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Definitions

• the present invention relates to a method for producing methacrylic acid, and more specifically to a method for producing methacrylic acid by decarboxylating mesaconic acid or an isomer thereof using protocatechuic acid decarboxylase.
• the present invention also relates to a variant of protocatechuate decarboxylase that can be used for the production, a DNA encoding the variant, a vector containing the DNA, and a host cell into which the DNA or the vector has been introduced.
• the present invention relates to a method for producing the modified product.
• the present invention relates to an agent for promoting the production of methacrylic acid, including the above-described modified product.
• Methacrylic acid is a corrosive liquid with a pungent odor.
• esters in the form of esters, it is widely used not only as a raw material for synthetic polymers such as acrylic resins, but also in paints, adhesives, and paint solvents.
• acrylic resin has high transparency and excellent weather resistance, so it is in high demand as a useful material to replace glass in a wide range of fields such as signboards, lighting equipment, automobile parts, and construction-related materials.
• Methods for chemically producing methacrylic acid derivatives include the ACH method via acetone cyanohydrin (ACH) using hydrocyanic acid and acetone as raw materials, and the C4 oxidation method using isobutylene or tert-butanol as raw materials. ing.
• ACH acetone cyanohydrin
• Non-Patent Document 1 a reaction has been suggested in which methacrylic acid is produced in one step from mesaconic acid derived from microorganisms (Patent Document 1).
• the enzyme involved in the production has not been clarified, and an efficient method for producing methacrylic acid has not yet been developed.
• the present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a method that can produce methacrylic acid from a carbon source.
• protocatechuate decarboxylase derived from multiple bacterial species has catalytic activity to decarboxylate mesaconic acid and produce methacrylic acid. Ta.
• the present invention provides the following aspects.
• a method for producing methacrylic acid comprising a step of decarboxylating mesaconic acid or an isomer thereof in the presence of protocatechuic acid decarboxylase.
• the decarboxylation is performed in a cell expressing protocatechuate decarboxylase, and includes the step of culturing the cell and collecting methacrylic acid produced in the cell and/or the culture.[1 The manufacturing method described in ].
• the protocatechuate decarboxylase is a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 8, 12 or 16, [1] or [2] The manufacturing method described in ].
• position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine.
• position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to the position is methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine.
• Position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this site is modified to methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine, and mesacon Protocatechuate decarboxylase having catalytic activity to produce methacrylic acid from the acid or its isomers.
• Position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position has been modified to methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine, and , protocatechuate decarboxylase that has been modified with at least one of the amino acids listed in (a) to (e) below (a) position 185 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position to valine, isoleucine, methionine, threonine, serine, asparagine, leucine, tyrosine, histidine, or glutamine.
• a method for producing protocatechuate decarboxylase which comprises the steps of culturing the host cell according to [10] and collecting the protein expressed in the host cell.
• a method for producing protocatechuate decarboxylase with enhanced catalytic activity for producing methacrylic acid from mesaconic acid or its isomer comprising protocatechuate decarboxylase at position 327 of the amino acid sequence set forth in SEQ ID NO: 2.
• a manufacturing method comprising the step of modifying the amino acid corresponding to the site to methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine.
• a method for producing protocatechuate decarboxylase with enhanced catalytic activity for producing methacrylic acid from mesaconic acid or its isomer comprising: position 327 of the amino acid sequence set forth in SEQ ID NO: 2; or the amino acid corresponding to the site is modified to methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine, and at least one of the following (a) to (e) is added.
• a manufacturing method including a step of modifying amino acids.
• An agent for decarboxylating mesaconic acid or its isomer and promoting the production of methacrylic acid which contains protocatechuate decarboxylase, a DNA encoding the protocatechuate decarboxylase, or a vector into which the DNA is inserted.
• the protocatechuate decarboxylase is a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, 8, 12 or 16, [14]. agent.
• position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine.
• position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is methionine, phenylalanine, asparagine, isoleucine, glutamine, serine, valine, threonine, tyrosine, or leucine. and has at least one amino acid listed in the following (a) to (e), the agent according to [14] (a) position 185 of the amino acid sequence set forth in SEQ ID NO: 2 or the site thereof The amino acid corresponding to valine, isoleucine, methionine, threonine, serine, asparagine, leucine, tyrosine, histidine, or glutamine.
• methacrylic acid from a carbon source.
