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

Abstract

The present application relates to a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR) including a ribosome-binding site (RBS) mutant sequence, 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 produces no toxic waste and is environmentally friendly, possessing thermoplastic and elastic properties. The most well-known PHA is poly-3-hydroxybutyrate (P3HB), which is polymerized using 3-hydroxybutyrate (3HB) 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] [Patent documents]

[0003] [Patent Document 1] US 10323261 B2 [Non-patent literature]

[0004] [Non-Patent Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-licensed document 2] 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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Non-licensed literature 9

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Non-licensed Document 11

[0005] The present inventors have confirmed that the 5' untranslated region (5'UTR) mutant sequence of the present application can be used to regulate the carbon flow in the TCA cycle, and in particular, that it can regulate the 4HB (4-hydroxybutyrate) content of polyhydroxyalkanoate (PHA)-producing microorganisms to provide PHA with the desired physical properties, thereby completing the present application. [Means for solving the problem]

[0006] One object of the present application is to provide a mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) comprising a sequence in which one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated.

[0007] Another object of the present application is a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, The mutant polynucleotide is a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, The mutant polynucleotide further comprises a nucleotide sequence encoding a mutant 5'UTR, which comprises a sequence in which any one or more nucleotides selected from the group consisting of nucleotides corresponding to positions 3, 4, 5, 7, 8, and 10 in the nucleotide sequence of SEQ ID NO: 1 are mutated.

[0008] Another object of the present application is to provide a microorganism for producing polyhydroxyalkanoates, comprising: a mutant polynucleotide encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR.

[0009] Another object of the present application is to provide a method for producing polyhydroxyalkanoate, which includes a step of culturing in a medium a microorganism for producing polyhydroxyalkanoate, the microorganism comprising: a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR. [Effects of the Invention]

[0010] 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

[0011] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application also 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 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.

[0012] One aspect of the present application provides a mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated.

[0013] 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.

[0014] In the present application, the 5'UTR can include 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.

[0015] 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 may include base substitution, addition, deletion, or inversion. As a representative example, the Shine-Dalgarno sequence in the 5'UTR is conserved, and the base sequence before and after it may include a sequence in which one or more bases are modified by substitution, addition, deletion, inversion, or the like.

[0016] The 5'UTR can include a ribosome binding site (RBS).

[0017] 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 near the 5' upstream side of the start codon ('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.

[0018] 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.

[0019] 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.

[0020] 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 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.

[0021] In one embodiment, the 5'UTR of the present application may include the base sequence of SEQ ID NO: 19 as the base sequence including the base sequence of SEQ ID NO: 1.

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

[0023] More specifically, the mutant 5'UTR of the present application may have one or more mutated bases at positions corresponding to the third to tenth bases from the 5' end in the base sequence of SEQ ID NO: 1.

[0024] More specifically, the mutant 5'UTR of the present application may include a polynucleotide in which one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated. For example, the mutant 5'UTR may include a polynucleotide in which one, two or more, three or more, four or more, five or more, or six bases selected from the above positions have been mutated.

[0025] For example, the term "mutation" used herein means that a specific base has been substituted with a base different from the base before substitution. That is, the mutant 5'UTR used herein may include a polynucleotide in which one or more bases selected from the group consisting of bases corresponding to the third, fourth, fifth, seventh, eighth, and tenth positions in the base sequence of SEQ ID NO: 1 have been substituted with different bases.

[0026] Specifically, the mutant 5'UTR of the present application can comprise a polynucleotide in which the T base corresponding to the third position in the nucleotide sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of G, C, and A. The mutant 5'UTR of the present application can comprise a polynucleotide in which the C base corresponding to the fourth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of A, G, and T. The mutant 5'UTR of the present application can comprise a polynucleotide in which the T base corresponding to the fifth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of G, C, and A. The mutant 5'UTR of the present application can comprise a polynucleotide in which the G base corresponding to the seventh position in the nucleotide sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of A, C, and T. The mutant 5'UTR of the present application can comprise a polynucleotide in which the G base corresponding to the eighth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of C, A, and T. Alternatively, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the 10th position in the base sequence of SEQ ID NO: 1 is substituted with a base selected from the group consisting of G, C, and A. The mutant 5'UTR of the present application may comprise a polynucleotide containing any one, or a combination of two or more, three or more, four or more, five or more, or six of the above-mentioned substitutions.

