Benzaldehyde manufacturing method

Abstract

Provided is a benzaldehyde production method. Benzaldehyde is produced via a benzaldehyde production method according to the present application, the method including a step for producing benzaldehyde by using amino acid deaminase (AAD), 4-hydroxymandelate synthase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC), said step being at least partially performed in the presence of catalase.

Description

[Technical Field]

[0001] The present invention relates to a method for producing benzaldehyde. [Background technology]

[0002] Benzaldehyde is the component responsible for the scent of almonds and apricot kernels, and is used as a flavoring agent in foods, beverages, perfumes, etc. Benzaldehyde is mainly produced by chemical synthesis.

[0003] Several enzymes involved in the biosynthesis of benzaldehyde are known. These enzymes include amino acid deaminase (AAD), 4-hydroxymandelate synthase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC) (Patent Document 1). A method for producing benzaldehyde from L-phenylalanine using microorganisms containing these enzymes has been reported (Patent Document 1).

[0004] Also, a method for producing benzaldehyde from D-phenylalanine using D-amino acid oxidase and peroxidase has been reported (Non-Patent Document 1). Non-Patent Document 1 discloses that the activity of D-amino acid oxidase is inhibited by hydrogen peroxide produced by the catalytic reaction of the enzyme, and that hydrogen peroxide is removed using catalase. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2017 / 122747 [Non-patent literature]

[0006] [Non-Patent Document 1] Krzysztof Okrasaa et al. In vitro bi-enzymatic synthesis of benzaldehyde from phenylalanine: practical and mechanistic studies. Journal of Molecular Catalysis B: Enzymatic 31 (2004) 97-101. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an efficient method for producing benzaldehyde. [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they found that benzaldehyde can be efficiently produced by carrying out at least a part of the production of benzaldehyde using amino acid deaminase, 4-hydroxymandelate synthase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase in the presence of catalase, and thus completed the present invention.

[0009] That is, the present invention can be exemplified as follows. [1] A method for producing benzaldehyde, comprising the steps of: The following step (A): (A) A process for producing benzaldehyde by utilizing four enzymes: amino acid deaminase (AAD), 4-hydroxymandelate synthase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC). Including, A method wherein at least a portion of step (A) is carried out in the presence of catalase. [2] The method, wherein the portion utilizes an HMAS. [3] The four enzymes are utilized in the form of at least one microorganism containing the enzymes, The method, wherein the at least one microorganism is a single microorganism that alone has the four enzymes, or a combination of multiple microorganisms that collectively have the four enzymes. [4] The method, wherein the AAD is an AAD that does not generate hydrogen peroxide. [5] The method, wherein benzaldehyde is produced from L-phenylalanine or a carbon source. [6] The step (A) includes the following steps (A1) and (A2): (A1) A process for producing benzoylformic acid by utilizing AAD, HMAS, and SMDH; (A2) A step of converting the benzoylformic acid produced in the step (A1) into benzaldehyde by using BFDC. The method comprising: [7] The step (A1) comprises the following step (1a), (1b), or (1c): (1a) culturing at least one microorganism in a medium containing a carbon source to produce and accumulate benzoylformic acid in the medium, wherein the at least one microorganism is a single microorganism that independently has AAD, HMAS, and SMDH and is capable of producing L-phenylalanine, or a combination of multiple microorganisms that collectively have AAD, HMAS, and SMDH and are capable of producing L-phenylalanine; (1b) culturing at least one microorganism in a medium containing L-phenylalanine to produce and accumulate benzoylformic acid in the medium, wherein the at least one microorganism is a single microorganism having AAD, HMAS, and SMDH alone, or a combination of multiple microorganisms having AAD, HMAS, and SMDH as a whole; (1c) A step of allowing AAD, HMAS, and SMDH to coexist with L-phenylalanine in a reaction solution, and generating and accumulating benzoylformic acid in the reaction solution. The method comprising: [8] The AAD, HMAS, and SMDH in the step (1c) are used in the form of bacterial cells of at least one microorganism having the AAD, HMAS, and SMDH; The method, wherein the at least one microorganism is a single microorganism that has AAD, HMAS, and SMDH alone, or a combination of multiple microorganisms that collectively have AAD, HMAS, and SMDH. [9] The step (A2) is the following step (2a) or (2b): (2a) culturing a microorganism having BFDC in a medium containing the benzoylformic acid produced in the step (A1) to produce and accumulate benzaldehyde in the medium; (2b) A step of allowing BFDC to coexist with the benzoylformic acid produced in the step (A1) in a reaction solution, and producing and accumulating benzaldehyde in the reaction solution. The method comprising:

[10] The method as described above, wherein the BFDC in step (2b) is utilized in the form of bacterial cells of a microorganism having the BFDC.

[11] The method as described above, wherein the bacterial cells are utilized in the form of a culture of the at least one microorganism, bacterial cells recovered from the culture, a processed product thereof, or a combination thereof.

[12] The method, wherein the catalase is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32; (b) a protein comprising an amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, which contains a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and which has catalase activity; (c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 and having catalase activity.

[13] The method, wherein the microorganism is a bacterium or yeast.

[14] The method as described above, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae or a coryneform bacterium.

[15] The method as described above, wherein the microorganism is a bacterium belonging to the genus Escherichia.

[16] The method, wherein the microorganism is Escherichia coli.

[17] The method further comprises the step of recovering the benzaldehyde. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described in detail below.

[0011] The method of the present invention is a method for producing benzaldehyde, comprising the steps of producing benzaldehyde using amino acid deaminase (AAD), 4-hydroxymandelate synthase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC), at least a portion of which is carried out in the presence of catalase. The four enzymes (AAD, HMAS, SMDH, and BFDC) are also collectively referred to as "benzaldehyde-producing enzymes." The benzaldehyde-producing enzymes and catalase are also collectively referred to as "target enzymes." Genes encoding the target enzymes are also collectively referred to as "target enzyme genes."

[0012] <1> Benzaldehyde-forming enzymes (AAD, HMAS, SMDH, and BFDC) Amino acid deaminase (AAD) is known as an enzyme that catalyzes the oxidative deamination of amino acids (e.g., EC 1.4.3.2 or EC 1.4.99.B3). The AAD used in the present invention uses at least L-phenylalanine as a substrate. As long as the AAD used in the present invention uses L-phenylalanine as a substrate, it may or may not use other amino acids as substrates. That is, in the present invention, "AAD" refers to a protein having the activity of catalyzing the reaction of oxidatively deaminating L-phenylalanine to produce phenylpyruvic acid. In the present invention, this activity is also referred to as "AAD activity." Specifically, "AAD activity" may refer to the activity of catalyzing the reaction of deaminating L-phenylalanine to produce phenylpyruvic acid in the presence of an electron acceptor. Examples of electron acceptors include oxygen and cytochrome b. When oxygen is used as the electron acceptor, hydrogen peroxide may be produced by oxidative deamination. When cytochrome b is used as an electron acceptor, reduced cytochrome b may be produced by oxidative deamination. That is, in one embodiment, "AAD activity" may refer to, for example, the activity of catalyzing the reaction of L-phenylalanine, water, and oxygen to produce phenylpyruvic acid, ammonia, and hydrogen peroxide. In another embodiment, "AAD activity" may refer to, for example, the activity of catalyzing the reaction of L-phenylalanine, water, and cytochrome b to produce phenylpyruvic acid, ammonia, and reduced cytochrome b. A gene encoding AAD is also referred to as an "AAD gene." "AAD" is sometimes called "amino acid oxidase" or "L-phenylalanine oxidase." Examples of AAD include AAD from various organisms, such as microorganisms.Examples of AAD include AAD from bacteria of the genus Providencia, such as Providencia rettgeri (WO2009 / 028338), AAD from bacteria of the genus Proteus, such as Proteus mirabilis (Massad G et al., Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of AAD. J. Bacteriol. 1995 Oct;177(20):5878-83), and AAD from various other organisms. Examples of Providencia rettgeri include the Providencia rettgeri AJ2770 (FERM BP-941) and IFO13501 strains. The nucleotide sequence of the AAD gene of Providencia rettgeri strain AJ2770 (FERM BP-941) (the modified nucleotide sequence used in the Examples) is shown in SEQ ID NO: 37, and the amino acid sequence of AAD encoded by the gene is shown in SEQ ID NO: 38. In particular, an AAD that does not produce hydrogen peroxide may be used as the AAD. For example, the AAD of Providencia rettgeri strain AJ2770 (FERM BP-941) may be an AAD that does not produce hydrogen peroxide. One type of AAD may be used, or two or more types of AAD may be used.

[0013] AAD activity can be measured, for example, by incubating the enzyme with a substrate (i.e., L-phenylalanine) in the presence of oxygen and measuring the enzyme- and substrate-dependent production of a product (i.e., phenylpyruvate) (Massad G et al., Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad. J Bacteriol. 1995 Oct;177(20):5878-83.). The production of phenylpyruvate can be measured, for example, by measuring the increase in absorbance at 614 nm due to the complexation of phenylpyruvate with iron ions (Ibid.).

[0014] 4-Hydroxymandelate synthase (HMAS) is known as an enzyme (e.g., EC 1.13.11.46) that catalyzes the oxidative decarboxylation of α-keto acids such as 4-hydroxyphenylpyruvic acid. The HMAS used in the present invention uses at least phenylpyruvic acid as a substrate. As long as the HMAS used in the present invention uses phenylpyruvic acid as a substrate, it may or may not use other α-keto acids, such as 4-hydroxyphenylpyruvic acid, as a substrate. That is, in the present invention, "HMAS" refers to a protein having the activity of catalyzing the reaction of oxidatively decarboxylating phenylpyruvic acid to produce (S)-mandelic acid. In the present invention, this activity is also referred to as "HMAS activity." Specifically, "HMAS activity" may refer to the activity of catalyzing the reaction of decarboxylating phenylpyruvic acid to produce (S)-mandelic acid in the presence of an electron acceptor. Examples of electron acceptors include oxygen. That is, "HMAS activity" may refer to, for example, the activity of catalyzing the reaction of producing (S)-mandelic acid and carbon dioxide from phenylpyruvic acid and oxygen. A gene encoding an HMAS is also referred to as an "HMAS gene." Examples of HMAS include HMAS from various organisms such as microorganisms.Specifically, HMAS include HMAS of Amycolatopsis bacteria such as Amycolatopsis orientalis and Amycolatopsis balhimycina, HMAS of Streptomyces bacteria such as Streptomyces coelicolor, Streptomyces toyocaensis, and Streptomyces rimosus, HMAS of Rhodococcus bacteria such as Rhodococcus rhodnii, HMAS of Actinoplanes teichomyceticus, Actinoplanes rectilinetus, and HMAS of Streptomyces spp. Examples of HMAS include HMAS from bacteria of the genus Actinoplanes, such as Actinoplanes rectilineatus and Actinoplanes subtropicus; HMAS from bacteria of the genus Kibdelosporangium, such as Kibdelosporangium aridum; HMAS from bacteria of the genus Nonomuraea, such as Nonomuraea coxensis; and HMAS from bacteria of the genus Herpetosiphon, such as Herpetosiphon aurantiacus, as well as HMAS from various other organisms. The nucleotide sequence of the HMAS gene from Actinoplanes teichomyceticus (codon usage optimized for expression in E. coli) is shown in SEQ ID NO: 39, and the amino acid sequence of the HMAS encoded by the gene is shown in SEQ ID NO: 40. One type of HMAS may be used, or two or more types of HMAS may be used. In one embodiment, the HMAS may exclude the HMAS of Kibdelosporangium aridum and Actinoplanes rectilineatus.In another embodiment, the HMAS may exclude HMAS of Amycolatopsis orientalis, Streptomyces coelicolor, Kibdelosporangium aridum, and Actinoplanes rectilineatus.

[0015] HMAS activity can be measured, for example, by incubating the enzyme with a substrate (i.e., phenylpyruvate) in the presence of oxygen and measuring the enzyme- and substrate-dependent production of a product (i.e., (S)-mandelic acid) (Sun Z et al., Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid. Microb Cell Fact. 2011 Sep 13;10:71.).

[0016] "(S)-mandelate dehydrogenase (SMDH)" refers to a protein having the activity of catalyzing the reaction of oxidizing (S)-mandelic acid to produce benzoylformic acid (e.g., EC 1.1.99.31). This activity is also referred to as "SMDH activity." Specifically, "SMDH activity" may refer to the activity of catalyzing the reaction of oxidizing (S)-mandelic acid to produce benzoylformic acid in the presence of an electron acceptor. Examples of electron acceptors include NAD. Examples of electron acceptors include artificial electron acceptors such as phenazine methosulfate (PMS) and dichloroindophenol (DCIP). A gene encoding SMDH is also referred to as an "SMDH gene." Examples of SMDH include SMDHs from various organisms, such as microorganisms. Specific examples of SMDH include the MdlB protein encoded by the mdlB gene of Pseudomonas bacteria such as Pseudomonas putida, and SMDHs from various other organisms. The nucleotide sequence of the mdlB gene (SMDH gene) of Pseudomonas putida is shown in SEQ ID NO: 41, and the amino acid sequence of the MdlB protein (SMDH) encoded by the same gene is shown in SEQ ID NO: 42. One type of SMDH may be used, or two or more types of SMDH may be used.

[0017] SMDH activity can be measured, for example, by incubating the enzyme with a substrate (i.e., (S)-mandelic acid) in the presence of NAD and measuring the enzyme- and substrate-dependent reduction of NAD (BS Al-Baharna and RY Hamzah, Aerobic metabolism of mandelates by Burkholderia cepacia ATTC 29351. Arab J. Biotech., Vol. 6, No. (1) Jan. (2003): 13-28.). SMDH activity can also be measured, for example, by incubating the enzyme with a substrate (i.e., (S)-mandelic acid) in the presence of phenazine methosulfate (PMS) and dichloroindophenol (DCIP) and measuring the enzyme- and substrate-dependent reduction of DCIP (Ibid.). SMDH activity can also be measured, for example, by incubating the enzyme with a substrate (i.e., (S)-mandelic acid) in the presence of potassium ferricyanide in sodium phosphate-citrate buffer and measuring the enzyme- and substrate-dependent reduction of potassium ferricyanide (Peng Wang et al., Immobilization of (S)-mandelate dehydrogenase and its catalytic performance on stereoselective transformation of mandelic acid. Journal of the Taiwan Institute of Chemical Engineers, Volume 45, Issue 3, May 2014, Pages 744-748).

[0018] "Benzoylformate decarboxylase (BFDC)" refers to a protein that catalyzes the reaction of decarboxylating benzoylformate to produce benzaldehyde (e.g., EC 4.1.1.7). This activity is also referred to as "BFDC activity." A gene encoding BFDC is also referred to as "BFDC gene." Examples of BFDC include BFDCs from various organisms, such as microorganisms. Specific examples of BFDC include the MdlC protein encoded by the mdlC gene of Pseudomonas bacteria, such as Pseudomonas putida, and BFDCs from various other organisms. The nucleotide sequence of the mdlC gene (BFDC gene) of Pseudomonas putida is shown in SEQ ID NO: 43, and the amino acid sequence of the MdlC protein (BFDC) encoded by the same gene is shown in SEQ ID NO: 44. One type of BFDC may be used, or two or more types of BFDC may be used.

[0019] BFDC activity can be measured, for example, by incubating the enzyme with a substrate (i.e., benzoylformate) and measuring the enzyme- and substrate-dependent production of a product (i.e., benzaldehyde) (Park, JK and Jung, JY, Production of benzaldehyde by encapsulated whole-cell benzoylformate decarboxylase, Enzyme Microb Technol, 30, 726-733, 2002.).

[0020] That is, each benzaldehyde-producing enzyme gene may be a gene having a known base sequence such as the base sequences exemplified above. Furthermore, each benzaldehyde-producing enzyme may be a protein having a known amino acid sequence such as the amino acid sequences exemplified above. Unless otherwise specified, the expression "having an (amino acid or base) sequence" means "including the (amino acid or base) sequence" and also encompasses the case where "consisting of the (amino acid or base) sequence."

[0021] The benzaldehyde-producing enzyme gene may be a variant of the benzaldehyde-producing enzyme gene exemplified above, so long as the original function is maintained. Similarly, the benzaldehyde-producing enzyme may be a variant of the benzaldehyde-producing enzyme exemplified above, so long as the original function is maintained. Such variants that maintain the original function are sometimes referred to as "conservative variants." Examples of conservative variants include homologs and artificially modified versions of the benzaldehyde-producing enzyme genes and benzaldehyde-producing enzymes exemplified above.

[0022] "Maintaining the original function" means that a gene or protein variant has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that a gene variant encodes a protein that maintains the original function. That is, "maintaining the original function" with respect to the AAD gene, HMAS gene, SMDH gene, and BFDC gene means that a gene variant encodes a protein that has AAD activity, HMAS activity, SMDH activity, and BFDC activity, respectively. Furthermore, "maintaining the original function" with respect to AAD, HMAS, SMDH, and BFDC means that a protein variant has AAD activity, HMAS activity, SMDH activity, and BFDC activity, respectively.

[0023] Examples of conservative variants are shown below.

[0024] A homolog of a benzaldehyde-producing enzyme gene or a homolog of a benzaldehyde-producing enzyme can be easily obtained from a public database, for example, by a BLAST search or a FASTA search using the nucleotide sequence of the above-exemplified benzaldehyde-producing enzyme gene or the amino acid sequence of the above-exemplified benzaldehyde-producing enzyme as a query sequence. Alternatively, a homolog of a benzaldehyde-producing enzyme gene can be obtained by PCR using, for example, the chromosome of an organism such as a bacterium or yeast as a template and oligonucleotides prepared based on the nucleotide sequence of the above-exemplified benzaldehyde-producing enzyme gene as primers.

[0025] As long as the original function is maintained, the benzaldehyde-producing enzyme gene may encode a protein having an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 38 for AAD, the amino acid sequence shown in SEQ ID NO: 40 for HMAS, the amino acid sequence shown in SEQ ID NO: 42 for SMDH, and the amino acid sequence shown in SEQ ID NO: 44 for BFDC). For example, the encoded protein may be extended or shortened at its N-terminus and / or C-terminus. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0026] The above-mentioned substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted with each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted with each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted with each other when the substitution site is a basic amino acid; Asp and Glu are substituted with each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted with each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Il Examples of such substitutions include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0027] Furthermore, the benzaldehyde-producing enzyme gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, as long as the original function is maintained.

