Novel acetoacetyl-coa reductase variant and use thereof

Abstract

The present application relates to: a novel protein having acetoacetyl-CoA reductase activity; a polynucleotide encoding the protein; a microorganism comprising the protein or the polynucleotide encoding the protein; and a method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), the method comprising a step for culturing the microorganism in a medium.

Description

Novel acetoacetyl-COA reductase variants and uses thereof

[0001] The present application relates to a method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising: a protein having novel acetoacetyl-CoA reductase activity; a polynucleotide encoding the protein; a microorganism comprising the protein or the polynucleotide encoding the protein; and a step of culturing the microorganism in a medium.

[0002]

[0003] Polyhydroxyalkanoate (PHA), which has recently been attracting attention as a biodegradable polymer material, is a natural polymer with a polyester structure that microorganisms accumulate as a carbon source and energy storage material when nutrients such as nitrogen and phosphorus are deficient in the presence of an excess carbon source.

[0004] Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable, and biocompatible polyesters with diverse applications. They can be synthesized from a variety of microorganisms. Depending on the number of carbon atoms constituting the monomer, PHAs can be classified into short-chain PHAs (SCL PHAs) and medium-chain PHAs (MCL PHAs). Short-chain PHAs are composed of monomers with 3 to 5 carbon atoms, while medium-chain PHAs are composed of monomers with 6 to 14 carbon atoms.

[0005] Polyhydroxybutyrate (PHB), a homopolymer composed of 3-hydroxybutyrate (3HB), is one of the most widely studied PHAs, but its stiffness and brittleness limit its processability. Poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(3HB-co-3HH), PHBH), a copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (3HH), exhibits decreased crystallinity and melting temperature with increasing 3-hydroxyhexanoate content, which can improve the processability, ductility, and impact properties of PHB. Since the characteristics of PHBH vary depending on the ratio of monomers, it is important to develop a technology that can control the ratio of monomers.

[0006] It has been reported that PHBH can be produced through various routes. When butyrate is provided, it can be incorporated into PHBH in the form of 3HH through the steps of butyryl-CoA, 3-Oxohexanoyl-CoA, and 3HH-CoA. The intermediate metabolites 3HB-CoA and 3HH-CoA are produced by beta-ketothiolase (BktB) and acetoacetyl-CoA reductase, but there is a difference in the activity of converting each substrate depending on the substrate specificity of each enzyme.

[0007] Meanwhile, in the case of beta-ketothiolase (BktB) derived from Cupriavidus necator, it has been reported that a strain expressing a bktB (M158A) mutant in which the 158th methionine is substituted with an alanine produces PHBH with an increased 3HH monomer content compared to a strain expressing wild-type bktB (Bonk BM et al., Biotechnol. Bioeng., 2018). However, research is still needed on a method to produce the desired PHBH by controlling the 3HH monomer content.

[0008]

[0009] The present application was completed by confirming the production of poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content by a mutant protein having acetoacetyl-CoA reductase activity.

[0010]

[0011] The present application provides a protein having acetoacetyl-CoA reductase activity, wherein the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine, or the amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine.

[0012] The present application also provides a polynucleotide encoding the protein.

[0013] The present application provides a microorganism comprising the protein or a polynucleotide encoding the protein.

[0014] The present application provides a method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), which comprises a step of culturing the above microorganism in a medium.

[0015]

[0016] When culturing a microorganism containing a protein having a modified acetoacetyl-CoA reductase activity of the present application, production of poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with an increased 3-hydroxyhexanoate (3HH) content is possible compared to a microorganism having an existing unmodified polypeptide.

[0017]

[0018] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied 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 scope of this application is not limited by the specific descriptions described below. Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which this application belongs and the contents of this application.

[0019] One aspect of the present application provides a protein having acetoacetyl-CoA reductase activity, wherein the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine, or the amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine.

[0020] Specifically, the protein may be an amino acid sequence having a sequence identity of 45% or more and less than 100% with at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 22.

[0021] Specifically, the protein may be composed of an amino acid sequence of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO: 24, but is not limited thereto.

[0022] In the present application, “a protein having acetoacetyl-CoA reductase activity in which an amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine” or “a protein having acetoacetyl-CoA reductase activity in which an amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine” may refer to an acetoacetyl-CoA reductase variant comprising one or more amino acid substitutions in the amino acid sequence of a parent acetoacetyl-CoA reductase having acetoacetyl-CoA reductase activity, and may be used interchangeably with terms such as “acetoacetyl-CoA reductase variant,” “variant,” or “variant polypeptide.”

[0023]

[0024] In this application, the term "acetoacetyl-CoA reductase" means an enzyme that converts acetoacetyl-CoA into 3-hydroxybutyryl-CoA.

[0025] In the present application, the term "parent acetoacetyl-CoA reductase" refers to an acetoacetyl-CoA reductase that is modified to produce an acetoacetyl-CoA reductase variant, variant or mutant polypeptide of the present application. Specifically, the parent acetoacetyl-CoA reductase or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, may include a variant or functional fragment thereof, but is not limited thereto as long as it is a polypeptide that has acetoacetyl-CoA reductase activity and can be a parent of a variant.