• Klebsiella pneumoniae Kp
• Bacillus sp. Bs is a graph showing the results of analyzing the catalytic activity for producing methacrylic acid of protocatechuate decarboxylase derived from Lactiplantibacillus pentosus (Lp) or Companilactobacillus farciminis (Cf).
• PDC protocatechuate decarboxylase
• Mesaconic acid which is a substrate in the present invention is a compound also called (2E)-2-methyl-2-butenedioic acid.
• isomers for example, citraconic acid ((2Z)-2-methyl-2-butenedioic acid, 2-methylmaleic acid), itaconic acid (2-propene-1, 2-dicarboxylic acid, 2-methylidenebutanedioic acid, 2-methylenesuccinic acid).
• these compounds can be purchased as commercial products, for example, as shown in the Examples below.
• methacrylic acid (2-methylprop-2-enoic acid) is obtained by eliminating (decarboxylation) one of the carboxy groups in these dicarboxylic acid compounds.
• the conditions for decarboxylating mesaconic acid or its isomers in the presence of PDC may be any conditions that promote the decarboxylation and produce methacrylic acid.
• the composition, pH of the reaction solution, reaction temperature, reaction time, etc. can be adjusted and set as appropriate. Further, in this case, only one type of PDC may be used, but two or more types may be used.
• the reaction solution to which PDC and its substrate mesaconic acid or its isomer are added is not particularly limited as long as it does not interfere with the reaction, but a buffer solution with a pH of 6 to 10 is preferred, and more preferably a buffer solution with a pH of 6 to 10.
• a buffer solution with a pH of 6 to 10 is preferred, and more preferably a buffer solution with a pH of 6 to 10.
• Examples include buffers with a pH of 6.5 to 9.5, more preferably buffers with a pH of 6 to 7 (eg, buffers containing potassium chloride and sodium phosphate).
• prFMN prenylated flavin mononucleotide
• prFMN ketimine prFMN iminium
• prFMN and its isomers have been described by Karl A.P. Payne et al. Nature, published June 25, 2015, volume 522, number 7557, pages 497-501).
• the reaction temperature is also not particularly limited as long as it does not interfere with the reaction, but it is usually 10 to 60°C, preferably 10 to 50°C (for example, 25 to 37°C). Furthermore, the reaction time is not particularly limited as long as it can produce methacrylic acid, but is usually 30 minutes to 7 days, preferably 12 hours to 2 days.
• methacrylic acid produced under such conditions can be isolated from other components and collected (recovered) by a known method.
• known methods are not particularly limited, but include, for example, solvent extraction methods (e.g., continuous liquid-liquid extraction), pervaporation methods, membrane filtration methods, membrane separation methods, reverse osmosis methods, electrodialysis methods, distillation, and crystallization methods.
• chromatography eg, gas chromatography, ion exchange chromatography, size exclusion chromatography, adsorption chromatography
• ultrafiltration e.g, gas chromatography, ion exchange chromatography, size exclusion chromatography, adsorption chromatography
• these methods for collecting methacrylic acid may be carried out independently, or may be carried out in multiple stages by appropriately combining them.
• methacrylic acid could be produced by culturing host cells transformed to express PDC.
• a method for producing methacrylic acid which includes a step of collecting the methacrylic acid.
• the "cell that expresses PDC” will be described later, and such cells may express only one type of PDC, but may also express two or more types of PDC.
• the culture conditions for the cells are as described below, it is preferable that mesaconic acid or its isomer, which serves as a substrate for PDC in the present invention, is added to the medium.
• the culture temperature can be changed as appropriate depending on the type of host cell used, but is usually 20 to 40°C, preferably 25 to 37°C.
• the "culture” refers to a medium containing proliferated cells, secreted products of the cells, metabolic products of the cells, etc., obtained by culturing host cells in a medium, Including their dilutions and concentrates.
• timing of collection may be adjusted as appropriate depending on the type of host cell used, and may be any time that allows methacrylic acid to be produced, but it is usually 30 minutes to 7 days, preferably 12 hours to 2 days. be.
• PDC Protocatechuic acid decarboxylase
• PDC is not particularly limited as long as it has an activity that catalyzes the decarboxylation reaction.
• PDC derived from Klebsiella pneumoniae (for example, UniProtKB-B9A9M6) Specified protein (typically an enzyme comprising the amino acid sequence set forth in SEQ ID NO: 2), Bacillus sp.
