Novel acetoacetyl-CoA reductase variants and their applications

Abstract

This application relates to a method for producing polyhydroxyalkanoate (PHA) comprising the steps of: a protein having novel acetoacetyl-CoA reductase activity; a polynucleotide encoding the protein; a microorganism containing the protein, the polynucleotide encoding the protein, or a vector containing the polynucleotide; and culturing the microorganism in a culture medium.

Description

Technical Field

[0001] This application relates to a protein having a novel acetoacetyl-CoA reductase activity; a polynucleotide encoding the protein; a microorganism containing the protein, the polynucleotide encoding the protein or a vector containing the polynucleotide; and a method for producing polyhydroxyalkanoate (PHA) including culturing the microorganism in a medium.

Background Art

[0002] In recent years, polyhydroxyalkanoate (PHA), which has been in the spotlight as a biodegradable polymer material, is a natural polymer material with a polyester-based structure that microorganisms accumulate as a carbon source and energy storage substance when nutrients such as nitrogen and phosphoric acid are lacking in the presence of an excessive carbon source.

[0003] P3HB (poly-3-hydroxybutyrate), a typically well-known polyhydroxyalkanoate, is a substance polymerized using 3HB (3-hydroxybutylate) monomers and has mechanical properties similar to those of polypropylene, a commercially available petroleum synthetic polymer. Since it is completely decomposed by microorganisms in nature, it has attracted attention as an environmentally friendly plastic raw material.

[0004] In relation to this, among polyhydroxyalkanoate-based polymers, the 3-hydroxybutyrate homopolymer (Poly(3-hydroxybutyrate), P(3HB)), which has been most widely studied by microorganisms, has a high degree of crystallinity, is brittle and rigid, and is decomposed near its melting point, so there are also limitations in its processability. In contrast, research is underway to produce PHA copolymers through blending with new materials or copolymerization with monomers.

[0005] Among these, the copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate (P(3HB-co-4HB)) exhibits soft properties depending on the 4-hydroxybutyrate content. In other words, P(3HB-co-4HB) exhibits a wide range of physical properties, from hard crystalline plastics to highly elastic rubber, depending on the monomer ratio, and is highly valued as a useful polymer material that can be applied to a variety of products.

[0006] Therefore, research is still needed to produce 3-hydroxybutyrate-4-hydroxybutyrate copolymers with various monomer proportions in order to expand the PHA portfolio. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453. [Non-Patent Document 2] Rice et al., 2000,Trends Genet.16:276-277 [Non-Patent Document 3] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. [Non-Patent Document 4] FMAsubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8 [Non-Patent Document 5] Pearson et al (1988) [Proc.Natl.Acad.Sci.USA 85]:2444 [Non-Patent Document 6] Devereux,J.,et al,Nucleic Acids Research 12:387(1984)

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Non-licensed literature 9

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[0008] This application relates to a protein variant having acetoacetyl-CoA reductase activity and its use for polyhydroxyalkanoate (PHA) production. [Means for solving the problem]

[0009] One object of this application is to provide a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0010] Another object of this application is to provide a polynucleotide encoding the protein.

[0011] Another object of this application is to provide a microorganism comprising the protein, a polynucleotide encoding the protein, or a vector comprising the polynucleotide.

[0012] Another object of this application is to provide a method for producing polyhydroxyalkanoate (PHA), comprising the step of culturing the microorganism in a culture medium.

[0013] Another object of the present application is to provide a composition for producing polyhydroxyalkanoate (PHA) comprising the protein; a polynucleotide encoding the protein; a microorganism comprising the protein, the polynucleotide encoding the protein or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

Advantages of the Invention

[0014] When culturing a microorganism containing the protein having acetoacetyl-CoA reductase activity of the present application, it is possible to produce polyhydroxyalkanoate (PHA) in a higher yield than a microorganism having an existing non-modified polypeptide. In addition, the variant of the present application is used for producing a 3-hydroxybutyrate-4-hydroxybutyrate copolymer having a high 3-hydroxybutyrate content.

Modes for Carrying Out the Invention

[0015] Specifically, it is as follows. On the one hand, each of the explanations and embodiments disclosed in the present application is also applicable to each other explanation and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, the category of the present application is not considered to be limited by the specific descriptions described below. Also, numerous papers and patent documents are referred to throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which the present application belongs and the content of the present application.

[0016] One aspect of the present application provides a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0017] Specifically, the protein may have an amino acid sequence that has 55% or more but less than 100% sequence identity with SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1.

[0018] Specifically, the protein is such that the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs: 54 to 56 is replaced by another amino acid, The amino acid corresponding to the 34th position in the amino acid sequence of SEQ ID NO: 52 is substituted with another amino acid, or The amino acid corresponding to the 36th position in the amino acid sequence of sequence number 53 may be substituted with another amino acid, but is not limited to this.

[0019] In this application, "a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid," "a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs. 54 to 56 is substituted with another amino acid," "a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 34th position of the amino acid sequence of SEQ ID NO: 52 is substituted with another amino acid," or "a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 36th position of the amino acid sequence of SEQ ID NO: 53 is substituted with another amino acid" can mean an acetoacetyl-CoA reductase variant that includes one or more amino acid substitutions in the amino acid sequence of an unmodified parent acetoacetyl-CoA reductase having acetoacetyl-CoA reductase activity, and can be used interchangeably with terms such as "acetoacetyl-CoA reductase variant," "mutant," or "mutant polypeptide."

[0020] In this application, the term "acetoacetyl-CoA reductase" refers to an enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA.

[0021] In this application, "parent acetoacetyl-CoA reductase" means an acetoacetyl-CoA reductase that is modified to produce the acetoacetyl-CoA reductase variant, mutant, or mutant polypeptide of this application. Specifically, the parent acetoacetyl-CoA reductase or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, a mature polypeptide thereof, or may include a variant or functional fragment thereof, but is not limited to any polypeptide that has acetoacetyl-CoA reductase activity and can serve as a parent of a variant.

[0022] In this 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 activity corresponding to acetoacetyl-CoA reductase and its activity is enhanced in microorganisms, thereby increasing the production capacity of polyhydroxyalkanoates (PHAs).

[0023] Specifically, the parent acetoacetyl-CoA reductase protein may include, for example, one amino acid sequence from SEQ ID NO: 1 and SEQ ID NOs. 50 to 56, or an amino acid sequence having 55% or more homology or identity with it, but is not limited thereto as long as it has acetoacetyl-CoA reductase activity. Specifically, the amino acid sequence may include one amino acid sequence from SEQ ID NO: 1 and SEQ ID NOs. 50 to 56; or an amino acid sequence having at least 55%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with and it is obvious that auxiliary proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the protein. The amino acid sequences of SEQ ID NO: 1 and SEQ ID NOs: 50 to 56 can be obtained from known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0024] Specifically, any one of the amino acids in SEQ ID NOs. 50 to 56 has a sequence identity of 55% or more but less than 100% with the amino acid sequence of SEQ ID NO. 1.

[0025] In this application, acetoacetyl-CoA reductase refers to the following organisms: Cupriavidus necator, Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16, Pseudomonas putida, P. putida, Alcaligenes latus, Azohydromonas lata, Azohydromonas australica, Allochromatium vinosum DSM 180, Azotobacter Beijerinckii, Pandoraea sp. B-6, Burkholderiaceae bacterium 16, or Candidatus acumiribacter phosphatis. This may also be an acetoacetyl-CoA reductase derived from Accumulibacterphosphatis, but is not limited to this.