[0026] In the present application, the parent acetoacetyl-CoA reductase may be a protein having acetoacetyl-CoA reductase activity encoded by the phaB gene, but is not particularly limited in type as long as it has an activity corresponding to acetoacetyl-CoA reductase and can produce polyhydroxyalkanoate. Specifically, the parent acetoacetyl-CoA reductase protein may include, for example, an amino acid sequence of any one of SEQ ID NO: 12 and SEQ ID NO: 22 or an amino acid sequence having 45% or more homology or identity therewith, but is not limited thereto as long as it has acetoacetyl-CoA reductase activity. Specifically, the amino acid sequence may be an amino acid sequence of any one of SEQ ID NO: 12 and SEQ ID NO: 22; Or it may include an amino acid sequence having at least 45%, 50%, 55%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 22. In addition, it is obvious that an auxiliary protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the protein. The amino acid sequence of any one of the SEQ ID NO: 12 and SEQ ID NO: 22 can be obtained from a known database such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0027] Specifically, the amino acid of the above sequence number 22 has a sequence identity of 45% or more and less than 100% with the amino acid sequence of the above sequence number 12. In the present application, the acetoacetyl-CoA reductase is selected from the group consisting of Cupriavidus necator, Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16, Bacillus megaterium, Pseudomonas putida (P. putida), Alcaligenes latus (Azohydromonas lata, Azohydromonas australica), Allochromatium vinosum DSM 180, Azotobacter Beijerinckii, Pandoraeasp. B-6, Burkholderiaceae bacterium16, or Candidatus accumulibacter. It may be an acetoacetyl-CoA reductase derived from Candidatus Accumulibacter phosphatis, specifically, but not limited to, an acetoacetyl-CoA reductase derived from Cupriavidus necator, Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16, or Bacillus megaterium.

[0028] The acetoacetyl-CoA reductase derived from Cupriavidus necator (Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16) presented as an example in the present application may be a polypeptide / protein comprising an amino acid sequence set forth in SEQ ID NO: 12, but is not limited thereto.

[0029] The acetoacetyl-CoA reductase derived from Bacillus megaterium presented as an example in the present application may be a polypeptide / protein comprising an amino acid sequence set forth in SEQ ID NO: 22, but is not limited thereto.

[0030]

[0031] In addition, the polynucleotide sequence encoding the above-mentioned parent acetoacetyl-CoA reductase may be a polynucleotide sequence encoding a protein capable of producing PHA in a microorganism by exhibiting the activity of acetoacetyl-CoA reductase. For example, it may be a polynucleotide sequence encoding an acetoacetyl-CoA reductase (SEQ ID NO: 12) derived from Cupriavidus necator, Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophusH16, or an acetoacetyl-CoA reductase (SEQ ID NO: 22) derived from Bacillus megaterium, but is not limited thereto. For example, any one of the base sequences of SEQ ID NO: 13 and SEQ ID NO: 23; Or it may be encoded by a polynucleotide having or including a base sequence having at least 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of the base sequences of SEQ ID NO: 13 and SEQ ID NO: 23, or consisting of or consisting essentially of such a base sequence, but is not limited thereto. The base sequence may be modified in the coding region due to the degeneracy of the codon, and various modifications may be made in the coding region within a range that does not change the amino acid sequence, taking into consideration the codon preferred in the organism that is to express the base sequence. In addition, it is obvious that a polynucleotide sequence encoding a protein having such homology or identity and exhibiting substantially the same or corresponding effect as the protein is also included within the scope of the present application, even if a polynucleotide sequence in which some sequences are deleted, modified, substituted or added is included.

[0032]

[0033] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Furthermore, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and / or C-terminus of the mature protein. The above term “variant” may be used interchangeably with terms such as variant, modification, variant polypeptide, variant protein, variant and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of variant.

[0034] Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in co-translational or post-translational protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0035]

[0036] As used herein, the term “acetoacetyl-CoA reductase variant,” “variant,” or “variant polypeptide” refers to a protein having one or more amino acids that differ from the amino acid sequence of a parent acetoacetyl-CoA reductase and having acetoacetyl-CoA reductase activity.

[0037] Specifically, the acetoacetyl-CoA reductase variant of the present application may be an acetoacetyl-CoA reductase variant in which the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine, or in which the amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine, but is not limited thereto.

[0038]

[0039] Even if the present application describes a “protein having an amino acid sequence described by a specific sequence number,” it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the present application, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the mutant protein, it does not exclude sequence additions that do not alter the function of the protein before or after the amino acid sequence, mutations that may occur naturally, silent mutations thereof, or conservative substitutions, and it is obvious that even if it has such sequence additions or mutations, it falls within the scope of the present application.

[0040] The "N-position" of the present application may include the N-position and an amino acid position corresponding to the N-position. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be any one of SEQ ID NO: 12 and SEQ ID NO: 22.

[0041] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid 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 protein.

[0042] For example, any amino acid sequence can be aligned with SEQ ID NO: 12, and each amino acid residue of the amino acid sequence can be numbered based on the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 12. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0043] 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 package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0044] In the present application, when the amino acid sequence of SEQ ID NO: 22 is aligned with the amino acid sequence of SEQ ID NO: 12, the amino acid corresponding to the 144th position of the amino acid sequence of SEQ ID NO: 22 corresponds to the 141st position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 12.