[0027] More specifically, the mutation of the present application may be any one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

[0028] In one embodiment, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the third position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base C corresponding to the fourth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the fifth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the seventh position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the eighth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. Alternatively, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the tenth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application can comprise a polynucleotide containing any one, two or more, three or more, four or more, five or more, or a combination of six of the substitutions described above.

[0029] As used herein, the term "corresponding position" refers to the base (nucleotide) at the recited position in a base sequence or polynucleotide, or a base that is similar, identical, or homologous to the recited base in a base sequence or polynucleotide. As used herein, "corresponding region" generally refers to an analogous or corresponding position in a related or reference polynucleotide.

[0030] In this application, specific numbering is used for base positions in the base sequences or polynucleotides used in this application. For example, by aligning the base sequences of the subject base sequence to be compared with those of the present application, it is possible to renumber the positions corresponding to the base positions of the polynucleotides of the present application.

[0031] More specifically, the polynucleotide encoding the mutant 5′UTR of the present application can comprise any one of the nucleotide sequences set forth in SEQ ID NO:2 to SEQ ID NO:18.

[0032] Furthermore, the polynucleotide encoding the mutant 5' UTR of the present application has homology or identity with any one of the sequences selected from SEQ ID NO: 2 to SEQ ID NO: 18, which is a portion of the polynucleotide encoding the wild-type 5' UTR. Each of the mutant sequences of SEQ ID NO: 2 to SEQ ID NO: 18 is fixed 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, or 99.5% or more relative to the entire sequence. The nucleic acid sequence may have or comprise a base sequence that is 7% or more, 99.9% or more, and less than 100% homologous or identical to any one of the sequences selected from SEQ ID NO: 2 to SEQ ID NO: 18, or may comprise, have, consist of, or essentially consist of a base sequence that is 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% homologous or identical to any one of the sequences selected from SEQ ID NO: 2 to SEQ ID NO: 18, but is not limited to these.

[0033] 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.

[0034] In one embodiment, the polynucleotide encoding the mutant 5'UTR of the present application may include a base sequence selected from SEQ ID NOs: 2 to 18, and may also include a base sequence selected from SEQ ID NOs: 20 to 36.

[0035] In this application, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or greater, which is a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds.

[0036] In the present application, 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 may 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 ability of the mutant polynucleotide may be increased, unchanged, or decreased compared to the polynucleotide before the mutation. The term "mutant polynucleotide" may be interchangeable with other terms such as variant, modification, mutated polynucleotide, mutated gene, mutation, and variant (in English, variant, modification, modified polynucleotide, modified gene, mutant, mutein, divergent, etc.), and is not limited thereto as long as it is used in the sense of mutation.

[0037] The variant polynucleotides may be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.

[0038] 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 also be determining a particular base in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference polynucleotide sequence.

[0039] 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").

[0040] 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.

[0041] As used herein, the terms "homology" or "identity" refer 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.

[0042] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing 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.

[0043] 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.

[0044] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in 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.

[0045] The polynucleotide of the present application may be modified in various ways in the coding region within a range that does not change the base sequence of the mutant gene comprising a polynucleotide encoding the mutant 5'UTR of the present application, taking into consideration codon degeneracy or preferred codons in an organism in which the mutant gene comprising a polynucleotide encoding the mutant 5'UTR of the present application is to be expressed.

[0046] Furthermore, the polynucleotides of the present application may include, without limitation, any sequences that can hybridize under stringent conditions to probes prepared from known gene sequences, for example, sequences complementary to all or part of the 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, conditions include conditions under which polynucleotides with 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, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions include washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for standard Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×X SSC, 0.1% SDS.

[0047] 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 the entire sequence, as well as substantially similar nucleic acid sequences.

[0048] 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.

[0049] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0050] 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.

[0051] By way of example, the target protein may be, but is not limited to, phosphoenolpyruvate carboxylase, and the mutant polynucleotide encoding the mutant 5'UTR of the present application can be used universally for the purpose of regulating the translation of polynucleotides encoding various target proteins into proteins.

