JP2024052995A - How vanillin is produced - Google Patents

Abstract

[Problem] To provide a mutant aromatic carboxylic acid reductase (ACAR) useful for producing vanillin, and a method for producing vanillin using the same. [Solution] Vanillin is produced using a mutant ACAR having mutations in amino acid residues such as C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, and I574. [Selected Figures] None

Description

The present invention relates to a method for producing vanillin.

Vanillin is the main component of vanilla scent and is used as a flavoring in foods, beverages, perfumes, etc. Vanillin is mainly produced by extraction from natural products or chemical synthesis.

There have also been attempts to produce vanillin using biotechnological techniques. For example, vanillin can be produced from vanillin precursors such as vanillic acid using microorganisms such as Corynebacterium glutamicum.

Vanillin can be produced using protocatechuic acid as an intermediate. That is, protocatechuic acid can be converted to vanillic acid by the action of O-methyltransferase (OMT). Vanillic acid can be converted to vanillin by the action of aromatic carboxylic acid reductase (ACAR).
In addition, isovanillin can be produced as a by-product using protocatechuic acid as an intermediate. That is, protocatechuic acid can be converted to isovanillic acid by the action of OMT. Isovanillic acid can be converted to isovanillin by the action of ACAR.

The objective of the present invention is to provide a mutant ACAR useful for producing vanillin and a method for producing vanillin using the mutant ACAR.

The present inventors discovered a mutation that improves the substrate specificity of ACAR for vanillic acid, and completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A mutant aromatic carboxylic acid reductase having a specific mutation in the amino acid sequence of a wild-type aromatic carboxylic acid reductase and having vanillin-producing activity,
The mutant aromatic carboxylic acid reductase, wherein the specific mutation is a mutation in one or more amino acid residues selected from the following:
C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, I574.
[2]
The mutant aromatic carboxylic acid reductase as described above, wherein the specific mutation comprises a mutation at G386.
[3]
The mutant aromatic carboxylic acid reductase, wherein the specific mutation comprises one or more mutations selected from the following:
C28A, C135 (A, V), R241 (K, Q), G249 (N, S, T), R252 (K, Q, T), W259 (F, Y), M265 (I, V), V270 (A, I), M271 (A, G, H, I, L, N, Q, R, S, T, V, Y), S274 (A, N), T275S, Q278D, Q310L, V312 (A, C, D, F, G, I, L, M, P, Q, S, T, Y), I353 (L, V), S356 (A, T), I376 (F, L, V), E377D, G378D, Y379 (F, L), S381 (A, T), T382S, A384 (C, F, G, H, I, K, L, N, Q, S, T), A385 (D, E, K, P, R), G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y), V387I, S388 (M, T), I389V, Y467 (F, H, W), I574 (A, C, F, G, L, L, M, N, P, T, V).
[4]
The mutant aromatic carboxylic acid reductase as described above, wherein the specific mutation comprises G386N.
[5]
The mutant aromatic carboxylic acid reductase, wherein the specific mutation comprises any one of the following mutations:
G386M/M271Y, G386M/S274N, G386M/I353F, G386M/I353L, G386N/M271Y, G386N/V312A, G386N/V312F, G386N/V312I, G386N/I353F, G386N/I353L, G386N/A384S, G386N/A385M, G386N/A385V, G386N/V387I, G386N/I574A, G386N/I574M, G386N/I574T.
[6]
The mutant aromatic carboxylic acid reductase, wherein the wild-type aromatic carboxylic acid reductase is an aromatic carboxylic acid reductase derived from a bacterium belonging to the genus Gordonia.
[7]
The mutant aromatic carboxylic acid reductase, wherein the wild-type aromatic carboxylic acid reductase is any one of the following proteins:
(a) a protein comprising the amino acid sequence set forth in SEQ ID NO:2;
(b) a protein comprising the amino acid sequence shown in SEQ ID NO:2, which contains a substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues;
(c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:2.
[8]
The mutant aromatic carboxylic acid reductase, wherein a by-product rate of isovanillin is 1.0% or less.
[9]
A method for producing vanillin, comprising the steps of:
converting vanillic acid to vanillin using the mutant aromatic carboxylic acid reductase.
[10]
The method, wherein the conversion comprises culturing a microorganism having the mutant aromatic carboxylic acid reductase in a medium containing vanillic acid, and producing and accumulating vanillin in the medium.
[11]
The method, wherein the conversion comprises allowing the mutant aromatic carboxylic acid reductase to act on vanillic acid in a reaction solution to produce and accumulate vanillin in the reaction solution.
[12]
The method as described above, wherein the mutant aromatic carboxylic acid reductase is utilized in the form of a bacterial cell of a microorganism having the mutant aromatic carboxylic acid reductase.
[13]
The method as described above, wherein the bacterial cells are used in the form of a culture medium of the microorganism, bacterial cells recovered from the culture medium, a processed product thereof, or a combination thereof.
[14]
The method, wherein the microorganism is a bacterium or a yeast.
[15]
The method, wherein the microorganism is a coryneform bacterium or a bacterium of the Enterobacteriaceae family.
[16]
The method as described above, wherein the microorganism is a bacterium of the genus Corynebacterium or Escherichia.
[17]
The method as described above, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.
[18]
The above method, wherein the microorganism has been modified so that the activity of phosphopantetheinyl transferase is increased as compared to an unmodified strain.
[19]
The above method, wherein the microorganism has been modified so that the activity of vanillic acid demethylase and/or alcohol dehydrogenase is reduced as compared to a non-modified strain.
[20]
The process further comprising recovering the vanillin.
[21]
A gene encoding the mutant aromatic carboxylic acid reductase.
[22]
A vector carrying the gene.
[23]
A microorganism having the gene.
[24]
The microorganism is a bacterium or a yeast.
[25]
The microorganism is a bacterium of the family Coryneform bacteria or Enterobacteriaceae.
[26]
The microorganism is a bacterium of the genus Corynebacterium or Escherichia.
[27]
The microorganism is Corynebacterium glutamicum or Escherichia coli.
[28]
The above microorganism, which has been modified so that the activity of phosphopantetheinyl transferase is increased as compared to a non-modified strain.
[29]
The above microorganism, which has been modified so that the activity of vanillic acid demethylase and/or alcohol dehydrogenase is reduced as compared to a non-modified strain.