[0045] That is, a person skilled in the art can align the amino acid sequence of SEQ ID NO: 22 with the amino acid sequence of SEQ ID NO: 12 through sequence alignment known in the art, and based on this, when each amino acid residue of the amino acid sequence is numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 12, it can be seen that the amino acid corresponding to the 141st position is substituted with alanine.

[0046]

[0047] In one embodiment, the acetoacetyl-CoA reductase variant of the present application may be, but is not limited to, a polypeptide in which valine, the amino acid corresponding to position 141 of SEQ ID NO: 12, is substituted with alanine.

[0048] In one embodiment, the acetoacetyl-CoA reductase variant of the present application may be, but is not limited to, a polypeptide in which isoleucine, an amino acid corresponding to position 144 of SEQ ID NO: 22, is substituted with alanine.

[0049]

[0050] Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 141st position of the amino acid sequence of SEQ ID NO. 12 of the present application or the 144th position of the amino acid sequence of SEQ ID NO. 22 in any amino acid sequence through sequence alignment known in the art, and even if not described separately in the present application, when "an amino acid at a specific position in a specific sequence number" is described, it is self-evident that it includes "an amino acid at a corresponding position" in any amino acid sequence.

[0051] In one embodiment, a protein (or acetoacetyl-CoA reductase variant) having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 141st position from the N-terminus of the amino acid sequence of SEQ ID NO: 12 of the present application or the 144th position from the N-terminus of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine may have the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 24, but is not limited thereto.

[0052]

[0053] For example, the variant of the present application may comprise or consist essentially of an amino acid sequence of any one of SEQ ID NO: 16 and SEQ ID NO: 24. Alternatively, the variant may comprise an amino acid sequence having at least 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with an amino acid sequence selected from the group consisting of the amino acid sequences of any one of SEQ ID NO: 16 and SEQ ID NO: 24.

[0054] For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present application at the N-terminus, C-terminus and / or within the amino acid sequence.

[0055] The term "conservative substitution" in this application refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Additionally, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Charged side chain amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged side chain amino acids can be further classified into nonpolar amino acids or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0056] In one embodiment, the acetoacetyl-CoA reductase variant of the present application may have an activity with expanded substrate specificity such that it can produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content compared to the wild type or unmodified polypeptide, but is not limited thereto.

[0057]

[0058] Another aspect of the present application provides a polynucleotide encoding a variant of the present application.

[0059] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0060] The polynucleotide encoding the acetoacetyl-CoA reductase variant of the present application may include, without limitation, any polynucleotide sequence encoding the acetoacetyl-CoA reductase variant of the present application. For example, the polynucleotide encoding the acetoacetyl-CoA reductase variant of the present application may be, but is not limited to, a polynucleotide sequence encoding the amino acid sequence of the acetoacetyl-CoA reductase variant of the present application.

[0061] For example, a polynucleotide encoding an acetoacetyl-CoA reductase variant of the present application may include, but is not limited to, a base sequence having at least 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the base sequence of either SEQ ID NO: 17 or SEQ ID NO: 25.

[0062] In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application. The term “stringent conditions” refers to conditions that enable 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; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 45% or more, 50% or more, 55% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions under which washing conditions of typical southern hybridization are performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0063] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0064] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0065] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0066] As used herein, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0067] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0068] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85: 2444]. Alternatively, 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) can be determined using 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 ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0069] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 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 terminal gaps. Accordingly, the term "homology" or "identity" as used herein refers to the relevance between sequences.

[0070]

[0071] Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0072] The term “vector” in this application may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0073] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0074] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0075] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism, so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can be located either within the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

[0076] Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0077]

[0078] Another aspect of the present application provides a microorganism comprising the protein or a polynucleotide encoding the protein.

[0079] Specifically, a microorganism is provided, comprising a protein having acetoacetyl-CoA reductase activity, wherein the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine, or an amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine, or a polynucleotide encoding the protein.

[0080] At this time, “a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine or the amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine” and “a polynucleotide encoding the protein” are as described above.

[0081] In one embodiment, the protein may be an amino acid sequence having a sequence identity of at least 45% and less than 100% with any one or more amino acid sequences selected from the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 22.

[0082] Specifically, the protein may be composed of an amino acid sequence of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO: 24.

[0083] In one embodiment, the microorganism of the present application may be a microorganism having the ability to produce polyhydroxyalkanoates (PHA), and specifically, may be a microorganism having the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)).

[0084] In this application, the term "polyhydroxyalkanoates (PHA)" refers to a type of biodegradable bioplastic synthesized by various microorganisms.

[0085] In the present application, the term “poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH), PHBH)” is one of polyhydroxyalkanoates and refers to a copolymer in which 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HH) are combined. Here, depending on the monomer ratio of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HH) included in poly-3-hydroxybutyrate-3-hydroxyhexanoate (PHBH), the processability, ductility, impact properties, etc. of PHBH may vary, but are not limited thereto. PHBH may include 3-hydroxyhexanoate (3HH) formed from butyrate through the steps of Butyryl-CoA, 3-Oxohexanoyl-CoA, and 3HH-CoA.