• PDC derived from Lactiplantibacillus pentosus for example, a protein specified by UniProtKB-A0A2A8FJC5, typically an enzyme comprising the amino acid sequence set forth in SEQ ID NO: 8
• PDC derived from Lactiplantibacillus pentosus for example, a protein specified by UniProtKB-A0A2S9VXY3
• protein typically an enzyme comprising the amino acid sequence set forth in SEQ ID NO: 12
• PDC derived from Companilactobacillus farciminis e.g., a protein specified by UniProtKB-A0A0H4LCR3, typically an enzyme comprising the amino acid sequence set forth in SEQ ID NO: 16
• An enzyme containing the described amino acid sequence can be used.
• proteins corresponding to "3,4-dihydroxybenzoate decarboxylase” or “Protocatechuate decarboxylase” on UNIPROT can be mentioned, and more specifically, Clostridium those derived from hydroxybenzoicum (e.g.
• UniProtKB-P86833 Enterobacter cloacae (e.g., UniProtKB-A0A7G3F3G5), Pseudopedobacter saltans (e.g., UniProtKB-A0A) 2W5F854), Gilliamella apicola-derived proteins (for example, proteins specified in UniProtKB-A0A1B9JW43) or Lactobacillus pentosus-derived substances (for example, proteins specified in UniProtKB-A0A241RRA2) can also be used.
• Enterobacter cloacae e.g., UniProtKB-A0A7G3F3G5
• Pseudopedobacter saltans e.g., UniProtKB-A0A 2W5F854
• Gilliamella apicola-derived proteins for example, proteins specified in UniProtKB-A0A1B9JW43
• Lactobacillus pentosus-derived substances for example
• the PDC according to the present invention may be a homolog of the PDC derived from the above bacteria such as Klebsiella pneumoniae.
• Homologues of PDC are not particularly limited as long as they have catalytic activity to produce methacrylic acid; , preferably 15% or more (for example, 16% or more, 17% or more, 18% or more, 19% or more), more preferably 20% or more (for example, 30% or more, 40% or more), It is more preferably 50% or more (e.g., 60% or more, 70% or more), and more preferably 80% or more (e.g., 85% or more, 86% or more, 87% or more, 88% or more, 89% or more).
• identity refers to the number of amino acids that match the decarboxylase of the present invention and the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16, relative to the total number of amino acids of the decarboxylase of the present invention.
• “Homology” refers to the ratio (%) of the number of amino acids similar to the decarboxylase according to the present invention and the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16 to the total number of amino acids in the decarboxylase according to the present invention. means.
• similar amino acids refers to a combination of amino acids with a BLOSUM62 substitution score greater than 0.
• the catalytic activity for producing methacrylic acid can be improved by substituting an amino acid at a specific site of PDC with another amino acid. Therefore, the PDC according to the present invention has one or more amino acid substitutions, deletions, additions, and/or "Proteins consisting of inserted amino acid sequences (PDC variants)" are also included.
• “plurality” is not particularly limited, but usually 2 to 300, preferably 2 to 250, more preferably 2 to 200, even more preferably 2 to 150, more preferably 2 to 100.
• the mutation introduced in PDC as long as the catalytic activity for producing methacrylic acid is improved compared to before the introduction, but for example, the mutation at position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or Examples include substitution of the amino acid corresponding to the site with another amino acid.
• corresponding site refers to amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). , refers to a site that is aligned with a specific site in the amino acid sequence set forth in SEQ ID NO:2 when aligned with the amino acid sequence set forth in SEQ ID NO:2 (for example, "SEQ ID NO:2)".
• position 327 of the amino acid sequence or the amino acid corresponding to the position refers to the amino acid at the same position as histidine at position 327 in the amino acid sequence set forth in SEQ ID NO: 2).
• substitution with another amino acid includes, for example, substitution with an amino acid other than histidine, and preferably with a polar neutral amino acid or a hydrophobic amino acid. Examples include substitution with amino acids.
• substitutions with such "polar neutral amino acids” include substitutions with asparagine, glutamine, serine, threonine, tyrosine, or cysteine, and more preferably substitutions with asparagine, glutamine, serine, threonine, or tyrosine. Can be mentioned. More preferred is substitution with asparagine or glutamine, and more preferred is substitution with asparagine.
• substitution with a "hydrophobic amino acid” include substitution with methionine, phenylalanine, isoleucine, valine, or leucine, and more preferably substitution with methionine or phenylalanine.
• mutations such as amino acid substitutions may be introduced at one or more other positions.
