Methane-utilizing bacteria that produce PHB and a method for producing PHB using the same

Abstract

This application relates to a methane-assimilating bacterium having PHB-producing ability with enhanced malate thiokinase activity compared to its endogenous activity; a method for producing PHB, comprising the step of culturing the methane-assimilating bacterium in a culture medium; a PHB-producing composition comprising the methane-assimilating bacterium, a culture of the methane-assimilating bacterium, a fermented product of the methane-assimilating bacterium, or a combination of two or more of these; and the use of the methane-assimilating bacterium for PHB production.

Description

Technical Field

[0001] This application relates to a methane-assimilating bacterium having a PHB production ability with enhanced malate thiokinase activity compared to the intrinsic activity; a method for producing PHB including the step of culturing the methane-assimilating bacterium in a medium; a composition for producing PHB including the methane-assimilating bacterium, a culture of the methane-assimilating bacterium, a ferment of the methane-assimilating bacterium, or a combination of two or more thereof; and the use of the methane-assimilating bacterium for PHB production.

Background Art

[0002] Methane is a non-carbon dioxide-based greenhouse gas. Although its concentration in the atmosphere is low, its global warming potential is about 84 times higher than that of carbon dioxide (CO2), accounting for about 15 - 20% of the causes of global warming. Methane-assimilating bacteria are methane-assimilating bacteria that utilize methane as a carbon source and an energy source, and their utility has emerged due to environmental and economic advantages. However, due to insufficient understanding of their physiological characteristics and metabolic regulation, the technical difficulty of research and industrialization using them is high.

[0003] Polyhydroxybutyrate (PHB), a type of biodegradable plastic, is a polymer substance accumulated as a carbon source and an energy storage substance when methane-assimilating bacteria are under conditions where nutrients such as nitrogen and phosphorus are limited while a large amount of carbon source is present. It has mechanical properties similar to various commercially available petroleum synthetic plastics and has attracted attention as a substitute for petroleum synthetic polymers due to its property of being completely decomposed by methane-assimilating bacteria in the natural environment.

[0004] In relation to this, Korean Registered Patent Publication No. 10 - 2202694 discloses a methanol-assimilating bacterial mutant in which the ftfL (formate-tetrahydrofolate ligase) gene is overexpressed and a method for producing PHB using the same. However, there is still a growing need for research on methods for efficiently producing PHB using methane-assimilating bacteria.

[0005] Therefore, research into increasing effective PHB production capacity is still needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Registered Patent Publication No. 10-2202694 [Patent Document 2] U.S. Registered Patent US 7662943 B2 [Patent Document 3] U.S. Registered Patent US 10584338 B2 [Patent Document 4] U.S. Registered Patent US 10273491 B2 [Non-patent literature]

[0007] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-Patent Document 2] Rice et al., 2000, Trends Genet. 16:276-277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-Patent Document 4] Devereux, J., et al, Nucleic Acids Research 12:387 (1984) [Non-Patent Document 5] Atschul, [S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-Patent Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-Patent Document 7] CARILLO et al.](1988) SIAM J Applied Math 48:1073

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Non - Patent Document 10

Non - Patent Document 11

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Summary of the Invention

Problems to be Solved by the Invention

[0008] The inventors of the present application completed the present application by confirming that a methane-assimilating bacterium with enhanced malate thiokinase activity compared to the intrinsic activity can produce PHB.

Means for Solving the Problems

[0009] One aspect of the present application provides a methane-assimilating bacterium having the ability to produce PHB, in which the activity of malate thiokinase is enhanced compared to the intrinsic activity.

[0010] In one specific example, the enhancement may be an enhancement of the copy number in the cell; a modification of the gene expression regulatory region; a modification of the nucleotide sequence encoding the start codon or the 5'UTR region; a modification of the amino acid sequence of the polypeptide; a modification of the polynucleotide sequence; the introduction of a foreign polypeptide or a foreign polynucleotide encoding the same; codon optimization of the polynucleotide; a structural modification of the polypeptide; or a combination thereof.

[0011] In one specific example, the malate thiokinase may be any one or more proteins selected from the group consisting of MtkA and MtkB.

[0012] As the methane-assimilating bacterium according to any one of the above-described specific examples, the MtkA protein may consist of the amino acid sequence of SEQ ID NO: 1, and the MtkB protein may consist of the amino acid sequence of SEQ ID NO: 2.

[0013] As the methane-assimilating bacterium according to any one of the above-described specific examples, the methane-assimilating bacterium may be a methane-assimilating bacterium of the genus Methylocystis sp.

[0014] As a methane-utilizing bacterium based on any one of the above-mentioned specific examples, the methane-utilizing bacterium may have increased PHB production capacity compared to a non-mute methane-utilizing bacterium.

[0015] Another aspect of this application provides a method for producing PHB, comprising the step of culturing a methane-utilizing bacterium having PHB-producing ability with enhanced malate thiokinase activity compared to its endogenous activity in a culture medium.