[0026] The acetoacetyl-CoA reductase derived from Cupriavidus necator (Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16), presented as an example in this application, may be a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 1, but is not limited thereto.

[0027] The acetoacetyl-CoA reductase derived from Pseudomonas putida (P. putida), presented as an example in this application, may be, but is not limited to, a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 50.

[0028] The acetoacetyl-CoA reductase derived from Alcaligenes latus (Azohydromonas lata, Azohydromonas australica), presented as an example in this application, may be a polypeptide / protein containing the amino acid sequence described in Sequence ID No. 51, but is not limited thereto.

[0029] The acetoacetyl-CoA reductase derived from Allochromatium vinosum DSM 180, presented as an example in this application, may be, but is not limited to, a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 52.

[0030] The acetoacetyl-CoA reductase derived from Azotobacter beijerinckii presented as an example in this application may be, but is not limited to, a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 53.

[0031] The acetoacetyl-CoA reductase derived from Pandoraea sp. B-6, presented as an example in this application, may be, but is not limited to, a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 54.

[0032] The acetoacetyl-CoA reductase derived from Burkholderiaceae bacterium 16, presented as an example in this application, may be a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 55, but is not limited thereto.

[0033] The acetoacetyl-CoA reductase derived from Candidatus Accumulibacter phosphatis presented as an example in this application may be a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 56, but is not limited thereto.

[0034] Furthermore, the polynucleotide sequence encoding the mother acetoacetyl-CoA reductase may also be a polynucleotide sequence encoding a protein that exhibits acetoacetyl-CoA reductase activity, and whose activity is enhanced in microorganisms, thereby improving the ability to produce polyhydroxyalkanoates (PHAs). For example, acetoacetyl-CoA reductase derived from Cupriavidus necator (Alcaligenes eutrophus, Ralstonia eutropha, Alcaligenes eutrophus H16) (SEQ ID NO: 1), acetoacetyl-CoA reductase derived from Pseudomonas putida (P. putida) (SEQ ID NO: 50), acetoacetyl-CoA reductase derived from Alcaligenes latus (Alcaligenes lata, Azohydromonas australica) (SEQ ID NO: 51), acetoacetyl-CoA reductase derived from Allochromatium vinosum DSM 180 (SEQ ID NO: 52), and Azotobacter beigerlinky (Azotobacter This may also be a polynucleotide sequence encoding acetoacetyl-CoA reductase from Beijerinckii (SEQ ID NO: 53), acetoacetyl-CoA reductase from Pandoraea sp. B-6 (SEQ ID NO: 54), acetoacetyl-CoA reductase from Burkholderiaceae bacterium 16 (SEQ ID NO: 55), or acetoacetyl-CoA reductase from Candidatus Accumulibacter phosphatis (SEQ ID NO: 56), but is not limited to these.For example, having or containing a nucleotide sequence that has homology or identity of 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 with the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70; or the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 64 The sequence may be encoded by a polynucleotide consisting of, or substantially composed of, a sequence of nucleotides having homology or identity of 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with the sequence of nucleotides corresponding to SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70; or a sequence of nucleotides corresponding to, SEQ ID NO: 2, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70; or a sequence of nucleotides having homology or identity of 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more. The sequence may be modified into a coding region due to codon degeneracy, and various modifications may be made to the coding region within the range that does not change the amino acid sequence, taking into consideration the preferred codons in the organism that intends to express the sequence. Furthermore, it is obvious that polynucleotide sequences having such homology or identity and encoding a protein that is substantially the same as or corresponding to the aforementioned protein, in which some sequences are deleted, modified, substituted, or added, are also included within the scope of this application.

[0035] In this application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence that differs from the amino acid sequence of the variant before the mutation, but in which the functions or properties are maintained. Such variants may 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 capabilities of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may include those in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain, are removed. Other variants may include those in which parts of a mature protein are removed from the N and / or C-terminus. The term "variant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and divergent (in English, these may be expressed as modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used in the sense of a mutated state.

[0036] Furthermore, the variants may include the deletion or addition of amino acids that have minimal effect on the polypeptide's properties and secondary structure. For example, the polypeptide can be conjugated with a protein N-terminal signal (or leader) sequence involved in protein transfer co-translationally or post-translationally. The polypeptide can also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0037] In this application, the terms "acetoacetyl-CoA reductase variant," "variant," or "mutant polypeptide" refer to a protein in which one or more amino acids differ from the amino acid sequence of the parent acetoacetyl-CoA reductase and which has acetoacetyl-CoA reductase activity.

[0038] Specifically, the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, an acetoacetyl-CoA reductase variant in which the amino acid corresponding to the 35th position of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 54 to SEQ ID NO: 56 is substituted with another amino acid, or the amino acid corresponding to the 34th position of the amino acid sequence of SEQ ID NO: 52 is substituted with another amino acid, or the amino acid corresponding to the 36th position of the amino acid sequence of SEQ ID NO: 53 is substituted with another amino acid.

[0039] The aforementioned "other amino acids" are not limited to any amino acid different from the amino acid before substitution. On the other hand, when the expression "a specific amino acid is substituted" is used in this application, it is self-evident that the substitution is made with an amino acid different from the amino acid before substitution, even without specifically stating that it is substituted with another amino acid.

[0040] Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of their residues.

[0041] Examples of this classification include: positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, aspartic acid) and uncharged amino acids (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). As yet another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As yet another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified by size and divided into five groups based on their relatively small volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, this is not necessarily the only classification.

[0042] For example, when it is stated that "the amino acid corresponding to position 35 in Sequence ID No. 1 has been substituted with another amino acid," it could mean, but is not limited to, the substitution with alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, tryptophan, valine, methionine, threonine, or leucine, excluding glycine.

[0043] Even if this application states "a protein having an amino acid sequence described by a specific sequence number," it is obvious that proteins having amino acid sequences with some sequences deleted, modified, substituted, conserved substituted, or added are also used in this application, as long as they have the same or corresponding activity as the protein consisting of the amino acid sequence of said sequence number. For example, if they have the same or corresponding activity as the mutant protein, this does not exclude the addition of sequences that do not change the function of the protein before or after the amino acid sequence, naturally occurring mutations, silent mutations, or conserved substitutions, and it is obvious that even if such sequences are added or mutated, they fall within the scope of this application.

[0044] The "position N" in this application may include the position N and the amino acid position corresponding to the position N. Specifically, it may include the amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a particular amino acid sequence. The particular amino acid sequence may be any one of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NOs. 50 to 56.

[0045] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at a corresponding position may determine a specific amino acid in a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0046] For example, any amino acid sequence can be aligned with Sequence ID No. 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the position of the amino acid residue in Sequence ID No. 1 and the corresponding numerical position of the amino acid residue. For example, a sequence alignment algorithm such as the one described in this application can be used to verify the position of amino acids, or the position where deformations such as substitutions, insertions, or deletions occur, by comparing them with a query sequence (also called a "reference sequence").

[0047] For such sorting, one can use, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) or the Needleman program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), but is not limited to these. Other sequence sorting programs and pairwise sequence comparison algorithms known in the industry can be used as appropriate.

[0048] In this application, when aligning the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53 with any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 54 to SEQ ID NO: 56, the amino acid corresponding to the 34th position of the amino acid sequence of SEQ ID NO: 52 and the amino acid corresponding to the 36th position of the amino acid sequence of SEQ ID NO: 53 correspond to the 35th position of the amino acid residue corresponding to the amino acid residue of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 54 to SEQ ID NO: 56.