The present invention provides a mutant ACAR that is useful for producing vanillin, and vanillin can be efficiently produced using the mutant ACAR.

<1> Mutant ACAR
The present invention provides aromatic carboxylic acid reductases (ACARs) having the "specific mutations" described herein.

"Aromatic carboxylic acid reductase (ACAR)" may refer to a protein having an activity that catalyzes a reaction in which an aromatic carboxylic acid is reduced to produce the corresponding aromatic aldehyde (EC 1.2.99.6, etc.). The activity is also referred to as "ACAR activity." A gene encoding ACAR is also referred to as "ACAR gene."

Specifically, the term "ACAR activity" may refer to the activity of catalyzing the reaction of reducing an aromatic carboxylic acid to the corresponding aromatic aldehyde in the presence of an electron donor and ATP. Examples of the electron donor include NADH and NADPH. Examples of the electron donor include, in particular, NADPH.

Examples of combinations of aromatic carboxylic acids and corresponding aromatic aldehydes include a combination of vanillic acid and vanillin, and a combination of isovanillic acid and isovanillin. ACAR activity in which vanillic acid is a substrate, i.e., activity that catalyzes the reaction of reducing vanillic acid to produce vanillin, is also referred to as "vanillin-producing activity". "Vanillin-producing activity" may specifically mean activity that catalyzes the reaction of reducing vanillic acid to produce vanillin in the presence of an electron donor and ATP. ACAR activity in which isovanillic acid is a substrate, i.e., activity that catalyzes the reaction of reducing isovanillic acid to produce isovanillin, is also referred to as "isovanillin-producing activity". "Isovanillin-producing activity" may specifically mean activity that catalyzes the reaction of reducing isovanillic acid to produce isovanillin in the presence of an electron donor and ATP.

ACAR activity (e.g., vanillin-producing activity or isovanillin-producing activity) can be determined by, for example, ATP
The ACAR activity can be measured by incubating the enzyme with a substrate (e.g., vanillic acid or isovanillic acid) in the presence of ATP and NADPH, and measuring the enzyme- and substrate-dependent oxidation of NADPH (modified from the method described in J. Biol. Chem. 2007, Vol. 282, No.1, p478-485). "A protein has ACAR activity" may mean that the protein has ACAR activity measured under at least one appropriate condition. The same applies to the activity of other proteins mentioned in this application.

ACAR with a "specific mutation" is also called "mutant ACAR." The gene encoding mutant ACAR is also called "mutant ACAR gene."

An ACAR that does not have a "specific mutation" is also referred to as a "wild-type ACAR." The gene encoding wild-type ACAR is also referred to as a "wild-type ACAR gene." Note that "wild-type" here is a convenient description to distinguish "wild-type" ACAR from "mutant" ACAR, and is not limited to those that are obtained naturally, so long as they do not have a "specific mutation." "ACAR does not have a 'specific mutation'" may mean that ACAR does not have a mutation selected as a "specific mutation." Wild-type ACAR may or may not have a mutation that was not selected as a "specific mutation," so long as it does not have a mutation selected as a "specific mutation."

