5apos of the gene encoding a phosphoenolpyruvate carboxylase; uTR variant sequences and uses thereof

Abstract

The present application relates to a variant gene encoding a phosphoenolpyruvate carboxylase comprising a variant 5 '-untranslated region (5' UTR), the variant gene comprising a ribosome binding site (RBS) variant sequence, a polyhydroxyalkanoate (PHA)-producing microorganism comprising the gene, and a PHA production method using the same.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a mutant gene encoding phosphoenolpyruvate carboxylase, which includes a mutant 5'-untranslated region (5'UTR), a microorganism producing polyhydroxyalkanoate (PHA) including the gene, and a method for producing PHA using the microorganism. BACKGROUND

[0002] Due to environmental pollution problems arising from the accumulation of petroleum-based non-degradable plastics, biodegradable bio-plastics that replace them are attracting attention. Polyhydroxyalkanoate (PHA) is a bio-plastic material that is a polyester-based natural polymer accumulated in microbial bodies. PHA is a biodegradable material that does not generate toxic waste and is considered an environmentally friendly polymer having properties such as thermoplasticity and elasticity. Among PHAs, the most representative known material is poly-3-hydroxybutyrate (P3HB), which is polymerized using 3-hydroxybutyrate (3HB) monomers. P3HB has physical properties similar to polypropylene, which is a synthetic resin used for automobile parts, home appliance parts, and the like. Because P3HB can be relatively easily synthesized by microorganisms, much commercial research has been conducted on its production. However, P3HB has limitations: it is brittle due to its high crystallinity, and is difficult to process because it is easily decomposed near its melting point. Therefore, research has been conducted on the production of PHA copolymers by blending with new materials or copolymerization of monomers. Among them, poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) copolymer is a material having elastomeric properties due to the addition of 4-hydroxybutyrate (4HB) monomers. The physical properties of PHA vary depending on the proportion of 4HB monomers added, and PHA can be used to produce various products. Therefore, it is attracting attention as a promising biodegradable high molecular material. SUMMARY

[0003] [PROBLEMS TO BE SOLVED BY THE INVENTION]

[0004] The present inventors found that the 5'-untranslated region (5'UTR) mutant sequence of the present disclosure can be used to control carbon flow of the TCA cycle, in particular, to control the 4-hydroxybutyrate (4HB) content of a microorganism producing polyhydroxyalkanoate (PHA), thereby providing PHA having desired physical properties, and completed the present disclosure.

[0005] [TECHNICAL SOLUTION]

[0006] An object of the present disclosure is to provide 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 nucleotide sequence of SEQ ID NO: 1 are mutated.

[0007] Another object of the present disclosure is to provide a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding a mutant 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 nucleotide sequence of SEQ ID NO: 1 are mutated.

[0008] Another object of the present disclosure is to provide a microorganism producing polyhydroxyalkanoate, the microorganism comprising: a mutant polynucleotide encoding a mutant 5' UTR 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 nucleotide sequence of SEQ ID NO: 1 are mutated; or a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding the mutant 5' UTR.

[0009] Another object of the present disclosure is to provide a method of producing polyhydroxyalkanoate, the method including the step of culturing a microorganism producing polyhydroxyalkanoate in a culture medium, the microorganism comprising: a mutant polynucleotide encoding a mutant 5' UTR 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 nucleotide sequence of SEQ ID NO: 1 are mutated; or a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding the mutant 5' UTR.

[0010] [Advantageous Effects]

[0011] The 5'-untranslated region (5' UTR) mutant sequence of the present disclosure can be used to control carbon flow of the TCA cycle, particularly to control the 4-hydroxybutyrate (4HB) content of a microorganism producing polyhydroxyalkanoate (PHA), thereby providing a PHA having desired physical properties. DETAILED DESCRIPTION

[0012] The present disclosure will be described in detail below. Meanwhile, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. Furthermore, the scope of the present disclosure is not limited by the specific descriptions described below. Furthermore, those skilled in the art will recognize, or be able to ascertain with no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that such equivalents fall within the scope of the present disclosure.

[0013] One aspect of the present disclosure 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 nucleotide sequence of SEQ ID NO: 1 are mutated.

[0014] As used herein, the term "5'-untranslated region (5' UTR)" refers to a region present at the 5'-end of an mRNA transcript but not translated into amino acids. Translation of mRNA into protein begins with the binding of a 30S ribosomal subunit to the 5' UTR. Specifically, when the 16S rRNA (16S ribosomal RNA) in the 30S ribosomal subunit binds to the RBS within the 5' UTR, the tRNA recognizes and binds to the start codon (AUG) of the mRNA, translation to protein begins. It is known that the ribosome binding site of the 5' UTR and the start codon are present at a distance of about 6 to 8 nucleotides.

[0015] In the present disclosure, the 5' UTR includes a sequence in which the sequence complementary to the 3' terminal sequence of the 16S rRNA, i.e., the Shine-Dalgarno sequence is conserved, and the sequences before and after the Shine-Dalgarno sequence are modified.

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

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

[0018] As used herein, the term "ribosome binding site (RBS)" refers to a region (structure) present inside an mRNA strand to which a ribosome can bind directly and which is capable of initiating translation, or a region (structure) of a DNA strand that results in the region being transcribed. Most prokaryotes have an RBS, which is a short sequence that allows the ribosome to easily recognize and bind to the mRNA, near the 5' upstream of the start codon (usually 'AUG') on the mRNA containing an open reading frame (ORF). The RBS includes a sequence complementary to the 3' terminal sequence of 16S rRNA, known as the Shine-Dalgarno sequence.

[0019] The mutant 5' UTR of the present disclosure can be a mutant 5' UTR in which one or more bases in the nucleotide sequence of SEQ ID NO: 1 of the wild-type 5' UTR are mutated.