[0016] In one specific example, the process may further include the step of recovering PHB from the cultured methane-utilizing bacteria, the culture of the methane-utilizing bacteria, the fermented product of the methane-utilizing bacteria, or the culture medium.

[0017] Another aspect of this application provides a PHB production composition comprising a methane-assimilating bacterium having PHB production ability with enhanced malate thiokinase activity compared to its endogenous activity, a culture of the methane-assimilating bacterium, a fermented product of the methane-assimilating bacterium, or a combination of two or more of these.

[0018] Another aspect of this application provides for the use of methane-utilizing bacteria with enhanced malate thiokinase activity compared to endogenous activity in PHB production. [Effects of the Invention]

[0019] The methane-utilizing bacteria described in this application, whose malate thiokinase activity is enhanced compared to its endogenous activity, can produce PHB in high yield and can be usefully utilized in the industrial production of PHB. [Brief explanation of the drawing]

[0020] [Figure 1] This figure shows the cell growth of the MtkAB-enhanced bacterial strain of this application. [Figure 2] This figure shows the PHB production capacity of the MtkAB-enhanced bacterial strain of this application. [Modes for carrying out the invention]

[0021] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not considered to be limited by the specific descriptions described below.

[0022] Furthermore, a person with ordinary skill in the art can recognize and confirm many equivalents of the specific embodiments of the present invention described in this application using only ordinary experiments. Such equivalents are intended to be included in this application.

[0023] As used in the specification and attached claims of this application, singular articles ("a," "an," and "the") refer to multiple subjects unless the context clearly indicates otherwise. Unless otherwise specified in the context, singular terms include plural forms, and plural terms include singular forms. In the specification and attached claims of this application, unless otherwise specified, the use of "or" is used to mean "and / or."

[0024] In this application, the term "about" may be presented before a specific number. As used in this application, "about" includes not only the exact number that follows, but also a range that is approximately that number or close to it. The context in which the number is presented can be considered to determine whether it is close to or approximately that specific number. For example, "about" may refer to a range of 10% to +10% of a number. Another example is that "about" may refer to a range of -5% to +5% of a given number. However, it is not limited to these examples.

[0025] In this application, terms such as "the first, second, third...", "i), ii), iii)...", or "(a), (b), (c), (d)..." are used to distinguish similar configurations, and these terms do not imply that they are performed sequentially or in order. For example, when the terms are used in relation to steps of a method, application, or analysis, there may be no time interval between these steps, they may occur simultaneously, or they may occur with intervals of seconds, minutes, hours, days, or months.

[0026] In this application, the term "consisting essentially of" means that the presence of the unspecified components is substantially unaffected by the presence of the unspecified components in the case that the features of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0027] In this application, the term "consisting of" means that the proportion of specific components(s) totals 100%. The components or features following the term "consisting of" may be essential or mandatory. In some specific examples, other optional or non-essential components may be excluded in addition to the components or features following "consisting of".

[0028] In this application, the term “comprising” means the presence of any of the features, stages, or components described below in the aforementioned term, and does not exclude the presence or addition of one or more features, stages, or components. In this application, any of the components or features described below in “comprising” may be essential or mandatory, but some specific examples may further include other optional or non-essential components or features.

[0029] One aspect of this application provides a methane-utilizing bacterium having PHB production ability, in which malate thiokinase activity is enhanced compared to its endogenous activity.

[0030] The enhanced malate thiokinase activity may be defined as the increased PHB production capacity of the methane-assimilating bacteria of this application compared to the production capacity of naturally occurring wild-type methane-assimilating bacteria or non-mutant methane-assimilating bacteria (e.g., methane-assimilating bacteria expressing polypeptides having wild-type malate thiokinase activity (e.g., the polypeptide of SEQ ID NO: 1 and / or SEQ ID NO: 2) or strains in which the malate thiokinase activity of this application is not enhanced compared to its endogenous activity, or before enhancement).

[0031] For example, the malate thiokinase activity can be measured by measuring PHB production capacity or yield, but is not limited to this.

[0032] In this application, the term "malate thiokinase" refers to a bacterial enzyme composed of two distinct subunits that forms malate-CoA from malate and CoA. The malate thiokinase in this application may, but is not limited to, one or more proteins selected from the group consisting of MtkA and MtkB. Specifically, the malate thiokinase in this application may be used to mean a complex of MtkA and MtkB, and can be used interchangeably with MtkAB, malate-CoA ligase, or succinate-CoA ligase.

[0033] Specifically, the malate thiokinase of this application may be a protein having the activity of the MtkA and MtkB proteins encoded by the mtkA and mtkB genes, respectively, but is not particularly limited in type as long as it has the activity corresponding to malate thiokinase. The MtkA and MtkB proteins encoded by the mtkA and mtkB genes, respectively, are publicly known in the art, and the amino acid and polynucleotide sequences of the MtkA and MtkB proteins can be obtained from publicly known databases, such as NCBI's GenBank, but is not limited thereto.

