5'UTR mutant sequences of the gene encoding phosphoenolpyruvate carboxylase and their uses

Abstract

The present application relates to a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR), a polyhydroxyalkanoate (PHA)-producing microorganism containing the gene, and a PHA production method using the same.

Description

[Technical Field]

[0001] The present application relates to a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR), a polyhydroxyalkanoate (PHA)-producing microorganism containing the gene, and a PHA production method using the same. [Background technology]

[0002] As environmental pollution caused by the accumulation of petroleum-based, persistent plastics becomes a growing concern, biodegradable bioplastics are gaining attention as an alternative. Polyhydroxyalkanoate (PHA), a bioplastic material, is a natural polyester polymer accumulated in microorganisms. It is not only biodegradable but also environmentally friendly, with thermoplastic and elastic properties, and no toxic waste is generated. The most well-known PHA is P3HB (poly-3-hydroxybutyrate), which is polymerized using 3HB (3-hydroxybutyrate) monomer. P3HB has similar physical properties to polypropylene, a synthetic resin used in automotive and home appliance components. It can be synthesized relatively easily by microorganisms, and numerous commercial production studies have been conducted. However, its high crystallinity makes it fragile, and it decomposes near its melting point, making it difficult to process. Therefore, research is underway to produce PHA copolymers through blending with new materials or copolymerization of monomers. Among them, P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer) is a material that has elastic properties due to the addition of 4HB (4-hydroxybutyrate) monomer. The physical properties of PHA change depending on the ratio of 4HB monomer added, and as this can be used to produce a variety of products, it is attracting attention as a promising biodegradable polymer material. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-patent document 2] EMBOSS:The European Molecular Biology Open Software Suite,Rice et al., 2000), Trends Genet. 16:276-277 [Non-licensed document 3] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444

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[0004] The present inventors have identified a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR) and confirmed that polyhydroxyalkanoate (PHA) is produced in a PHA-producing microorganism containing the gene, thereby completing the present application. [Means for solving the problem]

[0005] One object of the present application is to provide a mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) in which one or more bases have been mutated in the base sequence of SEQ ID NO:1.

[0006] Another object of the present application is to provide a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, wherein the mutant polynucleotide further comprises a nucleotide sequence encoding a mutant 5'UTR in which one or more nucleotides have been mutated in the nucleotide sequence of SEQ ID NO: 1.

[0007] Another object of the present application is to provide a microorganism for producing polyhydroxyalkanoate (PHA), comprising: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR.

[0008] Another object of the present application is to provide a method for producing PHA, which includes a step of culturing a PHA-producing microorganism in a culture medium, the microorganism including: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide including a base sequence encoding phosphoenolpyruvate carboxylase, which further includes a base sequence encoding the mutant 5'UTR.

[0009] Another object of the present application is to provide a PHA-producing microorganism comprising a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR; a culture medium in which the same has been cultured; or a composition for PHA production comprising a combination of two or more of them.

[0010] Another object of the present application is to provide use of a microorganism comprising a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR, for the production of PHA. [Effects of the Invention]

[0011] The 5' untranslated region (5'UTR) variant sequence of the present application can be utilized to regulate the carbon flow in the TCA cycle, and in particular, to regulate the 4HB (4-hydroxybutyrate) content of polyhydroxyalkanoate (PHA)-producing microorganisms, thereby providing PHA with desired physical properties. DETAILED DESCRIPTION OF THE INVENTION

[0012] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not intended to limit the scope of this application. Furthermore, those skilled in the art will be able to recognize or ascertain, using no more than routine experimentation, many equivalents to the specific aspects of the invention described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0013] One aspect of the present application provides a mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) in which one or more bases have been mutated in the base sequence of SEQ ID NO:1.