When a wild-type ACAR and a mutant ACAR are identical except for the presence or absence of a "specific mutation," the wild-type ACAR is also called "the wild-type ACAR corresponding to a mutant ACAR," and the mutant ACAR is also called "the mutant ACAR corresponding to a wild-type ACAR."

The following describes wild-type ACAR.

Wild-type ACAR may or may not have ACAR activity such as vanillin-producing activity or isovanillin-producing activity, so long as the corresponding mutant ACAR has vanillin-producing activity. Wild-type ACAR may typically have vanillin-producing activity and isovanillin-producing activity. Wild-type ACAR may have at least isovanillin-producing activity.

An example of a wild-type ACAR is ACAR from bacteria of the genus Gordonia (WO2018/079705). An example of a Gordonia bacterium is Gordonia effusa. The nucleotide sequence of the ACAR gene from Gordonia effusa is shown in SEQ ID NO: 1, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 2. That is, the wild-type ACAR gene may be, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 1. The wild-type ACAR may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 2. The expression "a gene or protein has a nucleotide sequence or an amino acid sequence" may mean that a gene or protein contains the nucleotide sequence or the amino acid sequence, unless otherwise specified, and may also include a case where a gene or protein consists of the nucleotide sequence or the amino acid sequence.

The wild-type ACAR gene may be a variant of the wild-type ACAR gene exemplified above (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 1) as long as the ACAR it encodes does not have a "specific mutation". Similarly, the wild-type ACAR may be a variant of the wild-type ACAR gene exemplified above (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 2) as long as the ACAR it encodes does not have a "specific mutation". That is, the term "wild-type ACAR gene" may include the wild-type ACAR gene exemplified above (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 1) as well as variants thereof. Similarly, the term "wild-type ACAR" may include the wild-type ACAR gene exemplified above (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 2) as well as variants thereof. Note that the gene identified in the derived biological species is not limited to the gene itself found in the biological species, but includes genes having the nucleotide sequence of the gene found in the biological species and variants thereof. Also, the protein identified in the derived biological species is not limited to the protein itself found in the biological species, but includes proteins having the amino acid sequence of the protein found in the biological species and variants thereof. These variants may or may not be found in the organism. That is, for example, "ACAR of Gordonia bacteria" is not limited to ACAR itself found in Gordonia bacteria, but includes proteins having the amino acid sequence of ACAR found in Gordonia bacteria and variants thereof. Variants include, for example, homologs and artificially modified forms of the genes and proteins exemplified above.

A homologue of the wild-type ACAR gene or a homologue of wild-type ACAR can be easily determined from a public database, for example, by a BLAST search or a FASTA search using the base sequence of the wild-type ACAR gene exemplified above or the amino acid sequence of the wild-type ACAR exemplified above as a query sequence. In addition, a homologue of the wild-type ACAR gene can be obtained, for example, by PCR using the chromosomes of various organisms as a template and oligonucleotides prepared based on the base sequence of the wild-type ACAR gene exemplified above as primers.

A wild-type ACAR gene may encode a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) have been substituted, deleted, inserted, and/or added, as long as the ACAR it encodes does not have a "specific mutation." For example, the encoded protein may have an extended or shortened N-terminus and/or C-terminus. Note that the above "one or several" may vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and may specifically be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

The above substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains the original function of the protein. 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 with a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His, or Lys, substitutions of Asn to Glu, Gln, Lys, His, or Asp, substitutions of Asp to Asn, Glu, or Gln, substitutions of Cys to Ser or Ala, substitutions of Gln to Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu to Gly, Asn, Gln, Lys, or Asp, substitutions of Gly to Pro, substitutions of His to Asn, Lys, Gln, Arg, or Tyr, substitutions of Il Examples of 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 that occur due to naturally occurring mutations (mutants or variants), such as those based on individual differences or species differences in the organism from which the gene is derived.

In addition, a wild-type ACAR 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 described above, so long as the ACAR it encodes does not have a "specific mutation."

In addition, the wild-type ACAR 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: 1), e.g., a complementary sequence to the entire or partial base sequence, as long as the ACAR it encodes does not have a "specific mutation.""Stringentconditions" may mean conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. One example of such a condition includes conditions under which DNAs with high identity, for example, DNAs with an identity of 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, hybridize with each other, but DNAs with lower identity do not hybridize with each other, or conditions for washing once, preferably 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1 x SSC, 0.1% SDS, preferably 60°C, 0.1 x SSC, 0.1% SDS, and more preferably 68°C, 0.1 x SSC, 0.1% SDS, which are the washing conditions for conventional Southern hybridization.