[0034] For example, the MtkA and MtkB proteins of this application may each contain the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or amino acid sequences having 60% or more homology or identity thereto, but are not limited thereto as long as they have malate thiokinase activity. Specifically, polypeptides having the MtkA and MtkB protein activity may each have, contain, consist of, or substantially consist of amino acid sequences having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1 and SEQ ID NO: 2. For example, the MtkA and mtkB proteins refer to proteins that are endogenously present in methane-utilizing bacteria, but are not limited to this. Specifically, they may be the MtkA protein consisting of the amino acid sequence of SEQ ID NO: 1 and the MtkB protein consisting of the amino acid sequence of SEQ ID NO: 2, both of which are endogenously present in methane-utilizing bacteria, but are not limited to this.

[0035] In this application, even if a polypeptide is described as "containing" an amino acid sequence described by a specific sequence number, a polypeptide "consisting of" an amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" an amino acid sequence described by a specific sequence number, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included in the scope of this application, provided that they have the same or corresponding activity as the protein consisting of the amino acid sequence of the said sequence number. For example, if they have the same or corresponding activity as the mutant protein, it is obvious that even if they have such additions or mutations, they still fall within the scope of this application, excluding additions of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions.

[0036] For example, the amino acid sequence may have additions of sequences that do not alter the function of the mutant polypeptide of this application, spontaneous mutations, silent mutations, or conserved substitutions at the N-terminus, C-terminus, and / or within it. For example, the polypeptide can be conjugated with an N-terminal signal (or leader) sequence of a protein involved in protein transfer co-translationally or post-translationally. The polypeptide can also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0037] In this application, the term "conservative substitution" means the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, aspartic acid) and uncharged amino acids (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). As yet another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As yet another example, valine, leucine, and isoleucine can be classified as branched amino acids.As another example, the 20 amino acids can be classified by size into five groups, starting with the smallest amino acid group: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, this is not necessarily the only classification. Typically, conservative substitutions have little to no effect on polypeptide activity.

[0038] Furthermore, the base sequence encoding the malate thiokinase may be a base sequence encoding a protein that exhibits malate thiokinase activity.

[0039] For example, the base sequences encoding the MtkA and MtkB proteins may be base sequences encoding proteins that exhibit malate thiokinase activity.

[0040] For example, the MtkA and MtkB proteins having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, may be encoded by polynucleotides having, containing, or consisting of, or substantially comprising, the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or nucleotide sequences that have homology or identity with SEQ ID NO: 3 and SEQ ID NO: 4, respectively, in amounts of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%. Furthermore, the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, can be obtained from known databases, such as NCBI's GenBank, but are not limited to these.

[0041] In this application, for example, a gene containing the base sequence of SEQ ID NO: 3 can be mixed with a polynucleotide containing the base sequence of SEQ ID NO: 3, a gene or polynucleotide having the base sequence of SEQ ID NO: 3, or a gene or polynucleotide consisting of the base sequence of SEQ ID NO: 3.

[0042] The polynucleotides of this application may be modified in various ways in the coding region, either due to codon degeneracy or considering preferred codons in organisms expressing the malate thiokinase of this application, as long as the amino acid sequence of the malate thiokinase of this application is not altered. Therefore, it is obvious that the polynucleotides may also be translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the malate thiokinase of this application or polypeptides homologous or identical thereto. For example, the polynucleotides of this application may be SEQ ID NO: 3 and / or SEQ ID NO: 4 or their degenerated sequences.

[0043] Other examples include, but are not limited to, having or including a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with SEQ ID NO: 3 and / or SEQ ID NO: 4, or consisting of or substantially comprising a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0044] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of this application and encode the malate thiokinase of this application.

[0045] In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0046] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally hybridizable under moderate to high stringent conditions, either in whole or at least about 50%, 60%, 70%, 80%, or 90% of the total length. It is obvious that hybridization also includes polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0047] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0048] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) unitary matrices (with values ​​of 1 for identity and 0 for non-identity), PAM Matrix (see disclosure in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation (1978)), Gribskov et al (1986) Nucl. Acids Res. 14:6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0049] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and the defined appropriate hybridization conditions may be determined by methods well known to those skilled in the art, within the scope of the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0050] In this application, the term "stringent condition" means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0051] The aforementioned hybridization requires that the two nucleotides have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with one another. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include isolated nucleic acid fragments that are complementary throughout their entire sequence, as well as substantially similar base sequences.