[0014] In this application, the term "5' untranslated region (5'UTR)" refers to a region that is present at the 5' end of an mRNA transcript but is not translated into amino acids. The process of mRNA translation into protein begins with the binding of the 30S subunit of the ribosome to the 5'UTR. Specifically, 16S rRNA (16S ribosomal RNA) in the 30S subunit of the ribosome binds to the RBS in the 5'UTR, and translation into protein begins when tRNA recognizes and binds to the start codon (AUG) of the mRNA. The ribosome binding site and the start codon in the 5'UTR are known to be located approximately 6 to 8 nucleotides apart.

[0015] In the present application, the 5'UTR may comprise a sequence complementary to the 3'-end sequence of 16S rRNA, i.e., a sequence in which the Shine-Dalgarno sequence is conserved and the sequences before and after it are regulated.

[0016] The "5'UTR sequence is modified" or "modified 5'UTR" refers to a modification of one or more bases in the base sequence constituting the 5'UTR, and such modifications can include base substitution, addition, deletion, or inversion. As a representative example, the Shine-Dalgarno sequence in the 5'UTR is conserved, and the base sequences before and after it can include sequences in which one or more bases are modified by substitution, addition, deletion, inversion, or the like.

[0017] The 5'UTR can include a ribosome binding site (RBS).

[0018] In this application, the term "ribosome binding site (RBS)" refers to a region (structure) within an mRNA chain to which a ribosome can directly bind and initiate translation, or a region (structure) of a DNA chain that initiates such a region upon transcription. Many prokaryotes possess an RBS, a short sequence located 5' upstream of the start codon (usually 'AUG') on the mRNA containing the open reading frame (ORF), that facilitates ribosome recognition and binding to the mRNA. The RBS contains a sequence complementary to the 3' end of 16S rRNA, known as the Shine-Dalgarno sequence.

[0019] The mutant 5'UTR of the present application may be a 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1, which is the wild-type 5'UTR.

[0020] In the present application, the nucleotide sequence of SEQ ID NO: 1 may be the 5' untranslated region (5'UTR) of a gene encoding phosphoenolpyruvate carboxylase. The 5'UTR of the present application may comprise, have, consist of, or essentially consist of the nucleotide sequence of SEQ ID NO: 1.

[0021] The nucleotide sequence of SEQ ID NO: 1 may include a nucleotide sequence having at least 70%, 75%, 76%, 80%, 84%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the nucleotide sequence set forth in SEQ ID NO: 1. It is also clear that nucleotide sequences having such homology or identity and exhibiting the efficacy of corresponding to the 5'UTR, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, are also included within the scope of the present application.

[0022] Specifically, the 5'UTR in the present application in which one or more bases have been mutated is one in which the RBS sequence within the 5'UTR has been mutated, and may be referred to interchangeably as a "mutated 5'UTR" or a "sequence containing a mutant RBS."

[0023] More specifically, the mutant 5'UTR of the present application may be one in which one or more bases at positions corresponding to positions 15 to 22 in the base sequence of SEQ ID NO: 1 have been mutated. As an example, the mutation may mean that a specific base has been substituted with a base different from the base before substitution.

[0024] More specifically, the mutant 5′UTR of the present application can comprise any one of the nucleotide sequences selected from SEQ ID NO: 2 to SEQ ID NO: 12.

[0025] Furthermore, the mutant 5'UTR of the present application has homology or identity with any one of the sequences selected from SEQ ID NOs: 2 to 12, and each of the mutant sequences of SEQ ID NOs: 2 to 12 is fixed compared to SEQ ID NO: 1 encoding the wild-type 5'UTR, and has a homology or identity of 70% or more, 75% or more, 76% or more, 80% or more, 84% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.9% or more, and less than 100% of the entire sequence. Alternatively, the nucleic acid sequence may comprise, have, consist of, or essentially consist of a nucleic acid sequence having a homology or identity of 70% or more, 75% or more, 76% or more, 80% or more, 84% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.9% or more, or less than 100% to any one sequence selected from SEQ ID NO: 2 to SEQ ID NO: 12, but is not limited thereto.