[0049] In other words, a person skilled in the art would see that, through sequence alignment known in the art, the variant of this application is obtained by aligning one of the amino acid sequences from SEQ ID NOs. 50 to 56 with SEQ ID NO. 1, and then numbering each amino acid residue in the amino acid sequence based on this alignment, referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO. 1, and finding that in all cases the amino acid corresponding to the 35th position is replaced by another amino acid.

[0050] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which glycine, the amino acid corresponding to position 35 of SEQ ID NO: 1, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0051] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, a polypeptide in which glycine, the amino acid corresponding to position 35 of SEQ ID NO: 1, is substituted with alanine.

[0052] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which glycine, the amino acid corresponding to the 35th position of SEQ ID NO: 50, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0053] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, a polypeptide in which glycine, the amino acid corresponding to the 35th position of SEQ ID NO: 50, is substituted with alanine.

[0054] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which glycine, the amino acid corresponding to the 35th position of SEQ ID NO: 51, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0055] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, a polypeptide in which glycine, the amino acid corresponding to the 35th position of SEQ ID NO: 51, is substituted with alanine.

[0056] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which histidine, the amino acid corresponding to the 34th position of SEQ ID NO: 52, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, glycine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0057] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which histidine, the amino acid corresponding to the 34th position of SEQ ID NO: 52, is substituted with alanine, but is not limited thereto.

[0058] The amino acid corresponding to the 34th position in the amino acid sequence of Sequence ID No. 52 may be the amino acid corresponding to the 35th position when the amino acid sequence of Sequence ID No. 52 is aligned with Sequence ID No. 1, and each amino acid residue in the amino acid sequence is numbered based on this alignment, referring to the numerical position of the amino acid residue corresponding to the amino acid residue in Sequence ID No. 1.

[0059] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which the amino acid threonine, corresponding to the 36th position of SEQ ID NO: 53, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, histidine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, glycine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0060] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, a polypeptide in which threonine, the amino acid corresponding to the 36th position of SEQ ID NO: 53, is substituted with alanine.

[0061] The amino acid corresponding to the 36th position in the amino acid sequence of Sequence ID No. 53 may be the amino acid corresponding to the 35th position when the amino acid sequence of Sequence ID No. 53 is aligned with Sequence ID No. 1, and each amino acid residue in the amino acid sequence is numbered based on this alignment, referring to the numerical position of the amino acid residue corresponding to the amino acid residue in Sequence ID No. 1.

[0062] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which serine, the amino acid corresponding to the 35th position of SEQ ID NO: 54, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, histidine, asparagine, glutamine, proline, threonine, tryptophan, phenylalanine, glycine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0063] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which serine, the amino acid corresponding to the 35th position of SEQ ID NO: 54, is substituted with alanine, but is not limited thereto.

[0064] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which the amino acid threonine, corresponding to the 35th position of SEQ ID NO: 55, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, histidine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, glycine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0065] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which threonine, the amino acid corresponding to the 35th position of SEQ ID NO: 55, is substituted with alanine, but is not limited thereto.

[0066] As one example, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which histidine, the amino acid corresponding to the 35th position of SEQ ID NO: 56, is selected from the group consisting of alanine, arginine, valine, leucine, methionine, isoleucine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, glycine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, and is not limited to this substitution.

[0067] As one example of any of the aforementioned embodiments, the acetoacetyl-CoA reductase variant of this application may be a polypeptide in which histidine, the amino acid corresponding to the 35th position of SEQ ID NO: 56, is substituted with alanine, but is not limited thereto.

[0068] As another example, the acetoacetyl-CoA reductase variant of this application may be a protein in which the amino acid corresponding to the 35th position in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, or the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs. 54 to 56 is substituted with alanine, or the amino acid corresponding to the 34th position in the amino acid sequence of SEQ ID NO: 52 is substituted with alanine, or the amino acid corresponding to the 36th position in the amino acid sequence of SEQ ID NO: 53 is substituted with alanine.

[0069] On the other hand, a person skilled in the art can identify the amino acid corresponding to the 35th position of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs. 54-56, the 34th position of the amino acid sequence of SEQ ID NO: 52, or the 36th position of the amino acid sequence of SEQ ID NO: 53 in any amino acid sequence through sequence alignment known in the art. It is self-evident that when "amino acids at a specific position in a particular SEQ ID NO" is described in this application, it includes "amino acids at the corresponding positions" in any amino acid sequence, even without further description.

[0070] As one concrete example, a protein having acetoacetyl-CoA reductase activity (or acetoacetyl-CoA reductase mutant) in which the amino acid corresponding to the 35th position of the amino acid sequence of Sequence ID No. 1 of this application is substituted with another amino acid may have an amino acid sequence having 55% or more but less than 100% sequence identity with Sequence ID No. 1 or the amino acid sequence of Sequence ID No. 1, but is not limited thereto.

[0071] Specifically, one amino acid from sequence numbers 50 to 56 has a sequence identity of 55% or more but less than 100% with the amino acid sequence of sequence number 1.

[0072] Specifically, the variants of this application include amino acid sequences having at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, and less than 100% homology or identity with SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1, in which the amino acid at position 35 from the N-terminus of any one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 54 to SEQ ID NO: 56 is substituted with another amino acid, in which the amino acid corresponding to position 34 from the N-terminus of the amino acid sequence of SEQ ID NO: 52 is substituted with another amino acid, or in which the amino acid corresponding to position 36 from the N-terminus of the amino acid sequence of SEQ ID NO: 53 is substituted with another amino acid. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also included in the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the variants of this application.

[0073] For example, the variants of this application may have and / or contain one of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NOs. 57 to 63, or may substantially consist of such amino acid sequences. Alternatively, they may include amino acid sequences having at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more, or less than 100% homology or identity with an amino acid sequence selected from the group consisting of one of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NOs. 57 to 63.

[0074] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the variant of this application, spontaneous mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within it.

[0075] In this application, the term “conservative substitution” means the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions 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. Furthermore, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further classified into nonpolar amino acids and 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 to no effect on the activity of the resulting polypeptide. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0076] As one example, the acetoacetyl-CoA reductase variant of this application may, but is not limited to, having enhanced acetoacetyl-CoA reductase activity. Furthermore, the variant of this application may, but is not limited to, having activity that increases polyhydroxyalkanoate (PHA) production capacity compared to a wild-type polypeptide having acetoacetyl-CoA reductase activity.

[0077] Another aspect of this application is to provide a polynucleotide encoding a variant of the present application.

[0078] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it means a polynucleotide fragment that codes for the aforementioned variant.

[0079] The polynucleotide encoding the acetoacetyl-CoA reductase variant of this application may include, without limitation, any polynucleotide sequence encoding the acetoacetyl-CoA reductase variant of this application. For example, the polynucleotide encoding the acetoacetyl-CoA reductase variant of this application may be, but is not limited to, a polynucleotide sequence encoding the amino acid sequence of the acetoacetyl-CoA reductase variant of this application.

[0080] The polynucleotides of this application can undergo various modifications to their coding regions, taking into account codon degeneracy or preferred codons in organisms that intend to express the variants of this application, while not altering the amino acid sequence of the variants. Therefore, it is obvious that the polynucleotides also include those that can be translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the variants of this application or polypeptides homologous or identical thereto.