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

[0021] The nucleotide sequence of SEQ ID NO: 1 can 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% or more homology or identity to the nucleotide sequence described in SEQ ID NO: 1. Furthermore, it is obvious that any nucleotide sequence having a deletion, modification, substitution, conservative substitution, or addition of certain sequences can also be included within the scope of the present disclosure, as long as the nucleotide sequence has such homology or identity and exhibits the efficacy corresponding to the 5' UTR.

[0022] In one embodiment, the 5' UTR of the present disclosure is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 1, and can include the nucleotide sequence of SEQ ID NO: 19.

[0023] In one embodiment, the 5' UTR of the present disclosure in which one or more bases are mutated can be used interchangeably with "mutant RBS", "sequence containing a mutant RBS", or "mutant 5' UTR" in which the RBS sequence in the 5' UTR is mutated.

[0024] More specifically, the mutant 5' UTR of the present disclosure can have a mutation in any one or more of the bases corresponding to positions 3-10 of the 5' end of the nucleotide sequence of SEQ ID NO: 1.

[0025] More specifically, the mutant 5' UTR of the present disclosure can include a polynucleotide in which any one or more of the bases selected from the group consisting of the bases corresponding to positions 3, 4, 5, 7, 8, and 10 of the nucleotide sequence of SEQ ID NO: 1 is mutated. Examples thereof can include a polynucleotide in which any one, two or more, three or more, four or more, five or more, or six or more of the bases selected from the bases corresponding to the above positions are mutated.

[0026] For example, the mutation of the present disclosure can mean that a particular base is replaced with a different base from the base before the replacement. In other words, the mutant 5' UTR of the present disclosure can include a polynucleotide in which any one or more of the bases selected from the group consisting of the bases corresponding to positions 3, 4, 5, 7, 8, and 10 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a different base.

[0027] Specifically, the mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 3 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of G, C, and A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base C corresponding to position 4 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of A, G, and T. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 5 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of G, C, and A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 7 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of A, C, and T. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 8 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of C, A, and T. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 10 of the nucleotide sequence of SEQ ID NO: 1 is replaced with a base selected from the group consisting of G, C, and A. The mutant 5' UTR of the present disclosure can include a polynucleotide including any one, or a combination of two or more, three or more, four or more, five or more, or six or more of the above replacements.

[0028] More specifically, the mutation of the present disclosure can be any one or more of the following replacements: G to A

[0029] i) replacing T with G;

[0030] ii) replacing C with A;

[0031] iii) replacing G with A; and

[0032] iv) replacing G with C.

[0033] In one embodiment, the mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 3 in the nucleotide sequence of SEQ ID NO: 1 is replaced with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base C corresponding to position 4 in the nucleotide sequence of SEQ ID NO: 1 is replaced with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 5 in the nucleotide sequence of SEQ ID NO: 1 is replaced with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 7 in the nucleotide sequence of SEQ ID NO: 1 is replaced with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 8 in the nucleotide sequence of SEQ ID NO: 1 is replaced with C. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 10 in the nucleotide sequence of SEQ ID NO: 1 is replaced with G. The mutant 5' UTR of the present disclosure can include a polynucleotide comprising any one of, or a combination of two or more, three or more, four or more, five or more, or six or more of the above-mentioned replacements.

[0034] As used herein, the term "corresponding to position" refers to a base (nucleotide) at a position listed in a nucleotide sequence or polynucleotide, or a base similar, identical, or homologous to a base listed in a nucleotide sequence or polynucleotide. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related polynucleotide or reference polynucleotide.

[0035] In the present disclosure, a specific numbering can be used for the position of a base in a nucleotide sequence or polynucleotide used in the present disclosure. For example, by aligning a target nucleotide sequence to be compared with a nucleotide sequence of the present disclosure, the position corresponding to the position of a base of a polynucleotide of the present disclosure can be renumbered.

[0036] More specifically, the polynucleotide encoding the mutant 5' UTR of the present disclosure can include a nucleotide sequence described by any one selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18.

[0037] Further, the polynucleotide encoding the mutant 5' UTR of the present disclosure can have or can include a nucleotide sequence having 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% homology or identity to the entire sequence of any one selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18, provided that the respective mutant sequence of SEQ ID NO: 2 to SEQ ID NO: 18 is fixed compared to the sequence of SEQ ID NO: 1 as a part of the polynucleotide encoding the wild-type 5' UTR; or it can include or can have, or can consist essentially of, but not limited to, a nucleotide sequence having 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% homology or identity to the entire sequence of any one selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18.

[0038] Further, it is obvious that any mutant 5' UTR having a deletion, modification, substitution, conservative substitution, or addition of certain sequences can also be included in the scope of the present disclosure, as long as the nucleotide sequence has such homology or identity and exhibits the efficacy corresponding to the mutant 5' UTR of the present disclosure.

[0039] In one embodiment, the polynucleotide encoding the mutant 5' UTR of the present disclosure can be a nucleotide sequence including the nucleotide sequence described by any one selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18, and can include the nucleotide sequence described by any one selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 36.

[0040] As used herein, the term "polynucleotide" is a nucleotide polymer in which nucleotide monomers are linked by covalent bonds into a long chain shape, and refers to a DNA or RNA chain having a predetermined or longer length.

[0041] As used herein, the term "mutant" refers to a polynucleotide having a nucleotide sequence different from that of the unmodified nucleotide sequence due to modification of one or more bases, but retaining a function or property. Such a mutant can be generally identified by modifying one or more bases of the nucleotide sequence of a polynucleotide and evaluating the property of the modified polynucleotide. In other words, the ability of a mutant polynucleotide can be increased, unchanged, or decreased compared to the polynucleotide before modification. The mutant polynucleotide can be used interchangeably with terms such as variant, modified, modified polynucleotide, modified gene, mutant, mutant protein, divergent, and the like, and is not limited thereto, as long as the other is a term having a variant meaning.