[0052] For example, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0053] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0054] In this application, the term "PHB (Polyhydroxybutyrate)" refers to a polymer of the polyhydroxyalkanoate series, and is a compound belonging to polyester as a polymer of 3-hydroxybutyrate. The PHB can be mixed with poly-3-hydroxybutyrate (Poly(3-hydroxybutyrate), P(3HB)), poly-3-hydroxybutanoate (poly-3-hydroxybutanoate, P3HA), and 3-hydroxybutyrate homopolymers.

[0055] In this application, the term "methanotrophic bacteria" refers to microorganisms that can grow using methane as an energy source, and includes wild-type methanotrophic bacteria and methanotrophic bacteria that have undergone natural or artificial genetic modification. These include methanotrophic bacteria in which specific mechanisms have been weakened or increased due to causes such as the insertion of external genes or the increase or inactivation of endogenous gene activity, and may also include methanotrophic bacteria that have undergone genetic modification for the production of a target polypeptide, protein, or product. In this application, "methanotrophic bacteria," "microorganism," and "strain" may be used interchangeably without limitation as they have the same meaning.

[0056] For example, the methane-utilizing bacteria of this application may be, but are not limited to, methane-utilizing bacteria (e.g., recombinant strains) in which malate thiokinase activity is enhanced compared to endogenous activity.

[0057] In this application, the term "methane-utilizing bacteria having PHB production ability" refers to a strain of methane-utilizing bacteria capable of producing PHB within the organism, and may include all methane-utilizing bacteria in which PHB production ability has been conferred from a parent strain that lacks PHB production ability, or methane-utilizing bacteria that inherently possess PHB production ability. PHB production ability may be conferred or enhanced through selective breeding.

[0058] In this application, the term "non-myxomycete methane-assimilating bacteria" does not exclude strains containing naturally occurring mutations in methane-assimilating bacteria, but rather means wild-type strains or natural strains themselves, or strains before their characteristics are altered by genetic mutations due to natural or artificial factors. The aforementioned "non-myxomycete methane-assimilating bacteria" may be used interchangeably with "pre-deformation strain," "pre-deformation methane-assimilating bacteria," "non-mutant strain," "non-myxomycete strain," "non-mutant methane-assimilating bacteria," "pre-mutant parent strain," "wild-type methane-assimilating bacteria," "reference methane-assimilating bacteria," or "standard methane-assimilating bacteria." In this application, non-myxomycete methane-assimilating bacteria means, but is not limited to, strains in which the malate thiokinase activity of this application is not enhanced compared to the endogenous activity, or before such enhancement occurs. Furthermore, in this application, the non-mutant methane-utilizing bacteria may be methane-utilizing bacteria containing an amino acid sequence consisting of SEQ ID NO: 1 and / or SEQ ID NO: 2 or a polynucleotide consisting of SEQ ID NO: 3 and / or SEQ ID NO: 4, but are not limited thereto.

[0059] For example, the methane-utilizing bacteria of this application may include all methane-utilizing bacteria in which malate thiokinase activity is enhanced compared to intrinsic activity and which are capable of producing the target PHB. For example, the methane-utilizing bacteria of this application are characterized by increased PHB production capacity due to enhanced malate thiokinase activity compared to intrinsic activity, and may be, but are not limited to, genetically modified methane-utilizing bacteria or recombinant methane-utilizing bacteria. Specifically, the recombinant strain with increased PHB production capacity may be, but are not limited to, a natural wild-type methane-utilizing bacterium or a non-mutated methane-utilizing bacterium with intrinsic malate thiokinase activity that has increased PHB production capacity.

[0060] As an example, a methane-assimilating bacterium capable of producing PHB is a methane-assimilating bacterial strain that can produce PHB within its body, and can include all methane-assimilating bacteria that inherently possess PHB-producing ability or parent strains that lack PHB-producing ability but have been given PHB-producing ability by the enhanced malate thiokinase activity described in this application. PHB-producing ability may be conferred or enhanced through selective breeding.

[0061] For example, the recombinant methane-assimilating bacteria having PHB production ability of this application may include all methane-assimilating bacteria that can be transformed through a vector to enhance the malate thiokinase activity of this application and produce PHB.

[0062] In this application, the term "increase" of polypeptide activity means that the activity of the polypeptide is enhanced compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement.

[0063] The aforementioned enhancement may include all those that exhibit activity not originally present, or that exhibit improved activity compared to the intrinsic activity or the activity before deformation.

[0064] For example, the "substance exhibiting activity not originally present" may be, but is not limited to, "introduction of a protein." The introduction of a protein means that a gene not originally present in the methane-utilizing bacterium is expressed within the bacterium, thereby exhibiting activity of a specific protein, or that the protein exhibits enhanced or improved activity compared to its intrinsic activity or activity before modification. For example, a polynucleotide encoding a specific protein may be introduced into the chromosome of the methane-utilizing bacterium, or a vector containing a polynucleotide encoding a specific protein may be introduced into the methane-utilizing bacterium, and its activity may be exhibited.

[0065] The aforementioned "intrinsic activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or non-mutated methane-assimilating bacterium before the trait change due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with "activity before deformation."

