Novel mutant polypeptides having propionaldehyde dehydrogenase activity and uses thereof

Abstract

The present application relates to a novel mutant polypeptide having propionaldehyde dehydrogenase activity, a polynucleotide encoding the mutant polypeptide, a microorganism containing the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide, and a method for producing 3HP-CoA or a derivative thereof, the method comprising culturing the microorganism in a culture medium.

Description

[Technical Field]

[0001] The present application relates to a novel mutant polypeptide having propionaldehyde dehydrogenase activity, a polynucleotide encoding the mutant polypeptide, a microorganism containing the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide, and a method for producing 3HP-CoA or a derivative thereof, the method comprising culturing the microorganism in a culture medium. [Background technology]

[0002] Acrylic acid (AA) is a highly reactive substance with double bonds and functional groups within its molecule. It is used as a monomer for polymer production through various chemical reactions and is produced for a variety of applications, including acrylics, fibers, and paints. High-purity glacial AA is primarily used in the production of superabsorbent polymers (SAPs), which are commercialized as moisture absorbents in disposable diapers and sanitary products. Currently, most AA is produced through petrochemical-based propylene oxidation. However, environmental and temperature issues have become a major concern in recent years, and consumer demand for bio-friendly chemical products has increased. This has led to a growing need for non-petroleum bio-based acrylic acid production.

[0003] In this regard, various attempts have been made to produce the precursor of AA, 3-hydroxypropionic acid (3HP), through genetic engineering and fermentation of microorganisms. For example, AA can be produced by conversion of lactic acid (LA) or 3HP, or by pyrolysis of poly-3-hydroxypropionic acid (P3HP). The latter method has the advantage of being a solid precursor, unlike 3HP, which is produced in a liquid state after purification.

[0004] 3HP is biologically produced via the 3HP-CoA-dependent and 3HP-CoA-independent pathways, using glycerol, a by-product of biodiesel production, as a carbon source. 3HP is a useful intermediate that can be converted into various compounds, including acrylic acid, acrylonitrile, methylacrylic acid, and 1,3-propanediol.

[0005] The 3HP-CoA dependent pathway is an enzymatic reaction in which 3HP-CoA is converted by the pduP enzyme as shown in Figure 1. The produced 3HP-CoA is then ultimately converted to AA, 3HP, P3HP, etc.

[0006] Research into the use of pduP for the production of 3HP has been reported in prior art documents (Non-Patent Document 1). However, most of the previous studies only disclose wild-type pduP from Salmonella typhimurium LT2 and Lactobacillus reuteri (Non-Patent Document 2, Patent Document 1), and do not disclose pduP mutants or their uses.

[0007] Therefore, there is still a need for research to effectively improve the productivity of 3HP-CoA or its derivatives. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 10-2016-0006030 [Patent Document 2] U.S. Patent No. 6,329,183 [Patent Document 3] U.S. Patent No. 6,593,116 [Non-patent literature]

[0009] [Non-Patent Document 1] Bioresource Technology, Volume103, Issue 1, January 2012, Pages 1-6 [Non-licensed document 2] Bioresour Technol. 2013Mar;131:548-51. [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0010] The problem to be solved by the present application is to provide a mutant polypeptide having novel propionaldehyde dehydrogenase activity, a polynucleotide encoding the mutant polypeptide, a microorganism containing the mutant polypeptide, the polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide, and a method for producing 3HP-CoA or a derivative thereof, the method comprising the steps of culturing the microorganism in a culture medium. [Means for solving the problem]

[0011] One aspect of the present application provides a mutant polypeptide having propionaldehyde dehydrogenase activity, in which the amino acid corresponding to position 272 of SEQ ID NO: 1 has been substituted with another amino acid.

[0012] In one embodiment, the mutant polypeptide may have the amino acid corresponding to position 272 of SEQ ID NO: 1 substituted with an amino acid selected from the group consisting of glycine, alanine, arginine, valine, leucine, methionine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid.

[0013] In another specific example, the mutant polypeptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:3 to SEQ ID NO:7 and SEQ ID NO:33 to SEQ ID NO:46.

[0014] Another aspect of the present application provides a polynucleotide encoding the variant polypeptide.

[0015] Yet another aspect of the present application provides a microorganism comprising at least one of the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, and a vector comprising the polynucleotide.

[0016] In one specific example, the microorganism may have an improved ability to produce 3HP-CoA or a derivative thereof, compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding it.

[0017] In the microorganism according to any of the above-mentioned embodiments, said microorganism may be an Escherichia microorganism.

[0018] In the microorganism according to any of the above-mentioned embodiments, the Escherichia microorganism may be Escherichia coli.

[0019] Yet another aspect of the present application provides a method for producing 3HP-CoA or a derivative thereof, comprising the step of culturing the microorganism in a medium.

[0020] In one embodiment, the method may further comprise the step of recovering a target substance from the cultured microorganism, a culture of the microorganism, a fermentation product of the microorganism, or the culture medium.

[0021] Yet another aspect of the present application provides a composition for producing 3HP-CoA or a derivative thereof, comprising the mutant polypeptide, the microorganism, or a combination thereof.