[0042] The mutant polynucleotide can be conjugated with other sequences or linkers for identification, purification, or synthesis.

[0043] As used herein, the term "corresponding to" refers to a base at a listed position in a polynucleotide, or a base similar, identical, or homologous to a base listed in a polynucleotide. Identifying a base at a corresponding position can be determining a particular base in a sequence that is referenced to a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related polynucleotide sequence or a reference polynucleotide sequence.

[0044] For example, any nucleotide sequence is aligned with SEQ ID NO: 1, and based on this, each base of the nucleotide sequence can be numbered with reference to the base of SEQ ID NO: 1 and the numerical position of the corresponding base. For example, a sequence alignment algorithm as described in the present disclosure can determine a base position or a position at which a modification such as substitution, insertion, or deletion occurs by comparing with a base position in a query sequence (also referred to as a "reference sequence").

[0045] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), and the like, but not limited thereto, and a sequence alignment program known in the art, a double sequence comparison algorithm, and the like can be appropriately used.

[0046] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms "homology and identity" can often be used interchangeably.

[0047] Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and can use the default gap penalties established by the program used. Essentially, a homologous or identical sequence will typically hybridize under moderately or highly stringent conditions to all or a portion of the sequence. Obviously, hybridization also includes hybridization to polynucleotides comprising degenerate codons or codons accounting for codon degeneracy.

[0048] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program, for example using the default parameters in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, homology, similarity or identity 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 software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), including GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J Mol 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, the BLAST or ClustalW of the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0049] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program (as described in Needleman et al. (1970), J Mol Biol. 48:443, as declared in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482). In summary, the GAP program can be defined as the value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include (1) a binary comparison matrix (including a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap, and an extra penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0050] In the polynucleotides of the present disclosure, various modifications can be made in the coding region, provided that the codon degeneracy or preferred codons in organisms intended to express the mutant genes including the polynucleotides encoding the mutant 5' UTRs of the present disclosure are considered without changing the nucleotide sequence of the mutant genes including the polynucleotides encoding the mutant 5' UTRs of the present disclosure.

[0051] In addition, the polynucleotide of the present disclosure can include a probe that can be prepared from a known gene sequence, for example, a sequence without limitation as long as it is a sequence capable of hybridizing to the complement of all or a part of the polynucleotide sequence of the present disclosure under stringent conditions. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 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, 9.50-9.51, 11.7-11.8). Examples thereof include conditions in which polynucleotides having a high degree of homology or identity, i.e., polynucleotides having 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 of homology or identity, hybridize to each other, and polynucleotides having a low degree of homology or identity do not hybridize to each other, or conditions in which the salt concentration and temperature are equivalent to 60°C, 1X SSC, 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS, and washing is performed once, specifically 2 to 3 times. This is a washing condition of general Southern hybridization.

[0052] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed 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 each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotide of the present disclosure can also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the entire sequence.

[0053] Specifically, the polynucleotide having homology or identity to the polynucleotide of the present disclosure can be detected using hybridization conditions including a Tm value of 55°C and a hybridization step under the above conditions. The Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0054] The appropriate stringency for hybridization of polynucleotides depends on the length and complementarity of the nucleotides, and variables are well known in the art (e.g., J. Sambrook et al., supra).

[0055] The mutant polynucleotide encoding the mutant 5' UTR of the present disclosure can regulate translation of a polynucleotide encoding a target protein into a protein operably linked thereto.

[0056] For example, the target protein can be phosphoenolpyruvate carboxylase, but is not limited thereto, and the mutant polynucleotide encoding the mutant 5' UTR of the present disclosure can be universally used for the purpose of regulating translation of a polynucleotide encoding various target proteins into a protein.

[0057] Another aspect of the present disclosure provides a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding a mutant 5' UTR, the nucleotide sequence encoding the mutant 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 nucleotide sequence of SEQ ID NO: 1 are mutated.

[0058] The mutant polynucleotide, the nucleotide sequence of SEQ ID NO: 1, the mutation, and the mutant 5' UTR are as described above.

[0059] The mutation of the present disclosure can be any one or more selected from the following substitutions:

[0060] i) substitution of T with G;

[0061] ii) substitution of C with A;

[0062] iii) substitution of G with A; and

[0063] iv) substitution of G with C.

[0064] In one embodiment, the mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 3 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base C corresponding to position 4 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 5 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 7 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 8 in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 10 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide including any one of, or a combination of two or more, three or more, four or more, five or more, or six or more of the above-mentioned substitutions. The description is as above.

[0065] As used herein, the term "phosphoenolpyruvate carboxylase" is an enzyme that facilitates the addition of bicarbonate and phosphoenolpyruvate to form oxaloacetate, which is a 4-carbon compound and inorganic phosphate, which has a major impact on carbon flow into the TCA cycle, and the expression level of the ppc gene encoding the enzyme can affect the 4-hydroxybutyrate (4HB) content of polyhydroxyalkanoates (PHAs) produced by a microorganism.

[0066] The amino acid sequence of the phosphoenolpyruvate carboxylase can be obtained from a known database such as NCBI GenBank, etc.

[0067] For example, the phosphoenolpyruvate carboxylase of the present disclosure can be derived from a microorganism, but is not limited thereto.

[0068] In the present disclosure, the phosphoenolpyruvate carboxylase can include, can have, or can consist of or can consist essentially of the amino acid sequence described by SEQ ID NO: 50.