[0026] Furthermore, it is obvious that mutant 5'UTRs having a base sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included within the scope of this application, as long as the base sequence has such homology or identity and exhibits the efficacy corresponding to the mutant 5'UTR of this application.

[0027] In this application, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or greater that is a polymer of nucleotides in which nucleotide units are linked in a long chain by covalent bonds.

[0028] As used herein, the term "mutant" refers to a polynucleotide in which one or more bases have been modified, resulting in a polynucleotide that differs from the base sequence of the mutant polynucleotide before the mutation but maintains its functions or properties. Such variants can generally be identified by modifying one or more bases in the base sequence of the polynucleotide and evaluating the properties of the modified polynucleotide. That is, the capabilities of the mutant polynucleotide may be increased, unchanged, or decreased compared to the polynucleotide before the mutation. The term "mutant polynucleotide" can be interchangeably used, including, but not limited to, variant, modification, mutated polynucleotide, mutated gene, mutation, and variant (in English, variant, modification, modified polynucleotide, modified gene, mutant, mutein, divergent, etc.), as long as it is used in the sense of mutation.

[0029] The variant polynucleotides can be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.

[0030] As used herein, the term "corresponding to" refers to a base at a recited position in a polynucleotide, or a base that is similar, identical, or homologous to a recited base in a polynucleotide. Identifying a base at a corresponding position may be determining a particular base in a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference polynucleotide sequence.

[0031] For example, any base sequence can be aligned with SEQ ID NO: 1, and based on this, each base in the base sequence can be numbered by referring to the numerical position of the base corresponding to the base in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application can identify the position of a base or the position where a variation such as a substitution, insertion, or deletion occurs compared to a query sequence (also referred to as a "reference sequence").

[0032] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16:276-277), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be used as appropriate.

[0033] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0034] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or a portion of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0035] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, e.g., 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 implemented 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), as implemented in the 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 Diego, 1994, and [CARILLO ET AL.] (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0036] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in Needleman et al. (1970), J Mol Biol. 48:443, as known in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0037] The polynucleotide of the present application may undergo various modifications in the coding region within a range that does not change the base sequence of the mutant gene containing the mutant 5'UTR of the present application, taking into account codon degeneracy or preferred codons in an organism in which the mutant gene containing the mutant 5'UTR of the present application is to be expressed.

[0038] Furthermore, the polynucleotides of the present application may include, without limitation, probes that can be prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to the entire or partial polynucleotide sequences of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as 70% or more, 75% or more, 76% or more, 80% or more, 84% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more, hybridize with each other, while polynucleotides having lower homology or identity do not hybridize with each other; and conditions under which washing is performed once, or specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, specifically 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0039] Hybridization requires that two nucleic acids have complementary sequences, even though 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 are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0040] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0041] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0042] The mutant polynucleotide encoding the mutant 5'UTR of the present application may regulate the translation into protein of a polynucleotide encoding a protein of interest operably linked thereto.

[0043] By way of example, the target protein may be, but is not limited to, phosphoenolpyruvate carboxylase. The polynucleotide encoding the mutant 5'UTR of the present application can be used for general purposes to regulate the translation of polynucleotides encoding various target proteins into proteins.

[0044] Another aspect of the present application provides a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, wherein the mutant polynucleotide further comprises a nucleotide sequence encoding a mutant 5'UTR in which one or more nucleotides have been mutated in the nucleotide sequence of SEQ ID NO: 1.

[0045] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, and the mutant 5'UTR are as described above.

[0046] In this application, the term "phosphoenolpyruvate carboxylase" refers to an enzyme that promotes the addition of bicarbonate to phosphoenolpyruvate to form the 4-carbon compound oxaloacetate and inorganic phosphate. This enzyme primarily affects the carbon flow into the TCA cycle, and the expression level of the ppc gene that encodes this enzyme affects the 4HB (4-hydroxybutyrate) content of polyhydroxyalkanoates (PHAs) produced by microorganisms.