[0086]

[0087] In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism that has a specific mechanism strengthened or weakened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, "microorganism" and "strain" may be used interchangeably without limitation with the same meaning.

[0088] In the present application, the term "microorganism producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH))" may include a prokaryotic or eukaryotic microbial strain capable of producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) within a living organism, a microorganism in which the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) is conferred on a parent strain that does not have the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), or a microorganism that inherently has the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)). The ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) can be conferred or enhanced by species improvement.

[0089] In one embodiment, the microorganism of the present application may be a microorganism that naturally has an acetoacetyl-CoA reductase variant or poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) production ability; or a microorganism that is introduced into a parent strain that does not have an acetoacetyl-CoA reductase variant or poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) production ability by introducing the variant of the present application or a polynucleotide encoding the same (or a vector including the polynucleotide) and / or is endowed with the poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) production ability, but is not limited thereto.

[0090] In one embodiment, the microorganism of the present application includes, but is not limited to, a microorganism in which a chromosomal gene encoding an acetoacetyl-CoA reductase variant is mutated and includes the sequence of the acetoacetyl-CoA reductase variant of the present application, and / or a microorganism in which a vector including a polynucleotide encoding the acetoacetyl-CoA reductase variant of the present application is introduced, thereby including the acetoacetyl-CoA reductase variant of the present application.

[0091] In this application, the term "unmodified microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain that is not introduced or before the acetoacetyl-CoA reductase variant described herein is introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain", "pre-modified microorganism", "unmutated strain", "unmodified microorganism", "unmutated microorganism", or "reference microorganism".

[0092]

[0093] The microorganism having the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may be, but is not limited to, a microorganism comprising at least one of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a microorganism modified to express the variant of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) expressing the variant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) having the activity of the variant of the present application.

[0094] For example, the strain of the present application is a cell or microorganism that is transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a variant of the present application, and expresses the variant of the present application, and the strain of the present application may include all microorganisms capable of producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), including the variant of the present application.

[0095] For example, the microorganism of the present application may be a recombinant strain that produces poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with an increased 3-hydroxyhexanoate (3HH) content by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism having the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), thereby expressing an acetoacetyl-CoA reductase variant. The recombinant strain producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content may be a microorganism that produces poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content compared to a natural wild-type microorganism or an acetoacetyl-CoA reductase-unmodified microorganism (e.g., a microorganism expressing wild-type acetoacetyl-CoA reductase or a microorganism that does not express the variant of the present application), but is not limited thereto. For example, the microorganism producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may be, but is not limited to, a microorganism producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) having an increased 3-hydroxyhexanoate (3HH) content compared to a microorganism comprising the polypeptide of any one of SEQ ID NO: 12 and SEQ ID NO: 22 or a polynucleotide encoding the same.

[0096] The microorganism of the present application may include any microorganism capable of expressing the acetoacetyl-CoA reductase variant of the present application by various known methods in addition to the introduction of the nucleic acid or vector.

[0097] For example, a microorganism producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content has a 3-hydroxyhexanoate (3HH) content of about 1% or more, about 5% or more, about 10% or more, about 50% or more, about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, or about 190% or more, compared to poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) produced by a parent strain or a non-transformed microorganism before mutation. It may be increased by about 200% or more, about 210% or more, about 220% or more, about 230% or more, about 240% or more, about 250% or more, about 260% or more, about 270% or more, about 280% or more, about 290% or more, about 300% or more, about 310% or more, about 320% or more, about 330% or more, about 340% or more, about 350% or more, about 360% or more, about 370% or more, about 380% or more, about 390% or more, or about 400% or more (the upper limit is not particularly limited and may be, for example, about 1000% or less, about 900% or less, about 800% or less, about 700% or less, about 600% or less, about 500% or less, or about 450% or less). As long as it has a positive increase compared to the production ability of the parent strain or the unmodified microorganism before mutation, it is not limited thereto. In another example, the recombinant strain producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) with increased 3-hydroxyhexanoate (3HH) content has a 3-hydroxyhexanoate (3HH) content of about 1.01 times or more, about 1.05 times or more, about 1.1 times or more, or about 1.It may be increased by, but is not limited to, 5 times or more, about 2 times or more, about 2.1 times or more, about 2.2 times or more, about 2.3 times or more, about 2.4 times or more, about 2.5 times or more, about 2.6 times or more, about 2.7 times or more, about 2.8 times or more, about 2.9 times or more, about 3 times or more, about 3.1 times or more, about 3.2 times or more, about 3.3 times or more, about 3.4 times or more, about 3.5 times or more, about 3.6 times or more, about 3.7 times or more, about 3.8 times or more, about 3.9 times or more, or about 4 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 9 times or less, about 8 times or less, about 7 times or less, about 6 times or less, about 5 times or less, or about 4.5 times or less). The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term “about,” but is not limited thereto.