[0066] Enhanced polypeptide activity compared to endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide has improved compared to what was originally present in the parental strain or non-mutant methane-utilizing bacterium before the trait change.

[0067] For example, the enhancement may be, but is not limited to, enhancements in which the activity or concentration of the corresponding protein is shown to be absent, or enhancements in which the activity or concentration is generally increased by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000%, or about 2000% or more, relative to the activity or concentration of the wild-type protein or the initial methane-utilizing strain of bacteria.

[0068] The enhancement of the polypeptide's activity can be achieved by introducing an exogenous polypeptide or by enhancing the activity of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by the enhancement of its activity level, expression level, or the amount of product excreted from the polypeptide.

[0069] The enhancement of the polypeptide activity can be achieved by applying various methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the methane-assimilating bacteria before modification. Specifically, this may involve, but is not limited to, the use of genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to ordinary technicians in this field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0070] Specifically, the enhancement of the activity of the polypeptide in this application is 1) Enhancement of the intracellular copy number of polynucleotides encoding polypeptides; 2) Modification of gene expression regulatory regions on chromosomes that encode polypeptides (e.g., mutation within the regulatory region, replacement with a more active sequence, or insertion of a more active sequence); 3) Modification of the nucleotide sequence encoding the start codon or 5'UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites, and deform or chemically modify them; 9) Regulation of the intracellular localization of polypeptides; or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0071] for example, The enhancement of the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing a vector into the host cell that is operably linked to the polynucleotide encoding the polypeptide and capable of replicating and functioning independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide may be introduced into the chromosomes of the host cell. The introduction into the chromosomes can be achieved by introducing a vector into the host cell that can insert the polynucleotide into the chromosomes of the host cell, but is not limited to this.

[0072] The replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to generate a sequence mutation or replacement with a sequence having stronger activity, in order to further enhance the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter, but is not limited to these.

[0073] Examples of well-known strong promoters include, but are not limited to, the cj1-cj7 promoter (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0074] The modification of the start codon or 5'UTR region of the polypeptide-encoding gene described in 3) above may, for example, be by substitution with another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited to these.

[0075] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that enhances activity, in order to enhance the activity of the polypeptide. Specifically, the replacement can be carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to these methods. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0076] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may also be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide into the host cell generates the polypeptide, thereby enhancing its activity.

[0077] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription and translation are enhanced in the host cell, or the codon of the exogenous polynucleotide may be optimized so that optimized transcription and translation occur in the host cell.

[0078] 8) Analyzing the tertiary structure of the polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0079] The intracellular positional regulation of polypeptides described in 9) above may involve targeting the polypeptide to a specific organelle or intracellular space within the cell. For example, it may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the polypeptide, but is not limited thereto.

[0080] Such enhancement of polypeptide activity may be achieved by increasing the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification methane-utilizing strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0081] For example, the recombinant methane-assimilating bacteria having PHB production ability of this application may include all methane-assimilating bacteria that can be transformed through a vector to enhance the malate thiokinase activity of this application and produce PHB.

[0082] For example, in the methane-utilizing bacteria of this application, the enhancement may be, but is not limited to, enhancement of the intracellular copy number; modification of the gene expression regulatory region; modification of the base sequence encoding the start codon or 5'UTR region; modification of the polypeptide amino acid sequence; modification of the polynucleotide sequence; introduction of a foreign polypeptide or a foreign polynucleotide encoding it; codon optimization of the polynucleotide; structural modification of the polypeptide; or a combination thereof.

[0083] For example, in the methane-utilizing bacteria of this application, the enhancement of the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by inserting the polynucleotide into an expression vector and introducing the expression vector into a host cell, thereby increasing the copy number. The polynucleotide may include, but is not limited to, intergenic regions between genes.

[0084] For example, the intergenic regions between the mtkA and mtkB genes encoding the MtkA and MtkB proteins, respectively, may have, contain, consist of, or substantially consist of the nucleotide sequence of Sequence ID No. 5, or may be polynucleotides.

[0085] For example, the mtkAB operon, which includes the intergenic regions between mtkA and mtkB genes and represents the entire mtkAB gene, may, but is not limited to, the nucleotide sequence of Sequence ID No. 6.

[0086] The vector of this application may comprise a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0087] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0088] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within the cell. The insertion of the polynucleotide into the chromosome can be performed by any method known in the art, such as homologous recombination, but is not limited to these methods. The method may further include a selection marker for confirming the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and may confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0089] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or methane-assimilating bacterium so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosomes of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced into and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may, but is not limited to, be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell.

[0090] In this application, the term “operatably linked” means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence directs the expression of a coding sequence. Thus, “operatably linked” includes a regulatory region of a functional domain known as a promoter, terminator, signal sequence, or enhancer region, or having a desired activity, being attached to or linked to a target (gene or polypeptide) so that its expression, secretion, or function can be regulated by the known or desired activity. For example, it means that a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the target mutant polypeptide of this application is functionally linked to the polynucleotide sequence.