[0047] The amino acid sequence of the phosphoenolpyruvate carboxylase can be obtained from a publicly known database such as NCBI Genebank.

[0048] By way of example, but not limitation, the phosphoenolpyruvate carboxylase of the present application may be derived from a microorganism.

[0049] In the present application, a phosphoenolpyruvate carboxylase may comprise, have, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:26.

[0050] In the present application, the amino acid sequence of SEQ ID NO: 26 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity thereto. It is also clear that proteins having such homology or identity and exhibiting efficacy corresponding to the protein comprising the amino acid sequence of SEQ ID NO: 26, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, are also included within the scope of the present application.

[0051] For example, the amino acid sequence may have additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally that do not change the function of the protein of the present application.

[0052] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0053] In the present application, the gene encoding phosphoenolpyruvate carboxylase may be the ppc gene.

[0054] The base sequence of the ppc gene can be obtained from a known database such as NCBI Genebank.

[0055] By way of example, but not limitation, the ppc gene of the present application may be derived from a microorganism.

[0056] A polynucleotide encoding the phosphoenolpyruvate carboxylase of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 27. As an example of the present application, the polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 27. Furthermore, the polynucleotide of the present application may consist of, or essentially consist of, the sequence of SEQ ID NO: 27. Specifically, the phosphoenolpyruvate carboxylase may be encoded by a polynucleotide set forth in the nucleotide sequence of SEQ ID NO: 27.

[0057] The polynucleotide of the present application may have various modifications in the coding region without changing the amino acid sequence of the phosphoenolpyruvate carboxylase, taking into consideration codon degeneracy or preferred codons in the organism in which the phosphoenolpyruvate carboxylase of the present application is to be expressed. Specifically, the polynucleotide of the present application may include a nucleotide sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homologous or identical to the sequence of SEQ ID NO: 27.

[0058] For purposes of this application, a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase of the present application can regulate the content of 4HB (4-hydroxybutyrate) monomer in polyhydroxyalkanoate (PHA) produced therefrom, compared to a polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase and further comprising a nucleotide sequence encoding a wild-type 5'UTR.

[0059] For example, the mutant polynucleotide containing a base sequence encoding the phosphoenolpyruvate carboxylase of the present application may be such that the 4HB monomer content of the PHA produced therefrom is adjusted to a range of 3 to 21% by weight based on the total weight of the PHA. For example, the mutant polynucleotide comprising a base sequence encoding the phosphoenolpyruvate carboxylase of the present application may produce a PHA having a 4HB monomer content of about 21% by weight or less, based on the total weight of the PHA, specifically, 20.3% by weight or less, 20% by weight or less, 19% by weight or less, 18.7% by weight or less, 18% by weight or less, 17% by weight or less, 16% by weight or less, 15% by weight or less, 14% by weight or less, 13.9% by weight or less, 13% by weight or less, 12% by weight or less, 11.6% by weight or less, 11% by weight or less, 10.7% by weight or less, 10% by weight or less, 9% by weight or less, 8.3% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, or 3.2% by weight or less, but is not limited thereto.

[0060] In this application, the term "polyhydroxyalkanoate (PHA)" refers to a polyester-based natural polymer that accumulates in microorganisms. It is a biodegradable material that does not generate toxic waste and is known as an environmentally friendly polymer with thermoplastic and elastic properties. The most representative PHA is P3HB (poly-3-hydroxybutyrate), which is polymerized using 3HB (3-hydroxybutyrate) monomer. There is also P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer, which is manufactured to have elastic properties by adding 4HB (4-hydroxybutyrate) monomer, but this is not limited thereto. In this application, the PHA may be specifically a PHA copolymer containing 4HB monomer and one or more additional monomers different from 4HB, or a PHA copolymer containing two, three, four, five, six, or more additional monomers different from each other. More specifically, a PHA copolymer containing 4HB monomer and one or more additional monomers different from 4HB may be used. The monomer that may be further contained as a copolymer may be one or more monomers selected from the group consisting of 2-hydroxybutyrate, lactic acid, glycolic acid, 3-hydroxybutyrate (3HB), 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HH), 3-hydroxyheptanoate (3HHep), 3-hydroxyoctanoate (3HO), 3-hydroxyoctanoate (3HN), 3-hydroxydecanoate (3HD), 3-hydroxyundecanoate (3HDd), 4-hydroxyvalerate (4HV), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HH).