[0052] Another aspect of the present application is a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, The mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th, and 10th positions in the base sequence of SEQ ID NO: 1 are mutated.

[0053] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutation, and the mutant 5'UTR are as described above.

[0054] The mutations of the present application may be any one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

[0055] In one embodiment, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the third position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base C corresponding to the fourth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the fifth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the seventh position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the eighth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. Alternatively, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the tenth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application can comprise a polynucleotide containing any one, two or more, three or more, four or more, five or more, or a combination of six of the above-described substitutions, as described above.

[0056] In this application, the term "phosphoenolpyruvate carboxylase" refers to an enzyme that promotes the addition of bicarbonate to phosphoenolpyruvate to form the four-carbon compound oxaloacetate and inorganic phosphate. The enzyme primarily affects the carbon flow rate in the TCA cycle, and the expression level of the ppc gene encoding this enzyme affects the 4HB (4-hydroxybutyrate) content of polyhydroxyalkanoates (PHAs) produced by microorganisms.

[0057] The amino acid sequence of the phosphoenolpyruvate carboxylase can be obtained from a publicly known database such as NCBI Genebank.

[0058] By way of example, but not limitation, the phosphoenolpyruvate carboxylase of the present application may be derived from a microorganism.

[0059] 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:50.

[0060] In the present application, the amino acid sequence of SEQ ID NO: 50 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 having an amino acid sequence 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, as long as the amino acid sequence has the same efficacy as the protein comprising the amino acid sequence of SEQ ID NO: 50.

[0061] 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 alter the function of the protein of the present application.

[0062] 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 the amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0063] In the present application, the gene encoding phosphoenolpyruvate carboxylase may be the ppc gene.

[0064] The base sequence of the ppc gene can be obtained from a known database such as NCBI Genebank.

[0065] By way of example, but not limitation, the ppc gene of the present application may be derived from a microorganism.

[0066] A polynucleotide encoding a phosphoenolpyruvate carboxylase of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 50. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 51. Furthermore, a polynucleotide of the present application may consist of or essentially consist of the sequence of SEQ ID NO: 51. Specifically, the phosphoenolpyruvate carboxylase may be encoded by a polynucleotide set forth in the nucleotide sequence of SEQ ID NO: 51.

[0067] The polynucleotide of the present application may have various modifications in the coding region within a range that does not change the amino acid sequence of the phosphoenolpyruvate carboxylase, taking into consideration codon degeneracy or codons preferred in the organism in which the phosphoenolpyruvate carboxylase of the present application is to be expressed. Specifically, the polynucleotide of the present application may comprise 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: 51.

[0068] For purposes of the present application, a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase of the present application may have a regulated 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.

[0069] For example, the mutant polynucleotide comprising 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 21.5 to 46% 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 be such that the 4HB monomer content of the PHA produced therefrom is about 21.5% by weight or more, specifically, 21.8% by weight or more, 22% by weight or more, 22.7% by weight or more, 23% by weight or more, 23.2% by weight or more, 24% by weight or more, 24.7% by weight or more, 25% by weight or more, 25.8% by weight or more, 26% by weight or more, 27% by weight or more, 28% by weight or more, 29% by weight or more, 29.1% by weight or more, based on the total weight of the PHA. The amount may be, but is not limited to, 30% by weight or more, 31% by weight or more, 32% by weight or more, 33% by weight or more, 33.5% by weight or more, 34% by weight or more, 35% by weight or more, 36% by weight or more, 36.5% by weight or more, 37% by weight or more, 38% by weight or more, 39% by weight or more, 40% by weight or more, 40.3% by weight or more, 41% by weight or more, 41.2% by weight or more, 41.7% by weight or more, 42% by weight or more, 43% by weight or more, 44% by weight or more, 44.5% by weight or more, 45% by weight or more, 45.2% by weight or more, or 46% by weight or more.