[0022] Yet another aspect of the present application provides the use of said microorganism for producing 3HP-CoA or a derivative thereof. [Effects of the Invention]

[0023] When a microorganism containing the mutant polypeptide of the present application having propionaldehyde dehydrogenase activity is cultured, 3HP-CoA or a derivative thereof can be produced in a higher yield than when a microorganism containing a conventional unmodified polypeptide is cultured. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram showing an example of a pathway for producing 3HP-CoA or a derivative thereof from glucose or glycerin according to the present invention. [Figure 2] Schematic diagram of the pFS-pduP(WT) plasmid. DETAILED DESCRIPTION OF THE INVENTION

[0025] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0026] One aspect of the present application provides a mutant polypeptide having propionaldehyde dehydrogenase activity, in which the amino acid corresponding to position 272 of SEQ ID NO: 1 has been substituted with another amino acid.

[0027] In the present application, the term "variant polypeptide having propionaldehyde dehydrogenase activity" refers to a variant polypeptide having propionaldehyde dehydrogenase activity that contains at least one amino acid substitution in the amino acid sequence of a polypeptide having propionaldehyde dehydrogenase activity.

[0028] In the present application, the term "variant polypeptide having propionaldehyde dehydrogenase activity" refers to a variant of a polypeptide having propionaldehyde dehydrogenase activity that contains at least one amino acid substitution in the parent sequence, which is the amino acid sequence of a polypeptide having propionaldehyde dehydrogenase activity.

[0029] In this application, "propionaldehyde dehydrogenase (CoA-acylatingpropionaldehyde dehydrogenase, PduP)" refers to an enzyme that converts 3-hydroxypropionaldehyde (3HPA) into 3-hydroxypropionyl-CoA (3HP-CoA).

[0030] The gene encoding the propionaldehyde dehydrogenase is pduP from Salmonella sp., Klebsiellapneumonia, Lactobacillus, specifically Salmonella enterica, but is not limited thereto.

[0031] Specifically, the propionaldehyde dehydrogenase protein includes, for example, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has propionaldehyde dehydrogenase activity. Specifically, the amino acid sequence includes SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto. The sequence of SEQ ID NO: 1 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the propionaldehyde dehydrogenase protein is derived from the genus Salmonella or Salmonella enterica, and more specifically, is a polypeptide / protein comprising the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto. Furthermore, it goes without saying that auxiliary proteins having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added are also included in the present application, as long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the protein.

[0032] Furthermore, the propionaldehyde dehydrogenase protein having the amino acid sequence of SEQ ID NO: 1 is encoded by a polynucleotide having, including, consisting of, or substantially consisting of the polynucleotide sequence of SEQ ID NO: 2, or a base sequence that has homology or identity to the polynucleotide sequence of SEQ ID NO: 2 of 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, and less than 100%, but is not limited to these.

[0033] In this application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been deleted. Other variants include variants in which a portion has been deleted from the N- and / or C-termini of a mature protein. The term "variant" is often used interchangeably with terms such as "mutated type," "modification," "mutated polypeptide," "mutated protein," "mutant," "mutein," and "divergent" (in English), but it may refer to any term that refers to a mutation.

[0034] Variants may also include deletions or additions of amino acids that minimally affect the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co- or post-translational protein transfer. The polypeptide may also be linked to other sequences or linkers that allow for identification, purification, or synthesis of the polypeptide.

[0035] The mutant polypeptide having propionaldehyde dehydrogenase activity of the present application is a mutant polypeptide having propionaldehyde dehydrogenase activity in which the amino acid corresponding to position 272 of SEQ ID NO: 1 has been substituted with another amino acid, but is not limited thereto.

[0036] The "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid has been substituted" in the present application means that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid has been substituted with another amino acid.

[0037] Amino acids are commonly classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0038] As an example of such classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids. Valine, leucine, and isoleucine are classified as branched-chain amino acids. As another example, the 20 amino acids are classified by size and divided into five groups in order of decreasing relative 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, the groups are not necessarily limited to these.

[0039] For example, "the amino acid corresponding to position 272 in SEQ ID NO: 1 is substituted with another amino acid" means that the amino acid is substituted with glycine, alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, valine, methionine, threonine, or leucine, excluding isoleucine, but is not limited to these.

[0040] Although the present application describes a "protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, as long as they have the same or corresponding activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions are not excluded, and it goes without saying that proteins having such sequence additions or mutations are also included in the present application, as long as they have the same or corresponding activity as the mutant protein.

[0041] In the present application, the "Nth position" includes the Nth position and an amino acid position corresponding to the Nth position. Specifically, it includes an amino acid position corresponding to any amino acid residue in a mature polypeptide represented by a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0042] "Corresponding to," as used herein, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. "Corresponding region," as used herein, generally refers to a similar or corresponding position in a related or reference protein.

[0043] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

[0044] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 4) can be used, but the present invention is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used as appropriate.

[0045] In one embodiment, the mutant polypeptide has the amino acid corresponding to position 272 of SEQ ID NO: 1 substituted with a residue selected from the group consisting of, but not limited to, glycine, alanine, arginine, valine, leucine, methionine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid.

[0046] In any of the above-described embodiments, the variant polypeptide provided by the present application may have the amino acid corresponding to position 272 from the N-terminus of SEQ ID NO: 1 substituted with an amino acid having a hydrophilic side chain (polar amino acid) selected from serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Specifically, the amino acid may be an amino acid selected from threonine, tyrosine, asparagine, and glutamine.

[0047] In any of the above-described embodiments, the variant polypeptide provided by the present application may have the amino acid corresponding to position 272 from the N-terminus of SEQ ID NO: 1 substituted with an uncharged amino acid (also referred to as a neutral amino acid) having an uncharged side chain selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Specifically, the amino acid may be selected from valine, threonine, tyrosine, asparagine, and glutamine.