[0091] In this application, the term "expression" includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0092] In this application, the term "expression vector" means a linear or cyclic nucleic acid molecule comprising a coding sequence and a regulatory sequence operably linked for its expression.

[0093] In this application, the term "regulatory sequence" means a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (of the same origin) or foreign (derived from another gene). Examples of regulatory sequences include leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and translation termination. The smallest unit of a regulatory sequence may include a promoter and transcription and translation termination sequences.

[0094] In relation to cells, polynucleotides, polypeptides, or vectors, the term “recombinant” in this application means that a cell, polynucleotide, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native polynucleotide or polypeptide, or that a cell has been derived from such a modified cell. Therefore, for example, a recombinant cell may express genes not found in the cell’s native (non-recombinant) form, or express native genes that are expressed, not expressed at all, or abnormally expressed.

[0095] For example, the methane-utilizing bacteria that produce the PHB may be methane-utilizing bacteria that intrinsically contain a protein consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0096] For example, the methane-utilizing bacteria that produce PHB may be methane-utilizing bacteria that intrinsically contain a polynucleotide sequence encoding a protein having at least 80% homology to the SEQ ID NO: 1 and / or SEQ ID NO: 2, the base sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4, or a base sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the base sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0097] The methane-utilizing bacteria of this application can include all methane-utilizing bacteria whose malate thiokinase activity has been enhanced compared to its endogenous activity by various known methods.

[0098] For example, the methane-utilizing bacteria with increased PHB production capacity in this application may be, but are not limited to, any methane-utilizing bacteria with increased PHB production capacity compared to non-myxomycete methane-utilizing bacteria. For example, the non-myxomycete methane-utilizing bacteria used as a comparison for the presence or absence of the increased PHB production capacity may be, but are not limited to, any Methylocystis OB3b strain.

[0099] For example, the methane-utilizing bacteria with increased PHB production capacity may have an increase of approximately 1% or more compared to the PHB production capacity of the parent strain before mutation or the non-mutant methane-utilizing bacteria. Specifically, this could be approximately 1% or more, approximately 2.5% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, or approximately 30% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 45% or less, or approximately 40% or less). However, it is not limited to these values ​​as long as it has a positive increase compared to the production capacity of the parent strain before mutation or the non-mutant methane-utilizing bacteria. As another example, the recombinant strain with increased PHB production capacity may have increased PHB production capacity by approximately 1.1 times or more, approximately 1.15 times or more, approximately 1.2 times or more, approximately 1.25 times or more, or approximately 1.3 times or more compared to the original parent strain or non-mutenant methane-assimilating bacteria (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, or approximately 1.4 times or less), but is not limited to these.

[0100] As an example, methane-utilizing bacteria having the ability to produce PHB include, as an example, the genera Methylocystis, Methylomonas, Methylobacter, Methylococcus, Methylomicrobium, Methylosphaera, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, and Methylohalobius. This may include methane-utilizing bacterial strains belonging to the genera Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylorubrum, Methyloparacoccus, Methylosinus, Methylocella, Methylocapsa, Methylofurula, Methylacidiphilum, and Methylacidimicrobium.

[0101] As a methane-utilizing bacterium based on any one of the specific examples mentioned above, the methane-utilizing bacterium of this application may be a microorganism of the genus Methylocystis. Specifically, the methane-utilizing bacterium of this application may be, but is not limited to, the Methylocystis OB3b strain.

[0102] Another aspect of this application provides a method for producing PHB, comprising the step of culturing a methane-utilizing bacterium having PHB-producing ability with enhanced malate thiokinase activity compared to its endogenous activity in a culture medium.

[0103] In this application, the term "cultivation" means growing the strain of this application under appropriately controlled environmental conditions. The cultivation process of this application can be carried out according to suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adapted and used by those skilled in the art depending on the selected microorganism. Specifically, the cultivation may be batch, continuous, and / or fed-batch.

[0104] In this application, the term "culture medium" means a substance mixed primarily with nutrients necessary for culturing the methane-utilizing bacteria of this application, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the strains of this application can be any culture medium used for culturing ordinary methane-utilizing bacteria without particular limitations. However, the methane-utilizing bacteria of this application can be cultured under aerobic conditions in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, with the temperature, pH, etc., adjusted.

[0105] In this application, methanol is primarily used as the carbon source due to the characteristics of methane-assimilating bacteria, but additionally, carbohydrates such as glucose, sucrose, lactose, fructose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine may be included. Furthermore, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0106] The nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0107] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to these methods.

[0108] Furthermore, during the cultivation of the methane-utilizing bacteria of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods.