[0098]

[0099] The microorganism of the present application may be a microorganism belonging to the genus Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacteria sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp., and Norcardia sp., or a microorganism belonging to the genus fungi or yeast, specifically a microorganism of the genus Escherichia, more specifically Escherichia coli (E. coli), but is not limited thereto.

[0100] Meanwhile, the Escherichia microorganism producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application includes a natural wild-type microorganism itself, an Escherichia microorganism in which the activity of a gene related to the poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) production mechanism is strengthened or weakened to increase the content of 3-hydroxyhexanoate (3HH) contained in poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), or an Escherichia microorganism in which the activity of an external gene is introduced or strengthened to have an improved content of 3-hydroxyhexanoate (3HH).

[0101]

[0102] In this application, the term "introduction" refers to a method of delivering a polynucleotide encoding the acetoacetyl-CoA reductase variant or a vector containing the same to a host cell. Such introduction can be easily performed according to a method commonly known in the art. Common methods include the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 method, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, transformation using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. The method for transforming the vector is not limited to the above examples, and any transformation or transfection method commonly used in the art can be used without limitation. In addition, the delivered polynucleotide can be integrated and located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. In addition, the polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide structure that includes all elements necessary for self-expression, but is not limited thereto. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the open reading frame (hereinafter abbreviated as "ORF") of the gene. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not particularly limited thereto.

[0103]

[0104] For example, the microorganism of the present application may be a microorganism having an ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) or a microorganism having an increased ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate, by additionally enhancing the activity of propionate CoA-transferase (Pct), beta-ketothiolase (BktB), PHA synthase (PhaC), or a combination thereof compared to the intrinsic activity, but is not limited thereto.

[0105] For example, the microorganism of the present application may be a microorganism that has the ability to produce poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) by additionally introducing any one of a polynucleotide encoding propionate CoA-transferase or a vector including the same; a polynucleotide encoding beta-ketothiolase or a vector including the same; a polynucleotide encoding PHA synthase or a vector including the same; or a combination thereof, but is not limited thereto.

[0106] The gene encoding the propionate CoA-transferase may be pct derived from Clostridium propionicum, but may be included without limitation if it is capable of expressing a protein having propionate CoA-transferase activity. The pct gene encoding the propionate CoA-transferase may have its sequence obtained from a known database such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the pct gene encoding the propionate CoA-transferase (SEQ ID NO: 26) derived from Clostridium propionicum may include, but is not limited to, the base sequence of SEQ ID NO: 27.

[0107] The gene encoding the above beta-ketothiolase may be bktB derived from Cupriavidus necator, but may be included without limitation if it is capable of expressing a protein having beta-ketothiolase activity. The sequence of the bktB gene encoding the above beta-ketothiolase can be obtained from a known database such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0108] The gene encoding the above PHA synthase may be phaC derived from Aeromonas caviae, but may be included without limitation if it is capable of expressing a protein having PHA synthase activity. The sequence of the phaC gene encoding the above PHA synthase can be obtained from a known database such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the phaC gene encoding the PHA synthase (SEQ ID NO: 30) derived from Aeromonas caviae may include, but is not limited to, the base sequence of SEQ ID NO: 31.

[0109]

[0110] Another aspect of the present application provides a method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising the step of culturing the microorganism of the present application in a medium.

[0111] Specifically, the method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may include, but is not limited to, a step of culturing a microorganism including the protein of the present application or a polynucleotide encoding the protein in a medium.

[0112] In this application, the term "cultivation" refers to growing the microorganism of this application under appropriately controlled environmental conditions. The culturing process of this application can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0113] In this application, the term "medium" refers to a material containing nutrients as a main component necessary for culturing the microorganism of this application, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of this application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0114] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; alcohols such as mannitol, sorbitol, glycerol, etc.; fats and oils or fractionated fats thereof such as palm oil or palm kernel oil (including palm olein, palm double olein, palm kernel oil olein, etc.), corn oil, palm oil, olive oil, soybean oil, rapeseed oil, etc.; fatty acids such as palmitic acid, stearic acid, linolenic acid, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugarcane bagasse, and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and other appropriate carbon sources can be used in a variety of ways without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0115] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0116] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0117] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection, but is not limited thereto.

[0118] 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.

[0119] Poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) produced by the culture of the present invention may be secreted into the medium or remain within the cell. In this case, the poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) produced may have an increased content of -hydroxyhexanoate (3HH) compared to a microorganism containing a protein having unmodified acetoacetyl-CoA reductase activity or a polynucleotide encoding the same.

[0120]

[0121] The method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may additionally include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0122] The method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may further include a step of recovering a target substance from the cultured microorganism, a culture of the microorganism, or the culture medium.

[0123] The above recovery step may be additionally included after the above culturing step.

[0124] The above recovery may be performed by collecting poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallization protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) can be recovered from a medium or microorganism using a suitable method known in the art.

[0125]

[0126] In addition, the method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0127]

[0128] Another aspect of the present application provides a composition for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising the protein; a polynucleotide encoding the protein; a microorganism comprising the protein or the polynucleotide encoding the protein; a culture of the microorganism; or a combination of two or more thereof.

[0129] The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing PHA, such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

[0130] In one specific example, each component present in the composition of the present application may be included in a microbiologically effective amount, or an amount that can be suitably present in the composition for production.