[0028] Furthermore, as long as the original function is maintained, the benzaldehyde-producing enzyme gene may be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned base sequence (e.g., the base sequence shown in SEQ ID NO: 37 for the AAD gene, the base sequence shown in SEQ ID NO: 39 for the HMAS gene, the base sequence shown in SEQ ID NO: 41 for the SMDH gene, and the base sequence shown in SEQ ID NO: 43 for the BFDC gene), such as a sequence complementary to all or part of the above-mentioned base sequence. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions is a condition under which DNAs with high identity, for example, DNAs with an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, while DNAs with lower identity do not hybridize with each other; or a condition in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0029] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0030] Furthermore, since codon degeneracy differs depending on the host, the benzaldehyde-producing enzyme gene may be one in which any codon has been replaced with an equivalent codon. That is, the benzaldehyde-producing enzyme gene may be a variant of the benzaldehyde-producing enzyme gene exemplified above due to the degeneracy of the genetic code. For example, the benzaldehyde-producing enzyme gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0031] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using the default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using the default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0032] HMAS further includes HMAS having a "specific mutation" (WO2017 / 122747). Furthermore, HMAS genes further include genes encoding HMAS having a "specific mutation". HMAS having a "specific mutation" is also referred to as a "mutant HMAS". Furthermore, a gene encoding a mutant HMAS is also referred to as a "mutant HMAS gene".

[0033] An HMAS that does not have the "specific mutation" is also referred to as a "wild-type HMAS." A gene encoding a wild-type HMAS is also referred to as a "wild-type HMAS gene." The term "wild-type" used here is a convenient description to distinguish "wild-type" HMAS from "mutant" HMAS, and is not limited to naturally occurring HMAS, but includes any HMAS that does not have the "specific mutation." Examples of wild-type HMAS include the HMAS exemplified above. Furthermore, conservative variants of the HMAS exemplified above are all included in wild-type HMAS, as long as they do not have the "specific mutation."

[0034] The mutant HMAS may be identical to a wild-type HMAS (e.g., the HMAS exemplified above or a conservative variant thereof) except for the "specific mutation." That is, the mutant HMAS may be a protein having the amino acid sequence of a wild-type HMAS except for the "specific mutation." Specifically, the mutant HMAS may be a protein having the amino acid sequence set forth in SEQ ID NO: 40 except for the "specific mutation." Furthermore, specifically, the mutant HMAS may be a protein having an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence set forth in SEQ ID NO: 40 except for the "specific mutation." Furthermore, specifically, the mutant HMAS may be a protein having an amino acid sequence that is 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 40 except for the "specific mutation."

[0035] In a conservative variant used as a wild-type HMAS, the conservative mutation may occur at a position other than the "specific mutation." In other words, a mutant HMAS may be a protein having an amino acid sequence that has the "specific mutation" in the amino acid sequence of the HMAS exemplified above, and further contains conservative mutations (e.g., substitution, deletion, insertion, and / or addition of one or several amino acids) at a position other than the "specific mutation."

[0036] A "specific mutation" may be a mutation that is effective for producing benzaldehyde, such as a mutation that increases the production of benzaldehyde in the method of the present invention. Such an increase in benzaldehyde production may be based, for example, on an increase in the production of (S)-mandelic acid by HMAS. Thus, a "specific mutation" may be a mutation that increases the production of (S)-mandelic acid by HMAS.

[0037] "Specific mutations" include mutations in amino acid residues corresponding to T2, M3, G5, Y18, A27, D35, E46, E180, A187, E191, V194, A199, D201, Q206, I217, D220, T222, G255, F319, G327, I336, K337, V343, and Q347. A "specific mutation" may be a mutation in one amino acid residue, or a combination of mutations in two or more amino acid residues. That is, a "specific mutation" may include or consist of a mutation in an amino acid residue corresponding to one or more amino acid residues selected from the group consisting of, for example, T2, M3, G5, Y18, A27, D35, E46, E180, A187, E191, V194, A199, D201, Q206, I217, D220, T222, G255, F319, G327, I336, K337, V343, and Q347.

[0038] In the above notation for identifying amino acid residues, the numbers indicate positions in the amino acid sequence shown in SEQ ID NO: 40, and the letters to the left of the numbers indicate the amino acid residues at each position in the amino acid sequence shown in SEQ ID NO: 40 (i.e., the amino acid residues at each position before modification). For example, "T2" indicates the T (Thr) residue at position 2 in the amino acid sequence shown in SEQ ID NO: 40.

[0039] In each of the above mutations, the amino acid residue after modification may be any amino acid residue other than the amino acid residue before modification. Specific examples of the amino acid residue after modification include K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln), other than the amino acid residue before modification. The amino acid residue after modification may be selected from those effective for producing benzaldehyde.

[0040] Specific examples of "specific mutations" include mutations corresponding to T2N, M3I, G5R, Y18F, A27V, D35G, E46Q, E180K, A187V, E191K, V194G, A199(S,V), D201N, Q206R, I217(L,V), D220(A,N), T222S, G255D, F319Y, G327(D,S), I336V, K337Q, V343M, and Q347L. That is, mutations in amino acid residues corresponding to T2, M3, G5, Y18, A27, D35, E46, E180, A187, E191, V194, A199, D201, Q206, I217, D220, T222, G255, F319, G327, I336, K337, V343, and Q347 are, for example, T2N, M3I, and G5, respectively. The mutation may be equivalent to R, Y18F, A27V, D35G, E46Q, E180K, A187V, E191K, V194G, A199(S,V), D201N, Q206R, I217(L,V), D220(A,N), T222S, G255D, F319Y, G327(D,S), I336V, K337Q, V343M, or Q347L. The "particular mutation" may include, or consist of, one or more mutations selected from the group consisting of, for example, T2N, M3I, G5R, Y18F, A27V, D35G, E46Q, E180K, A187V, E191K, V194G, A199(S,V), D201N, Q206R, I217(L,V), D220(A,N), T222S, G255D, F319Y, G327(D,S), I336V, K337Q, V343M, and Q347L.

[0041] In the above notation for specifying mutations, the numbers and the letters to their left have the same meanings as described above. In the above notation for specifying mutations, the letters to the right of the numbers indicate the amino acid residues after modification at each position. That is, for example, "T2N" indicates a mutation in which the T (Thr) residue at position 2 in the amino acid sequence shown in SEQ ID NO: 40 is substituted with an N (Asn) residue. Also, for example, "A199(S,V)" indicates a mutation in which the A (Ala) residue at position 199 in the amino acid sequence shown in SEQ ID NO: 40 is substituted with an S (Ser) residue or a V (Val) residue.

[0042] The combination of mutations is not particularly limited. Specific examples of the combination of mutations include M3I / A199S / G255D, Y18F / D220N, A27V / E191K, D35G / E46Q / T222S / I336V, E180K / I217V / D220N, A187V / I217V, A199V / I217V / K337Q, D201N / I217V, I217V / F319Y, D220A / Q347L, and A199V / Q206R / I217V / K337Q. A particular example of the combination of mutations is A199V / Q206R / I217V / K337Q. That is, a "specific mutation" may, for example, include a mutation corresponding to any of these combinations, or may consist of such mutations.

[0043] In the above notation for specifying combinations, the meanings of the numbers and the letters to the left and right of them are the same as those described above. In the above notation for specifying combinations, the combination of two or more mutations separated by " / " indicates a double mutation or multiple mutations. For example, "M3I / A199S / G255D" indicates a triple mutation of M3I, A199S, and G255D.

[0044] "A mutation corresponding to a mutation in the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 40" should be read as a mutation in the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 40. That is, for example, "a mutation corresponding to T2N" refers to a mutation in which the amino acid residue corresponding to T2 is substituted with an N (Asn) residue.

[0045] "Position X" in an amino acid sequence refers to the Xth position counting from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the 1st amino acid residue. The above-mentioned mutation positions indicate relative positions, and their absolute positions may vary due to deletion, insertion, addition, or the like of amino acid residues. For example, if one amino acid residue is deleted or inserted at a position N-terminal to position X in the amino acid sequence shown in SEQ ID NO: 40, the original amino acid residue at position X becomes the X-1th or X+1th amino acid residue, respectively, counting from the N-terminus, and is considered to be "the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 40."

[0046] The amino acid residues before the above mutations are typical examples and are not limited thereto. For example, the "amino acid residue corresponding to T2" may typically be a T (Thr) residue, but may not be a T (Thr) residue. In other words, when wild-type HMAS has an amino acid sequence other than SEQ ID NO: 40, the "amino acid residue corresponding to T2" may not be a T (Thr) residue. Therefore, "mutation corresponding to T2N" is not limited to a mutation in which the T (Thr) residue corresponding to T2 is replaced with an N (Asn) residue when the "amino acid residue corresponding to T2" is a T (Thr) residue, but also includes a mutation in which the amino acid residue corresponding to T2 is replaced with an N (Asn) residue when the "amino acid residue corresponding to T2" is a K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), P (Pro), F (Phe), W (Trp), C (Cys), M (Met), D (Asp), E (Glu), or Q (Gln) residue. This can also be applied mutatis mutandis to other mutations.

[0047] In the amino acid sequence of any HMAS, which amino acid residue corresponds to the "amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 40" can be determined by aligning the amino acid sequence of any HMAS with the amino acid sequence shown in SEQ ID NO: 40. Alignment can be performed using, for example, known genetic analysis software. Specific examples of such software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0048] The above descriptions regarding the benzaldehyde-producing enzyme gene and conservative variants of the benzaldehyde-producing enzyme can also be applied mutatis mutandis to any gene and protein such as a catalase gene and catalase.

[0049] <2> Catalase "Catalase" refers to a protein that has the activity of catalyzing the reaction of decomposing hydrogen peroxide to produce water and oxygen (e.g., EC 1.11.1.6). This activity is also called "catalase activity." A gene encoding catalase is also called "catalase gene." Examples of catalase include catalases from various organisms such as microorganisms. Specific examples of catalases include catalases from bacteria of the genus Escherichia, such as E. coli; catalases from bacteria of the genus Erwinia, such as Erwinia mallotivora and Erwinia tracheiphila; catalases from bacteria of the genus Pseudomonas, such as Pseudomonas putida, Pseudomonas entomophila, Pseudomonas parafulva, and Pseudomonas protegens; catalases from bacteria of the genus Shewanella, such as Shewanella oneidensis; catalases from bacteria of the genus Bacillus, such as Bacillus subtilis; catalases from bacteria of the genus Thermus, such as Thermus thermophilus; catalases from bacteria of the genus Rhodothermus, such as Rhodothermus marinus; catalases from bacteria of the genus Corynebacterium, such as C. glutamicum; and catalases from bacteria of the genus Micrococcus, such as Micrococcus lysodeikticus. Specific examples of catalases from E. coli include HPI and HPII. The nucleotide sequences of DNA fragments containing the catalase genes of these organisms are shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31, and the amino acid sequences of the catalases encoded by these genes are shown in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32. One type of catalase may be used, or two or more types of catalases may be used. In one embodiment, catalases may exclude catalases having the amino acid sequence of SEQ ID NO: 16 (e.g., catalases consisting of the amino acid sequence of SEQ ID NO: 16).

[0050] The catalase gene and catalase may be, for example, a gene having a known nucleotide sequence such as the exemplified nucleotide sequences above, and a protein having a known amino acid sequence such as the exemplified amino acid sequences above, respectively. Furthermore, the catalase gene and catalase may be conservative variants of the exemplified genes (e.g., genes having a known nucleotide sequence such as the exemplified nucleotide sequences above) and conservative variants of the exemplified proteins (e.g., proteins having a known amino acid sequence such as the exemplified amino acid sequences above), respectively. Specifically, for example, the catalase gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in a known amino acid sequence such as the exemplified amino acid sequences above, so long as the original function is maintained. Furthermore, for example, the catalase gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identical to the entire amino acid sequence of the exemplified amino acid sequences or known proteins, so long as the original function is maintained. With respect to a catalase gene, "maintaining the original function" means that a variant of the gene encodes a protein with catalase activity. Furthermore, with respect to catalase, "maintaining the original function" means that a variant of the protein has catalase activity. The above-mentioned descriptions regarding conservative variants of the benzaldehyde-producing enzyme gene and benzaldehyde-producing enzyme can be applied mutatis mutandis to such conservative variants of genes and proteins.

[0051] Catalase activity can be measured, for example, by incubating the enzyme with hydrogen peroxide and measuring the enzyme-dependent reduction of hydrogen peroxide. The reduction of hydrogen peroxide can be quantified, for example, as a decrease in absorbance at 240 nm. Hydrogen peroxide can also be quantified using a peroxidase such as horseradish peroxidase (HRP) and a colorimetric substrate.

[0052] <3> Production of benzaldehyde-forming enzyme and catalase The target enzymes (i.e., benzaldehyde-producing enzyme and catalase) may be commercially available or may be prepared by appropriate manufacturing. For example, commercially available catalase products include commercially available catalase preparations. Examples of commercially available catalase preparations include those used in the Examples.

[0053] The target enzymes can be produced, for example, by expressing the target enzyme genes (i.e., benzaldehyde-forming enzyme gene and catalase gene) encoding the enzymes in a host having the genes.

[0054] Alternatively, each target enzyme can be produced by, for example, expressing a target enzyme gene encoding the enzyme in a cell-free protein synthesis system.

[0055] The production of a target enzyme using a host carrying the target enzyme gene will be described in detail below.

[0056] <3-1>Host The host has a target enzyme gene. Specifically, the host has the target enzyme gene in an expressible manner. The host may or may not inherently have the target enzyme gene. For example, the host may not inherently have the target enzyme gene but may be modified to have the target enzyme gene. A host modified to have the target enzyme gene can be obtained by introducing the target enzyme gene into a host that does not have the target enzyme gene. Methods for introducing the gene will be described later. "Having the target enzyme gene" can also be referred to as "having the target enzyme."

[0057] The host may be modified to increase the activity of the target enzyme. Specifically, the host may be modified to increase the activity of the target enzyme compared to an unmodified strain. For example, a host that does not inherently have a target enzyme gene may be modified to increase the activity of the target enzyme. That is, by introducing a target enzyme gene into a host that does not inherently have a target enzyme gene, the target enzyme activity of the host can be increased (the target enzyme activity can be imparted to the host). Furthermore, for example, a host that inherently has a target enzyme gene may be modified to increase the activity of the target enzyme. Techniques for increasing the activity of a protein (enzyme, etc.) will be described below.

[0058] As the host, the following hosts can be used either as is or after appropriate modification (for example, by introducing a target enzyme gene or enhancing the target enzyme activity). That is, the host may be a modified strain derived from the following hosts.

[0059] The host is not particularly limited as long as it can express a functional target enzyme. Examples of the host include microorganisms, plant cells, insect cells, and animal cells. Examples of the host include, in particular, microorganisms. Examples of the microorganisms include bacteria and yeast. Examples of the microorganisms include, in particular, bacteria.

[0060] Examples of bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and Bacillus bacteria.

[0061] Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified as Enterobacteriaceae according to the classification used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Examples of bacteria belonging to the genus Escherichia include, but are not limited to, bacteria classified as Escherichia according to classifications known to microbiologists. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, pp. 2460-2488. Table 1. In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli. Examples of Escherichia coli include Escherichia coli K-12 strains such as the W3110 strain (ATCC 27325) and the MG1655 strain (ATCC 47076); the Escherichia coli K5 strain (ATCC 23506); the Escherichia coli B strain such as the BL21(DE3) strain; and derivatives thereof.Examples of bacteria of the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Examples of bacteria of the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of bacteria of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of bacteria of the genus Klebsiella include Klebsiella planticola. In recent years, bacteria belonging to the Enterobacteriaceae family have been reclassified into multiple families based on comprehensive comparative genome analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599). However, in the present invention, bacteria previously classified in the Enterobacteriaceae family are treated as bacteria belonging to the Enterobacteriaceae family.

[0062] Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0063] Specific examples of coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0064] Specific examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium molassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354

[0065] The genus Corynebacterium includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0066] Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

[0067] Examples of yeast include yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, the genus Candida, such as Candida utilis, the genus Pichia, such as Pichia pastoris, the genus Hansenula, such as Hansenula polymorpha, and the genus Schizosaccharomyces, such as Schizosaccharomyces pombe.

[0068] These strains can be obtained, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain has a corresponding accession number, which can be used to obtain the strain (see http: / / www.atcc.org / ). The accession numbers are listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0069] A target enzyme gene can be obtained by cloning from an organism that has the target enzyme gene. Nucleic acids such as genomic DNA or cDNA containing the gene can be used for cloning. Alternatively, a target enzyme gene can be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).

[0070] The obtained target enzyme gene may be used as is or after appropriate modification. For example, the obtained target enzyme gene can be appropriately modified to obtain its variant. Gene modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site in DNA by site-directed mutagenesis. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that amino acid residues at specific sites in the encoded protein include substitutions, deletions, insertions, and / or additions. Examples of site-specific mutagenesis include a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and a method using phage (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)).

[0071] A mutant HMAS gene can be obtained, for example, by modifying a wild-type HMAS gene so that the encoded HMAS has a "specific mutation." Such modification can be performed by known techniques such as site-directed mutagenesis. Alternatively, a mutant HMAS gene can be obtained without the intervention of a wild-type HMAS gene. A mutant HMAS gene may be obtained directly, for example, by chemical synthesis. The obtained mutant HMAS gene may be used as is or after further modification.

[0072] The method for introducing the target enzyme gene into the host is not particularly limited, as long as the target enzyme gene is retained in the host in an expressible manner. The target enzyme gene can be introduced into the host in the same manner as the gene introduction described below in the "Method for increasing protein activity."

[0073] Furthermore, if a host already has an HMAS gene in a chromosome or the like, the host can be modified to have a mutant HMAS gene by introducing a "specific mutation" into the HMAS gene present in the chromosome, etc. Mutations can be introduced into genes present in chromosomes, etc., by, for example, natural mutation, mutagenesis, or genetic engineering.

[0074] The host may have the ability to produce L-phenylalanine. A "host capable of producing L-phenylalanine" may refer to a host capable of biosynthesizing L-phenylalanine when cultured in a medium (e.g., a medium containing a carbon source). Thus, a "host capable of producing L-phenylalanine" may specifically refer to a host capable of biosynthesizing L-phenylalanine from a carbon source. The biosynthesized L-phenylalanine may be used as a raw material for benzaldehyde production. Thus, a "host capable of producing L-phenylalanine" may specifically refer to a host capable of biosynthesizing the amount of L-phenylalanine required as a raw material for benzaldehyde production. The biosynthesized L-phenylalanine may or may not accumulate as a product within the cell and / or in the medium, for example. That is, the biosynthesized L-phenylalanine may be immediately consumed. For example, the biosynthesized L-phenylalanine may be immediately converted to benzaldehyde or an intermediate thereof. Therefore, in one embodiment, L-phenylalanine producing capacity may be measured based on the production of benzaldehyde or its intermediates.