• Such mutations at other sites are preferably mutations at position 185 or the amino acid corresponding to the site, position 331 or the amino acid corresponding to the site, or position 298 or the amino acid corresponding to the site of the amino acid sequence set forth in SEQ ID NO:2. Substitution with another amino acid at at least one position selected from the amino acid at position 183 or corresponding to this position, and the amino acid at position 438 or corresponding to this position.
• Position 438 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to the position The amino acid corresponding to the site is changed to isoleucine, methionine, valine, or threonine. Note that such amino acid substitution at other sites is preferably combined with modifying position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this site to methionine.
• whether or not PDC has a catalytic activity to produce methacrylic acid can be determined by, for example, directly measuring the amount of methacrylic acid using gas chromatography-mass spectrometry (GC-MS), as shown in the Examples below. It can be determined by Furthermore, by comparing the amount in wild-type PDC (for example, PDC containing the amino acid sequence set forth in SEQ ID NO: 2, 8, 12, or 16), it is possible to determine whether the catalytic activity for producing methacrylic acid is higher than that in the PDC. It can also be determined whether or not.
• GC-MS gas chromatography-mass spectrometry
• the methacrylic acid according to the present invention has a catalytic activity for producing methacrylic acid that is twice or more (for example, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more), more preferably 10 times or more (for example, 20 times or more, 30 times or more, 40 times or more), and 50 times or more (For example, 60 times or more, 70 times or more, 80 times or more, 90 times or more), more preferably 100 times or more (for example, 110 times or more, 120 times or more, 130 times or more, 140 times or more) More preferably, it is 150 times or more (for example, 160 times or more, 170 times or more, 180 times or more, 190 times or more), and even more preferably 200 times or more (for example, 210 times or more, 220 times or more). It is more preferable.
• Other compounds may be added directly or indirectly to the PDC according to the present invention. Such addition is not particularly limited, and may be addition at the gene level or chemical addition. There is also no particular restriction on the site to be added, and it may be either the amino terminal (hereinafter also referred to as "N-terminus”) or the carboxyl terminal (hereinafter also referred to as "C-terminus”) of the PDC according to the present invention. It may be both. Addition at the gene level is achieved by using a DNA encoding the PDC of the present invention with DNA encoding another protein added in reading frame.
• polyhistidine (His-) tag proteins FLAG- Tag proteins for purification such as Tag Protein (registered trademark, Sigma-Aldrich) and glutathione-S-transferase (GST) are preferably used, and for the purpose of facilitating the detection of PDC according to the present invention, Detection tag proteins such as fluorescent proteins such as GFP and chemiluminescent proteins such as luciferase are preferably used. Chemical attachment may be covalent or non-covalent.
• covalent bond there are no particular restrictions on the "covalent bond,” and examples include an amide bond between an amino group and a carboxyl group, an alkylamine bond between an amino group and an alkyl halide group, a disulfide bond between thiols, a thiol group and a maleimide group, or an alkyl halide. Examples include thioether bonds with groups. Examples of “non-covalent bonds” include biotin-avidin bonds. Further, as the "other compound” that is chemically added in this way, for example, fluorescent dyes such as Cy3 and rhodamine are preferably used for the purpose of facilitating the detection of PDC according to the present invention. It will be done.
• the PDC according to the present invention may be used in combination with other components.
• Other components are not particularly limited and include, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protease inhibitors, and preservatives.
• DNA encoding the PDC according to the present invention will be explained. By introducing such DNA, it becomes possible to transform the host cell and produce the PDC according to the present invention in the cell, which in turn makes it possible to produce methacrylic acid.
• the DNA according to the present invention may be natural DNA, DNA in which mutations have been artificially introduced into natural DNA, or artificial DNA, as long as it encodes the PDC according to the present invention described above. It may be DNA consisting of a designed nucleotide sequence. Further, there are no particular limitations on its form, and in addition to cDNA, genomic DNA and chemically synthesized DNA are included. These DNAs can be prepared by those skilled in the art using conventional methods. For example, genomic DNA is extracted from Klebsiella pneumoniae, etc., a genomic library is created (plasmid, phage, cosmid, BAC, PAC, etc. can be used as a vector), and this is developed to extract the PDC gene.
• It can be prepared by performing colony hybridization or plaque hybridization using a probe prepared based on the nucleotide sequence (for example, the nucleotide sequence set forth in SEQ ID NO: 1, 7, 11, or 15). Alternatively, it can be prepared by creating primers specific to the PDC gene and performing PCR using the primers. In addition, for example, cDNA is synthesized based on mRNA extracted from Klebsiella pneumoniae, etc., and inserted into a vector such as ⁇ ZAP to create a cDNA library. It can be prepared by hybridization or plaque hybridization, or by PCR.