[0069] In the present disclosure, the amino acid sequence of SEQ ID NO: 50 can 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% or more homology or identity thereto. Furthermore, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution, conservative substitution, or addition of some sequences can also be included within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits a corresponding efficacy to a protein including the amino acid sequence of SEQ ID NO: 50.

[0070] Examples thereof include addition or deletion of sequences at the N-terminus, C-terminus, and / or inside of the amino acid sequence, naturally occurring mutations, silent mutations, or conservative substitutions that do not change the function of the protein of the present disclosure.

[0071] The term "conservative substitution" refers to substitution of one amino acid with another amino acid having similar structure and / or chemical properties. Such substitution of an amino acid typically occurs based on similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Generally, a conservative substitution can hardly affect or not affect the activity of a protein or polypeptide.

[0072] In the present disclosure, the gene encoding phosphoenolpyruvate carboxylase can be a ppc gene.

[0073] The nucleotide sequence of the ppc gene can be obtained from a known database such as Genebank of NCBI, etc.

[0074] For example, the ppc gene of the present disclosure can be derived from a microorganism, but is not limited thereto.

[0075] The polynucleotide encoding the phosphoenolpyruvate carboxylase of the present disclosure can include a nucleotide sequence encoding the amino acid sequence described by SEQ ID NO: 50. As an example of the present disclosure, the polynucleotide can have or can include the sequence of SEQ ID NO: 51. Furthermore, the polynucleotide of the present disclosure can consist of or essentially consist of the sequence of SEQ ID NO: 51. Specifically, the phosphoenolpyruvate carboxylase can be encoded by the polynucleotide described by the nucleotide sequence of SEQ ID NO: 51.

[0076] In the polynucleotide of the present disclosure, various modifications can be made in the coding region, provided that the amino acid sequence of the phosphoenolpyruvate carboxylase is not changed in consideration of codon degeneracy or preferred codons in an organism in which the phosphoenolpyruvate carboxylase of the present disclosure is intended to be expressed. Specifically, the polynucleotide of the present disclosure can include a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 51.

[0077] With respect to the purpose of the present disclosure, the mutant polynucleotide comprising the nucleotide sequence encoding the phosphoenolpyruvate carboxylase of the present disclosure can regulate the content of 4-hydroxybutyrate (4HB) monomer of polyhydroxyalkanoate (PHA) produced therefrom, as compared to the polynucleotide comprising the nucleotide sequence encoding the phosphoenolpyruvate carboxylase and further comprising the nucleotide sequence encoding the wild-type 5' UTR.

[0078] For example, the mutant polynucleotide comprising the nucleotide sequence encoding the phosphoenolpyruvate carboxylase of the present disclosure can regulate the content of 4HB monomer of PHA produced therefrom in the range of 21.5% by weight to 46% by weight based on the total weight of PHA. For example, with respect to the mutant polynucleotide comprising the nucleotide sequence encoding the phosphoenolpyruvate carboxylase of the present disclosure, the content of 4HB monomer of PHA produced therefrom can be 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, 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, but is not limited thereto.

[0079] As used herein, the term "polyhydroxyalkanoate (PHA)" is a natural polymer based on a polyester accumulated in microorganisms, and is a biodegradable material that does not generate toxic waste and is considered to be an eco-friendly polymer having properties such as thermoplasticity and elasticity. Among PHAs, the most representative known material is poly-3-hydroxybutyrate (P3HB) polymerized using 3-hydroxybutyrate (3HB) monomers. There is also a poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) copolymer, which is prepared by adding 4-hydroxybutyrate (4HB) monomers to have properties as an elastomer, but is not limited thereto. In the present disclosure, the PHA can specifically be a PHA copolymer including 4HB monomers, while also including one or more monomers different from 4HB, or also including two, three, four, five, six or more monomers different from each other. More specifically, as the monomers different from 4HB, the monomers included together with the 4HB monomers can also 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-hydroxynonanoate (3HN), 3-hydroxydecanoate (3HD), 3-hydroxyundecanoate (3HDd), 4-hydroxyvalerate (4HV), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HH).

[0080] In particular, since the physical properties of the PHA vary depending on the addition ratio of the 4HB monomer that can be used to produce various products, it is industrially useful when the ratio of the 4HB monomer can be adjusted.

[0081] Specifically, the crystallinity of the PHA can vary depending on the addition ratio of the 4HB monomer, and more specifically, as the content of the 4HB monomer increases, the crystallinity decreases, and the PHA exhibits amorphous properties rather than semi-crystalline properties, which can reduce processability. Therefore, it is important to maintain the processability of the PHA by maintaining an appropriate 4HB monomer content.

[0082] Accordingly, by adjusting the content of the 4HB monomer of the PHA in the range of 21.5 to 46 wt% based on the total weight of the PHA, the mutant polynucleotide including the nucleotide sequence encoding the phosphoenolpyruvate carboxylase of the present disclosure can produce a PHA having semi-crystalline properties and excellent processability.

[0083] Another aspect of the present disclosure provides a mutant polynucleotide encoding a mutant 5' UTR, the mutant polynucleotide encoding a mutant 5' UTR including 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 nucleotide sequence of SEQ ID NO: 1 are mutated; a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding the mutant 5' UTR; or a vector including the same.

[0084] The mutant polynucleotide, the nucleotide sequence of SEQ ID NO: 1, the mutation, and the mutant 5' UTR are as described above.

[0085] The vector can be an expression vector for expressing a target polynucleotide in a host cell, but is not limited thereto.