[0075] The host may be one that inherently has the ability to produce L-phenylalanine, or may be one that has been modified to have the ability to produce L-phenylalanine. A host having the ability to produce L-phenylalanine can be obtained, for example, by imparting the ability to produce L-phenylalanine to the above-mentioned host or by enhancing the L-phenylalanine-producing ability of the above-mentioned host.

[0076] Specific examples of methods for imparting or enhancing L-phenylalanine-producing ability are given below. Note that the modifications for imparting or enhancing L-phenylalanine-producing ability as exemplified below may be used alone or in appropriate combination.

[0077] Methods for imparting or enhancing L-phenylalanine-producing ability include methods for increasing the activity of an L-phenylalanine biosynthetic enzyme. That is, the host may be modified so that the activity of an L-phenylalanine biosynthetic enzyme is increased. The activity of one type of L-phenylalanine biosynthetic enzyme may be increased, or the activities of two or more types of L-phenylalanine biosynthetic enzymes may be increased. Methods for increasing the activity of a protein (enzyme, etc.) will be described later. The activity of a protein (enzyme, etc.) can be increased, for example, by increasing the expression of a gene encoding the protein.

[0078] The L-phenylalanine biosynthetic enzymes include 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (aroF, aroG, aroH), 3-dehydroquinate synthase (aroB), 3-dehydroquinate dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroK, aroL), 5-enolpyruvylshikimate-3-phosphate synthase (aroA), and chorismate synthase (chorismate synthase). These include biosynthetic enzymes common to aromatic amino acids, such as aroC (aroA synthase), chorismate mutase (pheA), prephenate dehydratase (pheA), and tyrosine amino transferase (tyrB). Examples of the gene encoding each enzyme are shown in parentheses after the enzyme name (the same applies below). Chorismate mutase and prephenate dehydratase may be encoded by the pheA gene as bifunctional enzymes. Expression of genes encoding several L-phenylalanine biosynthetic enzymes, such as DAHP synthase, 3-dehydroquinate synthase, and 3-dehydroquinate dehydratase, can be repressed by the tyrosine repressor tyrR encoded by the tyrR gene. Therefore, the activity of L-phenylalanine biosynthetic enzymes can be increased, for example, by reducing the activity of the tyrosine repressor tyrR. Additionally, some L-phenylalanine biosynthetic enzymes can be subject to feedback inhibition by aromatic amino acids such as L-phenylalanine. For example, the bifunctional chorismate mutase-prephenate dehydratase can be subject to feedback inhibition by L-phenylalanine.Therefore, the activity of an L-phenylalanine biosynthetic enzyme can be increased, for example, by using a gene encoding a mutant L-phenylalanine biosynthetic enzyme that is desensitized to such feedback inhibition.

[0079] Another method for imparting or enhancing L-phenylalanine-producing ability is to reduce the activity of enzymes involved in the by-production of substances other than L-phenylalanine. Such substances other than L-phenylalanine are also called "by-products." Examples of by-products include other aromatic amino acids such as L-tyrosine and L-tryptophan. Examples of enzymes involved in the by-production of L-tyrosine include the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (tyrA).

[0080] Specific examples of L-phenylalanine-producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197; WO03 / 044191), which is deficient in chorismate mutase-prephenate dehydrogenase and tyrosine repressor; E. coli AJ12741 (JP H05-344881), which is deficient in chorismate mutase-prephenate dehydrogenase and tyrosine repressor and has a mutant aroG gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase deficient in feedback inhibition, a mutant pheA gene encoding chorismate mutase-prephenate dehydratase deficient in feedback inhibition, and an aroL gene encoding shikimate kinase; Examples of L-phenylalanine-producing bacteria and parent strains for deriving them include E. coli K-12 (ATCC 55371; U.S. Patent No. 5,354,672), E. coli HW1089 (ATCC 55371; U.S. Patent No. 5,354,672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (U.S. Patent No. 4,407,952), which carry the pheA34 gene encoding feedback-deprived chorismate mutase-prephenate dehydratase.<W3110 (tyrA) / pPHAB> (FERM BP-3566), E. coli K-12<W3110 (tyrA) / pPHAD> (FERM BP-12659), E. coli K-12<W3110 (tyrA) / pPHATerm> (FERM BP-12662), E. coli K-12 AJ 12604<W3110 (tyrA) / pBR-aroG4, pACMAB> (FERM BP-3579) is also included (EP 488424 B1).Specific examples of L-phenylalanine-producing bacteria or parent strains for deriving them include strains belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or the yddG gene is enhanced (US2003-0148473A, US2003-0157667A, WO03 / 044192). Specific examples of L-phenylalanine-producing bacteria or parent strains for deriving them include Corynebacterium glutamicum strains BPS-13 (FERM BP-1777), K77 (FERM BP-2062), and K78 (FERM BP-2063) with reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity (EP 331145 A, JP H02-303495 A), as well as tyrosine-requiring strains of coryneform bacteria (JP H05-049489 A).

[0081] The gene and protein used to breed a host capable of producing L-phenylalanine may be, for example, a gene and protein having a known nucleotide sequence and amino acid sequence, respectively. Furthermore, the gene and protein used to breed a host capable of producing L-phenylalanine may be a conservative variant of a gene and protein having a known nucleotide sequence and amino acid sequence, respectively. Specifically, for example, the gene used to breed a host capable of producing L-phenylalanine may be a gene encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the known amino acid sequence of the protein, as long as the original function (i.e., enzymatic activity, etc.) is maintained. The above-mentioned descriptions regarding the benzaldehyde-producing enzyme gene and conservative variants of the benzaldehyde-producing enzyme can be applied mutatis mutandis to such conservative variants of genes and proteins.

[0082] The host may have one target enzyme gene or two or more target enzyme genes. For example, the host may have one, two, three, or all four genes selected from the four benzaldehyde synthase genes (AAD, HMAS, SMDH, and BFDC). For example, the host may have a catalase gene in addition to one, two, three, or all four genes selected from the four benzaldehyde synthase genes (AAD, HMAS, SMDH, and BFDC).

[0083] The host may have any other modifications as long as it is capable of producing the target enzyme.

[0084] The order of modifications to construct the host is not particularly limited.

[0085] <3-2> Microorganism of the present invention The host may be particularly configured as a microorganism (specifically, at least one microorganism) having the four benzaldehyde-producing enzymes (AAD, HMAS, SMDH, and BFDC). A microorganism (specifically, at least one microorganism) having the four benzaldehyde-producing enzymes (AAD, HMAS, SMDH, and BFDC) is also referred to as a "microorganism of the present invention."

[0086] "Having a benzaldehyde-producing enzyme" may mean that a functional benzaldehyde-producing enzyme is expressed and maintained. Specifically, "having a benzaldehyde-producing enzyme" may mean that a functional benzaldehyde-producing enzyme is expressed and maintained within the bacterial cell.

[0087] The microorganism of the present invention may be a single microorganism or a combination of multiple microorganisms. That is, in the present invention, the singular form "microorganism" (e.g., "a microorganism" or "the microorganism") may be interpreted as at least one microorganism, i.e., a single microorganism or a combination of multiple microorganisms, depending on the context.

[0088] When the microorganism of the present invention is a single microorganism, the single microorganism alone has all four benzaldehyde-producing enzymes.

[0089] When the microorganism of the present invention is a combination of multiple microorganisms, the multiple microorganisms as a whole possess all four benzaldehyde-producing enzymes. In other words, when the microorganism of the present invention is a combination of multiple microorganisms, each of the four benzaldehyde-producing enzymes is retained in at least one of the multiple microorganisms. Each benzaldehyde-producing enzyme may be retained in one of the multiple microorganisms, or in two or more of them. Each of the multiple microorganisms may possess one of the benzaldehyde-producing enzymes, or in two or more of them. The type of benzaldehyde-producing enzyme possessed by each of the multiple microorganisms is not particularly limited, as long as it is capable of producing benzaldehyde. The type of benzaldehyde-producing enzyme possessed by each of the multiple microorganisms can be appropriately determined depending on various conditions such as the embodiment of the production process (e.g., the configuration of substeps in the production process). For example, the microorganisms of the present invention may be a combination of four microorganisms each having one of the four benzaldehyde-producing enzymes, i.e., a combination of a microorganism having AAD, a microorganism having HMAS, a microorganism having SMDH, and a microorganism having BFDC. The multiple microorganisms may or may not be identical to each other except for the type of benzaldehyde-producing enzyme possessed by the multiple microorganisms. For example, the multiple microorganisms may or may not be microorganisms derived from the same genus, species, or strain.

[0090] The microorganism of the present invention may have catalase.

[0091] When the microorganism of the present invention is a single microorganism, the single microorganism may have catalase.

[0092] When the microorganism of the present invention is a combination of multiple microorganisms, any one or more of the multiple microorganisms may have catalase. Which of the multiple microorganisms has catalase is not particularly limited, as long as benzaldehyde production is improved. For example, a microorganism having at least HMAS may have catalase. Alternatively, for example, a microorganism used in coexistence with a microorganism having at least HMAS may have catalase.

[0093] The microorganism of the present invention may have the ability to produce L-phenylalanine.

[0094] When the microorganism of the present invention is a single microorganism, the single microorganism may have the ability to produce L-phenylalanine.

[0095] When the microorganism of the present invention is a combination of multiple microorganisms, any one or more of the multiple microorganisms may have the ability to produce L-phenylalanine. Which of the multiple microorganisms has the ability to produce L-phenylalanine is not particularly limited, as long as it is capable of producing benzaldehyde. For example, a microorganism having at least AAD may have the ability to produce L-phenylalanine. Alternatively, for example, a microorganism used in coexistence with a microorganism having at least AAD may have the ability to produce L-phenylalanine.

[0096] The microorganism of the present invention has the ability to produce benzaldehyde. "Microorganisms capable of producing benzaldehyde" refers to microorganisms capable of producing benzaldehyde. "Microorganisms capable of producing benzaldehyde" may be microorganisms capable of producing benzaldehyde by fermentation, bioconversion, or a combination thereof. That is, "microorganisms capable of producing benzaldehyde" may refer to microorganisms capable of producing benzaldehyde from a carbon source or L-phenylalanine. Specifically, "microorganisms capable of producing benzaldehyde" may refer to microorganisms capable of producing and accumulating benzaldehyde in a medium containing a carbon source when cultured in the medium. Furthermore, "microorganisms capable of producing benzaldehyde" may refer to microorganisms capable of producing and accumulating benzaldehyde in a medium or reaction solution when cultured in a medium containing L-phenylalanine, or when coexisting with or acting on L-phenylalanine in a reaction solution.

[0097] Benzaldehyde can be produced from L-phenylalanine by the action of four benzaldehyde-producing enzymes. Thus, the microorganism of the present invention (having four benzaldehyde-producing enzymes) may be capable of producing benzaldehyde from L-phenylalanine. For example, the microorganism of the present invention may be capable of producing benzaldehyde from L-phenylalanine by a process of producing benzaldehyde by chemical conversion, or may be capable of producing benzaldehyde from L-phenylalanine by a combination of a substep of producing a benzaldehyde intermediate by chemical conversion and a subsequent substep of converting the intermediate to benzaldehyde.

[0098] When the microorganism of the present invention has the ability to produce L-phenylalanine, the microorganism of the present invention may be capable of producing benzaldehyde from a carbon source. For example, the microorganism of the present invention may be capable of producing benzaldehyde from a carbon source by a step of producing benzaldehyde by fermentation, or may be capable of producing benzaldehyde from a carbon source by a combination of a substep of producing a benzaldehyde intermediate by fermentation and a subsequent substep of converting the intermediate to benzaldehyde.

[0099] The microorganism of the present invention may be capable of accumulating benzaldehyde in the medium or reaction solution to an extent that it can be recovered. For example, the microorganism of the present invention may be capable of accumulating benzaldehyde in the medium or reaction solution in an amount of 0.01 mM or more, 0.1 mM or more, or 1 mM or more.

[0100] When the microorganism of the present invention is a single microorganism, the single microorganism has the ability to produce benzaldehyde.

[0101] When the microorganism of the present invention is a combination of multiple microorganisms, the combination as a whole has the ability to produce benzaldehyde.

[0102] In addition, the microorganism of the present invention may have any other modifications as long as it is capable of producing benzaldehyde.

[0103] <3-3>Methods for increasing protein activity Below, methods for increasing protein activity (including gene introduction methods) are explained.

[0104] "Increased protein activity" may mean that the activity of the protein is increased compared to that of an unmodified strain. "Increased protein activity" may specifically mean that the activity of the protein per cell is increased compared to that of an unmodified strain. "Protein activity per cell" may mean the average activity of the protein per cell. An unmodified strain is also referred to as an "unmodified host" or "unmodified host strain." As used herein, "unmodified strain" may refer to a control strain that has not been modified to increase the activity of a target protein. Examples of unmodified strains include wild-type strains and parent strains. Specific examples of unmodified strains include the type strains of the species to which the host belongs. Specific examples of unmodified strains include the strains exemplified in the description of the host. That is, in one embodiment, the activity of the protein may be increased compared to that of a type strain (i.e., the type strain of the species to which the host belongs). In another embodiment, the activity of the protein may be increased compared to that of the C. glutamicum ATCC 13869 strain. In another embodiment, the activity of the protein may be increased compared to that of the C. glutamicum ATCC 13032 strain. In another embodiment, the activity of the protein may be increased compared to that of the E. coli K-12 MG1655 strain. "Increased protein activity" is also referred to as "enhanced protein activity." More specifically, "increased protein activity" may mean an increase in the number of molecules of the protein per cell and / or an increase in the function of the protein per molecule compared to a non-modified strain. In other words, the "activity" in "increased protein activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The "number of protein molecules per cell" may refer to the average number of molecules of the protein per cell. Furthermore, "increasing the activity of a protein" includes not only increasing the activity of a target protein in a strain that originally has the activity of that protein, but also imparting the activity of that protein to a strain that does not originally have the activity of that protein.Furthermore, as long as the resulting protein activity is increased, the activity of a suitable target protein may be imparted after reducing or eliminating the activity of a target protein that the host naturally possesses.

[0105] The degree of increase in protein activity is not particularly limited, as long as the protein activity is increased compared to that of an unmodified strain. For example, the protein activity may be increased by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to that of an unmodified strain. Furthermore, if the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein, and for example, the protein may be produced to an extent that its activity can be measured.

[0106] Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of the gene encoding the protein. "Increased gene expression" may mean that the expression of the gene is increased compared to that of an unmodified strain such as a wild-type strain or a parent strain. "Increased gene expression" may specifically mean that the expression level of the gene per cell is increased compared to that of an unmodified strain. "Expression level of the gene per cell" may mean the average expression level of the gene per cell. "Increased gene expression" may more specifically mean that the transcription level (mRNA level) of the gene is increased and / or the translation level (protein level) of the gene is increased. Note that "increased gene expression" is also referred to as "enhanced gene expression." Gene expression may be increased, for example, by 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more compared to an unmodified strain. Furthermore, "increasing gene expression" encompasses not only increasing the expression level of a target gene in a strain in which the gene is originally expressed, but also expressing the gene in a strain in which the gene is not originally expressed. In other words, "increasing gene expression" may mean, for example, introducing the gene into a strain that does not harbor the target gene and expressing the gene.

[0107] Increased gene expression can be achieved, for example, by increasing the copy number of the gene.

[0108] The copy number of a gene can be increased by introducing the gene into a host chromosome. Introduction of a gene into a chromosome can be achieved, for example, by homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Gene introduction methods that utilize homologous recombination include, for example, methods using linear DNA such as Red-driven integration (Datsenko, K. A., and Wanner, BL, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), methods using plasmids containing a temperature-sensitive replication origin, methods using conjugatively transferable plasmids, methods using suicide vectors that do not have a replication origin that functions in the host, and transduction methods using phages. Only one copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that exists in multiple copies on a chromosome. Examples of sequences present in multiple copies on chromosomes include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination can also be performed by targeting appropriate sequences on chromosomes, such as genes not required for the production of a target substance. Genes can also be randomly introduced into chromosomes using transposons or Mini-Mu (see JP-A-2-109985, US Pat. No. 5,882,888, EP805867B1).

[0109] The introduction of the target gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0110] The copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the copy number of the gene. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. A vector capable of autonomous replication within host cells can be used. The vector may be a multicopy vector. Furthermore, the vector may contain a marker such as an antibiotic resistance gene for the selection of transformants. The vector may also contain a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene Co., Ltd.), pTrc99A (Pharmacia), pPROK-based vectors (Clontech), pKK233-2 (Clontech), pET-based vectors (Novagen), pQE-based vectors (Qiagen), pCold TF DNA (TaKaRa), pACYC-based vectors, and the broad-host-range vector RSF1010.Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids having drug resistance genes improved from these; pCRY30 (JP 3-210184 A); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP 2-72876 A, U.S. Pat. No. 5,185,262 A); pCRY2 and pCRY pCG4 and pCG11 (Japanese Patent Application Laid-Open No. 57-183799); pPK4 (U.S. Pat. No. 6,090,597); pVK4 (Japanese Patent Application Laid-Open No. 9-322774); pVK7 (Japanese Patent Application Laid-Open No. 10-215883); pVK9 (WO2007 / 046389); pVS7 (WO2013 / 069634); and pVC7 (Japanese Patent Application Laid-Open No. 9-070291). Furthermore, specific examples of vectors capable of autonomous replication in coryneform bacteria include pVC7 variants such as pVC7H2 (WO2018 / 179834).

[0111] When a gene is introduced, it is sufficient that the gene can be expressed by the host. Specifically, it is sufficient that the gene is maintained so that it is expressed under the control of a promoter that functions in the host. A "promoter that functions in the host" may mean a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene. For example, a stronger promoter as described herein may be used as the promoter.

[0112] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator from another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0113] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0114] Furthermore, when two or more genes are introduced, it is sufficient that each gene is retained in an expressible state in the host. For example, all of the genes may be retained on a single expression vector, or all may be retained on a chromosome. Alternatively, each gene may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Alternatively, two or more genes may constitute an operon and be introduced. When "two or more genes are introduced," for example, genes encoding two or more proteins (e.g., enzymes) may be introduced, genes encoding two or more subunits that constitute a single protein complex (e.g., an enzyme complex) may be introduced, or a combination thereof may be introduced.

[0115] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene derived from the host or a gene derived from a heterologous species. The gene to be introduced can be obtained, for example, by PCR using primers designed based on the nucleotide sequence of the gene and the genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. The gene to be introduced may also be totally synthesized based on the nucleotide sequence of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as is or after appropriate modification. In other words, by modifying the gene, its variants can be obtained. Gene modification can be performed by known techniques. For example, site-directed mutagenesis can be used to introduce a desired mutation into a target site in DNA. In other words, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Site-directed mutagenesis methods include PCR-based methods (Higuchi, R., 61, in PCR Technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, gene variants may be totally synthesized.