[0048] In any of the above-mentioned examples, the mutant polypeptide having propionaldehyde dehydrogenase activity of the present application is a polypeptide in which the isoleucine amino acid corresponding to position 272 of SEQ ID NO: 1 is substituted with an amino acid selected from valine, threonine, tyrosine, asparagine, and glutamine, but is not limited thereto.

[0049] Meanwhile, a person skilled in the art can ascertain the amino acid in any amino acid sequence that corresponds to position 272 of the amino acid sequence of SEQ ID NO: 1 of the present application by sequence alignment known in the art. In the present application, it goes without saying that "an amino acid at a specific position in a specific SEQ ID NO" includes "an amino acid at a corresponding position" in any amino acid sequence, unless otherwise specified.

[0050] In one example, a variant polypeptide having propionaldehyde dehydrogenase activity of the present application has a sequence identity of 60% or more and less than 100% with the amino acid sequence of SEQ ID NO: 1, specifically, but not limited to, a sequence identity of 80% or more and less than 100%.

[0051] Specifically, the variant of the present application may be an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the amino acid sequence represented by SEQ ID NO: 1, in which the amino acid at position 272 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid. Needless to say, the present application also includes variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the variant of the present application.

[0052] In another specific example, the mutant polypeptide may have, include, or consist essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 7 and 33 to 46. Alternatively, the mutant polypeptide may comprise an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homologous or identical to an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3 to 7 and 33 to 46.

[0053] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0054] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids are further classified into those with electrically charged side chains and those with uncharged side chains, with charged amino acids including aspartic acid, glutamic acid, lysine, arginine, and histidine, and uncharged amino acids are further classified into nonpolar and polar amino acids, with nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting protein or polypeptide.

[0055] By way of example, but not limitation, the variant polypeptide of the present application having propionaldehyde dehydrogenase activity has enhanced propionaldehyde dehydrogenase activity. Furthermore, the variant of the present application has an activity that improves the ability to produce 3HP-CoA or a derivative thereof compared to a wild-type polypeptide having propionaldehyde dehydrogenase activity, but is not limited to this.

[0056] Another aspect of the present application provides a polynucleotide encoding the variant polypeptide.

[0057] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the above-mentioned variant.

[0058] The polynucleotide encoding the variant polypeptide having propionaldehyde dehydrogenase activity of the present application may be any polynucleotide sequence that encodes the variant polypeptide having propionaldehyde dehydrogenase activity of the present application. For example, the polynucleotide encoding the variant polypeptide having propionaldehyde dehydrogenase activity of the present application is a polynucleotide sequence that encodes the amino acid sequence of the variant polypeptide having propionaldehyde dehydrogenase activity of the present application, but is not limited thereto.

[0059] The polynucleotides of the present application can be modified in various ways in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the variant of the present application is to be expressed, as long as the amino acid sequence of the variant of the present application is not changed. Therefore, it goes without saying that polynucleotides that, due to codon degeneracy, are translated into a polypeptide consisting of the amino acid sequence of the variant of the present application, or a polypeptide having homology or identity thereto, are also included.

[0060] For example, the polynucleotide of the present application may be a nucleotide sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 2, in which the codon encoding isoleucine, the amino acid corresponding to position 272 of SEQ ID NO: 2, is substituted with a codon encoding an amino acid other than isoleucine, such as an amino acid selected from valine, threonine, tyrosine, asparagine, and glutamine, but is not limited thereto. Needless to say, the present application also includes variants having a polynucleotide sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of a variant polypeptide of the present application having propionaldehyde dehydrogenase activity.

[0061] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 5 and 6). For example, conditions include those under which polynucleotides with high homology or identity, e.g., polynucleotides having a homology or identity of 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, hybridize with each other, while polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0062] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0063] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0064] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 5).

[0065] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0066] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0067] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 7. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 4) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 8), BLASTP, BLASTN, and FASTA (Non-Patent Documents 9, 10, and 11)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0068] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 12. Briefly, the GAP program defines homology as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 13; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application indicates the relevance between sequences.

[0069] Yet another aspect of the present application provides a vector comprising the polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0070] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the target polypeptide in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable microorganism, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0071] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pFS45 vectors may be used.

[0072] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0073] In the present application, "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the microorganism. The transformed polynucleotide may be any polynucleotide that can be expressed in the microorganism, regardless of whether it is located within the chromosome of the microorganism or extrachromosomally. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the microorganism. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to sequences necessary for expression in the microorganism, but is not limited thereto.

[0074] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0075] Yet another aspect of the present application provides a microorganism comprising at least one of the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, and a vector comprising the polynucleotide.

[0076] In one embodiment, the microorganism of the present application may be a microorganism capable of producing 3HP-CoA or a derivative thereof.

[0077] In one embodiment, the microorganism of the present application may be a microorganism capable of producing a compound selected from the group consisting of 3HP-CoA, 3HP, P3HP, P(3HB-3HP), P(4HB-3HP), P(3HP-3HV), acrylic-CoA, acrylic acid, and combinations thereof.

[0078] In this application, "3-hydroxypropionyl-CoA (3HP-CoA)" is a useful intermediate that can be converted into various substances such as acrylic acid (AA), 3HP, and P3HP.