[0086] The vector of the present disclosure can include a DNA construct including a nucleotide sequence of a target polynucleotide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so that the target polynucleotide (i.e., a mutant polynucleotide further including a nucleotide sequence encoding a mutant 5' UTR of the present disclosure) can be expressed in a suitable host. The expression regulatory region can include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable RBS, and a sequence regulating termination of transcription and translation. The vector can be transformed into a suitable host cell, and then can replicate independently of the host genome or function, or can be integrated into the genome itself.

[0087] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a bacteriophage vector or a cosmid vector, pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as a plasmid vector. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0088] For example, a target polynucleotide can be inserted into a chromosome by a vector for intracellular chromosomal insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, homologous recombination, but is not limited thereto. The vector can further include a selection marker for confirming chromosomal insertion. The selection marker is used to select a vector-transformed cell, i.e., to confirm insertion of a target nucleic acid molecule, and can use a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface polypeptide. Only cells expressing the selection marker survive or exhibit a different phenotype expression in an environment treated with a selection agent, and thus transformed cells can be selected.

[0089] As used herein, the term "transformation" refers to introduction of a vector including a target polynucleotide into a host cell or microorganism so that the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be inserted into a chromosome of the host cell or located outside the chromosome as long as it can be expressed in the host cell. The polynucleotide includes DNA and / or RNA encoding a target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced and expressed in the host cell. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette can often include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide is introduced into a host cell in its own form and can be operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0090] In addition, the term "operably linked" means that a nucleotide sequence of a mutant polynucleotide further including a nucleotide sequence encoding a mutant 5' UTR is functionally linked to a promoter sequence that initiates and mediates expression of the mutant polynucleotide further including a nucleotide sequence encoding a mutant 5' UTR.

[0091] In the present disclosure, a nucleotide sequence encoding a mutant 5' UTR, i.e., a mutant polynucleotide encoding a mutant 5' UTR, can regulate translation of a polynucleotide encoding a target protein operably linked thereto into a protein. For example, the target protein can be phosphoenolpyruvate carboxylase, but is not limited thereto. It can be universally used for the purpose of regulating translation of a polynucleotide encoding various target proteins into a protein.

[0092] Another aspect of the present disclosure can provide a microorganism for producing a polyhydroxyalkanoate, the microorganism comprising: a mutant polynucleotide encoding a mutant 5' UTR, the mutant 5' UTR having a mutation in any one or more of the bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the nucleotide sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a nucleotide sequence encoding the mutant 5' UTR.

[0093] The mutations of the present disclosure can be any one or more selected from the following substitutions:

[0094] i) substitution of T with G;

[0095] ii) substitution of C with A;

[0096] iii) substitution of G with A; and

[0097] iv) substitution of G with C.

[0098] In one embodiment, the mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base T corresponding to position 3 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base C corresponding to position 4 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base T corresponding to position 5 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base G corresponding to position 7 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base G corresponding to position 8 in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. The mutant 5' UTR of the present disclosure can comprise a polynucleotide in which the base T corresponding to position 10 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can comprise a polynucleotide comprising any one, or a combination of two or more, three or more, four or more, five or more, or six or more of the substitutions described above.

[0099] The microorganism of the present disclosure can include any one or more selected from the group consisting of: a mutant polynucleotide encoding a mutant 5' UTR including 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 nucleotide sequence of SEQ ID NO: 1 are mutated; a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase further comprising a nucleotide sequence encoding the mutant 5' UTR; or a vector comprising the same.

[0100] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a particular mechanism is weakened or enhanced due to insertion of an exogenous gene or activity enhancement or inactivation of an endogenous gene, and it can be a microorganism including a genetic modification for expression of a target polynucleotide. The microorganism can be a microorganism of the genus Escherichia. Specifically, the microorganism can be Escherichia coli.

[0101] The strain of the present disclosure can have the ability to produce PHA.

[0102] The strain described in the present disclosure can be a recombinant strain prepared by introducing into a naturally wild-type microorganism, an unmodified microorganism, or a PHA-producing microorganism: a mutant nucleic acid molecule to be transcribed into a mutant 5' UTR of the present disclosure; or a mutant gene encoding phosphoenolpyruvate carboxylase comprising a mutant nucleic acid molecule to be transcribed into the mutant 5' UTR.

[0103] As used herein, the term "unmodified microorganism" does not exclude a strain including mutations that can occur naturally in a microorganism, and can be a wild-type strain or a natural strain itself, or can be a strain before its properties are changed due to genetic variation by natural or artificial factors. For example, the unmodified microorganism can be a strain into which a mutant polynucleotide encoding a mutant 5' UTR described in the present disclosure has not been introduced or has not yet been introduced; or a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase further comprising a nucleotide sequence encoding the mutant 5' UTR. The term "unmodified microorganism" can be used interchangeably with "strain before modification," "microorganism before modification," "unvaried strain," "unmodified strain," "unvaried microorganism," or "reference microorganism."

[0104] For the purposes of the present disclosure, the strain of the present disclosure can modulate the content of 4HB monomers of polyhydroxyalkanoates produced therefrom to a range of 21.5 wt% to 46 wt% based on the total weight of PHA compared to a strain expressing a gene encoding phosphoenolpyruvate carboxylase whose nucleic acid molecule is to be transcribed into a wild-type 5' UTR.

[0105] For another example, in the recombinant microorganism of the present disclosure, the activity of some proteins in the PHA biosynthetic pathway can be additionally enhanced or attenuated.

[0106] In the microorganism of the present disclosure, the mutant polynucleotide, the nucleotide sequence of SEQ ID NO: 1, the mutation, the mutant 5' UTR, and the PHA are as described in other embodiments.