[0116] When a protein functions as a complex consisting of multiple subunits, all or only some of the subunits may be modified, as long as the resulting protein activity is increased. That is, for example, when increasing protein activity by increasing gene expression, the expression of all or only some of the genes encoding the subunits may be enhanced. It is usually preferable to enhance the expression of all of the genes encoding the subunits. Furthermore, the subunits constituting the complex may be derived from a single organism or from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, genes encoding multiple subunits derived from the same organism may be introduced into a host, or genes derived from different organisms may be introduced into a host.

[0117] Increased gene expression can also be achieved by improving gene transcription efficiency. Increased gene expression can also be achieved by improving gene translation efficiency. Gene transcription efficiency and translation efficiency can be improved, for example, by modifying expression regulatory sequences. "Expression regulatory sequence" may be a general term for sites that affect gene expression. Examples of expression regulatory sequences include promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), and spacer regions between the RBS and the start codon. Expression regulatory sequences can be determined using promoter search vectors or genetic analysis software such as GENETYX. These expression regulatory sequences can be modified, for example, by a method using a temperature-sensitive vector or the Red-driven integration method (WO2005 / 010175).

[0118] The transcription efficiency of a gene can be improved, for example, by replacing the promoter of the gene on a chromosome with a stronger promoter. A "stronger promoter" may refer to a promoter that improves gene transcription compared to the wild-type promoter that originally exists. Examples of stronger promoters include known high-expression promoters such as the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter from Bifidobacterium, PR promoter, and PL promoter. Examples of stronger promoters that can be used in coryneform bacteria include the artificially engineered P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), the pta, aceA, aceB, adh, and amyE promoters that can be induced in coryneform bacteria with acetic acid, ethanol, pyruvate, and the like, and the cspB, SOD, and tuf (EF-Tu) promoters, which are strong promoters that are highly expressed in coryneform bacteria (Journal of Biotechnology 104 (2003) 311-323, Appl. Environ. Microbiol. 2005). Examples of such promoters include the P2 promoter (WO2018 / 079684), the P3 promoter (WO2018 / 079684), the F1 promoter (WO2018 / 179834), the lac promoter, the tac promoter, the trc promoter, and the F1 promoter. Furthermore, highly active versions of conventional promoters can be obtained by using various reporter genes to enhance promoter activity. For example, promoter activity can be enhanced by adjusting the -35 and -10 regions of the promoter region to resemble consensus sequences (International Publication No. 00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application 2006134574).Methods for evaluating promoter strength and examples of strong promoters are described in, for example, Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0119] Gene translation efficiency can be improved, for example, by replacing the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence. A "stronger SD sequence" refers to an SD sequence that improves mRNA translation compared to the native wild-type SD sequence. An example of a stronger SD sequence is the RBS of gene 10 from phage T7 (Olins PO et al., Gene, 1988, 73, 227-235). Furthermore, substitution, insertion, or deletion of several nucleotides in the spacer region between the RBS and the start codon, particularly in the sequence immediately upstream of the start codon (5'-UTR), is known to significantly affect mRNA stability and translation efficiency. Gene translation efficiency can also be improved by modifying these sequences.

[0120] The translation efficiency of a gene can also be improved by, for example, codon modification. For example, the translation efficiency of a gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. That is, the gene to be introduced may be modified to have optimal codons depending on the codon usage frequency of the host used. Codon substitution can be performed, for example, by site-directed mutagenesis, which introduces a desired mutation into a target site in DNA. Alternatively, a gene fragment with a substituted codon may be totally synthesized. Codon usage frequencies in various organisms are disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al., Nucl. Acids Res., 28, 292 (2000)).

[0121] Furthermore, increasing gene expression can also be achieved by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

[0122] The above-mentioned methods for increasing gene expression may be used alone or in any combination.

[0123] Modifications that increase protein activity can also be achieved by, for example, enhancing the specific activity of the protein. Enhancement of specific activity may also include desensitization to feedback inhibition. That is, when a protein is subject to feedback inhibition by metabolites, the activity of the protein can be increased by mutating the gene or protein in the host so that feedback inhibition is desensitized. Unless otherwise specified, "desensitization to feedback inhibition" may include complete desensitization of feedback inhibition and reduced feedback inhibition. Furthermore, "desensitized feedback inhibition" (i.e., reduced or eliminated feedback inhibition) is also referred to as "resistance to feedback inhibition." Proteins with enhanced specific activity can be obtained, for example, by searching various organisms. Highly active proteins can also be obtained by introducing mutations into existing proteins. The introduced mutations may be, for example, substitutions, deletions, insertions, and / or additions of one or several amino acids at one or several positions in the protein. Mutations can be introduced, for example, by site-directed mutagenesis, as described above. Mutations can also be introduced by, for example, mutagenesis. Examples of mutagenesis include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Random mutations can also be induced by directly treating DNA with hydroxylamine in vitro. Enhancement of specific activity can be used alone or in any combination with the above-mentioned methods for enhancing gene expression.

[0124] The transformation method is not particularly limited, and conventionally known methods can be used, such as the method reported for Escherichia coli K-12, in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), or the method reported for Bacillus subtilis, in which competent cells are prepared from growing cells and DNA is introduced into the cells (Duncan, CH, Wilson, GA and Young, FE, 1997, Gene 1: 153-167). Alternatively, recombinant DNA can be introduced into recipient cells by converting them into protoplasts or spheroplasts, which readily incorporate recombinant DNA, as has been reported for Bacillus subtilis, actinomycetes, and yeast (Chang, S. and Choen, SN, 1979, Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM, and Hopwood, OA, 1978, Nature 274: 398-400; Hinnen, A., Hicks, JB, and Fink, GR, 1978, Proc. Natl. Acad. Sci. USA 75: 1929-1933). Alternatively, an electric pulse method, as reported for coryneform bacteria (JP 2-207791), can be used.

[0125] The increase in protein activity can be confirmed by measuring the activity of the protein.

[0126] Increased protein activity can also be confirmed by confirming increased expression of the gene encoding the protein, which can be confirmed by confirming increased transcription of the gene or increased amount of protein expressed from the gene.

[0127] Increased gene transcription levels can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a wild-type strain or a non-modified strain such as the parent strain. Methods for assessing mRNA levels include Northern hybridization, RT-PCR, microarrays, and RNA-seq (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The mRNA level may be increased, for example, by 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more compared to that of a non-modified strain.

[0128] The increase in the protein amount can be confirmed by Western blotting using an antibody (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The protein amount (e.g., number of molecules per cell) may be increased by, for example, 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more compared to the unmodified strain.

[0129] The above-mentioned methods for increasing protein activity can be used to enhance the activity of any protein or enhance the expression of any gene.

[0130] <3-4> Methods for reducing protein activity Below, methods for reducing protein activity are explained.

[0131] "Decreased protein activity" may mean that the activity of the protein is reduced compared to that of an unmodified strain. "Decreased protein activity" may specifically mean that the activity of the protein per cell is reduced compared to that of an unmodified strain. "Protein activity per cell" may mean the average activity of the protein per cell. An unmodified strain is also referred to as an "unmodified host" or "unmodified host strain." As used herein, "unmodified strain" may refer to a control strain that has not been modified to reduce the activity of the target protein. Examples of unmodified strains include wild-type strains and parent strains. Specific examples of unmodified strains include type strains of the species to which the host belongs. Specific examples of unmodified strains include the strains exemplified in the description of the host. That is, in one embodiment, the activity of the protein may be reduced compared to that of a type strain (i.e., a type strain of the species to which the host belongs). In another embodiment, the activity of the protein may be reduced compared to that of the C. glutamicum ATCC 13869 strain. In another embodiment, the activity of the protein may be reduced compared to that of the C. glutamicum ATCC 13032 strain. In another embodiment, the activity of the protein may be reduced compared to that of the E. coli K-12 MG1655 strain. Note that "reduced protein activity" may also encompass a complete loss of protein activity. More specifically, "reduced protein activity" may mean a reduction in the number of molecules of the protein per cell and / or a reduction in the function of the protein per molecule compared to a non-modified strain. That is, the "activity" in "reduced protein activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The "number of protein molecules per cell" may refer to the average number of molecules of the protein per cell. Note that "reduced number of protein molecules per cell" may also encompass a complete absence of the protein.Furthermore, "reduced function per molecule of protein" may also include complete loss of function per molecule of the protein. The degree of reduction in protein activity is not particularly limited, as long as the protein activity is reduced compared to that of an unmodified strain. The protein activity may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0132] Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced gene expression" may mean that the expression of the gene is reduced compared to an unmodified strain such as a wild-type strain or a parent strain. "Reduced gene expression" may specifically mean that the expression level of the gene per cell is reduced compared to an unmodified strain. "Expression level of the gene per cell" may mean the average expression level of the gene per cell. "Reduced gene expression" may more specifically mean that the transcription level (mRNA level) of the gene is reduced and / or the translation level (protein level) of the gene is reduced. "Reduced gene expression" may also include cases where the gene is not expressed at all. Note that "reduced gene expression" is also referred to as "attenuated gene expression." Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0133] Decreased gene expression may be due to, for example, decreased transcription efficiency, decreased translation efficiency, or a combination thereof. Decreased gene expression can be achieved, for example, by modifying expression regulatory sequences such as the gene promoter, Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)), or the spacer region between the RBS and the start codon. When modifying an expression regulatory sequence, one or more bases, two or more bases, or three or more bases of the expression regulatory sequence are modified. Decreased gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" may refer to a promoter that weakens gene transcription compared to the native wild-type promoter. Examples of weaker promoters include inducible promoters. Examples of weaker promoters include the P4 promoter (WO2018 / 079684) and the P8 promoter (WO2018 / 079684). That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (e.g., in the absence of an inducer). Alternatively, a portion or all of the expression regulatory sequence may be deleted. Reduced gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Reduced gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces gene expression. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Furthermore, gene expression itself can be reduced, for example, by disrupting the gene as described herein.

[0134] Furthermore, a modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene that encodes the protein. "Disrupting a gene" may mean that the gene is modified so that it does not produce a protein that functions normally. "Not producing a protein that functions normally" may also include cases where no protein is produced from the gene at all, or cases where the gene produces a protein with reduced or lost function per molecule (e.g., activity or properties).

[0135] Gene disruption can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Gene deletion" may refer to the deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on a chromosome. The region to be deleted may be any region, such as the N-terminal region (i.e., the region encoding the N-terminal side of the protein), an internal region, or the C-terminal region (i.e., the region encoding the C-terminal side of the protein), as long as a reduction in protein activity can be achieved. Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of the coding region of the gene. Furthermore, it is preferable that the sequences before and after the deleted region do not match in reading frame. Reading frame mismatches can cause frameshifts downstream of the deleted region.

[0136] Gene disruption can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting one to two bases (frameshift mutation) (Journal of Biological Chemistry 272:8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

[0137] Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be anywhere in the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. Furthermore, it is preferable that the reading frames of the sequences before and after the insertion site do not match. A mismatch in the reading frame can cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for producing target substances.

[0138] Gene disruption may be carried out particularly so as to delete (delete) the amino acid sequence of the encoded protein. In other words, modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein (partial or entire region of the amino acid sequence), specifically by modifying the gene so that it encodes a protein from which the amino acid sequence (partial or entire region of the amino acid sequence) has been deleted. The term "deletion of the amino acid sequence of a protein" may refer to the deletion of a partial or entire region of the amino acid sequence of a protein. The term "deletion of the amino acid sequence of a protein" may also refer to the absence of the original amino acid sequence in the protein and may also encompass cases where the original amino acid sequence is changed to a different amino acid sequence. For example, a region that has been changed to a different amino acid sequence due to frameshifting may be considered a deleted region. While deletion of an amino acid sequence typically shortens the overall length of the protein, it may also be possible for the overall length of the protein to remain unchanged or to be extended. For example, deletion of a partial or entire region of the coding region of a gene can delete the region encoded by the deleted region in the amino acid sequence of the encoded protein. For example, by introducing a stop codon into the coding region of a gene, the region coded for by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. For example, by causing a frameshift in the coding region of a gene, the region coded for by the frameshift site can be deleted. The position and length of the region to be deleted in the deletion of an amino acid sequence can be determined mutatis mutandis from the explanation of the position and length of the region to be deleted in the deletion of a gene.

[0139] The above-described modification of a gene on a chromosome can be achieved, for example, by creating a disrupted gene modified so that it does not produce a normally functioning protein, transforming a host with recombinant DNA containing the disrupted gene, and inducing homologous recombination between the disrupted gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, the recombinant DNA can be easily manipulated by incorporating a marker gene according to the host's traits, such as its nutritional requirements. Examples of disrupted genes include genes lacking part or all of the coding region of a gene, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, and genes with transposons or marker genes inserted. Even if a protein encoded by a disrupted gene is produced, it will have a different three-dimensional structure from the wild-type protein, resulting in reduced or lost function. Such gene disruption by gene replacement using homologous recombination has already been established, and includes methods that use linear DNA, such as a method called "Red-driven integration" (Datsenko, K. A., and Wanner, BL Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), a method that combines the Red-driven integration method with an excision system derived from λ phage (Cho, E. H., Gumport, R. I., Gardner, J. F. J. Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), methods that use a plasmid containing a temperature-sensitive replication origin, methods that use a conjugatively transferable plasmid, and methods that use a suicide vector that does not have a replication origin that functions in the host (U.S. Patent No. 6,303,383, JP 05-007491 A).

[0140] Modifications that reduce the activity of proteins may also be performed by, for example, mutation treatments, such as X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0141] When a protein functions as a complex consisting of multiple subunits, all or only a portion of the subunits may be modified, as long as the activity of the protein is reduced as a result. That is, for example, all or only a portion of the genes encoding each of the subunits may be disrupted, etc. Furthermore, when a protein has multiple isozymes, the activities of all or only a portion of the isozymes may be reduced, as long as the activity of the protein is reduced as a result. That is, for example, all or only a portion of the genes encoding each of the isozymes may be disrupted, etc.

[0142] The above-mentioned methods for reducing protein activity may be used alone or in any combination.

[0143] The decrease in the activity of the protein can be confirmed by measuring the activity of the protein.

[0144] A decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.

[0145] The reduction in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, microarray, and RNA-seq (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0146] The reduction in the protein amount can be confirmed by Western blotting using an antibody (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The protein amount (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0147] Gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene, depending on the means used for disruption.

[0148] The above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein or the expression of any gene.

[0149] <3-5> Host culture The target enzyme can be expressed by culturing a host carrying the target enzyme gene.

[0150] The medium used is not particularly limited as long as it allows the host to grow and expresses a functional target enzyme. For example, a conventional medium used for culturing microorganisms such as bacteria and yeast can be used as the medium. The medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed. The types and concentrations of medium components may be appropriately determined depending on various conditions such as the type of host.

[0151] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, pulverized products, or purified products. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Because hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses such as glucose into xylose, for example, by providing the host with a pathway for converting hexoses to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0152] The concentration of the carbon source in the medium is not particularly limited as long as the host can grow and a functional target enzyme is expressed. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit the production of the target enzyme. The initial concentration of the carbon source in the medium may be, for example, usually 5 to 30 w / v%, preferably 10 to 20 w / v%. Additionally, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied in response to a decrease or depletion of the carbon source as the culture progresses. While the carbon source may be temporarily depleted as long as the target enzyme is ultimately produced, it may be preferable to carry out the culture so that the carbon source does not become depleted or does not continue to be depleted.

[0153] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or aqueous ammonia, which are used for pH adjustment, may also be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0154] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0155] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0156] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and soy protein hydrolysate. As other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0157] When an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, it is preferable to supplement the medium with such required nutrients.

[0158] The culture conditions are not particularly limited as long as the host can grow and a functional target enzyme can be expressed. The culture can be carried out under normal conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions can be appropriately set depending on various conditions such as the type of host. Furthermore, the expression of the target enzyme gene can be induced as necessary.

[0159] Culturing can be carried out using a liquid medium. For example, the host may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured in a liquid medium and then inoculated into the liquid medium for main culture. That is, the culture may be divided into a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may or may not be the same. The target enzyme only needs to be expressed in the main culture. The amount of the host contained in the medium at the start of the culture is not particularly limited. For example, a seed culture solution with an OD660 of 4 to 100 may be added at the start of the culture in an amount of 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, relative to the medium for main culture.

[0160] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination of these. The medium at the start of cultivation is also called the "initial medium." The medium supplied to a cultivation system (e.g., a fermenter) in fed-batch or continuous cultivation is also called the "fed-batch medium." Supplying a fed-batch medium to a cultivation system in fed-batch or continuous cultivation is also called "fed-batch." When cultivation is carried out separately into a seed culture and a main culture, the cultivation forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out by batch culture, or the seed culture may be carried out by batch culture and the main culture may be carried out by fed-batch or continuous culture.

[0161] In the present invention, various components such as a carbon source may be contained in the initial medium, the feed medium, or both. That is, various components such as a carbon source may be additionally supplied to the medium during the culture process, either alone or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium. Furthermore, the concentrations of each component contained in the initial medium may or may not be the same as the concentrations of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the types and / or concentrations of components contained in each feed medium may or may not be the same.

[0162] The culture can be carried out under aerobic conditions, for example. "Aerobic conditions" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 50% of the saturated oxygen concentration, preferably about 5%. The culture can be carried out, for example, by aerobic culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During culture, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the host is lost.

[0163] By culturing the host in this manner, a culture containing the target enzyme can be obtained. The target enzyme can be accumulated, for example, within the host's bacterial cells. The term "bacterial cells" can be appropriately interpreted as "cells" depending on the type of host. Depending on the host and / or the design of the target enzyme gene used, it may be possible to accumulate the target enzyme in the periplasm or secrete and produce the target enzyme outside the bacterial cells.

[0164] The target enzyme may be used in the method of the present invention as it is contained in the culture (specifically, the medium or bacterial cells), or may be purified from the culture (specifically, the medium or bacterial cells) before use. Purification can be carried out to any desired extent. That is, the target enzyme may be a purified target enzyme or a fraction containing the target enzyme. In other words, the target enzyme may be used in the form of a purified enzyme, in the form of such a fraction (i.e., in the form contained in such a fraction), or in a combination thereof. Such fractions are not particularly limited, as long as they contain the target enzyme so that it can act on its substrate. Such fractions include cultures of hosts harboring the target enzyme gene (i.e., hosts harboring the target enzyme), bacterial cells recovered from the cultures, culture supernatants recovered from the cultures, processed products thereof (e.g., bacterial cell disruption, bacterial cell lysates, bacterial cell extracts, and other processed bacterial products such as those described below), partially purified products thereof (i.e., crude products), and combinations thereof. Note that the term "purified target enzyme" may also encompass crude products. These fractions can be used alone or in appropriate combination.