[0109] In the culture described in this application, the culture temperature can be maintained at 27-37°C, specifically 30-33°C, and the culture can be performed for approximately 20-120 hours, but is not limited to this.

[0110] In this application, the term "culture" means a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific methane-utilizing bacterium in a culture medium, wherein the culture solution contains the specific methane-utilizing bacterium, and the culture filtrate substantially does not contain the specific methane-utilizing bacterium (substantially, this means excluding the specific methane-utilizing bacterium separated by filtration or the like, and does not mean that the methane-utilizing bacterium is completely excluded from the filtrate). The dosage form of the culture is not limited and may, for example, be a liquid, emulsion, or solid. Specifically, for the purposes of this application, the culture may contain PHB.

[0111] In this application, the term "fermentation" refers to a process in which organic matter is decomposed using enzymes possessed by methane-assimilating bacteria, but which is not a putrefaction reaction. Fermentation and putrefaction reactions proceed through similar processes, but if useful substances are produced as a result of the decomposition, it is called fermentation, and if foul odors are produced or harmful substances are created, it is called putrefaction.

[0112] In this application, the method for obtaining the fermented product from the strain is not particularly limited and can be obtained by methods commonly used in the art or a similar field.

[0113] In this application, the term "fermented product" includes not only the fermented substance itself, but also all kinds of substances including fermented products generated from the strain, such as the culture medium of the strain in which the strain and culture material coexist, the fermented product obtained by filtering the strain from the culture medium, the fermented product obtained by sterilizing the strain from the culture medium and filtering it, the extract obtained by extracting the fermented product or the culture medium containing it, the diluted solution and concentrate obtained by diluting the fermented product or its extract, the dried product obtained by drying the fermented product or its extract, and the lysate obtained by collecting and crushing the microbial cells of the strain.

[0114] In the method of this application, any culture conditions and methods known in the art are used for culturing methane-utilizing bacteria. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0115] The PHB produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0116] In one specific example, the PHB production method of this application may further include, for example, a step of preparing the methane-utilizing bacteria of this application, a step of preparing a culture medium for culturing the strain, or a combination thereof (in any order), before the culturing step.

[0117] The PHB production method of this application may further include a step of recovering a target substance, specifically PHB, from the cultured methane-assimilating bacteria, the culture of the methane-assimilating bacteria, the fermentate of the methane-assimilating bacteria, or the culture medium. The recovery step may further include a step after the culture step.

[0118] The aforementioned recovery may also involve collecting the target PHB using appropriate methods known in the art, such as batch, continuous, or fed-batch culture methods for culturing methane-utilizing bacteria as described in this application. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover the target substance, specifically PHB, from the culture medium or methane-utilizing bacteria using appropriate methods known in the art.

[0119] Furthermore, the PHB production method of this application may further include a purification step. The purification can be carried out using a suitable method known in the art. For example, if the PHB production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously or integrated into a single step, but are not limited thereto.

[0120] In the method of this application, the enhancement of malate thiokinase activity and PHB, etc., are as described in the other aspects above.

[0121] Another aspect of this application provides a PHB production composition comprising a methane-assimilating bacterium having enhanced malate thiokinase activity compared to its endogenous activity, a culture of the methane-assimilating bacterium, a fermented product of the methane-assimilating bacterium, or a combination of two or more of these.

[0122] The composition of this application may further contain any suitable excipients commonly used in PHB production compositions, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0123] As one specific example, each component present in the composition of this application may be included in a microbiologically effective amount or in an amount that can be adequately present in a production composition.

[0124] In the composition of this application, the enhancement of malate thiokinase activity and PHB, etc., are as described in the other aspects above.

[0125] Another aspect of this application provides for the use of methane-utilizing bacteria in PHB production, in which the malate thiokinase activity of this application is enhanced compared to its endogenous activity.

[0126] In the use of this application, the enhancement of malate thiokinase activity and PHB, etc., are as described in the other aspects above. [Examples]

[0127] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0128] Example 1. Production of MtkAB-enhanced strains and evaluation of PHB production capacity Example 1-1. Production of mktAB gene-enhanced recombinant vector To produce mtkAB (mtkA; SEQ ID NO: 1, mtkB; SEQ ID NO: 2) reinforced plasmids, DNA fragments amplified by PCR using vec_F and vec_R primers with pAWP89 (Appl Environ Microbiol. 2015 Mar;81(5):1775-1781) as a template, DNA fragments amplified by PCR using mtkA_F and mtkB_R primers with DNA synthesized from Methylosinus sp. C49 (NCBI Taxonomy ID:2699395) (SEQ ID NO: 6) as a template, and DNA fragments amplified by PCR using TrnB_F and TrnB_R primers with Escherichia coli K-12 genomic DNA as a template were prepared using the In-Fusion(R) HD cloning kit (Takara, Cat. No. The plasmid was cloned using 639650) and the final completed plasmid exhibited kanamycin resistance, and was named pME-01.