[0081] For example, the polynucleotides encoding the acetoacetyl-CoA reductase variants of this application may include, but are not limited to, the nucleotide sequences of SEQ ID NOs. 71 to 78; or nucleotide sequences having a homology or identity of 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more of the nucleotide sequences of SEQ ID NOs. 71 to 78. Furthermore, it is obvious that any polynucleotide sequence having such homology or identity and encoding the amino acid sequence of the acetoacetyl-CoA reductase variant of this application, including polynucleotide sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added, is also included within the scope of this application.

[0082] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application. The “stringent conditions” refer 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; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels of 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, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0083] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary throughout the entire sequence.

[0084] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0085] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0086] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0087] The homology or identity of sequences of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally hybridizable in whole or in part with other sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0088] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as described in Pearson et al (1988) [Proc.Natl.Acad.Sci.USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]):403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0089] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, the terms “homology” or “identity” as used in this application refer to the relevance between sequences.

[0090] Another aspect of this application is to provide a vector comprising the polynucleotide of this application. The vector may, but is not limited to, be an expression vector for expressing the polynucleotide in a microorganism.

[0091] In this application, the term “vector” may include a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) to express the target polypeptide in a suitable host. The regulatory 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 decoding. The vector, after being transformed into a suitable microorganism, can replicate or function independently of the host genome and integrate into the genome itself.

[0092] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0093] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within the cell. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is for selecting cells transformed with the vector, i.e., confirming the presence or absence of the target nucleic acid molecule insertion, and may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0094] In this application, the term "transformation" means 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 within the microorganism. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosome of the microorganism, as long as they can be expressed within the microorganism. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the microorganism in any form that allows for its expression. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to the sequence necessary for expression in the microorganism, but is not limited thereto.

[0095] Furthermore, the term "operably linked" in the foregoing means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0096] Another aspect of this application provides a microorganism comprising the protein, a polynucleotide encoding the protein, or a vector containing the polynucleotide.

[0097] Specifically, the present invention provides a microorganism comprising: a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding the protein; or a vector containing the polynucleotide.

[0098] As one concrete example, the protein may have an amino acid sequence that has 55% or more but less than 100% sequence identity with SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1.

[0099] As one concrete example, the protein may be one in which the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs: 54 to 56 is replaced with another amino acid, or the amino acid corresponding to the 34th position in the amino acid sequence of SEQ ID NO: 52 is replaced with another amino acid, or the amino acid corresponding to the 36th position in the amino acid sequence of SEQ ID NO: 53 is replaced with another amino acid.

[0100] As one concrete example, the protein may be one in which the amino acid corresponding to the 35th position in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, or the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs. 54 to 56 is replaced with alanine, or the amino acid corresponding to the 34th position in the amino acid sequence of SEQ ID NO: 52 is replaced with alanine, or the amino acid corresponding to the 36th position in the amino acid sequence of SEQ ID NO: 53 is replaced with alanine.

[0101] Specifically, the protein may consist of one of the amino acid sequences from SEQ ID NO: 3 and SEQ ID NOs: 57 to 63.

[0102] Specifically, the protein may have a sequence identity of 55% or more but less than 100% with one of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NOs: 57 to 63.

[0103] As one concrete example, the microorganism of this application may be a microorganism capable of producing polyhydroxyalkanoate (PHA).

[0104] As one example of any of the aforementioned embodiments, the microorganism of this application may be a microorganism capable of producing 3-hydroxybutyrate-4-hydroxybutyrate copolymer and / or poly-3-hydroxybutyrate.

[0105] In this application, the term "polyhydroxyalkanoates (PHA)" refers to a type of biodegradable bioplastic synthesized by various microorganisms.

[0106] In this application, the term "3-hydroxybutyrate-4-hydroxybutyrate copolymer (Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB))" refers to a polyhydroxyalkanoate (PHA) in which 3-hydroxybutyrate and 4-hydroxybutyrate are bonded together to form a biodegradable polyester.

[0107] In this application, the term "poly-3-hydroxybutyrate (Poly(3-hydroxybutyrate), P(3HB))" refers to a polymer of the polyhydroxyalkanoate series, and is a compound belonging to the polyester class as a polymer of 3-hydroxybutyrate. The poly-3-hydroxybutyrate can be used in combination with poly-3-hydroxybutanoate (poly-3-hydroxybutanoate, P3HA) and 3-hydroxybutyrate homopolymers.

[0108] In this application, the term "poly-4-hydroxybutyrate (Poly(4-hydroxybutyrate), P(4HB))" refers to a polymer of the polyhydroxyalkanoate series, and is a compound belonging to the polyester class as a polymer of 4-hydroxybutyrate. The poly-4-hydroxybutyrate can be mixed with poly-4-hydroxybutanoate (poly-4-hydroxybutanoate, P4HA) and 4-hydroxybutyrate homopolymers.

[0109] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and which are microorganisms in which a particular mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be microorganisms that have undergone genetic modification for the production of a desired polypeptide, protein, or product. In this application, "microorganism," "strain," and "microorganism" may be used interchangeably without limitation as they have the same meaning.

[0110] In this application, the term "microorganisms that produce polyhydroxyalkanoates (PHAs)" refers to prokaryotic or eukaryotic microbial strains capable of producing polyhydroxyalkanoates (PHAs) within their bodies, and may include all microorganisms in which the ability to produce polyhydroxyalkanoates (PHAs) has been conferred to a parent strain that lacks the ability to produce polyhydroxyalkanoates (PHAs), or microorganisms that inherently possess the ability to produce polyhydroxyalkanoates (PHAs). The ability to produce polyhydroxyalkanoates (PHAs) can be conferred or enhanced through selective breeding.

[0111] As one example, the microorganisms of this application may be microorganisms that naturally possess acetoacetyl-CoA reductase mutants or the ability to produce polyhydroxyalkanoates (PHAs); or microorganisms in which the mutant of this application or the polynucleotide encoding it (or a vector containing the polynucleotide) is introduced into a parent strain that does not possess acetoacetyl-CoA reductase mutants or the ability to produce polyhydroxyalkanoates (PHAs), and / or into which the ability to produce polyhydroxyalkanoates (PHAs) is conferred.

[0112] As one example, the microorganisms of this application include, but are not limited to, all microorganisms in which a chromosomal gene encoding the acetoacetyl-CoA reductase variant is mutated and contains the acetoacetyl-CoA reductase variant sequence of this application, and / or microorganisms containing the acetoacetyl-CoA reductase variant of this application by introducing a vector containing a polynucleotide encoding the acetoacetyl-CoA reductase variant of this application.

[0113] In this application, the term "non-myxomycete" means a strain that is either wild-type or naturally occurring, or a strain before its characteristics are altered by genetic mutation due to natural or artificial factors, and does not exclude strains containing naturally occurring mutations in microorganisms. For example, the non-myxomycete means a strain in which the acetoacetyl-CoA reductase mutant described herein has not been introduced, or before it has been introduced. The term "non-myxomycete" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxomycete strain," "non-mutant microorganism," or "reference microorganism."

[0114] The microorganisms having polyhydroxyalkanoate (PHA) production ability of this application may be, but are not limited to, microorganisms comprising one or more of the mutants of this application, the polynucleotides of this application, and vectors comprising the polynucleotides of this application; microorganisms modified to express the mutants or polynucleotides of this application; microorganisms expressing the mutants or polynucleotides of this application (e.g., recombinant strains); or microorganisms having mutant activity of this application (e.g., recombinant strains).