[0107] Another aspect of the present disclosure provides a method of producing PHA, the method comprising the step of culturing a polyhydroxyalkanoate-producing microorganism in a culture medium, the microorganism comprising: a mutant polynucleotide encoding a mutant 5' UTR, the mutant 5' UTR having a mutation in any one or more of the bases corresponding to positions 3, 4, 5, 7, 8, and 10 in the nucleotide sequence of SEQ ID NO: 1; or a mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a nucleotide sequence encoding the mutant 5' UTR.

[0108] The mutation of the present disclosure can be any one or more selected from the following substitutions:

[0109] i) substitution of T with G;

[0110] ii) substitution of C with A;

[0111] iii) substitution of G with A; and

[0112] iv) substitution of G with C.

[0113] In one embodiment, the mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 3 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base C corresponding to position 4 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 5 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 7 in the nucleotide sequence of SEQ ID NO: 1 is substituted with A. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base G corresponding to position 8 in the nucleotide sequence of SEQ ID NO: 1 is substituted with C. The mutant 5' UTR of the present disclosure can include a polynucleotide in which the base T corresponding to position 10 in the nucleotide sequence of SEQ ID NO: 1 is substituted with G. The mutant 5' UTR of the present disclosure can include a polynucleotide including any one of, or a combination of two or more, three or more, four or more, five or more, or six or more of the above substitutions.

[0114] The method of producing PHA of the present disclosure can include a step of culturing a microorganism in a culture medium, the microorganism including any one or more selected from the group consisting of: a mutant polynucleotide encoding a mutant 5' UTR including 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 nucleotide sequence of SEQ ID NO: 1 are mutated; a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase further including a nucleotide sequence encoding the mutant 5' UTR; or a vector including the same.

[0115] As used herein, the term "culturing" refers to culturing the microorganism of the present disclosure under appropriate controlled environmental conditions. The culturing process of the present disclosure can be performed according to suitable culture media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art according to the selected microorganism. Specifically, the culturing can be batch type, continuous type, and / or fed-batch type, but is not limited thereto.

[0116] As used herein, the term "medium" refers to a mixed substance containing nutrients required for culturing the microorganism of the present disclosure as a main component, and the medium provides nutrients, growth factors, etc. (including water) indispensable for survival and growth. Specifically, for the medium and other culture conditions for culturing the microorganism of the present disclosure, any medium can be used without particular limitation, as long as it is a medium generally used for culturing microorganisms. The microorganism of the present disclosure can be cultured in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc. under aerobic conditions, while controlling the temperature, pH, etc.

[0117] In the present disclosure, the carbon source includes 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.; amino acids such as glutamic acid, methionine, lysine, etc.; and the like. Natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugar cane residue, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used, and other carbon sources in appropriate amounts can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0118] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids (such as glutamic acid, methionine, and glutamine, etc.), peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its degradation products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0119] The phosphorus source can include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition to these compounds, amino acids, vitamins, and / or suitable precursors, etc. can also be contained. These components or precursors can be added to the medium in batches or continuously, but are not limited thereto.

[0120] During the cultivation of the microorganism of the present disclosure, the pH of the culture medium can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in an appropriate manner. During the cultivation, foaming can be inhibited by using an antifoaming agent such as a fatty acid polyethylene glycol ester. In addition, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain an aerobic state of the culture medium, or no gas or nitrogen gas, hydrogen gas, or carbon dioxide gas can be injected to maintain an anaerobic and microaerophilic state of the culture medium, but not limited thereto.

[0121] In the cultivation of the present disclosure, the cultivation temperature can be maintained at 20°C to 45°C, specifically at 25°C to 40°C, and the microorganism can be cultivated for about 10 hours to about 160 hours, but not limited thereto.

[0122] The PHA produced by the cultivation of the present disclosure can be secreted into the culture medium or retained in the cells.

[0123] The method for producing PHA of the present disclosure can further include, for example, a step of preparing the microorganism of the present disclosure before the cultivation step, a step of preparing a culture medium for cultivating the microorganism, or a combination (in any order) of these steps.

[0124] The method for producing PHA of the present disclosure can further include a step of recovering PHA from the culture medium according to the cultivation (culture medium on which cultivation is performed) or from the strain. The recovery step can be further included after the cultivation step.

[0125] According to the method for cultivating a microorganism of the present disclosure, for example, a batch, continuous, or fed-batch cultivation method, the recovery can collect the target PHA by means of a suitable method known in the art. For example, centrifugation, filtration, treatment with a crystallization protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various forms of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC, or a combination thereof can be used. The target PHA can be recovered from the culture medium or the microorganism by a suitable method known in the art.

[0126] The PHA production method of the present disclosure can further include a purification step. The purification can be performed by means of a suitable method known in the art. For example, when the method for producing PHA of the present disclosure includes a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously regardless of the order, or can be performed simultaneously or combined into one step, but not limited thereto.

[0127] For the purposes of the present disclosure, the mutant gene encoding phosphoenolpyruvate carboxylase, which is expressed by the strain of the present disclosure, can adjust the content of 4HB monomer of polyhydroxyalkanoate produced therefrom to a range of 21.5 to 46 wt% based on the total weight of PHA, compared to a gene encoding phosphoenolpyruvate carboxylase including a nucleic acid molecule to be transcribed into a wild-type 5'UTR.

[0128] In the method of the present disclosure, the mutant polynucleotide, the nucleotide sequence of SEQ ID NO: 1, the mutation, the mutant 5'UTR, the PHA, etc. are as described in the other embodiments described above.

[0129] Another aspect of the present disclosure provides a composition for producing PHA, the composition including a microorganism for producing PHA, the microorganism including a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide encoding the mutant 5'UTR including 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 nucleotide sequence of SEQ ID NO: 1 are mutated; or a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding the mutant 5'UTR; a culture medium in which the microorganism is cultured; or a combination of two or more thereof.