• a person skilled in the art can introduce a mutation to substitute another amino acid at position 327, etc., into the DNA thus prepared, if necessary, by using a known site-directed mutagenesis method. It can be carried out.
• Site-directed mutagenesis methods include, for example, the Kunkel method (Kunkel, T.A., Proc Natl Acad Sci USA, 1985, Vol. 82, No. 2, pp. 488-492), SOE (splicing-by-overlap- extension) - PCR method (Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R., Gene, 1989, 77 volumes, (pages 51-59).
• nucleotide sequence encoding PDC in which position 327 is substituted with another amino acid can, for example, artificially design a nucleotide sequence encoding PDC in which position 327 is substituted with another amino acid, and based on the sequence information, use an automatic nucleic acid synthesizer to create the present invention.
• the DNA according to the invention can also be chemically synthesized.
• the DNA according to the present invention is a PDC according to the present invention whose codons are optimized according to the type of the host cell. It can also take the form of encoding DNA.
• the present invention may also take the form of a vector into which the aforementioned DNA is inserted so that the DNA can be replicated within a host cell.
• the "vector" can be constructed based on a self-replicating vector, that is, a vector that exists as an independent entity outside the chromosome, and whose replication does not depend on the replication of the chromosome, for example, on the basis of a plasmid. Furthermore, when the vector is introduced into a host cell, it may be integrated into the host cell's genome and replicated together with the chromosome into which it has been integrated.
• Plasmids include Escherichia coli-derived plasmids (pET22, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), and Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.).
• Examples of phage DNA include ⁇ phages (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.).
• the host cell is insect-derived, insect virus vectors such as baculovirus, if plant-derived, animal virus vectors such as T-DNA, and if animal-derived, animal virus vectors such as retroviruses and adenovirus vectors can also be used. It can also be used as a vector for.
• procedures and methods for constructing vectors according to the present invention can be those commonly used in the field of genetic engineering. For example, in order to insert the DNA according to the present invention into a vector, the purified DNA is first cut with an appropriate restriction enzyme, inserted into the restriction enzyme site or multi-cloning site of an appropriate vector, and ligated to the vector. etc. will be adopted.
• the vector according to the present invention may be in the form of an expression vector containing the PDC according to the present invention encoded by the DNA in a state capable of being expressed in a host cell.
• the "expression vector” contains a DNA sequence that controls the expression and a transformed host cell. It is desirable to include genetic markers for selection. Examples of DNA sequences that control expression include promoters, enhancers, splicing signals, polyA addition signals, ribosome binding sequences (SD sequences), terminators, and the like.
• the promoter is not particularly limited as long as it exhibits transcriptional activity in the host cell, and can be obtained as a DNA sequence that controls the expression of a gene encoding a protein that is homologous or heterologous to the host cell. Furthermore, in addition to the DNA sequence that controls expression, it may also contain a DNA sequence that induces expression. Examples of DNA sequences that induce such expression include, when the host cell is a bacterium, the addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) to induce the expression of downstream genes. The lactose operon is an example of this.
• the genetic marker in the present invention may be appropriately selected depending on the method of selecting the transformed host cell, and for example, a gene encoding drug resistance or a gene complementary to auxotrophy can be used.
• DNA or vector according to the present invention may be used in combination with other components.
• Other components are not particularly limited and include, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, DNase inhibitors, and preservatives.
• the present invention provides an agent for decarboxylating mesaconic acid or its isomer and promoting the production of methacrylic acid, which comprises the above-mentioned PDC, a DNA encoding the PDC, or a vector into which the DNA is inserted. provide.
• Such an agent may be one containing the PDC according to the present invention, but it may also be used in combination with other components.
• Such other components are not particularly limited and include, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizing agents, buffers, protease inhibitors, DNase inhibitors, and preservatives.
• the present invention can also provide a kit containing such an agent.
• the above-mentioned agent may be contained in the form of a host cell described below into which the DNA of the present invention has been introduced and transformed.
• this book also includes mesaconic acid or its isomers, host cells for introducing the DNA of the present invention, media for culturing the host cells, and instructions for their use. It may be included in the kit of the invention.
• instructions for use are instructions for using the agent of the present invention in the above-mentioned method for producing methacrylic acid.