[0115] As an example, the strains of this application are cells or microorganisms transformed with a vector containing the polynucleotide encoding the polynucleotide encoding the variant of this application, and expressing the variant of this application. The strains of this application may include all microorganisms capable of producing polyhydroxyalkanoates (PHAs) including the variant of this application. For example, the microorganisms of this application may be naturally occurring wild-type microorganisms or recombinant strains in which the acetoacetyl-CoA reductase variant is expressed by introducing the polynucleotide encoding the variant of this application into a microorganism capable of producing polyhydroxyalkanoates (PHAs), thereby increasing the polyhydroxyalkanoate (PHA) production capacity. The recombinant strains with increased polyhydroxyalkanoate (PHA) production capacity may be, but are not limited to, naturally occurring wild-type microorganisms or acetoacetyl-CoA reductase-non-mutant microorganisms (e.g., microorganisms expressing wild-type acetoacetyl-CoA reductase or microorganisms not expressing the variant of this application), and may have increased polyhydroxyalkanoate (PHA) production capacity. As an example, the microorganisms exhibiting increased polyhydroxyalkanoate (PHA) production capacity in this application may be, but are not limited to, microorganisms exhibiting increased polyhydroxyalkanoate (PHA) production capacity compared to microorganisms containing the polypeptide of Sequence ID No. 1 or the polynucleotide encoding it.

[0116] The microorganisms of this application may include all microorganisms capable of expressing the acetoacetyl-CoA reductase variant of this application by various known methods, in addition to the introduction of the nucleic acid or vector described above.

[0117] For example, a microorganism with increased polyhydroxyalkanoate (PHA) production capacity may have an increase of approximately 1% or more compared to the polyhydroxyalkanoate (PHA) production capacity of the parent strain or non-mutant microorganism before mutation. Specifically, this could be approximately 1% or more, approximately 2% or more, approximately 2.5% or more, approximately 5% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 26% or more, approximately 27% or more, approximately 28% or more, approximately 29% or more, approximately 30% or more, approximately 31% or more, approximately 32% or more, or approximately 33% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 45% or less, or approximately 40% or less). However, it is not limited to these values ​​as long as it has a positive increase compared to the production capacity of the parent strain or non-mutant microorganism before mutation. As another example, the recombinant strains with increased polyhydroxyalkanoate (PHA) production capacity showed polyhydroxyalkanoate (PHA) production capacity of approximately 1.1 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.15 times or more, approximately 1.16 times or more, approximately 1.17 times or more, approximately 1.18 times or more, approximately 1.19 times or more, approximately 1.2 times or more, approximately 1.21 times or more, approximately 1.22 times or more, and approximately 1. The values ​​may increase by 0.23 times or more, approximately 1.24 times or more, approximately 1.25 times or more, approximately 1.26 times or more, approximately 1.27 times or more, approximately 1.28 times or more, approximately 1.29 times or more, approximately 1.30 times or more, approximately 1.31 times or more, approximately 1.32 times or more, or approximately 1.33 times or more (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, or approximately 1.4 times or less), but are not limited to these. The term "about" includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and includes all numbers in a range equivalent to or similar to the number following the term "about," but is not limited to these.

[0118] The microorganisms of this application may be, but are not limited to, microorganisms belonging to the genera Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp., and Norcardia sp., or fungi or yeasts, specifically Escherichia microorganisms, and more specifically Escherichia coli (E. coli).

[0119] On the other hand, the Escherichia microorganisms having polyhydroxyalkanoate (PHA) production ability described in this application include the naturally occurring wild-type microorganisms themselves, Escherichia microorganisms that have acquired improved polyhydroxyalkanoate (PHA) production ability by enhancing or weakening the activity of genes related to the polyhydroxyalkanoate (PHA) production mechanism, and Escherichia microorganisms that have acquired improved polyhydroxyalkanoate (PHA) production ability by introducing or enhancing the activity of external genes.

[0120] Furthermore, the microorganism of this application may have enhanced acetoacetyl-CoA reductase activity compared to the parent strain.

[0121] In this application, “enhancement” of polypeptide activity means that the activity of the polypeptide increases compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting activity that is improved compared to the endogenous activity or the activity before the mutation. “Endogenous activity” means the activity of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred, in cases where the trait has changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before the mutation.” “Enhancement,” “upregulation,” “overexpression,” or “increase” of polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the mutation occurred is improved.

[0122] The enhancement of the activity of the polypeptide can be achieved by applying various methods well known in the field, including, but not limited to, increasing the intracellular copy number of the gene encoding the mutant, introducing mutations into the gene expression regulatory sequence on the chromosome encoding the mutant, replacing the gene expression regulatory sequence on the chromosome encoding the mutant with a more potent sequence, replacing the gene encoding the protein on the chromosome with a mutated gene that increases the activity of the mutant, and introducing mutations into the gene on the chromosome encoding the mutant protein to enhance the activity of the mutant.

[0123] In this application, the term "transfer" means a method of transferring a polynucleotide encoding the acetohydroxy acid synthase variant or a vector containing the same to a host cell. Such transfer can be easily carried out by conventional methods of the art. Generally, these methods include the CaCl2 precipitation method, the Hanahan method which is an improved version of the CaCl2 method using a reducing agent called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, plasmofusion, 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. Furthermore, the transferred polynucleotide can be inserted into or located outside the chromosome of the host cell, as long as it can be expressed in the host cell. In addition, the polynucleotide can be introduced into the host cell in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a polynucleotide structure containing all the elements necessary for autonomous expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all operably linked to the open reading frame (ORF) of the gene. The expression cassette may also be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0124] As an example, the microorganism of this application may further be a microorganism in which the activity of one of the following, acetyl-CoA acetyltransferase, PHA synthase, or a combination thereof, is enhanced compared to its endogenous activity, thereby enabling it to produce polyhydroxyalkanoate (PHA).

[0125] As an example, the microorganism of this application may, but is not limited to, a microorganism that has been further modified to have the ability to produce polyhydroxyalkanoate (PHA) by introducing any one of the following: a polynucleotide encoding acetyl-CoA acetyltransferase or a vector containing the same; a polynucleotide encoding PHA synthase or a vector containing the same; or a combination thereof.

[0126] The gene encoding the CoA acetyltransferase may be PhaA derived from Cupriavidus necator, Ralstonia eutropha, Ralstonia solanacearum, Zoogloea ramigera, Delftia acidovorans, Bacillus megaterium, Crombacterium sp.USM2, Rhodobacter sphaeroides, Chromobacterium sp.USM2, Alcaligenes latus, Azohydromonas lata, Acidovax species, Burkholderia pseudomallei, Burkholderia vietnamiensis, or Burkholderia glumae, but is not limited to any gene capable of substantially expressing a protein with CoA acetyltransferase activity. The sequence of the PhaA gene encoding the CoA acetyltransferase can be obtained from known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the PhaA gene encoding the CoA acetyltransferase (SEQ ID NO: 79) derived from Cupriavidus necator may contain, but is not limited to, the nucleotide sequence of SEQ ID NO: 80.