As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and a DNA fragment containing the above-mentioned 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 hybridization include 50°C, 2×SSC, and 0.1% SDS.

In addition, since the degeneracy of codons differs depending on the host, the wild-type ACAR gene may be one in which any codon has been replaced with an equivalent codon. That is, the wild-type ACAR gene may be a variant of the wild-type ACAR gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type ACAR gene may be modified to have an optimal codon according to the codon usage frequency of the host used. An example of a codon-optimized wild-type ACAR gene is an ACAR gene having the base sequence shown in SEQ ID NO: 177, which is codon-optimized according to the codon usage of E. coli.

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 base sequences refers to the identity between base sequences calculated by blastn using the default scoring parameters (Match/Mismatch Scores = 1, -2; Gap Costs = Linear).

Below, we explain mutant ACAR.

The mutant ACAR has vanillin-producing activity. The mutant ACAR may or may not have isovanillin-producing activity.

Mutant ACAR has a "specific mutation" in the amino acid sequence of wild-type ACAR.

That is, the mutant ACAR may be, for example, a protein having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2. The mutant ACAR may also be, for example, a protein having an amino acid sequence with a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2, which further includes substitution, deletion, insertion, and/or addition of one or several amino acids at a position other than the "specific mutation", and which has vanillin-producing activity.

In other words, mutant ACAR may be a protein having the same amino acid sequence as wild-type ACAR, except for the "specific mutation". That is, mutant ACAR may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 2, except for the "specific mutation". Mutant ACAR may be, for example, a protein having an amino acid sequence including one or several amino acid substitutions, deletions, insertions, and/or additions in the amino acid sequence shown in SEQ ID NO: 2, except for the "specific mutation", and having vanillin-producing activity. Mutant ACAR may be, for example, a protein having an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2, except for the "specific mutation", and having vanillin-producing activity.

The mutant ACAR may contain other amino acid sequences in addition to the amino acid sequence of the mutant ACAR as exemplified above. The other amino acid sequence is also referred to as an "additional sequence." That is, the mutant ACAR may be a fusion protein with an additional sequence. The mutant ACAR may be expressed, for example, in a form containing an additional sequence (i.e., as a fusion protein with an additional sequence), and may ultimately lose part or all of the additional sequence. "The mutant ACAR contains an additional sequence" or "The mutant ACAR is a fusion protein with an additional sequence" means that the mutant ACAR finally obtained contains the additional sequence, unless otherwise specified. On the other hand, "The mutant ACAR is expressed in a form containing an additional sequence" or "The mutant ACAR contains an additional sequence at the time of expression" means that the mutant ACAR contains the additional sequence at least at the time of expression, unless otherwise specified, and does not necessarily mean that the mutant ACAR finally obtained contains the additional sequence. In other words, the mutant ACAR gene may contain a base sequence encoding the additional sequence in addition to the base sequence of the mutant ACAR gene as exemplified above. The same applies to wild-type ACAR and the wild-type ACAR gene. The additional sequence is not particularly limited as long as the mutant ACAR has vanillin-producing activity. The additional sequence can be appropriately selected depending on various conditions such as the purpose of use. Examples of the additional sequence include a peptide tag, a signal peptide (also called a signal sequence), and a recognition sequence for a protease. The additional sequence may be linked, for example, to the N-terminus or C-terminus of the mutant ACAR, or both. As the additional sequence, one type of amino acid sequence may be used, or two or more types of amino acid sequences may be used in combination.

Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, maltose binding protein (MBP), cellulose binding protein (CBP), thioredoxin (TRX), green fluorescent protein (GFP), horseradish peroxidase (HRP), alkaline phosphatase (ALP), and Fc regions of antibodies. Examples of His tags include 6xHis tags. Peptide tags can be used, for example, to detect and purify expressed mutant ACAR.

The signal peptide is not particularly limited as long as it functions in a host in which the mutant ACAR is expressed. Examples of the signal peptide include signal peptides recognized by the Sec secretory pathway and signal peptides recognized by the Tat secretory pathway. Specific examples of the signal peptides recognized by the Sec secretory pathway include signal peptides of cell surface proteins of coryneform bacteria. Specific examples of the signal peptides of cell surface proteins of coryneform bacteria include the PS1 signal sequence and PS2 (CspB) signal sequence of C. glutamicum (JP Patent Publication No. 6-502548) and the SlpA (CspA) signal sequence of C. stationis (JP Patent Publication No. 10-108675). Specific examples of signal peptides recognized by the Tat secretion pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013/118544). Signal peptides can be used, for example, for secretion production of mutant ACAR. When a mutant ACAR is secreted using a signal peptide, the signal peptide is cleaved during secretion, and a mutant ACAR without a signal peptide can be secreted outside the bacterial cell. That is, typically, the mutant ACAR finally obtained does not need to have a signal peptide.