[0165] The target enzyme may be used in the method of the present invention (e.g., conversion reaction) particularly in a form contained in bacterial cells. The bacterial cells may be used in the method of the present invention (e.g., conversion reaction) as they are contained in the culture (specifically, medium), or may be recovered from the culture (specifically, medium) and used in the method of the present invention (e.g., conversion reaction). The bacterial cells may also be subjected to appropriate treatment before being used in the method of the present invention (e.g., conversion reaction). That is, examples of bacterial cells include a culture of a host having a target enzyme gene (i.e., a host having a target enzyme), bacterial cells recovered from the culture, and processed products thereof. In other words, the bacterial cells may be used in the form of a culture of a host having a target enzyme gene (i.e., a host having a target enzyme), bacterial cells recovered from the culture, processed products thereof, or a combination thereof. Examples of processed products include bacterial cells (e.g., bacterial cells contained in a culture or bacterial cells recovered from a culture) that have been subjected to treatment. These forms of bacterial cells can be used alone or in appropriate combinations.

[0166] The method for recovering the bacterial cells from the culture medium is not particularly limited, and known methods can be used, for example. Such methods include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be used. These methods can be used alone or in appropriate combination. The recovered bacterial cells can be washed appropriately using an appropriate medium. The recovered bacterial cells can also be resuspended appropriately using an appropriate medium. Examples of media that can be used for washing and suspending include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

[0167] Examples of treatments for bacterial cells include immobilization on carriers such as acrylamide or carrageenan, freeze-thaw treatment, and treatment to increase membrane permeability. Membrane permeability can be increased by using, for example, a surfactant or organic solvent. These treatments can be used alone or in appropriate combination.

[0168] Furthermore, the target enzyme may be diluted or concentrated as appropriate before use in the method of the present invention.

[0169] The target enzymes may be produced individually, or two or more may be produced together. For example, two or more target enzymes can be produced together by expressing the genes for two or more target enzymes in a host having those genes.

[0170] Alternatively, a host carrying a gene for a target enzyme may be cultured and used as the target enzyme in the method of the present invention.

[0171] <4> Methods of the Invention The method of the present invention is a method for producing benzaldehyde using a benzaldehyde-producing enzyme and catalase. Specifically, the method of the present invention is a method for producing benzaldehyde, comprising a step of producing benzaldehyde using the benzaldehyde-producing enzyme, at least a part of which is carried out in the presence of catalase. This step is also referred to as the "production step."

[0172] The benzaldehyde-producing enzyme may be used for producing benzaldehyde in any of the forms described above. That is, the use of the benzaldehyde-producing enzyme includes any of the forms described above. The use form of the benzaldehyde-producing enzyme can be independently determined for each benzaldehyde-producing enzyme. The benzaldehyde-producing enzyme may be used, for example, in a form contained in host cells such as microbial cells. The benzaldehyde-producing enzyme may also be used, for example, in a form not contained in host cells, such as a purified enzyme. Furthermore, for example, a portion of the benzaldehyde-producing enzyme may be used in a form contained in host cells such as microbial cells, and the remainder may be used in a form not contained in host cells, such as a purified enzyme. The benzaldehyde-producing enzyme may particularly be used in the form of the microorganism of the present invention. That is, the use of the benzaldehyde-producing enzyme particularly includes the use of the microorganism of the present invention. Use of the microorganism of the present invention includes culturing the microorganism of the present invention and using the microbial cells of the present invention. That is, the microorganism of the present invention may be used as a benzaldehyde-producing enzyme by, for example, culturing the microorganism.Furthermore, the microorganism of the present invention may be used as a benzaldehyde-producing enzyme by, for example, culturing the microorganism.

[0173] Benzaldehyde can be produced, for example, by fermentation, bioconversion, or a combination thereof. That is, the production process may be carried out, for example, by fermentation, bioconversion, or a combination thereof. The embodiment of the production process can be appropriately set depending on various conditions, such as the mode of use of the benzaldehyde-producing enzyme. Specifically, the production process may be carried out, for example, by culturing the microorganism of the present invention, by using the cells of the microorganism of the present invention, or by a combination thereof. Specifically, the combination may be a combination of culturing some of the multiple microorganisms constituting the microorganism of the present invention and using the cells of the remaining multiple microorganisms. Benzaldehyde can be produced, for example, from a carbon source or L-phenylalanine.

[0174] <4-1> Fermentation method Benzaldehyde can be produced, for example, by fermentation using the microorganism of the present invention capable of producing L-phenylalanine. That is, one embodiment of the method of the present invention may be a method of producing benzaldehyde by fermentation using the microorganism of the present invention capable of producing L-phenylalanine. This embodiment is also referred to as a "fermentation method." Furthermore, the process of producing benzaldehyde by fermentation using the microorganism of the present invention capable of producing L-phenylalanine is also referred to as a "fermentation process."

[0175] The fermentation step can be carried out by culturing the microorganism of the present invention. Specifically, in the fermentation step, benzaldehyde can be produced from a carbon source. That is, the fermentation step may be, for example, a step of culturing the microorganism of the present invention in a medium (e.g., a medium containing a carbon source) and producing and accumulating benzaldehyde in the medium. That is, the fermentation method may be a method for producing benzaldehyde, comprising culturing the microorganism of the present invention in a medium (e.g., a medium containing a carbon source) and producing and accumulating benzaldehyde in the medium. In other words, the fermentation step may be, for example, a step of producing benzaldehyde from a carbon source using the microorganism of the present invention.

[0176] When the microorganism of the present invention is a combination of multiple microorganisms, the multiple microorganisms may or may not be cultured simultaneously. For example, the multiple microorganisms may be inoculated and cultured simultaneously, or may be inoculated and cultured separately, or in any combination at different times. The order and timing of culturing the multiple microorganisms are not particularly limited as long as benzaldehyde can be produced from a carbon source. For example, a microorganism having AAD, a microorganism having HMAS, a microorganism having SMDH, and a microorganism having BFDC may be inoculated in this order. When the microorganism of the present invention is a combination of multiple microorganisms, the phrase "culturing the microorganism of the present invention in a medium containing a carbon source" means culturing at least one microorganism selected from the multiple microorganisms in a medium containing a carbon source in a manner that allows benzaldehyde to be produced from a carbon source; however, it is not necessary to culture all of the multiple microorganisms in a medium containing a carbon source. That is, "culturing the microorganism of the present invention in a medium containing a carbon source" may mean, for example, culturing at least a microorganism capable of producing L-phenylalanine (which may be a microorganism having AAD) in a medium containing a carbon source, when the microorganism of the present invention is a combination of multiple microorganisms. That is, for example, by culturing a microorganism having AAD in a medium containing a carbon source, the carbon source may be consumed completely, producing an intermediate as described below, and then culturing another microorganism. In culturing the microorganism after the carbon source has been consumed, an additional carbon source (which may or may not be used as a raw material for producing benzaldehyde) may be used as appropriate.

[0177] The medium used is not particularly limited as long as the microorganism of the present invention can grow and benzaldehyde can be produced. The culture conditions are not particularly limited as long as the microorganism of the present invention can grow and benzaldehyde can be produced. The medium may contain components useful for the function of the target enzyme. Examples of such components include ascorbic acid and oxygen. Ascorbic acid and oxygen can be, for example, components useful for the function of HMAS. That is, at least in the part using HMAS, the culture may be carried out in the presence of ascorbic acid and oxygen. The description of the part using HMAS in the presence of catalase described below applies mutatis mutandis to the culture in the presence of ascorbic acid and oxygen in the part using HMAS. Oxygen may be supplied to the medium, for example, by carrying out the culture in an oxygen-containing atmosphere such as air. Components capable of forming salts may be used in their free form, as their salts, or as mixtures thereof. That is, unless otherwise specified, the term "component" used in the present invention for components capable of forming salts refers to the free form of the component, its salt, or a mixture thereof. With regard to salts of components capable of forming salts, the description of salts of L-phenylalanine can be applied mutatis mutandis. For example, ascorbic acid may be used in its free form, as a salt, or as a mixture thereof. That is, the term "ascorbic acid" in the present invention means ascorbic acid in its free form, its salt, or a mixture thereof, unless otherwise specified. Examples of salts include ammonium salt, sodium salt, and potassium salt. As a salt, one type of salt may be used, or two or more types of salts may be used in combination. With regard to the culture in the fermentation method, the description of the culture of the host described above (e.g., the description of the medium and culture conditions) can be applied mutatis mutandis, except that benzaldehyde is produced in the fermentation method.

[0178] By culturing the microorganism of the present invention in this manner, a culture containing benzaldehyde can be obtained.

[0179] The production of benzaldehyde can be confirmed by known techniques used for detecting or identifying compounds. Examples of such techniques include HPLC, UPLC, LC / MS, GC / MS, and NMR. These techniques can be used alone or in appropriate combinations. These techniques can also be used to determine the concentrations of various components present in the medium.

[0180] The produced benzaldehyde can be recovered appropriately. That is, the method of the present invention may further include a step of recovering benzaldehyde. This step is also referred to as a "recovery step." The recovery step may be a step of recovering benzaldehyde from the culture, specifically from the medium. The produced benzaldehyde can be recovered by a known method used for separating and purifying compounds. Examples of such methods include an ion exchange resin method, a membrane treatment method, a precipitation method, an extraction method, a distillation method, and a crystallization method. Specifically, benzaldehyde can be recovered by extraction with an organic solvent such as ethyl acetate or by steam distillation. These methods can be used alone or in appropriate combination.

[0181] When benzaldehyde precipitates in the medium, it can be recovered by, for example, centrifugation or filtration. Alternatively, the benzaldehyde precipitated in the medium may be isolated together with the benzaldehyde dissolved in the medium after crystallization.

[0182] The recovered benzaldehyde may contain, in addition to benzaldehyde, components such as bacterial cells, medium components, water, metabolic by-products of microorganisms, etc. The purity of the recovered benzaldehyde may be, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0183] <4-2> Material transformation method Benzaldehyde can also be produced, for example, by material conversion using a benzaldehyde-producing enzyme (e.g., the microorganism of the present invention). That is, another embodiment of the method of the present invention may be a method for producing benzaldehyde by material conversion using a benzaldehyde-producing enzyme (e.g., the microorganism of the present invention). This embodiment is also referred to as a "material conversion method." Furthermore, the step of producing benzaldehyde by material conversion using a benzaldehyde-producing enzyme (e.g., the microorganism of the present invention) is also referred to as a "material conversion step."

[0184] Specifically, in the material conversion step, benzaldehyde can be produced from L-phenylalanine. More specifically, in the material conversion step, benzaldehyde can be produced by converting L-phenylalanine to benzaldehyde using a benzaldehyde-producing enzyme (e.g., the microorganism of the present invention). That is, the material conversion step may be a step of converting L-phenylalanine to benzaldehyde using a benzaldehyde-producing enzyme (e.g., the microorganism of the present invention). In the material conversion step, L-phenylalanine, the substance before conversion, is also referred to as the "substrate," and benzaldehyde, the substance after conversion, is also referred to as the "product."

[0185] L-phenylalanine may be used in its free form, as a salt, or as a mixture thereof. That is, the term "L-phenylalanine" in the present invention means L-phenylalanine in its free form, its salt, or a mixture thereof, unless otherwise specified. Examples of salts include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt. As the salt, one type of salt may be used, or two or more types of salts may be used in combination.

[0186] L-phenylalanine may be commercially available or may be obtained by appropriate production. The method for producing L-phenylalanine is not particularly limited, and known methods can be used. L-phenylalanine can be produced, for example, by chemical synthesis, enzymatic methods, material conversion methods, fermentation methods, extraction methods, or a combination thereof. That is, L-phenylalanine can be produced, for example, by culturing a microorganism capable of producing L-phenylalanine (an L-phenylalanine-producing bacterium) and recovering the amino acid from the culture. The produced L-phenylalanine can be used in the method of the present invention either directly or after appropriate treatments such as concentration, dilution, drying, dissolution, fractionation, extraction, and purification. That is, as L-phenylalanine, for example, a purified product purified to the desired degree or a material containing L-phenylalanine can be used. The material containing L-phenylalanine is not particularly limited as long as it can be utilized by an AAD-synthesizing enzyme (e.g., a microorganism having AAD, such as the microorganism of the present invention). Specific examples of materials containing L-phenylalanine include cultures obtained by culturing L-phenylalanine-producing bacteria, culture supernatants separated from the cultures, and processed products such as concentrates (e.g., concentrates) and dried products thereof.

[0187] In one embodiment, the substance conversion step can be carried out, for example, by culturing the microorganism of the present invention. This embodiment is also referred to as the "first embodiment of the substance conversion method." That is, the substance conversion step may be, for example, a step of culturing the microorganism of the present invention in a medium containing L-phenylalanine and converting L-phenylalanine to benzaldehyde. Specifically, the substance conversion step may be a step of culturing the microorganism of the present invention in a medium containing L-phenylalanine and producing and accumulating benzaldehyde in the medium.

[0188] When the microorganism of the present invention is a combination of multiple microorganisms, the multiple microorganisms may or may not be cultured simultaneously. For example, the multiple microorganisms may be inoculated and cultured simultaneously, or may be inoculated and cultured separately, or in any combination at different times. The order and timing of culturing the multiple microorganisms are not particularly limited as long as the conversion of L-phenylalanine to benzaldehyde is successful. For example, a microorganism having AAD, a microorganism having HMAS, a microorganism having SMDH, and a microorganism having BFDC may be inoculated in this order. Note that, when the microorganism of the present invention is a combination of multiple microorganisms, "culturing the microorganism of the present invention in a medium containing L-phenylalanine" means culturing at least one microorganism selected from the multiple microorganisms in a medium containing L-phenylalanine in such a manner that the conversion of L-phenylalanine to benzaldehyde is successful; it is not necessary to culture all of the multiple microorganisms in a medium containing L-phenylalanine. That is, "culturing the microorganism of the present invention in a medium containing L-phenylalanine" may mean, for example, culturing at least a microorganism having AAD in a medium containing L-phenylalanine when the microorganism of the present invention is a combination of multiple microorganisms. That is, for example, by culturing a microorganism having AAD in a medium containing L-phenylalanine, the culture of another microorganism may be started after all L-phenylalanine has been converted into an intermediate as described below.

[0189] The medium used is not particularly limited as long as it contains L-phenylalanine, allows the microorganism of the present invention to grow, and benzaldehyde is produced. The culture conditions are also not particularly limited as long as the microorganism of the present invention can grow and benzaldehyde is produced. The medium may contain components useful for the function of the target enzyme. Examples of such components include ascorbic acid and oxygen. Ascorbic acid and oxygen can be, for example, components useful for the function of HMAS. That is, at least in the part utilizing HMAS, the culture may be carried out in the presence of ascorbic acid and oxygen. The description of the part utilizing HMAS in the presence of catalase described below can be applied mutatis mutandis to the culture in the first embodiment of the substance conversion method, except that in the first embodiment of the substance conversion method, the medium contains L-phenylalanine and benzaldehyde is produced.

[0190] L-phenylalanine may be present in the medium throughout the entire culture period, or only for a portion of the culture period. In other words, "culturing a microorganism in a medium containing L-phenylalanine" does not require that L-phenylalanine be present in the medium throughout the entire culture period. For example, L-phenylalanine may or may not be present in the medium from the start of culture. If L-phenylalanine is not present in the medium at the start of culture, L-phenylalanine is supplied to the medium after the start of culture. The timing of supply can be appropriately determined depending on various conditions, such as the culture time. For example, L-phenylalanine may be supplied to the medium after the microorganism of the present invention has grown sufficiently. In either case, additional L-phenylalanine may be supplied to the medium as appropriate. For example, additional L-phenylalanine may be supplied to the medium in response to a decrease or depletion of L-phenylalanine due to the production of benzaldehyde. The means for supplying L-phenylalanine to the medium is not particularly limited. For example, L-phenylalanine can be supplied to a medium by feeding a feed medium containing L-phenylalanine to the medium. Alternatively, for example, the microorganism of the present invention can be co-cultured with an L-phenylalanine-producing bacterium to cause the L-phenylalanine-producing bacterium to produce L-phenylalanine in the medium, thereby supplying L-phenylalanine to the medium. These supplying methods can be used alone or in appropriate combination. The L-phenylalanine concentration in the medium is not particularly limited, as long as the microorganism of the present invention can utilize L-phenylalanine as a raw material for benzaldehyde. The L-phenylalanine concentration in the medium may be, for example, 1 mM or more, 10 mM or more, or 30 mM or more, or 5 M or less, 2 M or less, or 1 M or less, or a combination thereof. L-phenylalanine may or may not be contained in the medium at the concentrations exemplified above throughout the entire culture period. For example, L-phenylalanine may be contained in the medium at the concentrations exemplified above at the start of culture, or may be supplied to the medium to achieve the concentrations exemplified above after the start of culture. When the culture is divided into a seed culture and a main culture, benzaldehyde should be produced at least during the main culture period.Therefore, L-phenylalanine only needs to be contained in the medium during at least the period of the main culture, i.e., the entire period of the main culture or a part of the period of the main culture. That is, L-phenylalanine may or may not be contained in the medium during the seed culture. In such cases, descriptions of the culture (e.g., "culture period (period of culture)" or "start of culture") can be interpreted as referring to the main culture.

[0191] In another embodiment, the substance conversion step can be carried out, for example, by utilizing a benzaldehyde-producing enzyme (e.g., microbial cells of the present invention). This embodiment is also referred to as the "second embodiment of the substance conversion method." That is, the substance conversion step may be, for example, a step of converting L-phenylalanine in a reaction solution to benzaldehyde by utilizing a benzaldehyde-producing enzyme (e.g., microbial cells of the present invention). Specifically, the substance conversion step may be a step of allowing a benzaldehyde-producing enzyme (e.g., microbial cells of the present invention) to coexist with L-phenylalanine in a reaction solution, thereby producing and accumulating benzaldehyde in the reaction solution. More specifically, the substance conversion step may be a step of allowing a benzaldehyde-producing enzyme (e.g., microbial cells of the present invention) to act on L-phenylalanine in the reaction solution, thereby producing and accumulating benzaldehyde in the reaction solution. The substance conversion step in the second embodiment of the substance conversion method (i.e., the step of converting L-phenylalanine in the reaction solution to benzaldehyde) is also referred to as a “conversion reaction.” The conversion reaction may be carried out particularly by utilizing cells of the microbial organism of the present invention.