[0079] In the present application, the term "3HP-CoA derivative" refers to a compound obtained by conversion from 3HP-CoA. Examples include, but are not limited to, 3HP, polyhydroxyalkanoates (PHA) homopolymers, PHA copolymers, PHA homopolymers or copolymers containing 3HP as a monomer, acrylic-CoA, and acrylic acid. 3HP-CoA can be easily converted into various 3HP-CoA derivatives by methods well known in the art.

[0080] In one embodiment, the 3HP-CoA derivative is, but is not limited to, 3HP, P3HP, P(3HB-3HP), P(4HB-3HP), P(3HP-3HV), acrylic-CoA, or acrylic acid.

[0081] In this application, "3-hydroxypropionic acid (3HP)" refers to a compound with the chemical formula C3H6O3 and a molecular weight of 90.078, which is used as a platform material for the synthesis of commercially important compounds, specifically, for the production of acrylic acid, acrylic esters, methyl acrylate, acrylamide, ethyl 3-hydroxypropionate, malonic acid, propiolactone, acrylonitrile, 1,3-propanediol, etc. 3-hydroxypropionic acid is also used in the synthesis of biodegradable polymers as one of the monomer units incorporated into polyhydroxyalkanoates (PHAs), and is further useful in fields such as superabsorbent polymers, plastics, and paints.

[0082] In the present application, a "PHA homopolymer" is a compound in which all monomer units are the same. For example, examples of PHA homopolymers include poly-3-hydroxyalkanoic acids such as poly-3-hydroxypropionic acid (P3HP), poly-3-hydroxybutyric acid (PHB), and poly-3-hydroxyvaleric acid; poly-4-hydroxyalkanoic acids such as poly-4-hydroxybutyric acid (P4HB) and poly-4-hydroxyvaleric acid (P4HV); and poly-5-hydroxyalkanoic acids such as poly-5-hydroxyvaleric acid (P5HV).

[0083] In this application, "poly-3-hydroxypropionic acid (P3HP)" refers to a compound that is a type of polyhydroxyalkanoate (PHA) homopolymer, in which all of the monomer units are 3-hydroxypropionic acid.

[0084] In this application, a "PHA copolymer" is a copolymer (containing two or more different monomer units) in which different monomers are randomly distributed in the polymer chain. Examples of PHA copolymers include poly-3-hydroxybutyrate-co-3-hydroxypropionic acid (P(3HB-3HP)), poly-4-hydroxybutyrate-co-3-hydroxypropionic acid (P(4HB-3HP)), poly-3-hydroxypropionic acid-co-3-hydroxyvaleric acid (P(3HP-3HV)), poly-3-hydroxypropionic acid-co-3-hydroxyhexanoic acid (P(3HB-3HH)), poly-3-hydroxybutyrate-co-3-hydroxyhexanoic acid (P(3HB-3HH)), poly-3-hydroxybutyrate-co-3-hydroxypropionic acid (P(4HB-3HP)), poly-3-hydroxybutyrate-co-3-hydroxypropionic acid (P(4HB-3HP)), poly-3-hydroxybutyrate-co-3-hydroxyvaleric acid (P(3HP-3HV)), poly-3-hydroxybutyrate-co-3-hydroxyhexanoic acid (P(3HB-3HH)), poly-3-hydroxybutyrate-co-3-hydroxybutanoic acid (P(4HB-3HP ... Examples include poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-4HB)), poly-3-hydroxybutyrate-co-4-hydroxyvalerate (P(3HB-4HV)), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(3HB-3HV)), poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(3HB-3HH)), and poly-3-hydroxybutyrate-co-5-hydroxyvalerate (P(3HB-5HV)).

[0085] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a desired polypeptide, protein, or product. In this application, the terms "strain" and "microorganism" are used interchangeably and are used interchangeably.

[0086] In this application, the term "microorganism capable of producing 3HP-CoA or a derivative thereof" refers to a prokaryotic or eukaryotic microbial strain that produces 3HP-CoA or a derivative thereof within the organism, and includes microorganisms in which the ability to produce 3HP-CoA or a derivative thereof has been imparted to a parent strain that is incapable of producing 3HP-CoA or a derivative thereof, and microorganisms that endogenously have the ability to produce 3HP-CoA or a derivative thereof. The ability to produce 3HP-CoA or a derivative thereof can be imparted or improved by breeding.

[0087] As an example, the microorganism of the present application may be a microorganism that naturally has the ability to produce a mutant polypeptide having propionaldehyde dehydrogenase activity, 3HP-CoA, or a derivative thereof, or a microorganism to which a mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce a mutant polypeptide having propionaldehyde dehydrogenase activity, 3HP-CoA, or a derivative thereof, and / or to which the ability to produce 3HP-CoA or a derivative thereof has been imparted, but is not limited to these.

[0088] As an example, the microorganism of the present application includes, but is not limited to, a microorganism comprising a variant polypeptide sequence having propionaldehyde dehydrogenase activity of the present application due to a mutation in a gene on a chromosome encoding the variant polypeptide having propionaldehyde dehydrogenase activity, and / or a microorganism comprising a variant polypeptide having propionaldehyde dehydrogenase activity of the present application due to the introduction of a vector comprising a polynucleotide encoding the variant polypeptide having propionaldehyde dehydrogenase activity of the present application.

[0089] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its characteristics are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which a mutant polypeptide having propionaldehyde dehydrogenase activity as used herein has not been introduced or has not been introduced. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "unmodified strain," "non-mutated microorganism," or "reference microorganism."