Specific examples of protease recognition sequences include the recognition sequence for Factor Xa protease and the recognition sequence for proTEV protease. The recognition sequence for a protease can be used, for example, to cleave an expressed mutant ACAR. Specifically, for example, when a mutant ACAR is expressed as a fusion protein with a peptide tag, a protease recognition sequence can be introduced at the junction between the mutant ACAR and the peptide tag, allowing the peptide tag to be cleaved from the expressed mutant ACAR using a protease to obtain a mutant ACAR that does not have a peptide tag.

The mutant ACAR gene is not particularly limited as long as it encodes the mutant ACAR as described above. In the present invention, the term "gene" is not limited to DNA, and may include any polynucleotide as long as it encodes a protein of interest. That is, the "mutant ACAR gene" may mean any polynucleotide encoding a mutant ACAR. The mutant ACAR gene may be DNA, RNA, or a combination thereof. The mutant ACAR gene may be single-stranded or double-stranded. The mutant ACAR gene may be single-stranded DNA or single-stranded RNA. The mutant ACAR gene may be double-stranded DNA, double-stranded RNA, or a hybrid chain consisting of a DNA chain and an RNA chain. The mutant ACAR gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the mutant ACAR gene contains RNA, the description of DNA such as the exemplified base sequence may be appropriately interpreted according to RNA. The form of the mutant ACAR gene can be appropriately selected according to various conditions such as its mode of use.

The following explains "specific mutations."

"Specific mutation" means a mutation that is useful for the production of vanillin. Specifically, "specific mutation" may mean a mutation that, when introduced into wild-type ACAR, confers properties suitable for the production of vanillin to the wild-type ACAR. "Specific mutation" includes a mutation that improves the substrate specificity of ACAR for vanillic acid. An example of a mutation that improves the substrate specificity of ACAR for vanillic acid is a mutation that reduces the isovanillin by-production rate (iVNN by-production rate) of ACAR. In other words, the mutant ACAR may exhibit a lower iVNN by-production rate than the corresponding wild-type ACAR. The iVNN by-production rate of mutant ACAR may be, for example, 99% or less, 97% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 7% or less, 5% or less, 3% or less, 2% or less, or 1% or less of the iVNN by-production rate of the corresponding wild-type ACAR. In addition, the iVNN by-production rate of mutant ACAR may be, for example, 5.0% or less, 4.5% or less, 4.0% or less, 3.5% or less, 3.0% or less, 2.5% or less, 2.0% or less, 1.5% or less, 1.0% or less, 0.5% or less, 0.3% or less, 0.2% or less, 0.1% or less, or 0. Mutations that result in iVNN by-production rates in these ranges include the mutations that result in iVNN by-production rates in these ranges in the Examples. A decrease in the iVNN by-production rate of ACAR may be achieved, for example, by increasing the Km of ACAR for isovanillic acid, by decreasing the Km of ACAR for vanillic acid, or a combination thereof. A decrease in the iVNN by-production rate of ACAR may be achieved, for example, by decreasing the specific activity of the isovanillin-producing activity of ACAR, by increasing the specific activity of the vanillin-producing activity of ACAR, or a combination thereof.

"Isovanillin by-production rate (iVNN by-production rate)" refers to the molar ratio of the amount of isovanillin produced to the amount of vanillin produced when ACAR is allowed to act on a mixture of vanillic acid and isovanillic acid in which the vanillic acid content:isovanillic acid content is 5:0.68 in molar ratio. Specifically, the iVNN by-production rate can be calculated by, for example, adding an appropriate amount of ACAR to the reaction buffer shown in Table 3 (for example, a cell-free extract equivalent to 50 μg of total protein per 500 μL of the reaction buffer).
The enzyme reaction can be carried out at a reaction temperature of 30° C. for an appropriate reaction time (for example, 60 minutes) and then measured.

"Specific mutations" include mutations at the following amino acid residues:
C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, I574.

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, for example, a mutation in one or more amino acid residues selected from these amino acid residues. A "specific mutation" may be, for example, a mutation in one amino acid residue selected from these amino acid residues, or a combination of mutations in two or more amino acid residues selected from these amino acid residues.