[0061] In particular, the physical properties of PHA change depending on the ratio of 4HB monomer added, which can be used to produce a variety of products, and therefore, if the ratio of 4HB monomer can be adjusted, it may be industrially useful.

[0062] Specifically, the degree of crystallinity (crystallinity) of PHA changes depending on the ratio of 4HB monomer added. More specifically, as the content of 4HB monomer increases, the degree of crystallinity decreases, causing the PHA to exhibit amorphous rather than semi-crystalline properties, which can result in poor processability. Therefore, it is important to maintain the appropriate content of 4HB monomer to maintain the processability of PHA.

[0063] In contrast, the mutant polynucleotide of the present application containing a base sequence encoding phosphoenolpyruvate carboxylase can produce a PHA with semi-crystalline properties and excellent processability by adjusting the 4HB monomer content of the PHA to 3 to 21 wt % based on the total weight of the PHA.

[0064] Another aspect of the present application is to provide a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases are mutated in the base sequence of SEQ ID NO: 1; a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, and further comprising a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0065] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, and the mutant 5'UTR are as described above.

[0066] The vector may be, but is not limited to, an expression vector for expressing a polynucleotide of interest in a host cell.

[0067] The vector of the present application may comprise a DNA construct comprising the nucleotide sequence of a polynucleotide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the polynucleotide of interest, i.e., a mutant polynucleotide further comprising a nucleotide sequence encoding a mutant 5'UTR of the present application, in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable RBS, and a sequence regulating the termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome or can be integrated into the genome itself.

[0068] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0069] For example, a polynucleotide of interest can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for verifying the presence or absence of the insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to verify the presence or absence of the insertion of the nucleic acid molecule of interest. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the transformed cells to be selected.

[0070] The term "transformation" as used herein refers to introducing a vector containing a polynucleotide of interest into a host cell or microorganism to allow the polynucleotide to be expressed in the host cell. A transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and / or RNA encoding a polypeptide of interest. The polynucleotide may be introduced in any form that can be introduced 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 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 operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0071] In addition, the term "operably linked" as used above means that a promoter sequence that initiates and mediates the expression of a mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR of the present application is functionally linked to the base sequence of the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

[0072] In the present application, the nucleotide sequence encoding the mutant 5'UTR, i.e., the mutant polynucleotide encoding the mutant 5'UTR, may regulate the translation of a polynucleotide encoding a target protein operably linked thereto, and the target protein may be, for example, phosphoenolpyruvate carboxylase, but is not limited thereto, and may be generally used for the purpose of regulating the translation of polynucleotides encoding various target proteins into proteins.

[0073] Another aspect of the present application is to provide a microorganism for producing polyhydroxyalkanoates, comprising: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, and further comprising a base sequence encoding the mutant 5'UTR.

[0074] The microorganism of the present application may comprise one or more selected from the following: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1 of the present application; a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0075] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including those in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also include a microorganism containing a genetic modification for expressing a desired polynucleotide. The microorganism may be a microorganism of the genus Escherichia. Specifically, the microorganism may be Escherichia coli.

[0076] The strain of the present application can have the ability to produce PHA.

[0077] The strain of the present application may be a recombinant strain into which a mutant nucleic acid molecule capable of being transcribed by the mutant 5'UTR of the present application has been introduced into a natural wild-type microorganism, an untransformed microorganism, or a microorganism that produces PHA; or a mutant gene encoding phosphoenolpyruvate carboxylase comprising a mutant nucleic acid molecule capable of being transcribed by the mutant 5'UTR of the present application.