[0070] The term "polyhydroxyalkanoate (PHA)" used in this application refers to a polyester-based natural polymer that accumulates in microorganisms. It is known as an environmentally friendly polymer that is not only biodegradable but also has thermoplastic and elastic properties without generating toxic waste. The most representative PHA is P3HB (poly-3-hydroxybutyrate), which is polymerized using 3HB (3-hydroxybutyrate) monomer, and P(3HB-co-4HB), which is manufactured with the addition of 4HB (4-hydroxybutyrate) monomer to have elastic properties. (Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymers are also available, but are not limited thereto. In the present application, the PHA specifically includes a 4HB monomer and may further include one or more monomers different from the 4HB, and may be a PHA copolymer including two, three, four, five, six or more different monomers. More specifically, examples of monomers that may be included as a monomer different from the 4HB while including the 4HB monomer include 2-hydroxybutyrate, lactic acid, glycolic acid, 3HB (3-hydroxybutyrate), 3HP (3-hydroxybutyrate), and the like. The monomer may be one or more monomers selected from the group consisting of 3HH (3-hydroxypropionate), 3HV (3-hydroxyvalerate), 3HH (3-hydroxyhexanoate), 3HHep (3-hydroxyheptanoate), 3HO (3-hydroxyoctanoate), 3HN (3-hydroxyoctanoate), 3HD (3-hydroxydecanoate), 3HDd (3-hydroxyundecanoate), 4HV (4-hydroxyvalerate), 5HV (5-hydroxyvalerate), and 6HH (6-hydroxyhexanoate).

[0071] 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, so it is industrially useful if the ratio of 4HB monomer can be adjusted.

[0072] 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.

[0073] In contrast, the mutant polynucleotide of the present application containing a base sequence encoding phosphoenolpyruvate carboxylase can produce PHA with excellent processability and semi-crystalline properties by adjusting the 4HB monomer content of the PHA to 21.5 to 46 wt % based on the total weight of the PHA.

[0074] Another aspect of the present application is to provide a mutant polynucleotide encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0075] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutation, and the mutant 5'UTR are as described above.

[0076] The vector may be, but is not limited to, an expression vector for expressing a polynucleotide of interest in a host cell.

[0077] 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 being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0078] 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.

[0079] 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 confirming the presence or absence of the insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the insertion of the nucleic acid molecule of interest. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0080] As used herein, the term "transformation" 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 within the host cell's chromosome 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 typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an autonomously 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.

[0081] 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.

[0082] 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, into a protein. For example, the target protein may be, but is not limited to, phosphoenolpyruvate carboxylase, and the nucleotide sequence may be generally used for the purpose of regulating the translation of polynucleotides encoding various target proteins into proteins.

[0083] Another aspect of the present application is to provide a microorganism for producing polyhydroxyalkanoates, comprising: a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

[0084] The mutations of the present application may be any one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

[0085] In one embodiment, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the third position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base C corresponding to the fourth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the fifth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the seventh position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the eighth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. Alternatively, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the tenth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application can comprise a polynucleotide containing any one, two or more, three or more, four or more, five or more, or a combination of six of the above-described substitutions, as described above.

[0086] The microorganism of the present application may comprise one or more selected from the group consisting of a mutant polynucleotide encoding a mutant 5'UTR, which comprises a sequence in which one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th, and 10th positions in the base sequence of SEQ ID NO: 1 have been mutated; a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0087] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including microorganisms 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 microorganisms containing genetic modifications for expression of a desired polynucleotide. The microorganism may be a microorganism of the genus Escherichia. Specifically, the microorganism may be Escherichia coli.

[0088] The strain of the present application can have the ability to produce PHA.

[0089] 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.

[0090] As used herein, 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, which further comprises 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."

[0091] For purposes of this application, the strain of this application can be adjusted so that the 4HB monomer content of the polyhydroxyalkanoate produced therefrom is 21.5 to 46 wt. % 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.

[0092] As another example, in the recombinant microorganism of the present application, the activity of some of the proteins in the PHA biosynthetic pathway may be further enhanced or weakened.

[0093] In the microorganism of the present application, the mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutation, the mutant 5'UTR and PHA, etc. are as described in the other aspects above.