"Specific mutations" particularly include a mutation at G386. That is, "specific mutations" may include, for example, a mutation at G386. "Specific mutations" may be, for example, a mutation at G386, or a combination of a mutation at G386 and a mutation at one or more other amino acid residues.

In the above notation for identifying amino acid residues, the numbers indicate positions in the amino acid sequence shown in SEQ ID NO:2, 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:2 (i.e., the amino acid residues at each position before modification). That is, for example, "G386" indicates the G (Gly) residue at position 386 in the amino acid sequence shown in SEQ ID NO:2.

In any wild-type ACAR, these amino acid residues each represent "the amino acid residue corresponding to the corresponding amino acid residue in the amino acid sequence shown in SEQ ID NO:2." That is, for example, "G386" in any wild-type ACAR represents the amino acid residue corresponding to the G (Gly) residue at position 386 in the amino acid sequence shown in SEQ ID NO:2.

Each of the above mutations may be a substitution of an amino acid residue. In each of the above mutations, the modified amino acid residue may be any amino acid residue other than the amino acid residue before modification. Specific examples of the modified amino acid residue include amino acid residues selected from 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 modified amino acid residue may be one that is effective for vanillin production (for example, one that reduces the rate of iVNN by-production of ACAR).

Specific examples of "specific mutations" include the following:
C28A, C135 (A, V), R241 (K, Q), G249 (N, S, T), R252 (K, Q, T), W259 (F, Y), M265 (I, V), V270 (A, I), M271 (A, G, H, I, L, N, Q, R, S, T, V, Y), S274 (A, N), T275S, Q278D, Q310L, V312 (A, C, D, F, G, I, L, M, P, Q, S, T, Y), I353 (L, V), S356 (A, T), I376 (F, L, V), E377D, G378D, Y379 (F, L), S381 (A, T), T382S, A384 (C, F, G, H, I, K, L, N, Q, S, T), A385 (D, E, K, P, R), G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y), V387I, S388 (M, T), I389V, Y467 (F, H, W), I574 (A, C, F, G, L, L, M, N, P, T, V).

That is, the "specific mutation" may include, for example, one or more mutations selected from these mutations. The "specific mutation" may be, for example, one mutation selected from these mutations, or a combination of two or more mutations selected from these mutations. The "specific mutation" may also be, for example, a combination of one or more mutations selected from these mutations and one or more other amino acid residues selected from C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, and I574.

In addition, mutations at C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, and I574 are, for example, C28A, C135 (A, V), R241 (K, Q), G249 (N, S, T), R252 (K, Q, T), W259 (F, Y), M265 (I, V), V270 (A, I), M271 (A, G, H, I, L, N, Q, R, S, T, V, Y), S274 (A, N), T275S, Q278D, Q310L, V312 (A, C, D, F, G, I, L, M, P, Q, S, T, Y), I353 (L, V), S356 (A, T), I376 (F, L, V), E377D, G378D, Y379 (F, L), S381 (A, T), T382S, A384 (C, F, G, H, I, K, L, N, Q, S, T), A385 (D, E, K, P, R), G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y), V387I, S388 (M, T), I389V, Y467 (F, H, W), and I574 (A, C, F, G, L, L, M, N, P, T, V).

A particular example of a mutation at G386 is G386N.

In the above notation for identifying mutations, the numbers and the letters to the left of them have the same meanings as described above. In the above notation for identifying mutations, the letters to the right of the numbers indicate the amino acid residues after modification at each position. That is, for example, "C28A" indicates a mutation in which the C (Cys) residue at position 28 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with an A (Ala) residue. Also, for example, "G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y)" indicates a mutation in which the G (Gly) residue at position 386 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y residue.

In any wild-type ACAR, these mutations each indicate "a mutation corresponding to the mutation in the amino acid sequence shown in SEQ ID NO: 2." In any wild-type ACAR, "a mutation corresponding to a mutation in which the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue" is to be read as "a mutation in which the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue." That is, for example, in any wild-type ACAR, "G386N" indicates a mutation in which the amino acid residue corresponding to the G (Gly) residue at position 386 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with an N (Asn) residue.

The combination of mutations is not particularly limited. Examples of the combination of mutations include a combination that includes a mutation at G386. Specific examples of the combination of mutations include the following combinations:
G386M/M271Y, G386M/S274N, G386M/I353F, G386M/I353L, G386N/M271Y, G386N/V312A, G386N/V312F, G386N/V312I, G386N/I353F, G386N/I353L, G386N/A384S, G386N/A385M, G386N/A385V, G386N/V387I, G386N/I574A, G386N/I574M, G386N/I574T.