[0127] The gene encoding the PHA synthase may be a PhaC gene derived from Cupriavidus necator, Ralstonia eutropha, Ralstonia solanacearum, Zoogloea ramigera, Delftia acidovorans, Bacillus megaterium, Crombacterium sp.USM2, Rhodobacter sphaeroides, Chromobacterium sp.USM2, Alcaligenes latus, Azohydromonas lata, Acidovax species, Burkholderia pseudomallei, Burkholderia vietnamiensis, or Burkholderia glumae, but is not limited to any gene capable of substantially expressing a protein with PHA synthase activity. The sequence of the PhaC gene encoding the PHA synthase can be obtained from known databases 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: 81) derived from Cupriavidus necator may contain, but is not limited to, the nucleotide sequence of SEQ ID NO: 82.

[0128] Another aspect of this application provides a method for producing polyhydroxyalkanoate (PHA), comprising the step of culturing the microorganism of this application in a culture medium.

[0129] Specifically, the polyhydroxyalkanoate (PHA) production method of this application may include, but is not limited to, a step of culturing a microorganism containing the mutant, polynucleotide, or vector of this application in a culture medium.

[0130] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected microorganisms. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0131] In this application, the term "culture medium" means a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which are indispensable for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application are not particularly limited and any culture medium used for culturing ordinary microorganisms can be used. However, the microorganisms of this application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions while adjusting the temperature, pH, etc.

[0132] For example, culture media for Escherichia strains can be found in the following reference: [Tao H, Bausch C, Richmond C, Blattner FR, Conway T 1999. Functional Genomics: Expression Analysis of Escherichia coli Growing on Minimal and Rich Media. J Bacteriol 181: Functional Genomics: Expression Analysis of Escherichia coli Growing on Minimal and Rich Media].

[0133] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0134] The nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0135] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these methods.

[0136] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but these are not limited to these.

[0137] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0138] The polyhydroxyalkanoate (PHA) produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0139] The polyhydroxyalkanoate (PHA) production method of this application may further include, for example, a step of preparing the microorganism of this application, a step of preparing a culture medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0140] The polyhydroxyalkanoate (PHA) production method of this application may further include a step of recovering polyhydroxyalkanoate (PHA) from the culture medium (pre-cultured medium) or the microorganism of this application. The recovery step may further include a step after the culture step.

[0141] The aforementioned recovery may involve collecting polyhydroxyalkanoates (PHAs) using appropriate methods known in the art, such as batch, continuous, or fed-batch culture methods, as described in this application. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover polyhydroxyalkanoates (PHAs) from culture media or microorganisms using appropriate methods known in the art.

[0142] Furthermore, the polyhydroxyalkanoate (PHA) production method of this application may further include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the polyhydroxyalkanoate (PHA) production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step, but are not limited thereto.

[0143] Another aspect of this application is to provide a composition for the production of polyhydroxyalkanoate (PHA) comprising the protein; a polynucleotide encoding the protein; a vector containing the polynucleotide; a microorganism containing the protein, the polynucleotide encoding the protein, or the vector containing the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

[0144] The composition of this application may further contain any suitable excipients commonly used in compositions for amino acid production, such excipients may be, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0145] As one specific example, each component present in the composition of this application may be included in a microbiologically effective amount or in an amount that can be adequately present in a production composition.

[0146] Another aspect of this application provides for the use of a microorganism comprising the amino acid sequence of SEQ ID NO: 1 or a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; or the microorganism comprising the protein, a polynucleotide encoding the protein, or a vector containing the polynucleotide, for the production of polyhydroxyalkanoates (PHAs). Examples

[0147] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0148] Example 1. Preparation of a bacterial strain expressing an acetoacetyl-CoA reductase mutant and confirmation of P(3HB) production ability - 1 Example 1-1. Vector preparation for the production of P(3HB) producing strains. To produce P(3HB)-producing Eschericha coli, a vector containing phaA, phaB, and phaC was prepared.

[0149] Specifically, using the pCL1920 vector (GenBank No. AB236930), we constructed the pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB vector, which contains the PuspA promoter (universal stress protein A promoter) (Prytz et al. 2003; Dyk et al. 1995; Nystrφm and Neidhardt 1992; 1994), enabling the expression of Cupriavidus necator-derived phaA (SEQ ID NO: 80), phaC (SEQ ID NO: 82), and phaB gene sequences (SEQ ID NO: 2) in Eschericha coli. Using primers for SEQ ID NOs. 4 and 5, we performed PCR based on the NCBI database reference sequence NZ_CP039287.1 to amplify the phaA, phaC, and phaB genes.

[0150] The 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 30 times. The PCR results were subjected to electrophoresis on a 1.0% agarose gel, followed by elution and purification of the bands. The pCL1920 vector (GenBank No. AB236930) was treated with XbaI and PstI restriction enzymes. The restriction enzyme-treated pCL1920 vector and the insertion DNA fragment amplified through the above PCR were ligated using an Infusion Cloning Kit, transformed into E. coli DH5α, and colonies were sorted in LB medium containing 75 mg / L of the antibiotic Spectinomycin.

[0151] Examples 1-2. Vector preparation for the production of P(3HB)-producing strains expressing acetoacetyl-CoA reductase mutants. Vectors were prepared to express acetoacetyl-CoA reductase mutants in bacterial strains. Specifically, sequence numbers 6, 7, 4, and 5 were used for introducing the M12S mutation of phaB; sequence numbers 8, 9, 4, and 5 for introducing the G35A mutation; sequence numbers 10, 11, 4, and 5 for introducing the N61D mutation; sequence numbers 12, 13, 4, and 5 for introducing the V62T mutation; sequence numbers 14, 15, 4, and 5 for introducing the F148Y mutation; sequence numbers 16, 17, 4, and 5 for introducing the I194M mutation; sequence numbers 18, 19, 4, and 5 for introducing the V198T mutation; sequence numbers 20, 21, 4, and 5 for introducing the E47L mutation; and sequence numbers 22, 23, 4, and 5 for introducing the T173S mutation. PCR was performed under the same conditions as in Example 1-1, based on the reference sequence NZ_CP039287.1 in the NCBI database. Primer sequence information is shown in Table 1.

[0152] [Table 1]

[0153] The phaA, phaC, and phaB mutant gene expression vectors obtained from the cloning results were named pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(M12S), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35A), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(N61D), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(V62T), and p CL-PuspA_phaA-PuspA_phaC-PuspA_phaB(F148Y), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(I194M), pCL-PuspA_phaA-PuspA_phaC- They were named PuspA_phaB(V198T), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(Q47L), and pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(T173S).

[0154] Examples 1-3. Preparation of acetoacetyl-CoA reductase mutant-expressing bacterial strains and confirmation of P(3HB) production ability. To evaluate the P(3HB) production capacity induced by the expression of acetoacetyl-CoA reductase mutants, E. coli with wild-type phaB introduced and E. coli with the phaB mutation introduced were prepared. Specifically, the plasmids prepared in Examples 1-1 and 1-2 were introduced into wild-type E. coli using the TSS method, and the samples were sorted in LB medium containing 75 mg / L of the antibiotic Spectinomycin (Proc Natl Acad Sci US A.1989 Apr;86(7):2172-5).

[0155] Next, the selected bacterial strains were subjected to a P(3HB) production capacity test. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of P(3HB) production medium with 5% glucose, and then cultured with shaking at 230 rpm for 5 hours at 37°C and 43 hours at 35°C. Subsequently, the P(3HB) concentration was analyzed by gas chromatography. The analyzed P(3HB) concentrations are shown in Table 2 below.