[0130] The composition of the present disclosure can further include any suitable excipient, which is generally used in the composition for producing PHA. Such an excipient can be, for example, a preservative, a humectant, a dispersant, a suspending agent, a buffer, a stabilizer, or an isotonic agent, etc., but is not limited thereto.

[0131] Another aspect of the present disclosure provides the use of a microorganism in the production of PHA, the microorganism including a mutant polynucleotide encoding a mutant 5'UTR, the mutant polynucleotide encoding the mutant 5'UTR including 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 nucleotide sequence of SEQ ID NO: 1 are mutated; or a mutant polynucleotide including a nucleotide sequence encoding phosphoenolpyruvate carboxylase, the mutant polynucleotide further including a nucleotide sequence encoding the mutant 5'UTR.

[0132] The mutant polynucleotide, the nucleotide sequence of SEQ ID NO: 1, the mutation, the mutant 5'UTR, the PHA, etc. are as described in the other embodiments described above.

[0133] [Mode for Invention]

[0134] Hereinafter, the present disclosure will be described in greater detail with reference to exemplary embodiments. However, the following exemplary embodiments are merely for illustrative purposes of the preferred embodiments of the present disclosure, and thus the scope of the present disclosure is not intended to be limited thereto. Meanwhile, technical matters not described in the present specification can be sufficiently understood and easily implemented by those skilled in the art or those skilled in the similar technical field of the present disclosure. In addition, in the present specification, many papers and patent documents are quoted and their origins are indicated. The entire contents disclosed in the quoted papers and patent documents are incorporated by reference into the present specification in order to more clearly describe the level of the technical field to which the present disclosure belongs and the contents of the present disclosure.

[0135] Example 1: Construction and screening of a library for introducing mutations into a ribosome binding site (RBS) of a ppc gene encoding phosphoenolpyruvate carboxylase

[0136] A template vector for constructing an RBS mutant library in a 5'UTR sequence of a ppc gene was prepared by the following method. The 5'-untranslated region (5'UTR) sequence of a wild-type ppc gene was amplified using the chromosome of E. coli MG1655 as a template and a primer pair of SEQ ID NOS: 37 and 38. The PCR condition was repeated for 25 cycles: denaturation at 95℃ for 20 seconds, annealing at 56℃ for 40 seconds, and extension at 72℃ for 30 seconds. For fluorescence-based screening, a fluorescent protein to be expressed downstream of a promoter was amplified using a primer pair of SEQ ID NOS: 39 and 40. The PCR condition was repeated for 25 cycles: denaturation at 95℃ for 20 seconds, annealing at 56℃ for 40 seconds, and extension at 72℃ for 1 minute. The template vector was constructed by cloning the two amplified products in an assembly manner and a pCL1920 vector treated with a BamH1 restriction enzyme (NEB).

[0137] In order to prepare an RBS mutant library of a ppc gene using the corresponding vector, a randomized sequence was amplified from the prepared template vector using a primer pair of SEQ ID NOS: 37 and 41. The PCR condition was repeated for 25 cycles: denaturation at 95℃ for 20 seconds, annealing at 56℃ for 40 seconds, and extension at 72℃ for 30 seconds. The remaining sequence of the template vector was amplified using a primer pair of SEQ ID NOS: 42 and 43, and the two amplified fragments were ligated using an Infusion cloning kit, and transformed into E. coli DH5α. The PCR condition was repeated for 25 cycles: denaturation at 95℃ for 20 seconds, annealing at 56℃ for 40 seconds, and extension at 72℃ for 3 minutes. In order to select the transformed strain, colonies were selected on an LB medium containing 75 mg / L spectinomycin. For DNA amplification, ProFi Taq PCR PreMix (Bioneer) was used. ProFi Taq PCR PreMix (Bioneer).

[0138] Single colonies obtained by transforming the constructed library vector into E. coli MG1655 were inoculated in a 96-well plate containing 500 μL of LB medium supplemented with 75 mg / L spectinomycin and incubated at 37°C for 16 hours. The saturated medium was diluted 1 / 100 with 500 μL of polyhydroxyalkanoate (PHA) production medium (US10323261 B2), and fluorescence was measured after the cells were fully saturated in the medium. At this time, the template vector having the 5'UTR sequence of the wild-type ppc gene was used as a control. Fluorescence and OD 600 were measured using a multi-label plate reader (PerkinElmer). 600 The measured fluorescence values were normalized by dividing by OD

[0139] As a result, a total of 17 strains having different expression levels were identified, the plasmids of each strain were isolated, each plasmid was named from vector verB.1 to vector verB.17, and sequencing was performed to identify the mutant sequences.

[0140] The plasmids used above are shown in Table 1 below, and the primer sequences are shown in Table 2 below.

[0141] The mutant sequences of the vectors verB.1 to verB.17 identified above are shown in Table 3 below.

[0142] [Table 1]

[0143]

[0144]

[0145] [Table 2]

[0146]

[0147] [Table 3]

[0148]

[0149]

[0150] Example 2: Construction of a strain introducing a mutant sequence and analysis of monomer content in PHA produced

[0151] A PHA-producing strain in which the mutant RBS sequence identified in Example 1 was introduced was prepared based on a poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) producing strain. As a parent strain, a P(3HB-co-4HB) producing strain in which the content of 4HB in P(3HB-co-4HB) was about 20% was used (US 10323261B2). First, the mutant sequence whose sequence was identified was amplified using the primer pair of SEQ ID NOS: 44 and 45, respectively. The PCR conditions were repeated for 25 cycles of denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 30 seconds. In order to perform gene recombination using a linear DNA fragment containing a selectable marker, the att-KanR-att DNA fragment was amplified using the primer pair of SEQ ID NOS: 46 and 47. The PCR conditions were repeated for 25 cycles of denaturation at 95°C for 20 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 2 minutes. In order to ligate the fragment with the previously amplified sequence, overlap PCR was performed using the primer pair of SEQ ID NOS: 48 and 49. The PCR conditions were repeated for 25 cycles of 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. After transforming the amplified DNA into the PHA-producing strain, a strain in which the RBS mutation was introduced was selected in a selection medium containing 50 mg / L kanamycin.