Combinations of mutations include, in particular, the following combinations:
G386M/M271Y, G386M/S274N, G386M/I353F, G386M/I353L, G386N/M271Y, G386N/V312A, G386N/V312F, G386N/V312I, G386N/I353F, G386N/I353L, G386N/A384S, G386N/A385M, G386N/V387I, G386N/I574A, G386N/I574M, G386N/I574T.

That is, a "specific mutation" may include, for example, any combination of these.

In the above notation for specifying the combination, the meaning of the number and the letters on the left and right of the number are the same as above. In the above notation for specifying the combination, the combination of two or more mutations separated by "/" indicates a double mutation or multiple mutations. That is, for example, "G386M/M271Y" indicates a double mutation of G386M and M271Y.

The positions of the amino acid residues mentioned in each of the above mutations are merely a description for convenience in identifying the amino acid residues to be modified, and do not necessarily indicate their absolute positions in wild-type ACAR. That is, the positions of the amino acid residues in each of the above mutations are relative positions based on the amino acid sequence shown in SEQ ID NO:2, and the absolute positions may vary due to deletion, insertion, or addition of amino acid residues. For example, in the amino acid sequence shown in SEQ ID NO:2, if one amino acid residue is deleted or inserted at a position N-terminal to position X, the original amino acid residue at position X becomes the X-1 or X+1 amino acid residue counting from the N-terminus, respectively, but 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:2." In addition, the amino acid residues before modification mentioned in each of the above mutations are merely a description for convenience in identifying the amino acid residues to be modified, and do not necessarily have to be conserved in wild-type ACAR. That is, if wild-type ACAR does not have the amino acid sequence shown in SEQ ID NO:2, the amino acid residues before modification mentioned in each of the above mutations may not be conserved. That is, each of the above mutations may also include a mutation in which the amino acid residue before modification mentioned in each of the above mutations is replaced with another amino acid residue (e.g., the modified amino acid residue mentioned in each of the above mutations) when the amino acid residue before modification is not preserved. For example, a "mutation at G386" is not limited to a mutation in which the amino acid residue corresponding to G386 is replaced with another amino acid residue when the amino acid residue corresponding to G386 is preserved (i.e., it is a G (Gly) residue), but may also include a mutation in which the amino acid residue corresponding to G386 is replaced with another amino acid residue when the amino acid residue is not preserved (i.e., it is not a G (Gly) residue). The amino acid residues before and after modification are selected so that they are not identical to each other.

In the amino acid sequence of any ACAR, which amino acid residue is "the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO:2" can be determined by aligning the amino acid sequence of the any ACAR with the amino acid sequence shown in SEQ ID NO:2. The alignment can be performed, for example, using known gene analysis software. Specific examples of the software include DNASIS made by Hitachi Solutions and GENETYX made 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).

<2> Production of mutant ACAR Mutant ACAR can be produced, for example, by expressing a mutant ACAR gene in a host carrying the gene.

Mutant ACAR can also be produced, for example, by expressing the mutant ACAR gene in a cell-free protein synthesis system.

The production of mutant ACAR using a host carrying a mutant ACAR gene is described in detail below.

<2-1> Host A host having a mutant ACAR gene can be obtained by introducing the mutant ACAR gene into a suitable host. "Introducing a mutant ACAR gene into a host" may also include modifying an ACAR gene, such as a wild-type ACAR gene, that the host has so that it encodes mutant ACAR. "Having a mutant ACAR gene" is also referred to as "having mutant ACAR."

The host is not particularly limited as long as it is capable of expressing a functional mutant ACAR. Hosts include microorganisms, plant cells, insect cells, and animal cells. Hosts in particular include microorganisms. Microorganisms in particular include bacteria and yeast. Microorganisms in particular include bacteria.

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

Examples of bacteria belonging to the Enterobacteriaceae family include bacteria belonging to the genera Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified into the Enterobacteriaceae family according to the classification method 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 into the genus Escherichia according to the classification known to microbiology experts. 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, p.
2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, DC). Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Examples of Escherichia coli include Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as BL21(DE3) strain; and derivatives thereof. Examples of bacteria belonging to the genus Enterobacter include
Examples of bacteria of the genus Pantoea 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.

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

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)
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

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 melassecola 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

The genus Corynebacterium also 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, etc. (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

Examples of the 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).

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

These strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is assigned, and the strains can be obtained by using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is listed in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository institution where each strain was deposited.