[0192] The benzaldehyde-producing enzymes may or may not be supplied to the reaction solution simultaneously. For example, when the microorganism of the present invention is a combination of multiple microorganisms, the cells of the multiple microorganisms may or may not be supplied to the reaction solution simultaneously. For example, the cells of the multiple microorganisms may be supplied to the reaction solution at different times, or in any combination at different times. The order and timing of supplying the cells of the multiple microorganisms to the reaction solution are not particularly limited, as long as the conversion of L-phenylalanine to benzaldehyde is successful. For example, AAD, HMAS, SMDH, and BFDC (e.g., cells of multiple microorganisms each having AAD, HMAS, SMDH, and BFDC) may be supplied to the reaction solution in that order. The phrase "allowing a benzaldehyde-generating enzyme to coexist with L-phenylalanine or act on L-phenylalanine in a reaction solution" means allowing at least one enzyme selected from benzaldehyde-generating enzymes to coexist with L-phenylalanine or act on L-phenylalanine in a reaction solution in such a way that conversion of L-phenylalanine to benzaldehyde is achieved, but it is not necessary to allow all benzaldehyde-generating enzymes to coexist with L-phenylalanine or act on L-phenylalanine in the reaction solution. In other words, the phrase "allowing a benzaldehyde-generating enzyme to coexist with L-phenylalanine or act on L-phenylalanine in a reaction solution" may mean, for example, allowing at least AAD to coexist with L-phenylalanine or act on L-phenylalanine in the reaction solution. For example, "allowing the cells of the microbial organism of the present invention to coexist with L-phenylalanine in a reaction solution or to act on L-phenylalanine" means, in the case where the microbial organism of the present invention is a combination of multiple microorganisms, allowing the cells of at least one microorganism selected from the multiple microorganisms to coexist with L-phenylalanine in a reaction solution or to act on L-phenylalanine in a manner that enables conversion of L-phenylalanine to benzaldehyde, and does not require allowing the cells of all of the multiple microorganisms to coexist with L-phenylalanine in the reaction solution or to act on L-phenylalanine.That is, "coexisting the cells of the microbial organism of the present invention with L-phenylalanine in a reaction solution or allowing them to act on L-phenylalanine" may mean, for example, that at least the cells of a microbial organism having AAD are allowed to coexist with L-phenylalanine in a reaction solution or allow them to act on L-phenylalanine when the microbial organism of the present invention is a combination of multiple microorganisms. That is, for example, after AAD (e.g., cells of a microbial organism having AAD) is allowed to coexist with L-phenylalanine in a reaction solution or allow it to act on L-phenylalanine, and all of the L-phenylalanine is converted to an intermediate as described below, another benzaldehyde-producing enzyme (e.g., cells of another microbial organism) may be supplied to the reaction solution.

[0193] The bacterial cells used in the conversion reaction are not particularly limited as long as they have the ability to produce benzaldehyde. The bacterial cells may or may not have the ability to grow.

[0194] The conversion reaction can be carried out in an appropriate reaction solution. Specifically, the conversion reaction can be carried out by coexisting a benzaldehyde-producing enzyme (e.g., cells of the microorganism of the present invention) and L-phenylalanine in an appropriate reaction solution. The conversion reaction can be carried out in a batch or column format. In the batch format, the conversion reaction can be carried out, for example, by mixing a benzaldehyde-producing enzyme (e.g., cells of the microorganism of the present invention) and L-phenylalanine in a reaction solution in a reaction vessel. The conversion reaction can be carried out stationary, or with stirring or shaking. In the column format, the conversion reaction can be carried out, for example, by passing a reaction solution containing L-phenylalanine through a column packed with an immobilized enzyme such as immobilized cells. Examples of the reaction solution include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

[0195] In addition to L-phenylalanine, the reaction solution may contain components other than L-phenylalanine as needed. Examples of components other than L-phenylalanine include oxygen, metal ions such as iron ions, ascorbic acid, thiamine pyrophosphate, buffers, and various other medium components. Ascorbic acid and oxygen may be useful components for the function of HMAS, for example. That is, at least in the part using HMAS, the conversion reaction may be carried out in the presence of ascorbic acid and oxygen. The description of the part using HMAS in the presence of catalase, described below, applies mutatis mutandis to the conversion reaction in the presence of ascorbic acid and oxygen in the part using HMAS. Oxygen may be supplied to the reaction solution by carrying out the conversion reaction in an oxygen-containing atmosphere such as air. Components capable of forming salts may be used in their free form, as their salts, or as mixtures thereof. That is, unless otherwise specified, the term "component" used in the present invention for components capable of forming salts refers to the free form of the component, its salt, or a mixture thereof. The salts of components capable of forming salts can be determined as described for L-phenylalanine salts. The types and concentrations of components contained in the reaction solution may be appropriately determined depending on various conditions, such as the properties of the benzaldehyde-forming enzyme and the type of host.

[0196] The conditions for the conversion reaction (such as dissolved oxygen concentration, pH of the reaction solution, reaction temperature, reaction time, and concentrations of various components) are not particularly limited as long as benzaldehyde is produced. The conversion reaction can be carried out, for example, under conditions typically used for enzyme-based substance conversion (e.g., substance conversion using purified enzymes or substance conversion using microbial cells such as resting cells). The conditions for the conversion reaction may be appropriately set depending on various conditions, such as the properties of the benzaldehyde-producing enzyme and the type of host. The conversion reaction can be carried out, for example, under aerobic conditions. "Aerobic conditions" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled, for example, to 1 to 50% of the saturated oxygen concentration, preferably about 5%. The pH of the reaction solution may be, for example, usually 6.0 to 10.0, preferably 6.5 to 9.0. The reaction temperature may be, for example, usually 15 to 50°C, preferably 15 to 45°C, and more preferably 20 to 40°C. The reaction time may be, for example, 5 minutes to 200 hours. In the case of a column method, the flow rate of the reaction solution may be, for example, such that the reaction time falls within the range of the reaction time exemplified above. The conversion reaction may also be carried out under culture conditions such as those typically used for culturing microorganisms such as bacteria and yeast. In this case, when microbial cells such as those of the microbial cell of the present invention are used, the microbial cells may or may not grow. That is, the description of the culture in the first embodiment of the substance conversion method may be applied mutatis mutandis to the conversion reaction in the second embodiment of the substance conversion method, except that the microbial cells may or may not grow. In this case, the culture conditions for obtaining the microbial cells may or may not be the same as the conditions for the conversion reaction. The concentration of L-phenylalanine in the reaction solution may be, for example, 1 mM or more, 10 mM or more, or 30 mM or more, or 5 M or less, 2 M or less, or 1 M or less, or a combination thereof. The density of the bacterial cells in the reaction solution, converted into optical density (OD) at 600 nm, may be, for example, 1 or more, 300 or less, or a combination thereof.

[0197] During the conversion reaction, the benzaldehyde-producing enzyme (e.g., cells of the microorganism of the present invention), L-phenylalanine, and other components may be additionally supplied to the reaction solution, either alone or in any combination. For example, L-phenylalanine may be additionally supplied to the reaction solution in response to a decrease or depletion of L-phenylalanine due to the production of benzaldehyde. These components may be supplied once or multiple times, or continuously.

[0198] There are no particular limitations on the means for supplying various components such as L-phenylalanine to the reaction solution. These components can be supplied to the reaction solution by directly adding them to the reaction solution. Alternatively, for example, a benzaldehyde-producing enzyme (e.g., cells of the microorganism of the present invention) can be coexisted with an L-phenylalanine-producing bacterium (e.g., co-cultured) to cause the L-phenylalanine-producing bacterium to produce L-phenylalanine in the reaction solution, thereby supplying L-phenylalanine to the reaction solution.

[0199] Furthermore, the reaction conditions may be uniform from the start to the end of the conversion reaction, or may change during the course of the conversion reaction. "The reaction conditions change during the conversion reaction" does not necessarily mean that the reaction conditions change over time, but also includes that the reaction conditions change spatially. "The reaction conditions change spatially" means that, for example, when a conversion reaction is carried out in a column, reaction conditions such as reaction temperature and enzyme density (e.g., bacterial cell density) vary depending on the position on the flow path.

[0200] By carrying out the substance conversion step in this manner, a culture or reaction solution containing benzaldehyde can be obtained. Confirmation of benzaldehyde production and recovery of benzaldehyde can both be carried out in the same manner as in the fermentation method described above. That is, the substance conversion method may further include a recovery step (e.g., a step of recovering benzaldehyde from the culture or reaction solution). The recovered benzaldehyde may contain other components in addition to benzaldehyde, such as the target enzyme, microbial cells, medium components, reaction solution components, water, and metabolic by-products of the microorganism. The purity of the recovered benzaldehyde may be, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more.

[0201] <4-3> Combining substeps The production process (e.g., fermentation process or conversion process) is not particularly limited, and may be carried out via the production of an intermediate. Examples of intermediates include phenylpyruvic acid, (S)-mandelic acid, and benzoylformic acid, which are products of reactions catalyzed by AAD, HMAS, and SMDH. In the sequence of phenylpyruvic acid, (S)-mandelic acid, and benzoylformic acid, the phenylpyruvic acid side is also referred to as "upstream" and the benzoylformic acid side is also referred to as "downstream." That is, the production process may include multiple steps involving the production of one or more intermediates. Each of the multiple steps included in such a production process is also referred to as a "substep." A production process may include, for example, two, three, or four substeps. Examples of the substeps in a production process include a fermentation substep and a conversion substep. Examples of the fermentation substep include a substep of producing an intermediate from a carbon source. The substance conversion substeps include a step of converting L-phenylalanine to an intermediate, a step of converting the intermediate to another downstream intermediate, and a step of converting the intermediate to benzaldehyde. The combination of the substeps in the production process is not particularly limited as long as benzaldehyde can be produced.

[0202] The production process may include, for example, a step of producing an intermediate from a carbon source and a step of converting the intermediate to benzaldehyde. That is, the production process may include, for example, a step of producing phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid from a carbon source and a step of converting phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid to benzaldehyde. Specifically, the production process may include, for example, a step of producing benzoylformic acid from a carbon source and a step of converting benzoylformic acid to benzaldehyde. Furthermore, the production process may include, for example, a step of producing intermediate A from a carbon source, a step of converting intermediate A to another downstream intermediate B, and a step of converting intermediate B to benzaldehyde. The production process may also include, for example, a step of producing phenylpyruvic acid from a carbon source, a step of converting phenylpyruvic acid to (S)-mandelic acid, a step of converting (S)-mandelic acid to benzoylformic acid, and a step of converting benzoylformic acid to benzaldehyde.

[0203] The production process may include, for example, a step of converting L-phenylalanine into an intermediate and a step of converting the intermediate into benzaldehyde. That is, the production process may include, for example, a step of converting L-phenylalanine into phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid, and a step of converting phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid into benzaldehyde. Specifically, the production process may include, for example, a step of converting L-phenylalanine into benzoylformic acid and a step of converting benzoylformic acid into benzaldehyde. Furthermore, the production process may include, for example, a step of converting L-phenylalanine into intermediate A, a step of converting intermediate A into another downstream intermediate B, and a step of converting intermediate B into benzaldehyde. The production process may also include, for example, the steps of converting L-phenylalanine to phenylpyruvic acid, converting phenylpyruvic acid to (S)-mandelic acid, converting (S)-mandelic acid to benzoylformic acid, and converting benzoylformic acid to benzaldehyde.

[0204] Similarly to the production process, each substep of the production process may include further substeps. The description of the production process and its substeps applies mutatis mutandis to the substeps and their further substeps. For example, the step of converting L-phenylalanine to benzoylformic acid may include the steps of converting L-phenylalanine to phenylpyruvic acid, converting phenylpyruvic acid to (S)-mandelic acid, and converting (S)-mandelic acid to benzoylformic acid. In each fermentation substep, the intermediate produced is also referred to as the "product." In each substance conversion substep, the substance before conversion is also referred to as the "substrate," and the substance after conversion is also referred to as the "product." For example, in the step of converting L-phenylalanine to an intermediate, L-phenylalanine is considered to be the substrate, and the intermediate is considered to be the product.

[0205] The means for carrying out the substeps of the production process can be set independently for each substep. The description of the means for carrying out the fermentation process can be applied mutatis mutandis to the means for carrying out each fermentation substep of the production process. The description of the means for carrying out the substance conversion substep can be applied mutatis mutandis to the means for carrying out each substance conversion substep of the production process. In this case, the "benzaldehyde-producing enzyme" used in the production process (e.g., "the microorganism of the present invention") can be read as the benzaldehyde-producing enzyme corresponding to each substep (e.g., a microorganism having the benzaldehyde-producing enzyme). Furthermore, "benzaldehyde," which is the product of the fermentation process, can be read as the product of each fermentation substep. Furthermore, "L-phenylalanine," which is the substrate of the substance conversion process, and "benzaldehyde," which is the product, can be read as the substrate and product of each substance conversion substep, respectively. In each substance conversion substep, the product produced in the immediately preceding substep is used as the substrate, except for L-phenylalanine. The substrate in each substance conversion substep may be used in its free form, its salt, or a mixture thereof. That is, the term "substrate" in the present invention means a substrate in its free form, its salt, or a mixture thereof, unless otherwise specified. The same description of L-phenylalanine salts can be applied to substrate salts.

[0206] Each substep of the production process is carried out using a benzaldehyde-producing enzyme corresponding to that substep (e.g., a microorganism having a benzaldehyde-producing enzyme corresponding to that substep). The "benzaldehyde-producing enzyme corresponding to that substep of the production process" used in each fermentation substep refers to a single enzyme or a series of enzymes that catalyze the conversion of L-phenylalanine to the product of that substep. Furthermore, the "benzaldehyde-producing enzyme corresponding to that substep of the production process" used in each substance conversion substep refers to a single enzyme or a series of enzymes that catalyze the conversion of the substrate to the product of that substep. That is, for example, the step of producing phenylpyruvic acid from a carbon source can be carried out using a microorganism having AAD (e.g., a microorganism having AAD and the ability to produce L-phenylalanine). Furthermore, for example, the step of producing (S)-mandelic acid from a carbon source can be carried out using a microorganism having AAD and HMAS (e.g., a microorganism having AAD and the ability to produce L-phenylalanine). Furthermore, for example, the step of producing benzoylformic acid from a carbon source can be carried out using a microorganism having AAD, HMAS, and SMDH (e.g., a microorganism capable of producing L-phenylalanine having AAD, HMAS, and SMDH). Furthermore, for example, the step of converting L-phenylalanine to phenylpyruvic acid, the step of converting phenylpyruvic acid to (S)-mandelic acid, the step of converting (S)-mandelic acid to benzoylformic acid, and the step of converting benzoylformic acid to benzaldehyde can be carried out using AAD (e.g., a microorganism having AAD), HMAS (e.g., a microorganism having HMAS), SMDH (e.g., a microorganism having SMDH), and BFDC (e.g., a microorganism having BFDC), respectively. Also, for example, the process of converting L-phenylalanine to phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid can be carried out using AAD (e.g., a microorganism having AAD), AAD and HMAS (e.g., a microorganism having AAD and HMAS), or AAD, HMAS, and SMDH (e.g., a microorganism having AAD, HMAS, and SMDH), respectively.Furthermore, for example, the step of converting phenylpyruvic acid, (S)-mandelic acid, or benzoylformic acid to benzaldehyde can be carried out using HMAS, SMDH, and BFDC (e.g., a microorganism having HMAS, SMDH, and BFDC), SMDH and BFDC (e.g., a microorganism having SMDH and BFDC), or BFDC (e.g., a microorganism having BFDC), respectively. For other substeps, the benzaldehyde-producing enzyme required for the substep (e.g., a microorganism having a benzaldehyde-producing enzyme) can be appropriately selected.

[0207] When a microorganism having multiple benzaldehyde-producing enzymes is used in a substep of the production process, the microorganism may be a single microorganism or a combination of multiple microorganisms. Regarding a microorganism having multiple benzaldehyde-producing enzymes, the explanation for the microorganism of the present invention (a microorganism having four benzaldehyde-producing enzymes) can be applied mutatis mutandis. That is, for example, a microorganism having AAD, HMAS, and SMDH may be a single microorganism having AAD, HMAS, and SMDH individually, or a combination of multiple microorganisms having AAD, HMAS, and SMDH as a whole.

[0208] The microorganisms used in one substep and another substep of the production process may be different microorganisms or the same microorganism. In other words, when a microorganism has a benzaldehyde-producing enzyme corresponding to one substep and a benzaldehyde-producing enzyme corresponding to another substep, the microorganism can be used in both substeps.

[0209] Each substep of the production process may be carried out, for example, by culturing a microorganism having the benzaldehyde-producing enzyme corresponding to that substep, or by using the cells of a microorganism having the benzaldehyde-producing enzyme corresponding to that substep. That is, each fermentation substep of the production process may be a step of culturing a microorganism having the benzaldehyde-producing enzyme corresponding to that substep and L-phenylalanine-producing ability in a medium containing a carbon source, and producing and accumulating a product in the medium. Furthermore, each substance conversion substep of the production process may be a step of culturing a microorganism having the benzaldehyde-producing enzyme corresponding to that substep in a medium containing a substrate, and producing and accumulating a product in the medium. Each substance conversion substep of the production process may be a step of causing the benzaldehyde-producing enzyme corresponding to that substep (e.g., cells of a microorganism having the benzaldehyde-producing enzyme corresponding to that substep) to coexist with the substrate in a reaction solution, and producing and accumulating a product in the reaction solution. All of the substeps of the production process may be carried out by culturing, or all of them may be carried out using the cells. Alternatively, some of the substeps may be performed by culturing, and the remaining substeps may be performed using bacterial cells. The conditions for performing each substep of the production process may be appropriately set depending on various conditions, such as the type of benzaldehyde-producing enzyme corresponding to the substep and the type of microorganism used in the substep.

[0210] The production process may include, for example, the following steps (A1) and (A2): (A1) A process for producing benzoylformic acid by utilizing AAD, HMAS, and SMDH; (A2) A step of converting the benzoylformic acid produced in the step (A1) into benzaldehyde by utilizing BFDC.

[0211] The step (A1) may include, for example, the following step (1a), (1b), or (1c): (1a) culturing at least one microorganism in a medium containing a carbon source to produce and accumulate benzoylformic acid in the medium, wherein the at least one microorganism is a single microorganism that independently has AAD, HMAS, and SMDH and is capable of producing L-phenylalanine, or a combination of multiple microorganisms that collectively have AAD, HMAS, and SMDH and are capable of producing L-phenylalanine; (1b) culturing at least one microorganism in a medium containing L-phenylalanine to produce and accumulate benzoylformic acid in the medium, wherein the at least one microorganism is a single microorganism having AAD, HMAS, and SMDH alone, or a combination of multiple microorganisms having AAD, HMAS, and SMDH as a whole; (1c) A step of allowing AAD, HMAS, and SMDH to coexist with L-phenylalanine in a reaction solution, and generating and accumulating benzoylformic acid in the reaction solution.