[0094] 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, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

[0095] The mutations of the present application may be any one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

[0096] In one embodiment, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the third position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base C corresponding to the fourth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the fifth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the seventh position in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5'UTR of the present application may comprise a polynucleotide in which the base G corresponding to the eighth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. Alternatively, the mutant 5'UTR of the present application may comprise a polynucleotide in which the base T corresponding to the tenth position in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5'UTR of the present application can comprise a polynucleotide containing any one, two or more, three or more, four or more, five or more, or a combination of six of the above-described substitutions, as described above.

[0097] The method for producing PHA of the present application may include culturing in a medium a microorganism containing one or more selected from the group consisting of a mutant polynucleotide encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th, and 10th positions in the base sequence of SEQ ID NO: 1 of the present application have been mutated; a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, which further comprises a base sequence encoding the mutant 5'UTR; or a vector comprising the same.

[0098] 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.

[0099] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily as ingredients, required for culturing the microorganism of the present application, and provides nutrients such as water and growth factors essential for survival and growth. 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. The microorganism of the present application can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0100] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and 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. Natural organic nutrient sources such as starch hydrolysates, 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 carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0101] 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 degradation products, and defatted soybean cake or its degradation products. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0102] 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, the present invention is not limited to these compounds.

[0103] During the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium in an appropriate manner to adjust the pH of the medium. During cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. 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.

[0104] 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.

[0105] The PHA produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0106] 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.

[0107] 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.

[0108] The recovery may involve collecting the desired PHA using an appropriate 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 an appropriate method known in the art.

[0109] 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. In one 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 is not limited thereto.

[0110] For the 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, which is expressed by the strain of the present application, may be such that the 4HB monomer content of the polyhydroxyalkanoate produced therefrom is adjusted to a range of 21.5 to 46% by weight, 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.

[0111] In the method of the present application, the mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutation, the mutant 5'UTR and PHA, etc. are as described in the other aspects above.

[0112] Another aspect of the present application is to provide a PHA-producing microorganism comprising: a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR; a culture medium in which the microorganism has been cultured; or a composition for PHA production comprising a combination of two or more of these.

[0113] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing PHA, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0114] Another aspect of the present application provides use of a microorganism comprising a mutant polynucleotide encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the base sequence of SEQ ID NO: 1 have been mutated; 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.

[0115] The mutant polynucleotide, the base sequence of SEQ ID NO: 1, the mutation, the mutant 5'UTR and PHA, etc. are as described in the other aspects above. [Example]

[0116] The present application will be described in more detail below with reference to examples. 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.

[0117] Example 1: Construction and screening of a library for introducing mutations into the RBS (ribosome-binding site) of the ppc gene encoding phosphoenolpyruvate carboxylase A template vector for constructing a mutation library of RBSs within the 5' UTR sequence of the ppc gene was constructed as follows. Using the chromosome of Escherichia coli MG1655 as a template, the 5' untranslated region (5' UTR) sequence of the wild-type ppc gene was amplified using the primer pair of SEQ ID NOs: 37 and 38. 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: 39 and 40. 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 and the pCL1920 vector treated with BamH1 restriction enzyme (NEB) were cloned by assembly method to prepare a template vector.

[0118] To construct a ppc gene RBS mutation library using this vector, the primer pair of SEQ ID NOs: 37 and 41 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: 42 and 43. The two amplified fragments were ligated using an Infusion Cloning Kit and then transformed into E. coli DH5α. 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. To screen for transformed strains, colonies were selected in LB medium containing 75 mg / L spectinomycin. AccuPower® ProFi Taq PCR PreMix (Bioneer) was used for DNA amplification.

[0119] The constructed library vector was transformed into E. coli MG1655, and the resulting single colony was inoculated into 500 μL of LB medium supplemented with 75 mg / L of spectinomycin in a 96-well plate 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 .

[0120] As a result, a total of 17 types of bacterial cells with different expression levels were identified, and the plasmids from each bacterial cell were isolated and named Vector verB.1 to Vector verB.17, after which the mutant sequences were sequenced to confirm their identity.

[0121] The plasmids used above are shown in Table 1 below, and the primer sequences are shown in Table 2 below.

[0122] The above-confirmed mutant sequences of Vector verB.1 to Vector verB.17 are shown in Table 3 below.