[0131]

[0132] Another aspect of the present application provides a use for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising the protein; a polynucleotide encoding the protein; and at least one microorganism comprising the protein or the polynucleotide encoding the protein.

[0133]

[0134] The present application will be described in more detail below through examples. However, the following examples are merely preferred embodiments intended to illustrate the present application and are therefore not intended to limit the scope of the present application. Furthermore, technical details not described herein can be readily understood and implemented by those skilled in the technical field of the present application or similar fields.

[0135]

[0136] Example 1: Production of a strain expressing an acetoacetyl-CoA reductase (PhaB) mutant and confirmation of PHBH production.

[0137]

[0138] Example 1-1: Construction of plasmid for production of PHBH producing strain

[0139]

[0140] To produce PHBH-producing Escherichia coli, a plasmid containing bktB and phaC and a plasmid containing pct and phaB were constructed.

[0141] Specifically, pACYC-PcysK_phaC-PsynB_bktB(M158A) containing PsynB (J23100; The iGEM Parts Registry) and PcysK promoter was constructed to enable expression of bktB(M158A) (SEQ ID NO: 28) from Cupriavidus necator and PHA synthase phaC (SEQ ID NO: 30) from Aeromonas caviae in Escherichia coli using pACYC177 (GenBank No. X06402). Each gene was obtained through gene synthesis and then amplified by PCR.

[0142] PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, repeated 28 times. The pACYC177 plasmid was treated with BamHI restriction enzyme. The inserted DNA fragment amplified through PCR and the restriction enzyme-treated plasmid were purified using LaboPass™PCR Purification Kit, ligated using Infusion Cloning Kit, and then transformed into E. coli DH5α. Colonies were selected on LB medium containing 50 mg / L of kanamycin antibiotic, and the sequence of the plasmid obtained through this was finally confirmed through sequencing.

[0143] The second plasmid, pCL-PsynB_pct-PuspA_phaB, was constructed using pCL1920 (GenBank No. AB236930) to contain the PsynB (J23100; The iGEM Parts Registry) and PuspA promoters so that the Clostridium propionicum-derived gene pct (propionate CoA-transferase) and Cupriavidus necatorphaB can be expressed in Escherichia coli. Each gene was obtained through gene synthesis and amplified by PCR. The PCR conditions were 28 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The pCL1920 plasmid was treated with EcoRI restriction enzyme. The inserted DNA fragment amplified by PCR and the plasmid treated with restriction enzymes were purified using the LaboPass™PCR Purification Kit, respectively, and ligated using the Infusion Cloning Kit, and then transformed into E. coli DH5α. Colonies were selected on LB medium containing 75 mg / L of spectinomycin antibiotic, and the sequence of the plasmid obtained through this was finally confirmed through sequencing. Finally, E. coliDH5α / pACYC-PcysK_phaC-PsynB_bktB(M158A) / pCL-PsynB_pct-PuspA_phaB strain (strain no. 1) was constructed, which expressed bktB (M158A) from Cupriavidus necator and PHA synthase phaC from Aeromonas caviae, as well as the pct and Cupriavidus necatorphaB genes from Clostridium propionicum.

[0144]

[0145] Example 1-2: Construction of a plasmid for constructing a PHBH-producing strain expressing an acetoacetyl-CoA reductase (PhaB) mutant.

[0146]

[0147] A vector was constructed to express acetoacetyl-CoA reductase (PhaB) mutants in a strain. Specifically, PCR was performed under the same conditions as Example 1-1 using SEQ ID NOs: 1, 2, 3, and 4 to introduce the S140A mutation in phaB, SEQ ID NOs: 1, 2, 4, and 5 to introduce the V141A mutation, and SEQ ID NOs: 1, 2, 4, and 6 to introduce the N142V mutation. Primer sequence information is shown in Table 1.

[0148] Primer base sequence SEQ ID NO: 15'-AGAAACGCTAGTTGGTTAATTAATCTAGAAGGAGGTTTTTACTATGACGCAAAGAATTGCATACGTTAC-3' SEQ ID NO: 25'-GCTAATGTTAACAATGCGAC'-3' SEQ ID NO: 35'-CGCATTGTTAACATTAGCgagAACGGCCAAAAGGGC-3' SEQ ID NO: 45'-GACCATGATTACGCCAAGCTTTCAACCCATGTGCAGGCCACCGTTC-3' SEQ ID NO: 55'-CGCATTGTTAACATTAGCTCAgcgAACGGCCAAAAGGGCCA-3' SEQ ID NO: 65'-CGCATTGTTAACATTAGCTCAGTGgtgGGCCAAAAGGGCCAGTTT-3'

[0149]

[0150] The plasmids expressing pct and each phaB mutation obtained as a result of cloning were named pCL-PsynB_pct-PuspA_phaB(S140A), pCL-PsynB_pct-PuspA_phaB(V141A), and pCL-PsynB_pct-PuspA_phaB(N142V), respectively.

[0151]

[0152] Example 1-3. Construction of a strain expressing an acetoacetyl-CoA reductase mutant and confirmation of PHBH production.