[0152] The primer sequences are shown in Table 4 below.

[0153] [Table 4]

[0154]

[0155] In order to evaluate the PHA-producing ability of the 17 strains in which the RBS mutation was introduced, they were inoculated in LB medium and cultured to be fully saturated. Thereafter, each strain was inoculated into a 250 ml baffled flask containing 25 ml of PHA production medium containing 5% glucose and cultured at 37°C for 5 hours at 230 rpm and at 35°C for 43 hours. After completion of the culture, the intracellular PHA content and the 4HB content in PHA were analyzed using gas chromatography. The specific culture conditions and analysis conditions refer to the previously used conditions (US 10323261B2).

[0156] The 4HB content of the PHA-producing strains in which the mutant sequence was introduced is shown in Table 5 below.

[0157] [Table 5]

[0158] Sequence name PHA (mg) 4HB content (wt%) Pppc 12.3 21.2％ Pppc_vB1 12.1 21.8％ Pppc_vB2 11.8 23.2％ Pppc_vB3 15.3 22.7％ Pppc_vB4 10 24.7％ Pppc_vB5 9.1 25.8％ Pppc_vB6 12.5 30.0％ Pppc_vB7 12.7 29.1％ Pppc_vB8 13.2 33.5％ Pppc_vB9 11.6 34.0％ Pppc_vB10 14.8 40.3％ Pppc_vB11 10.2 38.0％ Pppc_vB12 9.8 41.2％ Pppc_vB13 10.7 44.5％ Pppc_vB14 14.2 36.5％ Pppc_vB15 11.9 41.7％ Pppc_vB16 12 42.0％ Pppc_vB17 11.3 45.2％

[0159] As shown in Table 4, the 4HB content in PHA was 21.2% in the control strain having the wild-type RBS sequence, whereas the 4HB content in PHA was increased from 21.8% to 45.2% in the mutant strain having the mutant RBS sequence. Thus, it was confirmed that the content of PHA monomers can be effectively controlled by introducing the mutant RBS sequence into the strain producing PHA.

[0160] Among the 17 strains into which the mutant RBS sequence was introduced, the Pppc_vB8 strain was designated as CB02-6607 and deposited on February 17, 2023, at the International Depository Authority of Korea Microorganism Culture Collection (KCCM) under the Budapest Treaty, with the assigned deposit number KCCM13332P.

[0161] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are not restrictive but are illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by the description before them, and thus all changes and modifications falling within the boundaries and scope of the claims, or equivalents of these boundaries and scope, are included in the claims.

[0162] PCT / RO / 134 Table

Claims

1. A mutant polynucleotide encoding a mutant 5' untranslated region (5'UTR), comprising a sequence wherein one or more bases selected from the group consisting of bases at positions 3, 4, 5, 7, 8 and 10 of the nucleotide sequence corresponding to SEQ ID NO:1 are mutated.

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

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

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

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

6. The mutant polynucleotide of claim 1, wherein the mutant polynucleotide encoding the mutant 5'UTR regulates the translation of the polynucleotide encoding the target protein that is operatively linked to it.

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

8. The mutant polynucleotide of claim 2, wherein, compared with the polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase and further comprising a nucleotide sequence encoding the wild-type 5'UTR, the mutant polynucleotide comprising a nucleotide sequence encoding phosphoenolpyruvate carboxylase has its content of the 4-hydroxybutyrate (4HB) monomer of the polyhydroxyalkanoate produced therefrom adjusted to be in the range of 21.5% by weight to 46% by weight based on the total weight of PHA.

9. A microorganism for producing polyhydroxyalkanoates, the microorganism comprising: a mutant polynucleotide encoding a mutant 5'UTR, wherein one or more bases of the mutant polynucleotide encoding the mutant 5'UTR are mutated from the group consisting of bases at positions 3, 4, 5, 7, 8, and 10 of the nucleotide sequence corresponding to SEQ ID NO:1; or a mutant polynucleotide comprising a nucleotide sequence encoding a phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a nucleotide sequence encoding the mutant 5'UTR.

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

11. The microorganism according to claim 9, wherein the mutation is selected from any one or more of the following substitutions: i) Replace T with G; ii) Replace C with A; iii) Replace G with A; and iv) Replace G with C.

12. A method for producing polyhydroxyalkanoates, the method comprising the step of culturing microorganisms for producing polyhydroxyalkanoates in a culture medium, the microorganisms comprising: a mutant polynucleotide encoding a mutant 5'UTR, wherein one or more bases in the mutant 5'UTR are mutated from the group consisting of bases at positions 3, 4, 5, 7, 8, and 10 of the nucleotide sequence corresponding to SEQ ID NO:1; or a mutant polynucleotide comprising a nucleotide sequence encoding a phosphoenolpyruvate carboxylase, the mutant polynucleotide further comprising a nucleotide sequence encoding the mutant 5'UTR.

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

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

15. Use of a mutant polynucleotide in the production of polyhydroxyalkanoates, said mutant polynucleotide encoding a mutant 5'-untranslated region (5'UTR), wherein any one or more bases selected from the group consisting of bases at positions 3, 4, 5, 7, 8 and 10 of the nucleotide sequence corresponding to SEQ ID NO:1 are mutated.

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