[0123] [Table 1]

[0124] [Table 2]

[0125] [Table 3]

[0126] 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: 44 and 45. 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, repeated 25 times. A linear DNA fragment containing a selection marker was used for genetic recombination, and the att-KanR-att The DNA fragment was amplified using the primer pair of SEQ ID NOs: 46 and 47. 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, with this cycle repeated 25 times. To ligate this fragment to the previously amplified sequence, overlap PCR was performed using the primer pair of SEQ ID NOs: 48 and 49. 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, with this cycle 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.

[0127] The primer sequences are shown in Table 4 below.

[0128] [Table 4]

[0129] To evaluate the PHA production ability of 17 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).

[0130] The 4HB content of the PHA-producing strains into which each mutant sequence was introduced is shown in Table 5 below.

[0131] [Table 5]

[0132] As shown in Table 4, the 4HB content in PHA from the control strain with a wild-type RBS sequence was 21.2%, while the 4HB content in PHA from the mutant strain with a mutant RBS sequence increased from 21.8% to 45.2%. Therefore, it was confirmed that the PHA monomer content can be effectively controlled by introducing a mutant RBS sequence into a PHA-producing strain.

[0133] Of the 17 strains into which the mutant RBS sequence was introduced, the Pppc_vB8 strain was named CB02-6607 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, on February 17, 2023, and was assigned the deposit number KCCM13332P.

[0134] 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.

Claims

1. A mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th and 10th positions in the base sequence of SEQ ID NO: 1 have been mutated.

2. A mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, The mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR, which comprises a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th and 10th positions in the base sequence of SEQ ID NO: 1 are mutated.

3. 3. The mutant polynucleotide of claim 1 or 2, wherein the mutation is one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

4. The mutant polynucleotide according to claim 1 or 2, wherein the polynucleotide encoding the mutant 5'UTR comprises any one or more base sequences selected from SEQ ID NOs: 2 to 18.

5. The mutant polynucleotide according to claim 1 or 2, wherein the polynucleotide encoding the mutant 5'UTR comprises any one or more base sequences selected from SEQ ID NOs: 20 to 36.

6. The mutant polynucleotide of claim 1, wherein the mutant polynucleotide encoding the mutant 5'UTR regulates the translation of a polynucleotide encoding a target protein operably linked thereto into a protein.

7. The mutant polynucleotide according to claim 6 , wherein the target protein is phosphoenolpyruvate carboxylase.

8. 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 range of 21.5 to 46% by weight based on the total weight of PHA, compared to a polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase and further comprising a base sequence encoding a wild-type 5'UTR.

9. A microorganism for producing polyhydroxyalkanoates, comprising: a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th and 10th positions in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

10. The microorganism according to claim 9, wherein the content of 4HB monomer in the polyhydroxyalkanoate produced by the microorganism is adjusted to a range of 21.5 to 46 wt% based on the total weight of the PHA.

11. The microorganism of claim 9, wherein the mutation is one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

12. A method for producing polyhydroxyalkanoate, comprising the step of culturing in a medium a microorganism for producing polyhydroxyalkanoate, the microorganism comprising: a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to the 3rd, 4th, 5th, 7th, 8th, and 10th positions in the base sequence of SEQ ID NO: 1 have been mutated; or a mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

13. The method of claim 12, wherein the mutant polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase has a 4HB monomer content of polyhydroxyalkanoate produced therefrom adjusted to a range of 21.5 to 46% by weight based on the total weight of PHA, compared to a polynucleotide comprising a base sequence encoding phosphoenolpyruvate carboxylase and further comprising a base sequence encoding a wild-type 5'UTR.

14. 13. The method of claim 12, wherein the mutation is any one or more selected from the following: i) T is replaced by G; ii) C is replaced by A; iii) G is replaced by A; and iv) G is replaced by C.

15. Use of a mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR) comprising a sequence in which any one or more bases selected from the group consisting of bases corresponding to the third, fourth, fifth, seventh, eighth, and tenth positions in the base sequence of SEQ ID NO: 1 have been mutated in order to produce polyhydroxyalkanoates.

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