[0212] The step (A2) may include, for example, the following step (2a) or (2b): (2a) culturing a microorganism having BFDC in a medium containing the benzoylformic acid produced in the step (A1) to produce and accumulate benzaldehyde in the medium; (2b) A step of allowing BFDC to coexist with the benzoylformic acid produced in the step (A1) in a reaction solution, thereby producing and accumulating benzaldehyde in the reaction solution.

[0213] The AAD, HMAS, and SMDH in step (1c) may be used in the form of, for example, the cells of at least one microorganism having the AAD, HMAS, and SMDH. The at least one microorganism may be, for example, a single microorganism having AAD, HMAS, and SMDH alone, or a combination of multiple microorganisms having AAD, HMAS, and SMDH as a whole.

[0214] The BFDC in step (2b) may be used, for example, in the form of cells of a microorganism having the BFDC.

[0215] The substeps of the production process may or may not be carried out separately. That is, some or all of the substeps of the production process may be carried out simultaneously for part or all of the period. For example, substep A, which produces a certain product, and another substep B, which uses that product as a substrate, may be carried out separately, or may be carried out simultaneously for part or all of the period. That is, for example, substep A and substep B may be started simultaneously, or substep B may be started while substep A is in progress or after completion. For example, substep A and substep B can be started simultaneously by causing a benzaldehyde-producing enzyme corresponding to substep A and substep B (e.g., a microorganism having a benzaldehyde-producing enzyme corresponding to substep A and substep B) and a substrate for substep A to coexist in a reaction system (reaction solution or medium) at the start of substep A. Alternatively, for example, substep A can be initiated under conditions in which the benzaldehyde-producing enzyme corresponding to substep B (e.g., a microorganism having the benzaldehyde-producing enzyme corresponding to substep B) is not present in the reaction system, and substep B can be initiated by making the benzaldehyde-producing enzyme corresponding to substep B (e.g., a microorganism having the benzaldehyde-producing enzyme corresponding to substep B) present in the reaction system during or after substep A is completed. In addition, the product of substep A may or may not be recovered before use. That is, for example, the product of substep A may be recovered, and substep B may be carried out using the recovered product. The product of substep A may be used in substep B as is, or may be subjected to treatment such as concentration, dilution, drying, dissolution, fractionation, extraction, or purification as appropriate. The descriptions regarding substep A and substep B can be applied to any combination of consecutive substeps.

[0216] <4-4> Use of catalase In the method of the present invention, at least a part of the production process is carried out in the presence of catalase. By carrying out at least a part of the production process in the presence of catalase, benzaldehyde production can be improved. That is, by using catalase, benzaldehyde production can be improved compared to when catalase is not used. Examples of improvement in benzaldehyde production include an improvement in the amount of benzaldehyde produced and an improvement in the yield of benzaldehyde.

[0217] The improvement of benzaldehyde production by using catalase may be due to, for example, the decomposition of hydrogen peroxide in the medium or reaction solution by catalase. Hydrogen peroxide may be generated, for example, from ascorbic acid and oxygen. Ascorbic acid and oxygen may be used, for example, in combination with HMAS. The decomposition of hydrogen peroxide may, for example, increase the activity of HMAS. Specifically, for example, if the activity of HMAS is inhibited by hydrogen peroxide, the decomposition of hydrogen peroxide may reduce the inhibition of HMAS activity, thereby increasing the activity of HMAS. In other words, the improvement of benzaldehyde production may be due, for example, to the increase in HMAS activity. The increase in HMAS activity may, for example, improve the production of (S)-mandelic acid. The improvement in the production of (S)-mandelic acid may, for example, improve the production of benzoylformic acid. The improvement in the production of benzoylformic acid may, for example, improve the production of benzaldehyde. In other words, improved production of benzaldehyde may be due to improved production of intermediates to benzaldehyde, such as (S)-mandelic acid or benzoylformic acid.

[0218] That is, at least a part of the production process may include a portion in which HMAS is used. The portion in which HMAS is used may include the period in which HMAS is present in the culture medium or reaction solution. Specific examples of the period in which HMAS is present in the culture medium or reaction solution include the period in which a microorganism having HMAS is cultured in the culture medium and the period in which HMAS (e.g., cells of a microorganism having HMAS) is present in the reaction solution. Furthermore, the portion in which HMAS is used may include a substep in which HMAS is used. Particularly, the portion in which HMAS is used may include the period in which HMAS is present in the culture medium or reaction solution during the substep in which HMAS is used.

[0219] The amount of catalase used is not particularly limited as long as it improves benzaldehyde production. For example, the amount of catalase used, calculated in terms of catalase activity in the medium or reaction solution, may be 10 U / mL or more, 20 U / mL or more, 50 U / mL or more, 100 U / mL or more, 200 U / mL or more, 500 U / mL or more, 1000 U / mL or more, 2000 U / mL or more, 5000 U / mL or more, 500,000 U / mL or less, 200,000 U / mL or less, 100,000 U / mL or less, 50,000 U / mL or less, 20,000 U / mL or less, 10,000 U / mL or less, 5000 U / mL or less, 2000 U / mL or less, 1000 U / mL or less, or 500 U / mL or less, or any combination thereof that is consistent. Specifically, the amount of catalase used may be, for example, 10 to 100,000 U / mL, 100 to 50,000 U / mL, or 1,000 to 20,000 U / mL, calculated as catalase activity in the medium or reaction solution. 1 U of catalase activity is defined as the amount of enzyme required to decompose 1 μmol of hydrogen peroxide at pH 7.0 and 25°C for 1 minute. The amount of catalase used may be, for example, 1 or more, 300 or less, or a combination thereof, calculated as the optical density (OD) at 600 nm of the host cells containing catalase in the medium or reaction solution.

[0220] As long as catalase is used in at least a part of the production process, it may or may not be used in the other parts of the process. In other words, as long as catalase is used in at least a part of the production process, it may or may not be present in the medium or reaction solution in the other parts of the process.

[0221] When HMAS is used as at least a portion of the production process, catalase may be present in the culture medium or reaction solution throughout the entire period of the HMAS-using portion, or may be present in the culture medium or reaction solution for a portion of the period of the HMAS-using portion. The phrase "the HMAS-using portion is carried out in the presence of catalase" does not necessarily mean that catalase is present in the culture medium or reaction solution throughout the entire period of the HMAS-using portion. For example, catalase may be present in the culture medium or reaction solution for at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or even 100% of the period of the HMAS-using portion. For example, catalase may or may not be present in the culture medium or reaction solution from the start of the HMAS-using portion. If catalase is not present in the culture medium or reaction solution at the start of the HMAS-using portion, catalase is supplied to the culture medium or reaction solution after the start of the HMAS-using portion. The timing of supply can be determined appropriately depending on various conditions, such as the culture time and reaction time. For example, when a microorganism having an HMAS, such as the microorganism of the present invention, grows in the portion utilizing HMAS, catalase may be supplied to the culture medium or reaction solution after the microorganism having an HMAS, such as the microorganism of the present invention, has grown sufficiently. In either case, catalase may be additionally supplied to the culture medium or reaction solution as appropriate. The means for supplying catalase to the culture medium or reaction solution is not particularly limited. Catalase may or may not be contained in the culture medium or reaction solution at the concentration exemplified above throughout the entire period of the portion utilizing HMAS. For example, catalase may be contained in the culture medium or reaction solution at the concentration exemplified above at the start of the portion utilizing HMAS, or may be supplied to the culture medium or reaction solution after the start of the portion utilizing HMAS so as to achieve the concentration exemplified above.

[0222] Catalase may be used in any of the forms described above for the production of benzaldehyde. That is, the use of catalase includes any of the forms described above. Catalase may be used, for example, in a form contained in host cells such as microbial cells. Catalase may also be used in a form not contained in host cells, such as a purified enzyme. Catalase may be supplied to the culture medium or reaction solution separately from the benzaldehyde-producing enzyme, or may be supplied to the culture medium or reaction solution together with the benzaldehyde-producing enzyme. For example, by expressing a catalase gene and a benzaldehyde-producing enzyme gene in a host having those genes, catalase and the benzaldehyde-producing enzyme can be produced together and supplied to the culture medium or reaction solution together. Furthermore, when a host having a benzaldehyde-producing enzyme, such as the microorganism of the present invention, also contains catalase, at least a portion of the production process can be carried out in the presence of catalase by using the same host. For example, if a microorganism having at least HMAS or a microorganism used in coexistence with it has catalase, the production process can be carried out using this microorganism, and the part that utilizes HMAS can be carried out in the presence of catalase.

[0223] Catalase may or may not be used in combination with other components that decompose hydrogen peroxide, for example.

[0224] <5> Another aspect of the method of the present invention The method of the present invention is not limited to catalase, and can be carried out using any component that decomposes hydrogen peroxide.

[0225] That is, another embodiment of the method of the present invention is a method for producing benzaldehyde, comprising the step of producing benzaldehyde by utilizing amino acid deaminase (AAD), 4-hydroxymandelate synthase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC), at least a part of the step being carried out in the presence of a component that decomposes hydrogen peroxide.

[0226] The above-described description of the method of the present invention can be applied mutatis mutandis to this other embodiment, except that a component that decomposes hydrogen peroxide is used instead of catalase. Furthermore, the above-described description of the use of catalase in the method of the present invention can be applied mutatis mutandis to the use of a component that decomposes hydrogen peroxide in this other embodiment.

[0227] The component that decomposes hydrogen peroxide may be, for example, a component other than catalase. Examples of the component that decomposes hydrogen peroxide include manganese dioxide. Examples of the component that decomposes hydrogen peroxide also include peroxidases such as ascorbate peroxidase and peroxiredoxin.

[0228] The component that decomposes hydrogen peroxide may or may not be used in combination with catalase, for example. [Example]

[0229] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0230] Example 1: Expression of amino acid deaminase (AAD) The AAD-expressing strain (E. coli JM109 / pSFN-AAD) derived from Providencia rettgeri AJ2770, described in the benzaldehyde production method (WO2017 / 122747, Example 1), was cultured overnight at 25°C on an LB-amp (100 mg / L) plate. The resulting bacterial cells were inoculated into 100 mL of TB-amp (100 mg / L) (i.e., TB medium containing 100 mg / L ampicillin) and cultured with shaking at 25°C for 16 hours using a Sakaguchi flask. The resulting culture was used as the AAD culture medium.

[0231] Example 2: Expression of 4-hydroxymandelate synthase (HmaS) (1) Construction of quadruple mutant HmaS expression plasmid A DNA fragment containing the triple mutant hmaS At gene from Actinoplanes teichomyceticus (HmaS At, A199V / I217V / K337Q) was amplified by PCR using the plasmid pPC-hmaS At-His A199V / I217V / K337Q (SDatc) as a template. The first half of the fragment was amplified using primers RV (5'-CAGGAAACAGCTATGAC-3'; SEQ ID NO: 33) and Q206R-R (5'-GGCACTacgAACCACCTGGGAATCCAT-3'; SEQ ID NO: 34). The latter part was amplified using primers Q206R-F (5'-GTGGTTcgtAGTGCCGGTGGGGCTGTG-3'; SEQ ID NO: 35) and M4 (5'-GTTTTCCCAGTCACGAC-3'; SEQ ID NO: 36). PCR was performed using KOD-plus-ver.2 (Toyobo) under the following conditions. 1 cycle: 94℃, 2 min 25 cycles at 98°C for 10 seconds 60℃, 10 seconds 68℃, 60 seconds 1 cycle: 68℃, 60 seconds 4℃

[0232] Using the two resulting DNA fragments as templates, a DNA fragment containing the full-length quadruple mutant HmaS At gene was amplified by PCR using primers RV and M4. PCR was performed using KOD-plus-ver.2 (Toyobo) under the conditions described above.

[0233] The resulting approximately 1100 bp DNA fragment was digested with restriction enzymes NdeI and XhoI and ligated with pPC-hmaS At-His(SDatc) (WO 2017 / 122747, Example 8) similarly digested with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, and the target plasmid was extracted from the ampicillin-resistant strain. The resulting plasmid was designated pPC-hmaS At-His A199V / Q206R / I217V / K337Q (SDatc). This plasmid expresses the quadruple mutant HmaS At (A199V / Q206R / I217V / K337Q) with a His-tag at the C-terminus. The nucleotide sequence of the quadruple mutant hmaS At-His gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 2.

[0234] (2) Expression of the quadruple mutant HmaS The above-mentioned Actinoplanes teichomyceticus-derived quadruple mutant hmaS gene-containing plasmid pPC-hmaS At-His A199V / Q206R / I217V / K337Q (SDatc) was introduced into E. coli JM109, and transformants containing the plasmid were isolated from ampicillin-resistant strains. These strains were cultured overnight at 25°C on LB-amp (100 mg / L) plates. The resulting cells were inoculated into 100 mL of TB-amp (100 mg / L) and cultured at 37°C with shaking in a Sakaguchi flask for 16 hours. The resulting culture was designated the HmaS At culture medium.

[0235] Example 3: Expression of (S)-mandelate dehydrogenase (MdlB (SMDH)) The plasmid pET22-mdlB containing the mdlB gene from Pseudomonas putida, as described in the benzaldehyde production method (WO2017 / 122747, Example 3), was introduced into E. coli JM109(DE3), and a transformant containing the plasmid was obtained from ampicillin-resistant strains. This strain was cultured overnight at 25°C on an LB-amp (100 mg / L) plate. The resulting bacterial cells were inoculated into 100 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg / L ampicillin and cultured with shaking at 37°C in a Sakaguchi flask for 16 hours. The resulting culture was used as the MdlB culture medium.

[0236] Example 4: Expression of benzoylformate decarboxylase (MdlC(BFDC)) An MdlC-expressing strain (E. coli BL21(DE3) / pET22-mdlC) derived from Pseudomonas putida, as described in the benzaldehyde production method (WO2017 / 122747, Example 4), was cultured overnight at 25°C on an LB-amp (100 mg / L) plate. The resulting bacterial cells were inoculated into 100 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg / L ampicillin, and cultured with shaking at 37°C for 16 hours in a Sakaguchi flask. The resulting culture was used as the MdlC culture medium.

[0237] Example 5: Synthesis of benzaldehyde from L-Phe with the addition of catalase (1)Analysis conditions Benzaldehyde was quantified by HPLC analysis under the following analytical conditions. Mobile phase A: 10mM KH2PO4, 10mM K2HPO4 Mobile phase B: acetonitrile Flow rate: 1.0 ml / min Column temperature: 40℃ Detection: UV 210 nm Column: CAPCELL PAK MGII, 4.6 x 150 mm, 3 μm (Shiseido) Gradient: 0-2 min (B: 2%), 2-16 min (B: 2-50%), 16.1-20 min (B: 2%)

[0238] (2) Preparation of concentrated solution Ten mL of the AAD culture medium obtained in Example 1 was centrifuged, 8 mL of the supernatant was removed, and the bacterial cells were suspended in the remaining culture supernatant to obtain a 5-fold concentrated culture medium. This was used in the reaction as an AAD concentrate. Similarly, 5-fold concentrates were also prepared from the HmaS At culture medium, MdlB culture medium, and MdlC culture medium obtained in Examples 2 to 4, and these were used in the reaction as HmaS At concentrate, MdlB concentrate, and MdlC concentrate.

[0239] (3) Benzaldehyde synthesis reaction One mL of the reaction mixture containing 50 mM L-Phe, 0.01 mM ferrous sulfate, 10 mM trisodium citrate, 30 mM sodium ascorbate, 1 mM thiamine pyrophosphate chloride, 1 mM magnesium sulfate, 100 mM potassium phosphate buffer (pH 7.0), 0.02 mL of AAD concentrate, 0.1 mL of HmaS At concentrate, 0.02 mL of MdlB concentrate, and 20 μL of catalase or water was placed in a test tube and shaken at 25°C. After 20 hours, the reaction mixture was centrifuged, and 0.49 mL of the supernatant was mixed with 0.01 mL of MdlC concentrate and shaken in a test tube at 25°C. After 4 hours, 0.1 mL of the reaction mixture was mixed with 1 mL of reaction stop solution (1% phosphoric acid, 50% ethanol), and the supernatant was subjected to HPLC analysis. The amount of catalase added and the amount of benzaldehyde produced are shown in Table 1. When any of the catalases was added, an improvement in the amount of benzaldehyde produced was observed.

[0240] [Table 1]

[0241] Example 6: Expression of catalase (Cat) (1) Construction of an E. coli-derived Cat HPI (Cat 1) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and E. coli-derived Cat HPI (GenBank accession number: NP_418377). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 3) was obtained in which the gene was linked downstream of the phoC promoter. This DNA fragment was inserted into the EcoRI-HindIII site of pMW218 (Takara Bio) to construct a Cat 1 expression plasmid. The constructed plasmid, designated pMW-cat1, was introduced into E. coli JM109. Transformants harboring the plasmid were isolated from kanamycin-resistant strains and used as Cat 1 expression strains. The amino acid sequence of E. coli-derived Cat HPI (Cat 1) is shown in SEQ ID NO: 4.

[0242] (2) Construction of an E. coli-derived Cat HPII (Cat 2) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and E. coli-derived Cat HPII (GenBank accession number: AML01688). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 5) was obtained in which the gene was linked downstream of the phoC promoter. A Cat 2-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of E. coli-derived Cat HPII (Cat 2) is shown in SEQ ID NO: 6.

[0243] (3) Construction of a strain expressing Thermus thermophilus Cat (Cat 3) We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Thermus thermophilus-derived Cat (GenBank accession number: AAS82214). We obtained a DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 7) in which the gene was linked downstream of the phoC promoter. A Cat 3-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Thermus thermophilus-derived Cat (Cat 3) is shown in SEQ ID NO: 8.

[0244] (4) Construction of a strain expressing Cat 4 from Rhodothermus marinus We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Rhodothermus marinus-derived Cat (GenBank accession number: ACY49648), and obtained a DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 9) in which the gene was linked downstream of the phoC promoter. A Cat 4-expressing strain was then obtained as in (1). The amino acid sequence of Rhodothermus marinus-derived Cat (Cat 4) is shown in SEQ ID NO: 10.

[0245] (5) Construction of a Corynebacterium glutamicum Cat (Cat 5) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Corynebacterium glutamicum-derived Cat (GenBank accession number: BAV22054). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 11) in which the gene was linked downstream of the phoC promoter was obtained. A Cat 5-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Corynebacterium glutamicum-derived Cat (Cat 5) is shown in SEQ ID NO: 12.