[0078] In the present application, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism refers to a strain before or without the introduction of a mutant polynucleotide encoding a mutant 5' UTR described herein; or a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, further comprising a nucleotide sequence encoding the mutant 5' UTR. The term "non-modified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0079] For purposes of this application, the strain of this application may be adjusted so that the 4HB monomer content of the polyhydroxyalkanoate produced therefrom is 3 to 21% by weight, based on the total weight of PHA, compared to a strain expressing a gene encoding phosphoenolpyruvate carboxylase containing a nucleic acid molecule that can be transcribed with a wild-type 5'UTR.

[0080] As another example, the recombinant microorganism of the present application may additionally have the activity of some proteins in the PHA biosynthetic pathway enhanced or attenuated.

[0081] In the microorganism of the present application, the mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutant 5'UTR and PHA, etc. are as described in the other aspects above.

[0082] Another aspect of the present application provides a method for producing PHA, comprising culturing in a medium a PHA-producing microorganism comprising: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR.

[0083] The method for producing PHA of the present application may include culturing in a medium a microorganism containing one or more selected from the following: a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1 of the present application; a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0084] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0085] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily as components, required for culturing the microorganism of the present application, and provides nutrients such as water essential for survival and growth, as well as growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application are not particularly limited as long as they are media used for culturing conventional microorganisms, and the microorganism of the present application can be cultured under aerobic conditions while controlling the temperature, pH, etc. in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0086] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other suitable carbon sources may also be used without limitation. These carbon sources may be used alone or in combination, and are not limited thereto.

[0087] Examples of the nitrogen source 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 such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0088] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0089] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0090] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0091] The PHA produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0092] The PHA production method of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0093] The method for producing PHA of the present application may further include a step of recovering PHA from the culture medium (the medium in which the culture was performed) or the strain. The recovery step may be performed after the culturing step.

[0094] The recovery may involve collecting the desired PHA using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used, and the desired PHA can be recovered from the medium or the microorganism using a suitable method known in the art.

[0095] Furthermore, the PHA production method of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. For example, when the PHA production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously in any order, or can be performed simultaneously or integrated into one step, but are not limited thereto.

[0096] For purposes of this application, the mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant nucleic acid molecule that can be transcribed with a mutant 5'UTR expressed by the strain of this application can be adjusted so that the 4HB monomer content of the polyhydroxyalkanoate produced therefrom is 3 to 21 wt% based on the total weight of PHA, compared to a gene encoding phosphoenolpyruvate carboxylase containing a nucleic acid molecule that can be transcribed with a wild-type 5'UTR.

[0097] In the method of the present application, the mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutant 5'UTR and PHA, etc. are as described in the other aspects above.

[0098] Another aspect of the present application is to provide a composition for producing PHA, comprising: a microorganism for producing PHA, which comprises a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases have been mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, further comprising a base sequence encoding the mutant 5'UTR; a culture medium in which the microorganism has been cultured; or a combination of two or more of them.

[0099] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing PHAs, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0100] Another aspect of the present application provides use of a microorganism comprising a mutant polynucleotide encoding a mutant 5'UTR in which one or more bases are mutated in the base sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR, for the production of PHA.

[0101] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutant 5'UTR and PHA, etc. are as described in the other embodiment above.

[0102] The present application will be described in more detail through examples below. However, the following examples are merely preferred embodiments for illustrating the present application and are not intended to limit the scope of the present application. Meanwhile, technical matters not described herein can be fully understood and easily implemented by those of ordinary skill in the technical field of the present application or a similar technical field. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which the present application pertains and the contents of the present application.

