidi, isoprene production was measured using a 1 L bioreactor.

[0126] Specifically, the methanotrophs were cultured using a 1 L benchtop stirred tank reactor (CNS21-M05-001; BIOCNS, Daejeon, Korea) equipped with a ceramic microsparger, and isoprene production was measured after capturing using a cooled dodecane trap. The culture was performed at 37°C and pH 6.9 by inoculating 0.5 L of 3 X NMS medium and periodically supplying 3.76 M KNO3, 250 g / L CaCl2, and phosphate solution (130 g / L KH2PO4 & 310 g / L Na2HPO4● 7H2O). Initially, a mixed gas of methane:air = 5:5 was supplied at 0.04 vvm, and then increased to methane:air = 2:8, 0.25 vvm. Gene expression was induced 18 h after cell inoculation by adding 5 μM phenol. Isoprene captured in the cooled dodecane trap was analyzed by GC-MS, and total production was measured by steady-state calculations.

[0127] As a result of the measurement, the isoprene production level of the methanogenic bacteria with optimized isoprene production was confirmed to be approximately 3.0 g / L, which is significantly higher than that of the existing isoprene-producing methanogenic bacteria (Fig. 6).

[0128]

[0129] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

[0130]

[0131] Sequence number 1: VF Primer

[0132] attgtcgggaagatgcg

[0133]

[0134] Sequence number 2: PtIspS-idi-R Primer

[0135] tacgttacctcctcaggatcttagcgttcgaacggca

[0136]

[0137] Sequence number 3: PtIspS-idi-F Primer

[0138] gatcctgaggaggtaacgtatg

[0139]

[0140] Sequence number 4: idi-R Primer

[0141] tcacgcatcttcccgacaatttattaagctgggtaaatgcaga

[0142]

[0143] Sequence number 5: idi-VR Primer

[0144] ttatttaagctgggtaaatgcaga

[0145]

[0146] SEQ ID NO: 6: idi-dxs-IF Primer

[0147] catttacccagcttaaataagtgacccaacagcaagaac

[0148]

[0149] Sequence number 7: dxs-R Primer

[0150] tcacgcatcttcccgacaattcagtgtttgtccttggc

[0151]

[0152] SEQ ID NO: 8: ScIDI1-IF1 Primer

[0153] agatcctgaggaggtaacgtatgactgccgacaacaatag

[0154]

[0155] SEQ ID NO: 9: ScIDI1-IR3 Primer

[0156] tcacgcatcttcccgacaatttatagcattctatgaatttgcctgtc

[0157]

[0158] SEQ ID NO: 10: RBS-PmoB1-VF Primer

[0159] gtgacccaacagcaagaac

[0160]

[0161] SEQ ID NO: 11: ScIDI1-IR4 Primer

[0162] agttcttgctgttgggtcacttatagcattctatgaatttgcctgtc

[0163]

[0164] Sequence number 12: VR Primer

[0165] ttagcgttcgaacggca

[0166]

[0167] Sequence number 13: IspH-IF Primer

[0168] ttctgccgttcgaacgctaagtggcatcaatccaacacc

[0169]

[0170] SEQ ID NO: 14: PtIspH-IR1 Primer

[0171] tcacgcatcttcccgacaatttaggccacctgcagg

[0172]

[0173] SEQ ID NO: 15: RcIspH-IR1 Primer

[0174] tcacgcatcttcccgacaatttaggccagctgcagg

[0175]

[0176] SEQ ID NO: 16: PtIspH-IR2 Primer

[0177] tacgttacctcctcaggatcttaggccacctgcagg

[0178]

[0179] SEQ ID NO: 17: RcIspH-IR2 Primer

[0180] tacgttacctcctcaggatcttaggccagctgcagg

[0181]

[0182] SEQ ID NO: 18: PtIspH-IR3 Primer

[0183] agttcttgctgttgggtcacttaggccacctgcagg

[0184]

[0185] SEQ ID NO: 19: RcIspH-IR3 Primer

[0186] agttcttgctgttgggtcacttaggccagctgcagg

[0187]

[0188] SEQ ID NO: 20: idi (E. coli) Protein Sequence

[0189] MQTEHVILLNAQGVPTGTLEKYAAHTADTLHLAFSSWLFNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGVEITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDLADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK

[0190]

[0191] SEQ ID NO: 21: idi (S. cerevisiae) Protein Sequence

[0192] MTADNNSMPHGAVSSYAKLVQNQTPEDILEEFPEIIPLQQRPNTRSSETSNDESGETCFSGHDEEQIKLMNENCIVLDWDDNAIGAGTKKVCHLMENIEKGLLHRAFSVFIFNEQGELLLQQRATEKITFPDLWTNTCCSHPLCIDDELGLKGKLDDKIKGAITAAVRKLDHELGIPEDETKTRGKFHFLNRIHYMAPSNEPWGEHEIDYILFYKINAKENLTVNPNVNEVRDFKWVSPNDLKTMFADPSYKFTPWFKIICENYLFNWWEQLDDLSEVENDRQIHRML