[0090] A microorganism capable of producing 3HP-CoA or a derivative thereof of the present application may be a microorganism containing at least one of a variant of the present application, a polynucleotide of the present application, and a vector containing a polynucleotide of the present application; a microorganism modified to express a variant of the present application or a polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of a variant of the present application, but is not limited to these.

[0091] For example, the strain of the present application is a cell or microorganism transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expressing the variant of the present application. The strain of the present application may be any microorganism that contains the variant of the present application and produces 3HP-CoA or a derivative thereof. For example, the microorganism of the present application may be a recombinant strain in which a mutant polypeptide having propionaldehyde dehydrogenase activity is expressed by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism capable of producing 3HP-CoA or a derivative thereof, thereby improving the ability to produce 3HP-CoA or a derivative thereof. The recombinant strain with improved ability to produce 3HP-CoA or a derivative thereof is a microorganism that has improved ability to produce 3HP-CoA or a derivative thereof compared to a natural wild-type microorganism or a microorganism without modified propionaldehyde dehydrogenase (e.g., a microorganism expressing wild-type propionaldehyde dehydrogenase or a microorganism not expressing the variant of the present application), but is not limited thereto. For example, but not limited to, a microorganism of the present application with improved ability to produce 3HP-CoA or a derivative thereof is a microorganism with improved ability to produce 3HP-CoA or a derivative thereof compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding it. For example, but not limited to, an unmodified microorganism that is a target strain for comparison to determine whether or not the ability to produce 3HP-CoA or a derivative thereof is improved is the MBX184 strain (Patent Document 2).

[0092] The microorganisms of the present application include all microorganisms that express the mutant polypeptides of the present application having propionaldehyde dehydrogenase activity by various known methods other than the introduction of the nucleic acid or vector.

[0093] For example, a microorganism with improved 3HP-CoA or derivative thereof production ability has an improved 3HP-CoA or derivative thereof production ability of at least about 1%, specifically at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, or at least about 55% (the upper limit is not particularly limited, for example, at most about 200%, at most about 150%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 65%, or at most about 60%) compared to the parent strain or unmodified microorganism before mutation. However, any increase in + value compared to the production ability of the parent strain or unmodified microorganism is acceptable. In other examples, the recombinant strain with improved 3HP-CoA or derivative thereof production ability has an improved 3HP-CoA or derivative thereof production ability of about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.05-fold or more, about 1.1-fold or more, about 1.15-fold or more, about 1.20-fold or more, about 1.25-fold or more, about 1.30-fold or more, about 1.35-fold or more, about 1.40-fold or more, about 1.45-fold or more, about 1.50-fold or more, or about 1.55-fold or more (there is no particular upper limit, and for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, or about 1.6-fold or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0094] The microorganisms of the present application are microorganisms belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hyphomonas, Chromobacterium, and Norcardia, or fungi or yeasts, specifically microorganisms of the genus Escherichia, and more specifically Escherichia coli (E. coli), but are not limited thereto.

[0095] Meanwhile, the Escherichia microorganisms of the present application that are capable of producing 3HP-CoA or a derivative thereof include all of the following: natural wild-type microorganisms themselves; Escherichia microorganisms whose ability to produce 3HP-CoA or a derivative thereof has been improved by strengthening or weakening the activity of a gene involved in the mechanism of production of 3HP-CoA or a derivative thereof; and Escherichia microorganisms whose ability to produce 3HP-CoA or a derivative thereof has been improved by introducing or strengthening the activity of an exogenous gene.

[0096] As a specific example, the Escherichia microorganism of the present application capable of producing 3HP-CoA or a derivative thereof has genes related to the P3HP production mechanism, specifically glycerol dehydratase and PHA synthase, more specifically glycerol dehydratase derived from Klebsiella pneumoniae and PHA synthase derived from Ralstonia eutropha introduced therein, but is not limited thereto. As another example, the Escherichia microorganism capable of producing 3HP-CoA or a derivative thereof of the present application uses 1,3-propanediol as a carbon source, or has a gene involved in the mechanism of 1,3-propanediol production, specifically 1,3-propanediol dehydrogenase, alcohol dehydrogenase, or aldehyde reductase, more specifically 1,3-propanediol dehydrogenase derived from Klebsiella pneumoniae, introduced therein, or has enhanced activity of alcohol dehydrogenase or aldehyde reductase derived from Escherichia coli, but is not limited thereto. Patent Document 2 is incorporated herein by reference in its entirety.

[0097] Furthermore, the microorganism of the present application may have enhanced propionaldehyde dehydrogenase activity compared to the parent strain.

[0098] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity prior to modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change.

[0099] Various methods well known in the art can be applied to enhance the activity of the polypeptide, including, but not limited to, increasing the intracellular copy number of the gene encoding the mutant, introducing a mutation into the expression regulatory sequence of the gene on the chromosome encoding the mutant, replacing the expression regulatory sequence of the gene on the chromosome encoding the mutant with a sequence with stronger activity, replacing the gene encoding the protein on the chromosome with a gene mutated to improve the activity of the mutant, and introducing a mutation into the gene on the chromosome encoding the mutant protein so as to enhance the activity of the mutant.