[0156] [Table 2]

[0157] As a result, the P(3HB) production capacity of the acetoacetyl-CoA reductase G35A mutant-expressing strain increased by 4% compared to the wild-type phaB-expressing strain, confirming that position 35 is an important location for P(3HB) production.

[0158] Example 2. Residual mutation experiment of acetoacetyl-CoA reductase mutant (G35) Example 2-1. Vector Production In Example 1, we confirmed that the 35th position of acetoacetyl-CoA reductase is an important position for polyhydroxyalkanoate (PHA) production. Therefore, we decided to create a mutant strain based on wild-type E. coli that expresses a mutant protein in which the 35th amino acid of acetoacetyl-CoA reductase is substituted with another amino acid, and to check whether or not there is an increase in P(3HB) production capacity.

[0159] Specifically, PCR was performed using the same method as in Example 1-1, with sequence numbers 24, 25, and 4, 5 to introduce the coding sequences for phaA, phaC, and phaB encoding the acetoacetyl-CoA reductase G35H variant; sequence numbers 26, 27, and 4, 5 to introduce the G35V variant coding sequence; and sequence numbers 28, 29, and 4, 5 to introduce the G35S variant coding sequence. Primer sequence information is shown in Table 3.

[0160] [Table 3]

[0161] Cloning was performed using the same method as in Example 1, and the vectors into which the phaB mutation obtained from the cloning results were introduced were named pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35H), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35V), and pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35S), respectively.

[0162] Example 2-2. Preparation of acetoacetyl-CoA reductase mutant-expressing bacterial strains and confirmation of P(3HB) production ability. To confirm the P(3HB) production ability of acetoacetyl-CoA reductase mutant-expressing strains, phaB containing the mutation and phaA and phaC were expressed together in wild-type E. coli. The vectors containing the phaB mutation prepared in Example 2-1 (pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35H), pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35V), and pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB(G35S)) were introduced into wild-type E. coli using the TSS method, and the samples were sorted in LB medium containing 75 mg / L of the antibiotic Spectinomycin (Proc Natl Acad Sci US A.1989 Apr;86(7):2172-5).

[0163] Next, the selected bacterial strains were subjected to a P(3HB) production capacity test. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of P(3HB) production medium with 5% glucose, and then cultured with shaking at 230 rpm for 5 hours at 37°C and 43 hours at 35°C. Subsequently, the P(3HB) concentration was analyzed by gas chromatography. The analyzed P(3HB) concentrations are shown in Table 4 below.

[0164] [Table 4]

[0165] As a result, we confirmed that a mutant of acetoacetyl-CoA reductase in which the 35th glycine position was substituted with alanine significantly increased P(3HB) production capacity. On the other hand, when the 35th glycine position of acetoacetyl-CoA reductase was substituted with histidine, valine, and serine, respectively, we confirmed that the P(3HB) yield was at a level equivalent to or lower than that of the wild-type phaB-expressing strain. This suggests that only the substitution of the 35th amino acid with alanine is significant in increasing P(3HB) production capacity.

[0166] Example 3. Production of acetoacetyl-CoA reductase mutant-expressing bacterial strain and confirmation of P(3HB) production ability - 2 Example 3-1. Vector Production In addition to the phaB gene derived from Cupriavidus necator confirmed in Example 1, we also decided to investigate the effects of a mutation in which the amino acid at the position corresponding to the 35th position of the Cupriavidus necator-derived phaB gene in Example 1-1 is substituted with alanine on phaB genes derived from other alien species. Specifically, to enable expression in Eschericha coli, we amplified phaB genes from seven species (Pseudomonas putida, Alcaligenes latus, Allochromatium vinosum DSM 180, Azotobacter beijerinckii, Pandoraea sp.B-6, Burkholderiaceae bacterium 16, Candidatus Accumulibacter phosphatis) that have 55% or more homology and structural similarity to the phaB gene encoding acetoacetyl-CoA reductase from Cupriavidus necator in Example 1, as well as phaB genes from each of these seven species. These phaB genes contained a mutation in which the amino acid at the position corresponding to the 35th position of the phaB gene from Cupriavidus necator in Example 1-1 was replaced with alanine. These phaB genes each encode acetoacetyl-CoA reductase mutants (sequence numbers 57 to 63, respectively). Each gene containing the mutation was subjected to PCR testing (Appl Environ Microbiol. 2013 Oct;79(19):6134-9), and the primer sequence information is shown in Table 5.

[0167] [Table 5]

[0168] The PCR conditions consisted of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, repeated 30 times. The PCR results were subjected to electrophoresis on a 1.0% agarose gel, followed by elution and purification of the bands. The pCL1920 vector was treated with EcoRV and HindIII restriction enzymes. The restriction enzyme-treated pCL1920 vector and the insertion DNA fragment amplified through the above PCR were ligated using an Infusion Cloning Kit, transformed into E. coli DH5α, and colonies were sorted in LB medium containing 75 mg / L of the antibiotic Spectinomycin. Vectors into which mutations were introduced into the phaA and phaC genes obtained from cloning results and phaB derived from exotic species (Pseudomonas putida, Alcaligenes latus, Allochromatium vinosum DSM 180, Azotobacter beijerinckii, Pandoraea sp.B-6, Burkholderiaceae bacterium 16, Candidatus Accumulibacter phosphatis) were respectively named pCL-PuspA_phaA-PuspA_phaC-PuspA_Ppu_phaB (G35A), pCL-PuspA_phaA-PuspA_phaC-PuspA__Aau_phaB (G35A), pCL-PuspA_phaA-PuspA_phaC-PuspA_Avi_phaB (H34A), and pCL-PuspA_phaA-PuspA_phaC-PuspA_Abe_phaB (T36A), pCL-PuspA_phaA-PuspA_phaC-PuspA_P_phaB (S35A), pCL-PuspA_phaA-PuspA_phaC_PuspA_Bba_phaB (T35A), and pCL-PuspA_phaA_PuspA_phaC_PuspA_Cac_phaB (H35A).

[0169] Example 3-2. Preparation of acetoacetyl-CoA reductase mutant-expressing bacterial strains and confirmation of P(3HB) production ability. Similar to Examples 1-2 above, wild-type Escherichia coli was used to express phaA, phaC, and phaB including the mutation. Vectors containing the respective phaB mutations prepared in Example 3-1 for wild-type E. coli (pCL-PuspA_phaA-PuspA_phaC-PuspA_Ppu_phaB (G35A), pCL-PuspA_phaA-PuspA_phaC-PuspA_Aau_phaB (G35A), pCL-PuspA_phaA-PuspA_phaC-PuspA_Avi_phaB (H34A), pCL-PuspA_phaA-PuspA_phaC-PuspA_Abe_phaB (T36A), pCL-PuspA_phaA-PuspA_phaC-PuspA_P_phaB (S35A), pCL-PuspA_phaA-PuspA_phaC-PuspA_Bba_phaB) (T35A) and pCL-PuspA_phaA-PuspA_phaC-PuspA_Cac_phaB (H35A)) were introduced using the TSS method and sorted in LB medium containing 75 mg / L of spectinomycin antibiotic (Proc Natl Acad Sci US A.1989 Apr;86(7):2172-5).

[0170] Next, the selected bacterial strains were subjected to a P(3HB) production capacity test. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of P(3HB) production medium with 5% glucose, and then cultured with shaking at 230 rpm for 5 hours at 37°C and 43 hours at 35°C. The P(3HB) concentration was analyzed by gas chromatography. The test results are shown in Table 6 below.