[0193]

[0194] 서열번호 22: idi (E. coli) Gene Sequence

[0195] atgcaaacggaacacgtcattttattgaatgcacagggagttcccacgggtacgctggaaaagtatgccgcacacacggcagacacccgcttacatctcgcgttctccagttggctgtttaatgccaaaggacaattattagttacccgccgcgcactgagcaaaaaagcatggcctggcgtgtggactaactcggtttgtgggcacccacaactgggagaaagcaacgaagacgcagtgatccgccgttgccgttatgagcttggcgtggaaattacgcctcctgaatctatctatcctgactttcgctaccgcgccaccgatccgagtggcattgtggaaaatgaagtgtgtccggtatttgccgcacgcaccactagtgcgttacagatcaatgatgatgaagtgatggattatcaatggtgtgatttagcagatgtattacacggtattgatgccacgccgtgggcgttcagtccgtggatggtgatgcaggcgacaaatcgcgaagccagaaaacgattatctgcatttacccagcttaaataa

[0196]

[0197] 서열번호 23: idi (S. cerevisiae) Gene Sequence

[0198] atgactgccgacaacaatagtatgccccatggtgcagtatctagttacgccaaattagtgcaaaaccaaacacctgaagacattttggaagagtttcctgaaattattccattacaacaaagacctaatacccgatctagtgagacgtcaaatgacgaaagcggagaaacatgtttttctggtcatgatgaggagcaaattaagttaatgaatgaaaattgtattgttttggattgggacgataatgctattggtgccggtaccaagaaagtttgtcatttaatggaaaatattgaaaagggtttactacatcgtgcattctccgtctttattttcaatgaacaaggtgaattacttttacaacaaagagccactgaaaaaataactttccctgatctttggactaacacatgctgctctcatccactatgtattgatgacgaattaggtttgaagggtaagctagacgataagattaagggcgctattactgcggcggtgagaaaactagatcatgaattaggtattccagaagatgaaactaagacaaggggtaagtttcactttttaaacagaatccattacatggcaccaagcaatgaaccatggggtgaacatgaaattgattacatcctattttataagatcaacgctaaagaaaacttgactgtcaacccaaacgtcaatgaagttagagacttcaaatgggtttcaccaaatgatttgaaaactatgtttgctgacccaagttacaagtttacgccttggtttaagattatttgcgagaattacttattcaactggtgggagcaattagatgacctttctgaagtggaaaatgacaggcaaattcatagaatgctataa

[0199]

[0200] SEQ ID NO: 24: ispS (P. trichocarpa) Protein Sequence

[0201] MACSVSTENVSFTETETETRRSANYEPNSWDYDYLLSSDTDESIEVYKDKAKKLEAEVRREINNEKAEFLTLLELIDNVQRLGLGYRFESDIRRALDRFVSSGGFDAVTKTSLHATALSFRLLRQHGFEVSQEAFSGFKD QNGNFLENLKEDIKAILSLYEASFLALEGENILDEAKVFAISHLKSEKIGKDLAEQVNHALEPLHRRTQRLEAVLSIEAYRKKEDADQVLLELAILDYNMIQSVYQRDLRETSRWWRRVGLATKLHFARDRLIESF YWAVGVAFEPQYSDCRNSVAKMFVTIIDDIYDVYGTLDELELFTNAVERWDVNAIDDLPDYMKLCFLALYNTINEIAYDNLKEKGENILPYLTKAWADLCNAFLQEAKWLYNKSTTPFDDDYFGNAWKSSSGPLQLVFA YFAVVQNIKKEEIEINLQKYHDIISRPSHIFRLCNDLASASAEIARGETANSVSCYMRTKGISEELATESVMNLIDETWKKMNKEKLGGSLFAKPFVETAINLARQSHCTYHNGDAHTSPDELTRKRVLSVITEPILPFER

[0202]

[0203] SEQ ID NO: 25: ispS (P. trichocarpa) Gene Sequence

[0204] ctgactacatgaaactgtgcttcctggcgctgtataacctatcaacgagatcgcgtatgataacctgaaagaaaaaggtgaaacattctgccgtatctgaccaaagcctgggccgacctgtaacgcattcctgcaggaggccaaatgctgtacaataagt ctactcctactttcgacgattacttcggtaacgcttggaaatctagctctggcccgctgcaactggtctcgcctatttcgggtagtgcaaaacatcaaaaaggaagagatcgagaatctgcagaaatatcacgacattatctcccgcccgagccacatctctcg cctgtgtaacgacctggcctcgcatccgcagaaattgcacgcggcgaaccgccaactccgtatcctgctatatgcgtaccaaggcatcgaagactgctaccgaatccgtgatgaacctgatcgatgaaacttggaagaagatgaacaaagaaaact gggcggttctctgttcgccaaccattcgttgaaaccgcgattaacctggcgcgccaatctcactgcacctatcataacggtgacgcacacacccccggatgaactgacccgtaagcgtgtgctgtcgttaccgaaccaattgccgttcgaacgctaa