[0100] In the present application, "transfection" refers to a method for delivering a polynucleotide encoding the acetohydroxyacid synthase variant or a vector containing the same into a host cell. Such introduction can be easily achieved using methods commonly used in the art. Commonly used methods include the CaCl precipitation method, the Hanahan method, which uses DMSO (dimethyl sulfoxide) as a reducing agent to improve the efficiency of the CaCl method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. The method for transforming the vector is not limited to these examples and may be any transformation or transfection method commonly used in the art. Furthermore, the delivered polynucleotide may be inserted into or extrachromosomally located within the host cell, as long as it is expressed in the host cell. Furthermore, the polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide structure containing all elements necessary for its own expression, but is not limited thereto. Typically, the expression cassette contains a promoter operably linked to the open reading frame (ORF) of the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may 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 sequences necessary for expression in the host cell, but is not limited thereto.

[0101] Yet another aspect of the present application provides a method for producing 3HP-CoA or a derivative thereof, comprising culturing the microorganism of the present application in a medium.

[0102] Specifically, the method for producing 3HP-CoA or a derivative thereof of the present application includes, but is not limited to, culturing a microorganism containing at least one of the variants of the present application, the polynucleotides of the present application, and the vectors of the present application in a medium.

[0103] The term "culturing" as used herein means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0104] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0105] In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; amino acids such as glutamic acid, methionine, and lysine; glycerin; and propanediol. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used as long as it is present in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0106] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0107] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0108] Furthermore, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during cultivation of the microorganism of the present application. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0109] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0110] The 3HP-CoA or a derivative thereof produced by the culture of the present invention is secreted into the medium or remains intracellularly.

[0111] In one embodiment, the method for producing 3HP-CoA or a derivative thereof of the present application may further include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), e.g., before the culturing step.

[0112] In one embodiment, the method for producing 3HP-CoA or a derivative thereof of the present application may further include a step of recovering 3HP-CoA or a derivative thereof from the culture medium (the medium in which the culture was carried out) or the microorganism of the present application. The recovery step may be further included after the culture step.

[0113] The recovery may involve collecting 3HP-CoA or a derivative thereof using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used. 3HP-CoA or a derivative thereof can be recovered from the medium or the microorganism using a suitable method known in the art.

[0114] The method for producing 3HP-CoA or a derivative thereof of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the method for producing 3HP-CoA or a derivative thereof of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously in any order, simultaneously, or integrated into a single step, but are not limited thereto.

[0115] Yet another aspect of the present application provides a composition for producing 3HP-CoA or a derivative thereof, comprising a variant polypeptide of the present application, a polynucleotide encoding the variant polypeptide, a vector comprising the polynucleotide, a microorganism comprising at least one of a variant polypeptide of the present application, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide, a culture of the microorganism, or a combination of at least two thereof.

[0116] The compositions of the present application may further comprise any suitable excipients commonly used in compositions for producing 3HP-CoA or its derivatives, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0117] In one embodiment, each component present in the compositions of the present application is included in a microbiologically effective amount, or in any suitable amount in the production composition.

[0118] Yet another aspect of the present application provides the use of a mutant polypeptide of the present application or in a microorganism of the present application for the production of 3HP-CoA or a derivative thereof. [Example]

[0119] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0120] Construction of a DNA library encoding propionaldehyde dehydrogenase mutated by artificial mutation In this example, a vector library was constructed to obtain propionaldehyde dehydrogenase mutants as follows. Error-prone PCR was performed on the pduP gene (SEQ ID NO: 2), which encodes propionaldehyde dehydrogenase (SEQ ID NO: 1) derived from Salmonella enterica, to obtain pduP gene mutants (1,395 bp) with randomly introduced base substitution mutations. Error-prone PCR was performed using the Genemorph II Random Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Primer 1 (SEQ ID NO: 8) and Primer 2 (SEQ ID NO: 9) in Table 1 were used as a template.

[0121] [Table 1]

[0122] The PCR conditions were as follows: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, with 30 cycles of introducing 0 to 2 mutations per 1 Kb of amplified gene fragment.

[0123] The amplified gene fragment was introduced into the pFS45 plasmid (Patent Document 2) so that it was expressed as a single operon under the control of the trc promoter, along with glycerol dehydratase from Klebsiella pneumoniae and PHA synthase from Ralstonia eutropha. The PCR-amplified fragment was ligated to the pFS45 plasmid as a template using Primer 3 (SEQ ID NO: 10) and Primer 4 (SEQ ID NO: 11) in Table 2, using the In-Fusion HD Cloning Kit (Takara Bio Inc., Kusatsu, Japan), and then transformed into Escherichia coli DH5α. The pFS45 plasmid used as a template contained the lacI gene, ampicillin resistance gene (ampR), and origin of replication (Ori) derived from pSE380 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0124] [Table 2]

[0125] After transformation, 20 colonies were selected by plating on LB solid medium containing 100 mg / L ampicillin. Plasmids were then isolated and sequenced, confirming that mutations had been introduced at different positions at a frequency of 0.9 mutations / Kb. Plasmids were extracted from approximately 10,000 transformed E. coli colonies and designated the pFS-pduP(mt) library.

[0126] Additionally, a plasmid containing the wild-type pduP gene was constructed for use as a control. The wild-type pduP gene was synthesized using Primer 1 (SEQ ID NO: 8) and Primer 2 (SEQ ID NO: 9) and used as a template for PCR under the same conditions as above to construct a plasmid in the same form as above. The constructed plasmid was designated pFS-pduP(WT). It is shown in Figure 2. [Example]

[0127] Construction of a propionaldehyde dehydrogenase mutant library and selection of strains with improved P3HP production In this example, to prepare a mutant strain library, the wild-type E. coli LS5218 strain MBX184 (Patent Document 3) was used as the parent strain, and the pFS-pduP(mt) library prepared in Example 1 was transformed into the parent strain by homologous chromosome recombination. Approximately 5,000 colonies were obtained by smearing the resulting culture on LB solid medium containing ampicillin (100 mg / L), and each colony was designated 184 / pFS-pduP(mt)-1 to 184 / pFS-pduP(mt)-5000. A control strain was also prepared by transforming the pFS-pduP(WT) vector prepared in Example 1, and designated 184 / pFS-pduP(WT).