• the instructions include, for example, information regarding the experimental method and experimental conditions of the production method of the present invention, the agent of the present invention, etc. (for example, information such as a vector map showing the nucleotide sequence of the vector, etc., the PDC according to the present invention). sequence information, the origin and properties of the host cell, information on the culture conditions of the host cell, etc.).
• cells expressing PDC according to the present invention include cells that naturally express PDC (e.g., Klebsiella pneumoniae, Bacillus sp., Lactiplantibacillus pentosus, Companilactobacillus farciminis, Clos tridium hydroxybenzoicum, Enterobacter cloacae, Pseudopedobacter saltans, Gilliamella apicola, Lactobacillus pentosus).
• PDC e.g., Klebsiella pneumoniae, Bacillus sp., Lactiplantibacillus pentosus, Companilactobacillus farciminis, Clos tridium hydroxybenzoicum, Enterobacter cloacae, Pseudopedobacter saltans, Gilliamella apicola, Lactobacillus pentosus.
• it may be a host cell (transformant) into which the DNA or vector according to the present invention has been introduced.
• the host cells into which the DNA or vector according to the present invention is introduced are not particularly limited, and include, for example, microorganisms (E. coli, Saccharomyces cerevisiae, fission yeast, Bacillus subtilis, actinomycetes, filamentous fungi, etc.), plant cells, insect cells, and animal cells.
• microorganisms E. coli, Saccharomyces cerevisiae, fission yeast, Bacillus subtilis, actinomycetes, filamentous fungi, etc.
• plant cells insect cells
• animal cells it is preferable to use microorganisms as host cells from the viewpoint that they exhibit high growth in a relatively inexpensive medium in a short time, and can contribute to the production of methacrylic acid with high productivity.
• Escherichia coli it is more preferable to use Escherichia coli.
• the host cell into which the DNA or vector according to the present invention is introduced induces prenylation of flavin mononucleotide (FMN) and produces prFMN or its isomer that contributes to improved productivity of methacrylic acid
• the cells retain flavinprenyltransferase.
• Methods for introducing microorganisms such as E. coli include heat shock method, electroporation method, spheroplast method, and lithium acetate method
• methods for introducing into plant cells include methods using Agrobacterium
• methods for introducing insect cells include the particle gun method, methods using baculovirus and electroporation
• methods for introducing into animal cells include the calcium phosphate method, lipofection, and electroporation.
• the DNA etc. introduced into the host cell in this way may be maintained in the host cell by being randomly inserted into the genomic DNA, or may be maintained by homologous recombination, or may be maintained by vectors. For example, it can be replicated and maintained as an independent entity outside of its genomic DNA.
• ⁇ Method for producing PDC variants of the present invention As shown in Examples below, by culturing host cells into which DNA encoding the PDC variant of the present invention has been introduced, a PDC variant can be produced in the host cell.
• the present invention provides a method for producing a PDC variant comprising the steps of culturing a host cell into which a DNA encoding the PDC variant of the present invention or a vector containing the DNA has been introduced, and collecting the protein expressed in the host cell.
• a manufacturing method can also be provided.
• the conditions for "cultivating host cells” may be any conditions that allow the host cells to produce the PDC variant of the present invention, and those skilled in the art will be able to , temperature, whether or not air is added, oxygen concentration, carbon dioxide concentration, pH of the medium, culture temperature, culture time, humidity, etc. can be adjusted and set as appropriate.
• Such a medium may contain anything that can be assimilated by host cells, such as carbon sources, nitrogen sources, sulfur sources, inorganic salts, metals, peptones, yeast extracts, meat extracts, casein hydrolysates, serum, etc. Listed as inclusions.
• such a medium may contain, for example, IPTG for inducing the expression of the DNA encoding the PDC variant of the present invention, or an antibiotic (for example, ampicillin) corresponding to the drug resistance gene that can be encoded by the vector according to the present invention. ) or a nutrient (eg, arginine, histidine) corresponding to a gene that complements the auxotrophy that can be encoded by the vector of the present invention.
• the host cells are collected from the culture medium by filtration, centrifugation, etc., and the collected host cells are Processing by dissolution, grinding, pressure crushing, etc., and further, ultrafiltration, salting out, solvent precipitation such as ammonium sulfate precipitation, chromatography (e.g., gel chromatography, ion exchange chromatography, affinity chromatography), etc.
• solvent precipitation such as ammonium sulfate precipitation
• chromatography e.g., gel chromatography, ion exchange chromatography, affinity chromatography
• examples include methods for purifying and concentrating proteins expressed in host cells.