[0129] The primer sequences used for vector preparation are as follows:

[0130] [Table 1]

[0131] Examples 1-2. Production of a PHB-producing strain with enhanced mktAB gene. Electroporation was used to introduce the pME-01 vector prepared in Example 1-1 into the Methylosinus trichosporium OB3b (KCTC12760) strain. Strains into which Plasmid was introduced were selected using NMS solid medium containing 5 μg / mL kanamycin. The OB3b strain into which the control group pAWP89 was introduced was named OB3b-C, and the OB3b strain into which the experimental group pME-01 was introduced was named Ob3b-M.

[0132] For culturing OB3b, NMS (Biotechnology for Biofuels volume 12,234 (2019)) was used as the culture medium, provided with 5 μM CuSO4. For solid culture, 30% of the air was removed using a vacuum desiccator, and then replenished with a gas mixture of methane and carbon dioxide in a 9:1 ratio. For liquid culture, a sealed flask was used to provide a culture medium at a level of 3-10% of the flask volume. After thoroughly replacing the internal air with a gas mixture of Air:methane:carbon dioxide in a 70:30:3 ratio, the culture was performed at 270 rpm. The culture temperature was 35°C.

[0133] Examples 1-3. Evaluation of PHB production capacity of strains with enhanced mktAB gene. To measure the PHB production capacity of the OB3b-C and OB3b-M strains prepared in Examples 1-2, PHB production was confirmed using NMS2 medium, in which the KNO3 concentration was adjusted to 5 mM in NMS medium.

[0134] The initial inoculation concentration was OD 0.2. After 24 hours of culture, the gas in the flask was changed twice a day, and the cells were cultured for a total of 72 hours. Finally, 15 ml of culture medium was harvested to obtain cells, which were then freeze-dried and the dry cell weight was measured. PHB content was confirmed by analyzing PHB via gas chromatography.

[0135] Specifically, to confirm cell growth, a 0.2 mL sample was taken at the gas exchange point, and the absorbance was measured at 600 nm to confirm the cell concentration (Figure 1). Then, the final culture medium was used for PHB content analysis (Figure 2 and Table 2).

[0136] [Table 2]

[0137] As shown in Figure 1, in the case of OD600 values, it was confirmed that OB3b-M showed slightly higher values ​​than the control group OB3b-C after 30 hours. As shown in Table 2 and Figure 2 above, in the case of PHB content, it was confirmed that the OB3b-M strain had a PHB content of 41.9%, which is approximately 1.4 times higher than the control group strain (OB3b-C) at 29.5%. In other words, it was confirmed that enhancing the expression of the mtkAB gene in methane-utilizing bacteria increases PHB production capacity while maintaining the growth capacity of the methane-utilizing bacteria.

[0138] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A methane-assimilating bacterium with enhanced malate thiokinase activity compared to its endogenous activity, possessing the ability to produce polyhydroxybutyrate (PHB).

2. The enhancement is an enhancement of the copy number within the cell; modification of the gene expression regulatory region; modification of the base sequence encoding the start codon or 5'UTR region; modification of the amino acid sequence of the polypeptide; modification of the polynucleotide sequence; introduction of a foreign polypeptide or a foreign polynucleotide encoding it; codon optimization of the polynucleotide; structural modification of the polypeptide; or a combination thereof, as described in claim 1, for a methane-assimilating bacterium having PHB production ability.

3. The methane-assimilating bacterium having PHB production ability according to claim 1, wherein the malate thiokinase is one or more proteins selected from the group consisting of MtkA and MtkB.

4. The methane-assimilating bacterium having PHB production ability according to claim 3, wherein the MtkA protein consists of the amino acid sequence of SEQ ID NO: 1 and the MtkB protein consists of the amino acid sequence of SEQ ID NO:

2.

5. The methane-utilizing bacterium is a methane-utilizing bacterium of the genus Methylocystis sp., as described in claim 1, which has PHB production ability.

6. The methane-assimilating bacteria described above have increased PHB production capacity compared to non-methane-assimilating bacteria, as described in any one of claims 1 to 5.

7. A method for producing PhB, comprising the step of culturing a methane-assimilating bacterium having PhB-producing ability with enhanced malate thiokinase activity compared to its endogenous activity in a culture medium.

8. The method for producing PHB according to claim 7, further comprising the step of recovering PHB from the cultured methane-utilizing bacteria, the culture of the methane-utilizing bacteria, the fermented product of the methane-utilizing bacteria, or the culture medium.

9. A PHB production composition comprising a methane-assimilating bacterium having PHB production ability with enhanced malate thiokinase activity compared to its endogenous activity, a culture of the methane-assimilating bacterium, a fermented product of the methane-assimilating bacterium, or a combination of two or more of these.

10. Use of methane-utilizing bacteria with enhanced malate thiokinase activity compared to endogenous activity for PHB production.

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