[0103] Example 1: Construction and screening of a library for introducing mutations into the 5' untranslated region (5'UTR) of the ppc gene encoding phosphoenolpyruvate carboxylase A template vector for constructing a 5' UTR mutation library of the ppc gene was constructed as follows. Using the chromosome of Escherichia coli MG1655 as a template, the 5'-UTR sequence of the wild-type ppc gene was amplified using the primer pair of SEQ ID NOs: 13 and 14. PCR conditions included denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 30 seconds, repeated 25 times. For fluorescence-based screening, a fluorescent protein expressed under a promoter was amplified using the primer pair of SEQ ID NOs: 15 and 16. PCR conditions included denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 1 minute, repeated 25 times. The two amplified products were then cloned into the pCL1920 vector, which had been treated with BamH1 restriction enzyme (NEB), using the assembly method to construct a template vector.

[0104] To construct a 5'UTR mutation library of the ppc gene using this vector, the primer pair of SEQ ID NOs: 13 and 17 was used to amplify the randomized sequence from the template vector. PCR conditions included denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 30 seconds, repeated 25 times. The remaining sequence of the template vector was amplified using the primer pair of SEQ ID NOs: 18 and 19, and the two amplified fragments were cloned using the assembly method to obtain a library vector. PCR conditions included denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 3 minutes, repeated 25 times. E. coli DH5α was used for cloning, and LB medium containing 75 mg / L spectinomycin was used for strain selection. AccuPower® ProFi Taq PCR PreMix (Bioneer) was used for DNA amplification.

[0105] The constructed library vector was transformed into E. coli MG1655, and the resulting single colony was inoculated into a 96-well plate containing 500 μL of LB medium supplemented with 75 mg / L of spectinomycin and cultured at 37°C for 16 hours. The saturated culture was diluted 1 / 100 and inoculated into 500 μL of polyhydroxyalkanoate (PHA) production medium (US 10323261 B2). After the cells reached full saturation in the culture medium, fluorescence was measured. A template vector containing the 5'-UTR sequence of the wild-type ppc gene was used as a control. Fluorescence and OD were measured. 600 was measured using a multilabel plate reader (PerkinElmer), and the measured fluorescence value was OD 600 It was normalized by dividing by .

[0106] As a result, a total of 11 types of bacterial cells with different expression levels were identified, and the plasmids from each bacterial cell were isolated and named Vector ver. 1 to Vector ver. 11. After that, the mutant sequences were confirmed by sequencing.

[0107] The plasmids used above are shown in Table 1 below, and the primer sequences are shown in Table 2 below. The mutated sequences of Vector ver. 1 to Vector ver. 11 confirmed above are shown in Table 3 below.

[0108] [Table 1]

[0109] [Table 2]

[0110] [Table 3]

[0111] Example 2: Construction of mutant sequence-introduced strains and analysis of monomer content in the produced PHA A PHA-producing strain was constructed by introducing the mutant RBS sequence confirmed in Example 1 into a P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)-producing strain. A P(3HB-co-4HB)-producing strain that produces 4HB at a level of approximately 20% in P(3HB-co-4HB) was used as the parent strain (US 10,323,261 B2). First, the mutant sequences whose sequences were confirmed were amplified using the primer pair of SEQ ID NOs: 20 and 21. The PCR conditions were denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 30 seconds, and were repeated 25 times. To perform genetic recombination using a linear DNA fragment containing a selection marker, att-Kan was used. RThe -att DNA fragment was amplified using the primer pair of SEQ ID NOs: 22 and 23. The PCR conditions were denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 2 minutes, repeated 25 times. To ligate this fragment to the previously amplified sequence, overlap PCR was performed using the primer pair of SEQ ID NOs: 24 and 25. The PCR conditions were denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 2 minutes and 15 seconds, repeated 25 times. The amplified DNA was transformed into a PHA-producing strain, and strains with RBS mutations were selected using a selection medium containing 50 mg / L kanamycin. AccuPower® ProFi Taq PCR PreMix (Bioneer) was used for DNA amplification.

[0112] The primer sequences are shown in Table 4 below.