[0246] (6) Construction of a Bacillus subtilis-derived Cat (Cat 6) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Bacillus subtilis-derived Cat (GenBank accession number: NP_388762). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 13) was obtained in which the gene was linked downstream of the phoC promoter. The Cat 6 expression strain was then obtained in the same manner as in (1). The amino acid sequence of Bacillus subtilis-derived Cat (Cat 6) is shown in SEQ ID NO: 14.

[0247] (7) Construction of a Pseudomonas syringae Cat (Cat 7) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Pseudomonas syringae-derived Cat (GenBank accession number: AAC61659). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 15) was obtained in which the gene was linked downstream of the phoC promoter. A Cat 7-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Pseudomonas syringae-derived Cat (Cat 7) is shown in SEQ ID NO: 16.

[0248] (8) Construction of a Pseudomonas putida Cat (Cat 8) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Pseudomonas putida-derived Cat (GenBank accession number: WP_012313921). We obtained a DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 17) in which the gene was linked downstream of the phoC promoter. A Cat 8-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Pseudomonas putida-derived Cat (Cat 8) is shown in SEQ ID NO: 18.

[0249] (9) Construction of an expression strain expressing the PelB signal sequence from E. coli and Cat 9 from Pseudomonas putida We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter, the E. coli-derived PelB signal sequence, and the signal-sequence-deleted Pseudomonas putida-derived Cat, resulting in a DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 19) in which the gene was linked downstream of the phoC promoter. A Cat 9-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Pseudomonas putida-derived Cat (Cat 9) with the E. coli-derived PelB signal sequence added is shown in SEQ ID NO: 20.

[0250] (10) Construction of a Shewanella oneidensis Cat (Cat 10) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding the Enterobacter aerogenes-derived phoC promoter and Shewanella oneidensis-derived Cat (GenBank accession number: NP_716358). A DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 21) in which the gene was linked downstream of the phoC promoter was obtained. A Cat 10-expressing strain was then obtained in the same manner as in (1). The amino acid sequence of Shewanella oneidensis-derived Cat (Cat 10) is shown in SEQ ID NO: 22.

[0251] (11) Construction of a Pseudomonas entomophila Cat (Cat 11) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding Pseudomonas entomophila-derived Cat (GenBank accession number: WP_011533980), and obtained a plasmid containing the gene. This DNA fragment was digested with NdeI and SacI to obtain a DNA fragment containing the cat 11 gene (codon usage optimized for expression in E. coli; SEQ ID NO: 23).

[0252] Using pMW218-cat3 obtained in (3) above as a template, a DNA fragment containing the cat3 gene, phoC promoter, and its upstream region was amplified by PCR using primers pMW218_delNdeI (5'-ggaattcattaATgcacagatgaaaacggtg-3') and RV (5'-CAGGAAACAGCTATGAC-3'). PCR was performed using KOD-plus-ver.2 (Toyobo) under the following conditions. 1 cycle: 94℃, 2 min 25 cycles at 98°C for 10 seconds 55℃, 10 seconds 68℃, 90 seconds 1 cycle: 68℃, 90 seconds 4℃

[0253] The resulting approximately 1500 bp DNA fragment was digested with VspI and EcoRI and ligated with pMW218 digested with NdeI and EcoRI. The ligation solution was transformed into E. coli JM109, and the desired plasmid was extracted from the kanamycin-resistant strain. The resulting plasmid was designated pMW-delNdeI-cat3.

[0254] The Cat11 gene was digested with NdeI and SacI and inserted into the NdeI-SacI site of pMW218-delNdeI-cat3 to construct a Cat11 expression plasmid in which the Cat11 gene was ligated downstream of the Enterobacter aerogenes-derived phoC promoter. The constructed plasmid was designated pMW-cat11 and introduced into E. coli JM109. Transformants containing the plasmid were isolated from kanamycin-resistant strains and used as Cat11-expressing strains. The amino acid sequence of Pseudomonas entomophila-derived Cat (Cat11) is shown in SEQ ID NO: 24.

[0255] (12) Construction of a Pseudomonas parafulva Cat (Cat 12) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding Cat from Pseudomonas parafulva (GenBank accession number: WP_039579465), and obtained a plasmid and DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 25) containing the gene. The Cat 12-expressing strain was then obtained as described in (11). The amino acid sequence of Cat from Pseudomonas parafulva (Cat 12) is shown in SEQ ID NO: 26.

[0256] (13) Construction of a Pseudomonas protegens Cat (Cat 13) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding Pseudomonas protegens-derived Cat (GenBank accession number: WP_053153995), and obtained a plasmid and DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 27) containing the gene. The Cat 13-expressing strain was then obtained as described in (11). The amino acid sequence of Pseudomonas protegens-derived Cat (Cat 13) is shown in SEQ ID NO: 28.

[0257] (14) Construction of an Erwinia mallotivora Cat (Cat 14) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding Cat (GenBank accession number: WP_034939749) derived from Erwinia mallotivora, and obtained a plasmid and DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 29) containing the gene. The Cat 14 expression strain was then obtained as described in (11). The amino acid sequence of Cat (Cat 14) derived from Erwinia mallotivora is shown in SEQ ID NO: 30.

[0258] (15) Construction of an Erwinia tracheiphila Cat (Cat 15) expression strain We commissioned Eurofins Genomics to synthesize a gene encoding Erwinia tracheiphila-derived Cat (GenBank accession number: KKF37821), and obtained a plasmid and DNA fragment (codon usage optimized for expression in E. coli; SEQ ID NO: 31) containing the gene. The Cat 15-expressing strain was then obtained as described in (11). The amino acid sequence of Erwinia tracheiphila-derived Cat (Cat 15) is shown in SEQ ID NO: 32.

[0259] (16) Construction of pMW218(Ctrl) strain Plasmid pMW218 (Takara Bio) was introduced into E. coli JM109, and transformants carrying pMW218 were selected from kanamycin-resistant strains and designated as Ctrl strains, which do not express catalase.

[0260] (17) Preparation of culture medium for Cat expression strain The Cat 1-expressing strain was cultured overnight at 25°C on an LB-Km (25 mg / L) plate. The resulting cells were inoculated into 4 mL of TB-Km (25 mg / L) (i.e., TB medium containing 25 mg / L kanamycin) and cultured in a test tube with shaking at 30°C for 16 hours. The resulting culture was designated as Cat 1 culture. Cat 2 to 15 cultures and Ctrl culture were obtained in the same manner.

[0261] Example 7: Synthesis of benzaldehyde from L-Phe by adding culture medium of catalase-expressing strain (1)Analysis conditions The analysis was carried out in the same manner as in Example 5.

[0262] (2) Preparation of concentrated solution Ten mL of the AAD culture obtained in Example 1 was centrifuged, 8 mL of the supernatant was removed, and the bacterial cells were suspended in the remaining culture supernatant to obtain a 5-fold concentrated culture. This was used in the reaction as the AAD concentrate. Similarly, 5-fold concentrates were also prepared from the HmaS At culture, MdlB culture, and MdlC culture obtained in Examples 2 to 4, and used in the reaction as the HmaS At concentrate, MdlB concentrate, and MdlC concentrate. 1 mL of the Cat 1 culture obtained in Example 6 was centrifuged, 0.8 mL of the supernatant was removed, and the bacterial cells were suspended in the remaining culture supernatant to obtain a 5-fold concentrated culture. This was used in the reaction as the Cat 1 concentrate. Similarly, 5-fold concentrates were also prepared from the Cat 2 to 15 cultures and the Ctrl culture, and used in the reaction as the Cat 2 to 15 concentrate and the Ctrl concentrate.

[0263] (3) Benzaldehyde synthesis reaction One mL of the reaction mixture containing 50 mM L-Phe, 0.01 mM ferrous sulfate, 10 mM trisodium citrate, 30 mM sodium ascorbate, 1 mM thiamine pyrophosphate chloride, 1 mM magnesium sulfate, 100 mM potassium phosphate buffer (pH 7.0), 0.02 mL of AAD concentrate, 0.1 mL of HmaS At concentrate, 0.02 mL of MdlB concentrate, and 0.1 mL of Cat 1-15 concentrate or Ctrl concentrate was placed in a test tube and shaken at 25°C. After 20 hours, the reaction mixture was centrifuged, and 0.49 mL of the supernatant was mixed with 0.01 mL of MdlC concentrate and shaken at 25°C in a test tube. After 4 hours, 0.1 mL of the reaction mixture was mixed with 1 mL of reaction stop solution (1% phosphoric acid, 50% ethanol), and the supernatant was subjected to HPLC analysis. The Cat concentrates (concentrated culture solutions of catalase-expressing strains) added and the amount of benzaldehyde produced are shown in Table 2. When any Cat concentrate was added, an increase in the amount of benzaldehyde produced was observed. In particular, when Cat concentrates other than Cat 7 concentrate were added, a significant increase in the amount of benzaldehyde produced was observed.

[0264] [Table 2]

[0265] Example 8: Deactivation of HMAS by hydrogen peroxide (1) Purification of HMAS Ten mL of the HmaS At culture obtained in Example 2 was centrifuged to collect cells, which were then suspended in 2 mL of xTractor Buffer (Takara Bio). The cells were then disrupted by allowing the suspension to stand at room temperature for 20 minutes. Cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as the soluble fraction. The soluble fraction was applied to a HisTALON Superflow Cartridges (Takara Bio, CV = 1 mL) His-tag protein purification column equilibrated with a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole, allowing the protein to adsorb to the carrier. Proteins that did not adsorb to the support (non-adsorbed proteins) were washed away with a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The adsorbed proteins were then eluted with a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 150 mM imidazole at a flow rate of 1.5 mL / min. The eluted fractions were collected and concentrated using an Amicon Ultra-15 30k (Millipore). The concentrate was diluted with 20 mM Tris-HCl (pH 7.6) to obtain purified HmaS At-His enzyme.

[0266] (2) HMAS activity measurement A 0.2 mL reaction mixture containing 10 mM sodium phenylpyruvate, 0.01 mM ferrous sulfate, 10 mM trisodium citrate, 10 mM sodium ascorbate, 100 mM Tris-HCl (pH 7.0), 0.02 mg / mL HmaS At-His purified enzyme, and 0 to 1 mM hydrogen peroxide was placed in a 1.5 mL tube and incubated at 25°C. A similar reaction was performed without HmaS At-His. After 15 minutes, 0.05 mL of the reaction mixture was mixed with 0.2 mL of a reaction stop solution (1% phosphoric acid) and subjected to HPLC analysis. HPLC analysis was performed as in Example 5, and the amount of enzymatically produced mandelic acid was quantified. Table 3 shows the amount of hydrogen peroxide added and the amount of mandelic acid produced, relative to the amount of mandelic acid produced without hydrogen peroxide, taken as 100%. The amount of mandelic acid produced decreased when hydrogen peroxide was added at 0.01 to 1 mM. This indicates that hydrogen peroxide inhibits HMAS activity, suggesting that benzaldehyde can be efficiently produced by using HMAS in the presence of catalase.

[0267] [Table 3] [Industrial Applicability]

[0268] According to the present invention, benzaldehyde can be produced efficiently.

[0269] <Description of Sequence Listing> SEQ ID NO: 1: Nucleotide sequence of the quadruple mutant HmaS gene of Actinoplanes teichomyceticus SEQ ID NO: 2: Amino acid sequence of quadruple mutant HmaS from Actinoplanes teichomyceticus SEQ ID NO: 3: Nucleotide sequence of the Cat HPI gene of Escherichia coli SEQ ID NO: 4: Amino acid sequence of Cat HPI of Escherichia coli SEQ ID NO: 5: Nucleotide sequence of the Cat HPII gene of Escherichia coli SEQ ID NO: 6: Amino acid sequence of Cat HPII from Escherichia coli SEQ ID NO: 7: Nucleotide sequence of the Cat gene of Thermus thermophilus SEQ ID NO: 8: Amino acid sequence of Cat from Thermus thermophilus SEQ ID NO: 9: Nucleotide sequence of the Cat gene of Rhodothermus marinus SEQ ID NO: 10: Amino acid sequence of Cat from Rhodothermus marinus SEQ ID NO: 11: Nucleotide sequence of the Cat gene of Corynebacterium glutamicum SEQ ID NO: 12: Amino acid sequence of Cat from Corynebacterium glutamicum SEQ ID NO: 13: Nucleotide sequence of the Cat gene of Bacillus subtilis SEQ ID NO: 14: Amino acid sequence of Cat from Bacillus subtilis SEQ ID NO: 15: Nucleotide sequence of the Cat gene of Pseudomonas syringiae SEQ ID NO: 16: Amino acid sequence of Cat from Pseudomonas syringiae SEQ ID NO: 17: Nucleotide sequence of the Cat gene of Pseudomonas putida SEQ ID NO: 18: Amino acid sequence of Cat from Pseudomonas putida SEQ ID NO: 19: Nucleotide sequence of the Cat gene of Pseudomonas putida SEQ ID NO: 20: Amino acid sequence of Cat from Pseudomonas putida SEQ ID NO: 21: Nucleotide sequence of the Cat gene of Shewanella oneidensis SEQ ID NO: 22: Amino acid sequence of Cat from Shewanella oneidensis SEQ ID NO: 23: Nucleotide sequence of the Cat gene of Pseudomonas entomophila SEQ ID NO: 24: Amino acid sequence of Cat from Pseudomonas entomophila SEQ ID NO: 25: Nucleotide sequence of the Cat gene of Pseudomonas parafulva SEQ ID NO: 26: Amino acid sequence of Cat from Pseudomonas parafulva SEQ ID NO: 27: Nucleotide sequence of the Cat gene of Pseudomonas protegens SEQ ID NO: 28: Amino acid sequence of Cat from Pseudomonas protegens SEQ ID NO: 29: Nucleotide sequence of the Cat gene of Erwinia mallotivora SEQ ID NO: 30: Amino acid sequence of Cat from Erwinia mallotivora SEQ ID NO: 31: Nucleotide sequence of the Cat gene of Erwinia tracheiphila SEQ ID NO: 32: Amino acid sequence of Cat from Erwinia tracheiphila SEQ ID NOs: 33 to 36: Primers SEQ ID NO: 37: Nucleotide sequence of the AAD gene of Providencia rettgeri AJ2770 SEQ ID NO: 38: Amino acid sequence of AAD of Providencia rettgeri AJ2770 SEQ ID NO: 39: HmaS gene of Actinoplanes teichomyceticus SEQ ID NO: 40: HmaS of Actinoplanes teichomyceticus SEQ ID NO: 41: Nucleotide sequence of the SMDH gene of Pseudomonas putida SEQ ID NO: 42: Amino acid sequence of SMDH from Pseudomonas putida SEQ ID NO: 43: Nucleotide sequence of the BFDC gene of Pseudomonas putida SEQ ID NO: 44: Amino acid sequence of BFDC of Pseudomonas putida

Claims

1. A method for producing benzaldehyde, comprising the steps of: The following step (A): (A) Four enzymes: amino acid deaminase (AAD), 4-hydroxymandelic acid synthase (4-MSA), A process for producing benzaldehyde from L-phenylalanine or a carbon source by utilizing (S)-mandelate dehydrogenase (HMAS), (S)-mandelate dehydrogenase (SMDH), and benzoylformate decarboxylase (BFDC). Including, A method in which catalase is provided in at least a portion of step (A), and at least a portion of step (A) is carried out in the presence of catalase.

2. The method of claim 1 , wherein the portion is a portion utilizing HMAS.

3. The four enzymes are utilized in the form of at least one microorganism containing the enzymes, The method of claim 1 or 2, wherein the at least one microorganism is a single microorganism that alone has the four enzymes, or a combination of multiple microorganisms that collectively have the four enzymes.

4. The method according to any one of claims 1 to 3, wherein the AAD is an AAD that does not generate hydrogen peroxide.

5. The step (A) comprises the following steps (A1) and (A2): (A1) A process for producing benzoylformic acid by utilizing AAD, HMAS, and SMDH ； (A2) A step of converting the benzoylformic acid produced in the step (A1) into benzaldehyde by using BFDC. The method according to any one of claims 1 to 4, comprising:

6. The step (A1) comprises the following step (1a), (1b), or (1c): (1a) A step of culturing at least one microorganism in a medium containing a carbon source and producing and accumulating benzoylformic acid in the medium, wherein the at least one microorganism independently produces AAD, HMAS, The microorganism may be a single microorganism having AAD, HMAS, and SMDH and capable of producing L-phenylalanine, or a plurality of microorganisms as a whole having AAD, HMAS, and SMDH and capable of producing L-phenylalanine. a combination of a number of microorganisms; (1b) culturing at least one microorganism in a medium containing L-phenylalanine to produce and accumulate benzoylformic acid in the medium, wherein the at least one microorganism is a single microorganism having AAD, HMAS, and SMDH alone, or a combination of multiple microorganisms having AAD, HMAS, and SMDH as a whole; (1c) AAD, HMAS, and SMDH were allowed to coexist with L-phenylalanine in a reaction mixture, and A step of generating and accumulating zoylformic acid in the reaction solution. The method of claim 5 , comprising:

7. The AAD, HMAS, and SMDH in the step (1c) are used in the form of bacterial cells of at least one microorganism having the AAD, HMAS, and SMDH; the at least one microorganism is a single microorganism having AAD, HMAS, and SMDH alone; or a combination of multiple microorganisms that collectively have AAD, HMAS, and SMDH The method according to claim 6 .

8. The step (A2) comprises the following step (2a) or (2b): (2a) culturing a microorganism having BFDC in a medium containing the benzoylformic acid produced in the step (A1) to produce and accumulate benzaldehyde in the medium; (2b) A step of allowing BFDC to coexist with the benzoylformic acid produced in the step (A1) in a reaction solution, and producing and accumulating benzaldehyde in the reaction solution. The method according to any one of claims 5 to 7, comprising:

9. The method according to claim 8, wherein the BFDC in step (2b) is used in the form of a bacterial cell of a microorganism having the BFDC.

10. The method according to any one of claims 7 to 9, wherein the bacterial cells are utilized in the form of a culture of the at least one microorganism, bacterial cells recovered from the culture, a processed product thereof, or a combination thereof.

11. The method according to any one of claims 1 to 10, wherein the catalase is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32; (b) a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32, and having catalase activity; (c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 and having catalase activity.

12. The method according to any one of claims 3 to 11, wherein the microorganism is a bacterium or a yeast.

13. The method according to any one of claims 3 to 12, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae or a coryneform bacterium.

14. The method according to any one of claims 3 to 13, wherein the microorganism is a bacterium of the genus Escherichia.

15. The method according to any one of claims 3 to 14, wherein the microorganism is Escherichia coli.

16. The method according to any one of claims 1 to 15, further comprising recovering the benzaldehyde.

Images