• the PDC variant of the present invention has the purified tag protein added thereto, it can also be purified and collected using a substrate to which the tag protein is adsorbed.
• these purification and concentration methods may be carried out independently or may be carried out in multiple stages by appropriately combining them.
• the PDC variant of the present invention is not limited to the above-mentioned biological synthesis, but can also be produced using the DNA, etc. of the present invention and the cell-free protein synthesis system.
• Such cell-free protein synthesis systems are not particularly limited, but include, for example, wheat germ-derived, Escherichia coli-derived, rabbit reticulocyte-derived, and insect cell-derived synthesis systems.
• those skilled in the art can also chemically synthesize the PDC variant of the present invention using a commercially available peptide synthesizer or the like.
• the present invention also provides a method for producing PDC with enhanced catalytic activity for producing methacrylic acid, in which position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is substituted with other It is also possible to provide a production method that includes a step of modifying an amino acid (for example, the above-mentioned polar neutral amino acid or hydrophobic amino acid) and, in some cases, further modifying an amino acid at another site.
• an amino acid for example, the above-mentioned polar neutral amino acid or hydrophobic amino acid
• PDC with enhanced catalytic activity for producing methacrylic acid refers to a PDC that is produced by introducing a mutation into position 327 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position, and in some cases, By further introducing a mutation into the amino acid at the other site, it means a PDC that has a higher catalytic activity for producing methacrylic acid than before the introduction.
• the objects of comparison are usually PDCs derived from various organisms such as the above-mentioned Klebsiella pneumoniae and natural variants thereof.
• Modification to other amino acids in PDC can be performed by modifying the encoding DNA.
• Modification of DNA refers to such DNA modification using methods known to those skilled in the art, such as site-directed mutagenesis and chemical synthesis of DNA based on modified sequence information. It is possible to implement it as appropriate using the following methods. Furthermore, “modification to other amino acids” can also be carried out using a chemical peptide synthesis method, as described above.
• Protocatechuic acid decarboxylase (hereinafter also referred to as "PDC") is originally known to have a catalytic activity related to a reaction in which catechol is produced through a decarboxylation reaction using protocatechuic acid (PCA) as a substrate.
• PCA protocatechuic acid
• the present inventors have now verified the possibility that PDC has catalytic activity for a reaction in which methacrylic acid is produced through decarboxylation using mesaconic acid as a substrate, as described below.
• the verification targeted PDCs derived from the following hosts.
• “homology” in the table below indicates the homology between the amino acid sequence of PDC derived from Klebsiella pneumoniae and those derived from other hosts.
• plasmid vector capable of expressing protocatechuic acid decarboxylase a wild type (Klebsiella pneumoniae, Bacillus sp., Lactiplantibacillus pentosus or Companilactobacillus farciminis) encoding it is prepared. (SEQ ID NO: 1, 7, 11 or 15, respectively)
• SEQ ID NO: 1, 7, 11 or 15, respectively The nucleotide sequence described in SEQ ID NO: 3, 9, 13, or 17 was modified in the form of a polyhistidine tag fused to the C-terminus of the nucleotide sequence described in SEQ ID NO: 3, 9, 13, or 17, respectively. ).
• DNA consisting of the modified nucleotide sequence was chemically synthesized according to a conventional method. Then, the DNA thus prepared and the pET22b(+) vector (manufactured by Novagen) are ligated by the Gibson Assembly method (using the kit NEBuilder HiFi DNA Assembly Master Mix (registered trademark) of New England Biolabs). by A plasmid vector (PDC vector) capable of expressing the wild-type PDC in E. coli was prepared.
• PDC vector plasmid vector capable of expressing the wild-type PDC in E. coli was prepared.
• a gene encoding flavin prenyltransferase (nucleotide sequence set forth in SEQ ID NO: 5) was amplified from Escherichia coli (K-12) strain using the Polymerase Chain Reaction method, and DNA was amplified using the pColADuet vector. (manufactured by Novagen) by the Gibson Assembly method to prepare a plasmid vector (UbiX vector) capable of expressing the wild-type UbiX in E. coli.
• the transformant was cultured for 6 hours in LB medium supplemented with ampicillin and kanamycin. Note that the growth of the transformant reaches a plateau after 6 hours of culture (preculture).
• the collected E. coli culture solution was centrifuged at 150,000 rpm for 15 minutes, and 200 ⁇ L of the supernatant was collected in a 1.5 mL tube.