[0153]

[0154] In order to confirm the change in 3HH content in PHBH according to the expression of acetoacetyl-CoA reductase (PhaB) mutants, E. coli into which the wild-type phaB gene was introduced and E. coli into which the phaB mutants were introduced were constructed. Specifically, wild-type E. coli was transformed with pACYC-PcysK_phaC-PsynB_bktB (M158A) constructed in Example 1-1 and each phaB mutant expression plasmid constructed in Example 1-2 by the TSS method, and selection was performed on LB medium containing two kinds of antibiotics, Kanamycin (50 mg / L) and Spectinomycin (75 mg / L). The strains produced in this way are E. coliDH5α / pACYC-PcysK_phaC-PsynB_bktB(M158A) / pCL-PsynB_pct-PuspA_phaB mutant strains (strain numbers 2, 3, and 4) that express bktB (M158A) derived from Cupriavidus necator and PHA synthase phaC derived from Aeromonas caviae, and the pct gene derived from Clostridium propionicum, Cupriavidus necatorphaB mutant.

[0155] Next, the P(3HB-co-3HH) production ability test was performed on the above-mentioned strain. 7 mL of PHBH production medium containing 10 g / L glucose and 4 g / L butyric acid was dispensed into 50 mL bioreactor tubes (SPL, Korea), and each strain was inoculated. The tubes were then cultured with shaking at 200 rpm for 5 hours at 37°C and 25 hours at 30°C. The PHBH concentration was then analyzed by GC (gas chromatography). The results are shown in Table 2 below.

[0156]

[0157] Strain NumberPlasmid 1Plasmid 23HB (mg)3HH (mg)3HHContent (%)1pACYC-PcysK_phaC-PsynB_bktB(M158A)pCL-PsynB_pct-PuspA_phaB1.450.38212pCL-PsynB_pct-PuspA_phaB( S140A)0.000.0003pCL-PsynB_pct-PuspA_phaB(V141A)0.150.59794pCL-PsynB_pct-PuspA_phaB(N142V)0.370.038

[0158]

[0159] As a result, it was confirmed that in the strain expressing the acetoacetyl-CoA reductase V141A mutant, 3HB monomers decreased and 3HH monomers increased compared to the wild-type phaB expression strain, and overall, the 3HH content increased by approximately 3.8 times. This confirms that the acetoacetyl-CoA reductase V141A mutant can increase the 3HH monomer ratio.

[0160]

[0161] Example 2. Confirmation of PHBH production ability according to the substituted residue of acetoacetyl-CoA reductase mutant (V141).

[0162]

[0163] In the above Example 1, it was confirmed that the 141st position of acetoacetyl-CoA reductase is an important position for introduction of 3HH monomer, and thus an E. coli strain expressing a mutant protein in which the 141st amino acid of acetoacetyl-CoA reductase is substituted with glycine was produced and confirmed.

[0164] Specifically, PCR was performed using sequence numbers 1, 2, 4, and 7 to introduce the V141G mutation of phaB in the same manner as in Example 1-2. Primer sequence information is shown in Table 3.

[0165]

[0166] Primer base sequence SEQ ID NO: 75'-CGCATTGTTAACATTAGCTCAggcAACGGCCAAAAGGGCCA-3'

[0167]

[0168] The plasmid expressing the pct and phaB mutants obtained as a result of cloning was named pCL-PsynB_pct-PuspA_phaB(V141G). In order to confirm the change in 3HH content in PHBH according to the expression of the acetoacetyl-CoA reductase (PhaB) mutant, E. coli introduced with the phaB mutant was constructed using the same method as in Example 1-3, and a P(3HB-co-3HH) production ability test was performed. As a result, it was found that PHBH was not produced in the strain introduced with the phaB(V141G) mutant. This suggests that the 3HH content can be increased only when amino acid 141 is substituted with alanine.

[0169]

[0170] Example 3. Production of strains expressing acetoacetyl-CoA reductase mutants derived from other alien species and confirmation of PHBH production ability.

[0171]

[0172] In addition to the phaB gene derived from Cupriavidus necator confirmed in Example 1 above, we also wanted to determine whether a mutation in which the amino acid at the position corresponding to the 141st position of phaB derived from Cupriavidus necator is substituted with alanine is effective for phaB genes derived from other alien species. Specifically, the evaluation was conducted on phaB derived from Bacillus megaterium, and in the case of phaB derived from Bacillus megaterium, the 144th amino acid (isoleucine) corresponds to the 141st amino acid position of phaB derived from Cupriavidus necator.

[0173]

[0174] Example 3-1. Construction of a plasmid expressing acetoacetyl-CoA reductase derived from Bacillus megaterium

[0175]

[0176] Plasmids expressing wild-type phaB and I144A mutant phaB derived from B. megaterium were constructed using the same method as in Examples 1-1 and 1-2. Specifically, pCL1920 (GenBank No. AB236930) was used to construct pCL-PsynB_pct-PuspA_Bme_phaB, which includes the C. propionicum-derived gene pct (propionate CoA-transferase), PsynB (J23100; The iGEM Parts Registry), and PuspA promoter so that B. megaterium phaB can be expressed in E. coli.