[0205]

[0206] 서열번호 26: ispH (P. trichocarpa) Protein Sequence

[0207] MGGDDSTSSVSLESEFDAKVFRHNLTRSKNYNRRGFGHKEETLELMNREYTSDIIKKLKENGYEYTWGNVTVKLAEAYGFCWGVERAVQIAYEARKQFPDDKIWIT NEIIHNPTVNKRLEEMEVENVPVEEGKQFEVVNGGDVVILPAFGAAVDEMLLSKNVQIVDTTCPWVSKVWTTVEKHKGDYTSIIHGKYAHEETVATASFAGK YIIVKDMKEAMYVCDYILGGELNGSSSTREEFLEKFKNAVSKGFDPDSDLVKLGIANQTTMLKGETEDIGKLVERIMMRKYGVENVNDHFISFNTICDATQERQDA MYKLVEEKLDMLVVGGWNSSNTSHLQEIAEHHGIPSYWIDSEQRIGPGNKIAYKLNHGELVEKENWLPQGPITIGVTSGASTPDKVVEDALIKVFDIKRDEALQVA

[0208]

[0209] 서열번호 27: ispH (R. communis) Protein Sequence

[0210] MPFSIRCSAAAASDDESSSASVAMDSDFDAKVFRHNLTRSKNYNRRGFGHKEETLQLMSQEYTSDIIKTLKENGNEYTWGNVTVKLAEAYGFCWGVERAVQIAYEARKQ FPDEKIWITNEIIHNPTVNKRLEEMNVENIPLEEGRKQFEVVNNGDVVILPAFGAAVDEMLTLSNKNVQIVDTTCPWVSKVWNTVEKKKGDYTSIIHGKYSHEETIAT ASFAGTYIIVKNMKEAMYVCDYILGGQLNGSSSTKEEFLKKFKNAVSKGFDPDVDLVKVGIANQTTMLKGETEDIGKLVEKTMQRYGVENVNDHFISFNTICDATQER QDAMFNLVEEKLDLVIGWNSSNTSHLQEIAELRGIPSYWIDSEQRIGPGNRIAYKLNHGELVEKENFLPEGPITIGVTSGASTPDKVVEDALVKVFDIKREEALQLA

[0211]

[0212] 서열번호 28: ispH (P. trichocarpa) Gene Sequence

[0213] cctcgcacctgcaggagatcgccgagcaccacggcatcccgtcgtactggatcgactcggagcagcgcatcggcccgggcaacaagatcgcctacaagctgaaccacggcgagctggtggagaaggagaactggctgccgcagggcccgatcaccatcggcgtgacctcgggcgcctcgaccccggacaaggtggtggaggacgccctgatcaaggtgttcgacatcaagcgcgacgaggccctgcaggtggcctaa

[0214]

[0215] 서열번호 29: ispH (R. communis) Gene Sequence

[0216] tgatcctggtgatcggcggctggaactcgtcgaacacctcgcacctgcaggagatcgccgagctgcgcggcatcccgtcgtactggatcgactcggagcagcgcatcggcccgggcaaccgcatcgcctacaagctgaaccacggcgagctggtggagaaggagaacttcctgccggagggcccgatcaccatcggcgtgacctcgggcgcctcgaccccggacaaggtggtggaggacgccctggtgaaggtgttcgacatcaagcgcgaggaggccctgcagctggcctaa

[0217]

[0218] 서열번호 30: dxs (M. capsulatus) Protein Sequence

[0219] MTETKRYALLEADHPAALRNLPEDRLPELAEELRGYLLESVSRSGGHLAAGLGTVELTIALHYVFNTPEDKLVWDVGHQAYPHKILTGRRARLPTIRKKGGSAFPNRAESPYDCFGVGHSSTSISALGMAVAAALERPIHAVAIIGDGGLTGGMAFEALNHAGTLDANLLIILNDNEMSISPNVGALNNYLAKILSGKFYSSVRESGKHLLGRHMPGVWELARRAEEHVKGMVAPGTLFEELGFNYFGPIDGHLDTLITTLRNLRDQKGPRFLHVVTRKGKGGYAPAEKDPVAYHGVGAFDLDADELPKSKPGTP SYTEVFGQWLCDMAARDRRLLGITPAMREGSGLVEFSQRFPDRYFDVGIAEQHAVTFAAGQASEGYKPVVAIYSTFLQRAYDQLIHDVALQNLPVLFAIDRAGLVGPDGPTHAGSFDLSFMRCIPNMLIMAPSDENECRQMLYTGFIHDGPAAVRYPRGRGPGVRPEETMTAFPVGKGEVRLRGKGTAILAFGTPLAAALAVGERIGATVANMRFVKPLDEALILELAATHDRIVTVEENAIAGGAGSAVGEFLAAQHCGIPVCHIGLKDEFLDQGTREELLAIAGLDQAGIARSIDAFIQATAAADKPRRARGQAKDKH