• Methacrylic acid was extracted from the E. coli culture solution into ethyl acetate by adding 20 ⁇ L of 1M hydrogen chloride and 200 ⁇ L of ethyl acetate, and performing a shaking operation at 2,000 rpm for 1 hour. After centrifuging the extracted sample at 150,000 rpm for 15 minutes, the upper layer of ethyl acetate was collected, and methacrylic acid was analyzed using a gas chromatography mass spectrometer. The results obtained are shown in Figure 1. In addition, in FIG. 1, in the chromatogram obtained by the said gas chromatography mass spectrometry, the area value (area value) of the peak derived from methacrylic acid is shown.
• amino acids present in the substrate binding site of these enzymes are almost the same, but the difference is that the amino acid corresponding to alanine at position 185 of the Kp-derived amino acid sequence (amino acid sequence described in SEQ ID NO: 2) is Among the four types, the one derived from Bs with the highest catalytic activity was valine (position 187 in the amino acid sequence shown in SEQ ID NO: 8). Therefore, it is suggested that this difference may affect the activity.
• Example 2 Preparation and evaluation of protocatechuate decarboxylase variants>
• the present inventors introduced a large number of mutations accompanied by amino acid substitutions at various positions in PDC using the methods shown below.
• a modified version of PDC was prepared. Then, the catalytic activity of these modified products regarding the production of methacrylic acid using mesaconic acid as a substrate was evaluated.
• PDC into which each mutation has been introduced can be expressed in E. coli in a form in which a polyhistidine tag is fused to its C-terminus, according to the protocol of the Gibson Assembly method.
• a plasmid vector (PDC variant vector) was prepared.
• the vectors prepared as described above (5 ⁇ g of PDC variant vector and 5 ⁇ g of UbiX vector) were introduced into Escherichia coli C41 (DE3) strain (manufactured by Lucigen Corporation, 100 ⁇ L) by the heat shock method, and each PDC variant and A transformant co-expressing UbiX was prepared.
• the collected E. coli culture solution was centrifuged at 150,000 rpm for 15 minutes, and 200 ⁇ L of the supernatant was collected in a 1.5 mL tube.
• Methacrylic acid was extracted from the E. coli culture solution into ethyl acetate by adding 20 ⁇ L of 1M hydrogen chloride and 200 ⁇ L of ethyl acetate, and performing a shaking operation at 2,000 rpm for 1 hour. After centrifuging the extracted sample at 150,000 rpm for 15 minutes, the upper layer of ethyl acetate was collected, and methacrylic acid was analyzed using a gas chromatography mass spectrometer.
• Table 3 shows the relative value of the amount of methacrylic acid produced in each PDC variant with respect to the wild type PDC, which was calculated based on the obtained peak area.
• substitution of methionine, phenylalanine, isoleucine, valine, or leucine at position 327 also results in at least a two-fold improvement.
• methionine or phenylalanine the catalytic activity was improved by more than 60 times (the amount of methacrylic acid produced when the substrate mesaconic acid was charged at 5 g/L was 2.5 g/L compared to the wild type). 1 mg/L, whereas H327M: 202 mg/L).
• Example 3 For the PDC single mutant (H327M) that showed the highest catalytic activity in Example 2, additional amino acid substitutions were introduced at other sites, and methacrylic acid was analyzed for these PDC double mutants in the same manner as above. I did it. The results obtained are shown in Table 4.
• Position 185 is changed to valine, isoleucine, methionine, threonine, serine, asparagine, leucine, tyrosine, histidine, or glutamine
• Position 331 is changed to valine or isoleucine
• Position 298 is changed to valine, glutamine , modification to isoleucine, alanine or leucine
• modification of position 183 to leucine, tyrosine, glutamic acid or methionine or
• Modification of position 438 to isoleucine, methionine, valine, or threonine.
• the catalytic activity is improved by more than 100 times compared to wild-type PDC.
• the amount of methacrylic acid produced when the substrate mesaconic acid was charged at 5 g/L was 2.1 mg/L for the wild type, 202 mg/L for H327M, and 525 mg/L for H327M/A185V). ).
• methacrylic acid can be produced by using protocatechuic acid decarboxylase. Furthermore, according to the present invention, methacrylic acid can be produced by biosynthesis rather than chemical synthesis, so there is less burden on the environment. Therefore, the present invention is extremely useful in producing raw materials for various synthetic polymers such as paints, adhesives, and acrylic resins.

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