[0177] Introduction of the I144A mutation into B. megateriumphaB was performed by PCR using sequence numbers 8, 9 and 10, 11 in the same manner as in Example 1-2. The corresponding primer sequence information is shown in Table 4.

[0178]

[0179] Primer base sequence SEQ ID NO: 85'-AGAAACGCTAGTTGGTTAATTAATCTAGAAGGAGGTTTTTACTATGACAACATTACAAGGTAAAGTAG-3' SEQ ID NO: 95'-TGATGAAATATTGATAACACGACC-3' SEQ ID NO: 105'-GGTCGTGTTATCAATATTTCATCAgcgATTGGTCAAGCGGGCGGATTTG-3' SEQ ID NO: 115'-GACCATGATTACGCCAAGCTTTTACATGTATAAGCCGCCGTTAATG-3'

[0180]

[0181] The plasmids expressing pct and each phaB mutation obtained as a result of cloning were named pCL-PsynB_pct-PuspA_Bme_phaB and pCL-PsynB_pct-PuspA_Bme_phaB(I144A), respectively.

[0182]

[0183] Example 3-2. Production of a strain expressing an acetoacetyl-CoA reductase mutant and confirmation of PHBH production.

[0184]

[0185] In the same manner as in Example 1-3, an E. coli / pACYC-PcysK_phaC-PsynB_bktB(M158A) / pCL-PsynB_pct-PuspA_Bme_phaB strain (strain no. 5) into which the wild-type phaB gene was introduced and an E. coli / pACYC-PcysK_phaC-PsynB_bktB(M158A) / pCL-PsynB_pct-PuspA_Bme_phaB(I144A) strain (strain no. 6) into which the phaB mutant was introduced were constructed. Specifically, wild-type E. E. coli was transformed with pACYC-PcysK_phaC-PsynB_bktB(M158A) constructed in Example 1-1 and the wild-type phaB expression plasmid pCL-PsynB_pct-PuspA_Bme_phaB or the phaB mutant expression plasmid pCL-PsynB_pct-PuspA_Bme_phaB(I144A) constructed in Example 3-1 by the TSS method, and selection was performed on LB medium containing two types of antibiotics, Kanamycin (50 mg / L) and Spectinomycin (75 mg / L). Subsequently, the P(3HB-co-3HH) production ability test was performed on the constructed strain. 7 mL of PHBH production medium containing 10 g / L glucose and 4 g / L butyric acid was dispensed into 50 mL bioreactor tubes (SPL, Korea), and each strain was inoculated. The tubes were then cultured with shaking at 200 rpm for 5 h at 37°C and 25 h at 33°C. The PHBH concentration was then analyzed by GC (gas chromatography). The results are shown in Table 5 below.

[0186]

[0187] Strain numberPlasmid 1Plasmid 23HB (mg)3HH (mg)3HH content (%)5pACYC-PcysK_phaC-PsynB_bktB(M158A)pCL-PsynB_pct-PuspA_Bme_phaB0.750.29286pCL-PsynB_pct-PuspA_Bme_phaB(I144A)0.090.1866

[0188]

[0189] As a result, it was confirmed that the 3HH ratio in PHBH increased approximately 2.4-fold from 28% to 66% in the strain introduced with the mutant (I144A) in which the 144th amino acid of phaB from B. megaterium was substituted with alanine compared to the strain introduced with the wild-type phaB. However, the overall PHBH production decreased in the strain introduced with the I144A mutant compared to the wild-type. In summary, this means that the amino acid at the position corresponding to the 141st amino acid of phaB from C. necator is an important position for the 3HH content in PHBH.

[0190] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A protein having acetoacetyl-CoA reductase activity, wherein the amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 12 is substituted with alanine, or the amino acid corresponding to position 144 of the amino acid sequence of SEQ ID NO: 22 is substituted with alanine.

2. In the first paragraph, the protein is an amino acid sequence having a sequence identity of 45% or more and less than 100% with at least one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO:

22.

3. In the first paragraph, the protein is composed of an amino acid sequence of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO:

24.

4. A polynucleotide encoding a protein of any one of claims 1 to 3.

5. A microorganism comprising a protein according to any one of claims 1 to 3 or a polynucleotide encoding the protein.

6. In the fifth paragraph, the microorganism produces poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)) having an increased 3-hydroxyhexanoate (3HH) content compared to a microorganism comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 12 or 22 or a polynucleotide encoding the same.

7. In the fifth paragraph, the microorganism is a microorganism of the genus Escherichia.

8. In the 7th paragraph, the microorganism of the genus Escherichia is Escherichia coli.

9. A method for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising a step of culturing the microorganism of clause 5 in a medium.

10. A method according to claim 9, further comprising a step of recovering a target substance from the cultured microorganism, a culture of the microorganism, or the culture medium.

11. Use for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising any one of the proteins of claims 1 to 3; a polynucleotide encoding the protein; and at least one microorganism comprising the protein or the polynucleotide encoding the protein.

12. A composition for producing poly-3-hydroxybutyrate-3-hydroxyhexanoate (P(3HB-co-3HH)), comprising the microorganism of paragraph 5; a culture of the microorganism; or a combination thereof.