[0220]

[0221] SEQ ID NO: 31: dxs (M. capsulatus) Gene Sequence

[0222] gcatcacgccggcgatgcgcgaaggctccgggctggtggaattctcacagcgcttcccggatcgctatttcgacgtcggcatcgccgagcagcatgccgtgacattcgccgcgggacaggccagcgaaggttacaagccggtggtggccatctactcgaccttcctccagcgcgcctatgaccagttgatccacgacgtcgccctgcagaacctgccggtgctgttcgccatcgaccgcgctggtctggtcggaccggacggcccgacccatgccggcagcttcgatctgagcttcatgcgctgcatccccaacatgctgatcatggcgccctcggatgaaaacgaatgcaggcagatgctctataccggcttcatccatgacggcccggcggccgtgcgttatccgcgcggcagaggccccggcgtcaggccggaggagacgatgactgccttccccgtcggcaaaggagaggtgcgcctccgcggcaagggaaccgccatcctggccttcggcacccccctggccgccgcactcgcggtcggcgagcggatcggcgccacggtggccaacatgcgtttcgtgaaacccctggatgaggcgcttatcctggagttggcagccacccacgaccgcatcgtgaccgtcgaggaaaacgccatcgccggcggtgccggcagtgcggtcggcgaattcctcgctgcccagcactgcggcattccggtctgccacatcggtctgaaggacgaattcctcgaccagggcacccgggaggaactcctggccatcgccggcctcgatcaggccggcatcgcccgatcgatcgacgcattcatccaggccacggctgccgcagacaaaccacggcgggcccgcggccaggccaaggacaaacactga

Claims

1. A transformant into a methanotroph, wherein a base sequence encoding exogenous isoprene synthase, a base sequence encoding exogenous isopentenyl diphosphate isomerase, and a base sequence encoding exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are introduced.

2. In claim 1, A transformant, wherein the exogenous isopentenyl diphosphate isomerase is derived from yeast.

3. In claim 2, The above yeast is a transformant belonging to the genus Saccharomyces.

4. In claim 3, A transformant, wherein the yeast of the genus Saccharomyces is Saccharomyces cerevisiae.

5. In claim 1, A transformant, wherein the exogenous isopentenyl diphosphate isomerase comprises the amino acid sequence of SEQ ID NO:

20.

6. In claim 1, A transformant, wherein the exogenous isopentenyl diphosphate isomerase is encoded by the base sequence of SEQ ID NO:

21.

7. In claim 1, A transformant wherein the exogenous isoprene synthase is derived from a plant of the genus Populus.

8. In claim 7, A transformant, wherein the plant of the above genus Poplar is Populus trichocarpa.

9. In claim 1, A transformant, wherein the exogenous isoprene synthase comprises the amino acid sequence of sequence number 22.

10. In claim 1, A transformant, wherein the exogenous isoprene synthase is encoded by the base sequence of SEQ ID NO:

23.

11. In claim 1, The base sequence encoding the exogenous isoprene synthase, the base sequence encoding the exogenous isopentenyl diphosphate isomerase and the base sequence encoding the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase are P o A transformant operably linked to a promoter.

12. In claim 1, A transformant, wherein the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is derived from Ricinus communis.

13. In claim 1, A transformant, wherein the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase comprises the amino acid sequence of SEQ ID NO:

24.

14. In claim 1, A transformant, wherein the exogenous 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is encoded by the base sequence of SEQ ID NO:

25.

15. In claim 1, A transformant, wherein the transformant has further introduced a base sequence encoding exogenous 1-deoxy-D-xylulose 5-phosphate synthase.

16. In claim 15, A transformant, wherein the base sequence encoding the exogenous 1-deoxy-D-xylulose 5-phosphate synthase is introduced downstream of the base sequence encoding the exogenous isopentenyl diphosphate isomerase.

17. In claim 15, A transformant, wherein the exogenous 1-deoxy-D-xylulose 5-phosphate synthase comprises the amino acid sequence of SEQ ID NO:

26.

18. In claim 1, The above methanogenic bacteria is a transformant, Methylococcus capsulatus Bath.

19. A composition for producing isoprene comprising a transformant of any one of claims 1 to 18.

20. A method for producing isoprene, comprising the step of culturing a transformant of any one of claims 1 to 18 in the presence of methane.

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