[0128] Approximately 5,000 colonies were inoculated into 350 μL of LB liquid medium containing 100 mg / L ampicillin and cultured in a 96-well plate at 37°C and 1,000 rpm for 16 hours. LB liquid medium was prepared by adjusting the concentration of LB broth (Merk, Darmstadt, Germany) to 25 g / L. Each culture was then centrifuged (2,000 × g, 10 minutes) to remove the supernatant and resuspended in 350 μL of selective medium. The selective medium was prepared to contain 2.5 g of LB broth powder, 50 mmol of phosphate buffer (pH 7), 10 g of glycerol, 2 g of glucose, 5 nmol of coenzyme B-12, 100 μg of ampicillin, and 0.1 mmol of isopropyl β-D-1-thiogalactopyranoside (IPTG) per liter, and then aliquoted into a 96-well plate. The resuspended culture was cultured at 30°C and 1,000 rpm for approximately 24 hours, after which strains predicted to have improved P3HP production were selected based on absorbance measured at 600 nm. This was based on the idea that when propionaldehyde dehydrogenase is weakened, its substrate, 3-hydroxypropionaldehyde (3HPA), accumulates, inhibiting growth. As P3HP production increases, P3HP granules stored within the cells increase the cellular optical density (OD). Mutant strains showing an absorbance increase of 20% or more compared to the control strain were selected. As shown in Table 3, 184 / pFS-pduP(mt)-2101 exhibited a 26.5% increase in absorbance compared to the control, 184 / pFS-pduP(WT), and was therefore selected. The other colonies showed absorbance values ​​that were either comparable to or lower than the control.

[0129] [Table 3] [Example]

[0130] Confirmation of P3HP production ability of strains selected from a propionaldehyde dehydrogenase mutant library and confirmation of pduP gene mutation To confirm whether the P3HP production ability of the 184 / pFS-pduP(mt)-2101 strain obtained in Example 2 was improved compared to the control 184 / pFS-pduP(WT) strain, the two strains were each inoculated into 100 mL of LB liquid medium containing ampicillin (100 mg / L) and cultured in a 500 mL baffled flask at 37°C and 225 rpm for 16 hours. LB liquid medium was prepared by adjusting the concentration of LB broth (Merck, Darmstadt, Germany) to 25 g / L. Each culture was then centrifuged (2000 × g, 10 minutes) to remove the supernatant, and the culture was then resuspended in 100 mL of production medium. The production medium was prepared to contain 2.5 g of LB broth powder, 50 mmol of phosphate buffer (pH 7), 10 g of glycerol, 2 g of glucose, 5 nmol of coenzyme B-12, 100 μg of ampicillin, and 0.1 mmol of isopropyl β-D-1-thiogalactopyranoside (IPTG) per liter, and then dispensed into 500 mL baffled flasks. The resuspended culture was grown at 30°C and 225 rpm for approximately 48 hours, after which the absorbance was measured at 600 nm. 30 mL of the culture was centrifuged and the supernatant was removed.

[0131] The collected cells were washed once with distilled water, then lyophilized and placed in 2 mL of butanol dissolution medium for the butanol dissolution reaction (110°C, 3 h). The butanol dissolution medium was prepared by mixing n-butanol and concentrated aqueous hydrogen chloride solution in a 9:1 volume ratio. Benzoic acid was added at 2 mg / mL as an internal standard. The water-soluble components were removed by layer separation using 3 mL of distilled water after the reaction. The organic layer was then subjected to GC (gas chromatography) analysis. The sample volume was 1 μL, the split ratio was 1:50, and the flow rate was 2 mL / min. The GC column was an SPB-1 fused silica capillary GC column (30 m, 0.32 mm ID, 0.25 μm film; Merck, Darmstadt, Germany). The temperature was increased at 80°C for 2 min, increasing by 10°C per minute until 250°C was reached, and then the temperature was maintained at 250°C for 2 min. β-Propiolactone was used as a standard substance for quantifying P3HP.

[0132] As a result of the analysis, as shown in Table 4, the selected strain 184 / pFS-pduP(mt)-2101 was confirmed to have a 47% increase in the content of P3HP produced and a 67% increase in the titer compared to the control strain 184 / pFS-pduP(WT).

[0133] [Table 4]

[0134] Next, to confirm the random mutations introduced into the propionaldehyde dehydrogenase gene of one of the selected strains, the nucleotide sequence of the pduP gene was analyzed using primer 5 (SEQ ID NO: 12) and primer 6 (SEQ ID NO: 13) in Table 5.

[0135] [Table 5]

[0136] After analyzing the nucleotide sequence of the isolated mutant pduP gene, the nucleotide sequence of the mutant pduP gene was confirmed by comparing it with the wild-type pduP gene sequence of SEQ ID NO: 2. This confirmed the amino acid sequence of the mutated propionaldehyde dehydrogenase protein. As shown in Table 6, the mutated propionaldehyde dehydrogenase protein of the selected 184 / pFS-pduP(mt)-2101 strain was confirmed to have a mutation of the 272nd amino acid from isoleucine to threonine.