[0113] [Table 4]

[0114] To evaluate the PHA production ability of 11 strains containing RBS mutations, they were inoculated into LB medium and cultured until fully saturated. Each strain was then inoculated into a 250 ml corner-baffled flask containing 25 ml of PHA production medium containing 5% glucose and cultured at 37°C for 5 hours and 35°C for 43 hours with shaking at 230 rpm. After the culture was completed, the intracellular PHA content and the 4HB content in the PHA were analyzed using gas chromatography. The specific culture and analysis conditions were based on those previously used (US 10323261 B2). The 4HB content of the PHA-producing strains into which each mutant sequence was introduced is shown in Table 5 below.

[0115] [Table 5]

[0116] As shown in Table 5, the control strain with a wild-type RBS sequence had a 4HB content of 21.2% in PHA, while the mutant strain with a mutant RBS sequence reduced the 4HB content in PHA from 20.3% to 3.2%. Therefore, it was confirmed that the introduction of a mutant RBS sequence into a PHA-producing strain can effectively control the PHA monomer content.

[0117] Of the 11 strains into which the mutant RBS sequence was introduced, the Pppc_v10 strain was named CB02-6588 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, on December 9, 2022, and was assigned the deposit number KCCM13300P.

[0118] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

[0119] JPEG0007792977000006.jpg227170

Claims

1. A mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) comprising a nucleotide sequence selected from SEQ ID NOs: 2 to 12, wherein the mutant polynucleotide encoding the mutant 5'UTR regulates protein translation of a polynucleotide operably linked thereto that encodes a target protein, and the target protein is phosphoenolpyruvate carboxylase.

2. A mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, The mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR, A mutant polynucleotide, wherein the mutant 5'UTR comprises a base sequence selected from SEQ ID NOs: 2 to 12.

3. 3. The mutant polynucleotide of claim 2, wherein the mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase has a 4HB (4-hydroxybutyrate) monomer content of polyhydroxyalkanoate produced therefrom adjusted to a content range of 3 to 21 wt% based on the total weight of PHA, compared to a polynucleotide comprising the base sequence encoding phosphoenolpyruvate carboxylase and further comprising a base sequence encoding a wild-type 5'UTR, and the polyhydroxyalkanoate comprises 4HB (4-hydroxybutyrate) as a monomer.

4. A polyhydroxyalkanoate-producing microorganism of the genus Escherichia coli, comprising the mutant polynucleotide according to any one of claims 1 and 2, wherein the polyhydroxyalkanoate contains 4HB (4-hydroxybutyrate) as a monomer.

5. The microorganism according to claim 4, wherein the content of 4HB monomer in the polyhydroxyalkanoate produced by the microorganism is adjusted to be in the range of 3 to 21 wt % based on the total weight of the PHA.

6. A method for producing a polyhydroxyalkanoate, comprising a step of culturing in a medium a polyhydroxyalkanoate-producing microorganism of the genus Escherichia coli, which contains the mutant polynucleotide according to any one of claims 1 and 2, wherein the polyhydroxyalkanoate contains 4HB (4-hydroxybutyrate) as a monomer.

7. The method of claim 6, wherein the mutant polynucleotide comprising the base sequence encoding phosphoenolpyruvate carboxylase has a 4HB monomer content of the polyhydroxyalkanoate produced therefrom adjusted to a content range of 3 to 21% by weight based on the total weight of PHA, compared to a polynucleotide comprising the base sequence encoding phosphoenolpyruvate carboxylase and further comprising a base sequence encoding a wild-type 5'UTR.

8. A composition for PHA production comprising a microorganism for PHA production of the genus Escherichia coli containing the mutant polynucleotide according to any one of claims 1 and 2, a medium in which the microorganism is cultured, and the medium containing the microorganism, or a combination of two or more of them, wherein the PHA contains 4HB (4-hydroxybutyrate) as a monomer.

9. 3. Use of a microorganism of the genus Escherichia coli containing the mutant polynucleotide according to claim 1 or 2 for producing a PHA, wherein the PHA contains 4HB (4-hydroxybutyrate) as a monomer.

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