[0171] [Table 6]

[0172] As a result, as shown in Table 6 above, when strains expressing phaB containing amino acid mutations derived from each alien species were cultured, it was confirmed that the P(3HB) yield increased compared to when wild-type phaB was expressed. Therefore, it was confirmed that when the amino acid corresponding to the 35th amino acid of acetoacetyl-CoA reductase expressed by phaB derived from Cupriavidus necator was substituted with alanine in phaB derived from various alien species, the activity of the enzyme expressed by phaB was enhanced, and the P(3HB) production capacity increased.

[0173] Example 4. Production of acetoacetyl-CoA reductase mutant-expressing bacterial strains and confirmation of P(3HB-co-4HB) production ability. Example 4-1. Vector Production To construct a vector capable of inserting the acetoacetyl-CoA reductase G35A mutant coding sequence phaB (G35A) into the gene, the pSKH vector (KR 10-2006-0137650 A) was used. Specifically, PCR was performed using sequence numbers 44 and 45 with pCL-PuspA_phaA-PuspA_phaC-PuspA_phaB (G35A) prepared in Example 1-1 as the template. To include a region with the same sequence as the intended insertion site, PCR was performed using sequence numbers 46, 47, 48, and 49 based on E. coli genomic DNA. Primer sequence information is shown in Table 7.

[0174] [Table 7]

[0175] The 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 30 times. The PCR results were subjected to electrophoresis on a 1.0% agarose gel, the bands were eluted and purified, and the pSKH vector was treated with EcoRV restriction enzyme. The restriction enzyme-treated pSKH vector and the insertion DNA fragment amplified through the above PCR were ligated using an Infusion Cloning Kit, and then transformed into E. coli DH5α. Colonies were sorted in LB medium containing 50 mg / L of Kanamycin antibiotic. The phaB gene expression vector obtained from the cloning results was named pSKH-PuspA-phaB(G35A).

[0176] Example 4-2. Production of acetoacetyl-CoA reductase mutant-expressing bacterial strains and confirmation of P(3HB-co-4HB) production ability. To confirm the change in the ratio of 3HB to 4HB through the increase of 3HB by introducing the mutant phaB(G35A) gene into a P(3HB-co-4HB) producing strain, we used a previously produced P(3HB-co-4HB) producing strain (Strain 13) (US 10323261 B1). Based on the P(3HB-co-4HB) producing strain (Strain 13), we transformed the pSKH-PuspA-phaB(G35A) produced in Example 3-1 using the electropulse method. The produced strain was named Escherichia coli CB02-6589 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depositary under the Budapest Convention, on December 9, 2022, receiving the depositary number KCCM13301P.

[0177] Next, a P(3HB-co-4HB) production capacity test was performed on the P(3HB-co-4HB) producing strain CB02-6589 into which the aforementioned mutant phaB(G35A) had been introduced. The concentrations and 4HB content of the analyzed P(3HB-co-4HB) are shown in Table 8 below.

[0178] [Table 8]

[0179] As a result, it was confirmed that in the case of the acetoacetyl-CoA reductase mutant (G35A) expressing strain, 3HB increased due to enhancement of the 3HB biosynthesis pathway compared to the parent strain, resulting in an approximately 8% increase in 3HB content. Furthermore, it was confirmed that the P(3HB-co-4HB) yield increased by 4.6% compared to the parent strain due to enhancement of acetoacetyl-CoA reductase enzyme activity.

[0180] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0181] [Table 9]

Claims

1. A protein having acetoacetyl-CoA reductase activity, wherein the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

2. The aforementioned protein is If the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs: 54-56 is substituted with another amino acid, The amino acid corresponding to the 34th position in the amino acid sequence of sequence number 52 is substituted with another amino acid, or The protein according to claim 1, wherein the amino acid corresponding to the 36th position in the amino acid sequence of sequence number 53 is substituted with another amino acid.

3. The aforementioned protein is The amino acid corresponding to the 35th position in the amino acid sequence of Sequence ID No. 1 is substituted with alanine, The amino acid corresponding to the 35th position in any one of the amino acid sequences of Sequence ID 50, Sequence ID 51, and Sequence IDs 54 to 56 is substituted with alanine, The amino acid corresponding to the 34th position in the amino acid sequence of Sequence ID No. 52 is substituted with alanine, or The protein according to claim 1, wherein the amino acid corresponding to the 36th position in the amino acid sequence of Sequence ID No. 53 is substituted with alanine.

4. The protein according to claim 1, wherein the protein consists of one amino acid sequence from SEQ ID NO: 3 and SEQ ID NOs: 57 to 63.

5. The protein according to claim 4, wherein the protein has an amino acid sequence that has 55% or more and less than 100% sequence identity with any one of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 57 to 63.

6. A polynucleotide encoding a protein according to any one of claims 1 to 5.

7. A microorganism comprising: a protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding the protein; or a vector containing the polynucleotide.

8. The aforementioned protein is If the amino acid corresponding to the 35th position in any one of the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NOs: 54-56 is substituted with another amino acid, The amino acid corresponding to the 34th position in the amino acid sequence of sequence number 52 is substituted with another amino acid, or The microorganism according to claim 7, wherein the amino acid corresponding to the 36th position in the amino acid sequence of sequence number 53 is substituted with another amino acid.

9. The aforementioned protein is The amino acid corresponding to the 35th position in the amino acid sequence of Sequence ID No. 1 is substituted with alanine, The amino acid corresponding to the 35th position in any one of the amino acid sequences of Sequence ID 50, Sequence ID 51, and Sequence IDs 54 to 56 is substituted with alanine, The amino acid corresponding to the 34th position in the amino acid sequence of Sequence ID No. 52 is substituted with alanine, or The microorganism according to claim 7, wherein the amino acid corresponding to the 36th position in the amino acid sequence of Sequence ID No. 53 is substituted with alanine.

10. The microorganism according to claim 7, wherein the protein consists of the amino acid sequence of SEQ ID NO: 3 and one of the amino acid sequences of SEQ ID NOs: 57 to 63.

11. The microorganism according to claim 7, wherein the protein has 55% or more and less than 100% sequence identity with any one amino acid sequence of Sequence ID No. 3 and Sequence ID Nos. 57 to 63.

12. The microorganism according to claim 7, wherein the microorganism has increased polyhydroxyalkanoate (PHA) production capacity compared to a microorganism containing one polypeptide or polynucleotide encoding any one of Sequence ID No. 1 and Sequence ID No. 50 to No.

56.

13. The microorganism according to claim 7, wherein the microorganism is a microorganism of the genus Escherichia.

14. The microorganism according to claim 13, wherein the Escherichia genus microorganism is Escherichia coli.

15. A method for producing polyhydroxyalkanoate (PHA), comprising the step of culturing the microorganism described in claim 7 in a culture medium.

16. The method according to claim 15, further comprising the step of recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

17. A protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; Polynucleotide encoding the aforementioned protein; A microorganism comprising the protein, a polynucleotide encoding the protein, or a vector containing the polynucleotide; A composition for the production of polyhydroxyalkanoate (PHA) comprising a culture of the aforementioned microorganism; or a combination of two or more of these.

18. A protein having acetoacetyl-CoA reductase activity in which the amino acid corresponding to the 35th position of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 55% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; or A microorganism comprising the protein, a polynucleotide encoding the protein, or a vector containing the polynucleotide, Use for the production of polyhydroxyalkanoates (PHAs).

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