[0137] [Table 6] [Example]

[0138] Construction of vectors containing DNA encoding propionaldehyde dehydrogenase with amino acid substitutions at the same mutation site and comparison of P3HP production ability In order to confirm the effect of the mutation position in the mutated propionaldehyde dehydrogenase protein confirmed in Example 3, a vector containing a mutation in which the amino acid at position 272 was substituted with an amino acid other than isoleucine or threonine was constructed.

[0139] First, to prepare the vector, PCR was performed using the synthesized wild-type pduP gene as a template and primer 1 (SEQ ID NO: 8), primer 7 (SEQ ID NO: 14), primer 8 (SEQ ID NO: 15), and primer 2 (SEQ ID NO: 9) in Table 7 to amplify a DNA fragment of approximately 830 bp and a DNA fragment of approximately 600 bp, respectively. The PCR conditions were 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds.

[0140] [Table 7]

[0141] The gene fragment amplified as described above was ligated to the PCR-amplified pFS45 plasmid site using Primer 3 (SEQ ID NO: 10) and Primer 4 (SEQ ID NO: 11) using the In-Fusion HD Cloning Kit (Takara Bio Inc., Kusatsu, Japan), as in Example 1. The resulting fragment was then transformed into Escherichia coli DH5α, yielding a plasmid vector containing a mutation in which the 272nd amino acid of propionaldehyde dehydrogenase had been replaced with glycine. The resulting plasmid was designated pFS-pduP(I272G).

[0142] Similarly, 17 additional plasmids containing mutations in which the 272nd amino acid of propionaldehyde dehydrogenase was substituted with an amino acid other than isoleucine, threonine, or glycine were constructed using primers containing the nucleotide sequence encoding the substituted amino acid. The primer sequences used are shown in Table 8. The substituted amino acid is indicated in each primer.

[0143] [Table 8]

[0144] MBX184 was transformed by homologous chromosomal recombination using all 18 plasmids prepared as described above as a parent strain, and the transformed strains were smeared on LB solid medium containing ampicillin (100 mg / L) to prepare strains carrying mutant propionaldehyde dehydrogenase. The constructed plasmids and the names of the introduced strains are shown in Table 9. Each of the plasmids listed in Table 9 contains a gene encoding a propionaldehyde dehydrogenase mutant inserted into it so that it is expressed as a single operon under the control of the trc promoter together with glycerol dehydratase derived from Klebsiella pneumoniae and PHA synthase derived from Ralstonia eutropha, as in the pFS-pduP(WT) plasmid in Example 1. It also contains the lacI gene, ampicillin resistance gene (ampR), and replication origin (Ori) derived from pSE380 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0145] [Table 9]

[0146] To evaluate the P3HP-producing ability of strains containing the prepared propionaldehyde dehydrogenase mutants, all 20 strains, including the control strain 184 / pFS-pduP(WT) and the 184 / pFS-pduP(mt)-2101 strain, were cultured in the same manner as in Example 3, and their P3HP-producing ability was analyzed. The results are shown in Table 10.

[0147] [Table 10]

[0148] As a result, it was confirmed that all novel mutations in which the 272nd amino acid of propionaldehyde dehydrogenase was replaced with an amino acid selected from valine, threonine, tyrosine, asparagine, and glutamine increased the amount of P3HP produced compared to the control group.

[0149] Specifically, the 184 / pFS-pduP(I272N) strain, in which the 272nd amino acid of propionaldehyde dehydrogenase was substituted with asparagine, produced 55% more P3HP and 104% more P3HP than the control strain 184 / pFS-pduP(WT).

[0150] Therefore, it was confirmed that the 272nd position of propionaldehyde dehydrogenase is an important position for P3HP production.

[0151] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. A mutant polypeptide having propionaldehyde dehydrogenase activity, in which the amino acid corresponding to position 272 of SEQ ID NO: 1 has been substituted with another amino acid.

2. The mutant polypeptide of claim 1, wherein the amino acid corresponding to position 272 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of glycine, alanine, arginine, valine, leucine, methionine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid.

3. The mutant polypeptide of claim 1, wherein the mutant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 7 and SEQ ID NO: 33 to SEQ ID NO:

46.

4. A polynucleotide encoding the mutant polypeptide of any one of claims 1 to 3.

5. A microorganism comprising at least one of the mutant polypeptide according to any one of claims 1 to 3, a polynucleotide encoding said mutant polypeptide, and a vector comprising said polynucleotide.

6. The microorganism according to claim 5, wherein the microorganism has improved ability to produce 3HP-CoA or a derivative thereof, compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

7. The microorganism according to claim 5 , wherein the microorganism is an Escherichia microorganism.

8. The microorganism according to claim 7, wherein the Escherichia microorganism is Escherichia coli.

9. A method for producing 3HP-CoA or a derivative thereof, comprising the step of culturing the microorganism according to claim 5 in a medium.

10. The method of claim 9, further comprising the step of recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

11. 10. A composition for producing 3HP-CoA or a derivative thereof, comprising the mutant polypeptide of claim 1, or a microorganism comprising at least one of the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, and a vector comprising the polynucleotide, or a combination thereof.

12. 10. Use of the mutant polypeptide of claim 1, or a microorganism comprising at least one of said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, and a vector comprising said polynucleotide, for the production of 3HP-CoA or a derivative thereof.

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