A mutant ACAR gene can be obtained, for example, by modifying a wild-type ACAR gene so that the encoded ACAR has a "specific mutation." The wild-type ACAR gene that is the source of modification can be obtained, for example, by cloning from an organism having a wild-type ACAR gene or by chemical synthesis. A mutant ACAR gene can also be obtained without going through a wild-type ACAR gene. A mutant ACAR gene may be obtained directly, for example, by chemical synthesis. The obtained mutant ACAR gene may be used as is or after further modification. For example, a mutant ACAR gene of a certain embodiment may be modified to obtain a mutant ACAR gene of another embodiment.

Genetic modification can be performed by known techniques. For example, a desired mutation can be introduced into a target site of DNA by site-specific mutagenesis. That is, for example, a coding region of a gene can be modified by site-specific mutagenesis so that the encoded protein contains substitutions, deletions, insertions, and/or additions of amino acid residues at specific sites. Examples of site-specific mutagenesis include a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and a method using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

There are no particular limitations on the method for introducing the mutant ACAR gene into the host. The mutant ACAR gene only needs to be retained in the host in an expressible manner. The mutant ACAR gene can be introduced into the host in the same manner as the introduction of genes described later in the "Method for increasing protein activity."

In addition, if the host already has an ACAR gene, such as a wild-type ACAR gene, in a chromosome or the like, the host can be modified to have a mutant ACAR gene by modifying the ACAR gene so that it encodes a mutant ACAR. Modification of the ACAR gene present in a chromosome or the like can be carried out, for example, by natural mutation, mutation treatment, or genetic engineering.

The host may have any properties as long as it is capable of producing mutant ACAR.

The host may be modified, for example, to have properties useful for the production of vanillin. Specifically, the host may be modified, for example, to have properties useful for the production of vanillin from vanillic acid. Such modifications include those described in WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, and US2019-0161776A for imparting or enhancing vanillin production ability. Specific examples of such modifications include increasing the activity of phosphopantetheinylating enzymes, increasing the activity of the vanillic acid uptake system, decreasing the activity of vanillic acid demethylase, and decreasing the activity of alcohol dehydrogenase (ADH). In particular, such modifications include increasing the activity of phosphopantetheinylating enzymes, decreasing the activity of vanillic acid demethylase, and decreasing the activity of alcohol dehydrogenase. These modifications can be used alone or in appropriate combination. The order of modifications for constructing a host is not particularly limited.

The host may be modified to increase the activity of a phosphopantetheinylating enzyme. ACAR can become an active enzyme by being phosphopantetheinylated (J. Biol. Chem. 2007, Vol. 282, No.1, p478-485). Therefore, the activity of ACAR can be increased by increasing the activity of an enzyme that catalyzes the phosphopantetheinylation of proteins (also called a "phosphopantetheinylating enzyme"). An example of a phosphopantetheinylating enzyme is phosphopantetheinyl transferase (PPT).

"Phosphopantetheinyl transferase (PPT)" may refer to a protein having an activity of catalyzing a reaction of phosphopantetheinylating ACAR in the presence of a phosphopantetheinyl group donor. This activity is also referred to as "PPT activity." A gene encoding a PPT is also referred to as a "PPT gene." An example of a phosphopantetheinyl group donor is coenzyme A (CoA). An example of a PPT is the EntD protein encoded by the entD gene. Examples of PPTs such as EntD proteins include those from various organisms. A specific example of a PPT is the EntD protein of E. coli, such as E. coli K-12 MG1655 strain. The nucleotide sequence of the entD gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 3, and the amino acid sequence of the EntD protein encoded by the gene is shown in SEQ ID NO: 4. Specific examples of PPT include the PPT of Nocardia brasiliensis, the PPT of Nocardia farcinica IFM10152 (J. Biol. Chem. 2007, Vol. 282, No. 1, pp. 478-485), and the PPT of C. glutamicum (App. Env. Microbiol. 2009, Vol. 75, No. 9, pp. 2765-2774). Examples of C. glutamicum include the above-mentioned strains such as the C. glutamicum ATCC 13032 strain and the ATCC 13869 strain.

The host may be modified to increase the activity of the vanillic acid uptake system. The "vanillic acid uptake system" may mean a protein having the function of taking up vanillic acid from outside the cell into the cell. This activity is also called "vanillic acid uptake activity." A gene encoding the vanillic acid uptake system is also called "vanillic acid uptake system gene." An example of the vanillic acid uptake system is the VanK protein encoded by the vanK gene (M. T. Chaudhry, et al., Microbiology, 2007. 153:857-865). Examples of vanillic acid uptake systems such as the VanK protein include those found in various organisms such as coryneform bacteria. A specific example of the vanillic acid uptake system is the VanK protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.

The host may be modified so that the activity of vanillate demethylase is reduced. "Vanillate demethylase" may refer to a protein having an activity of catalyzing a reaction of demethylating vanillic acid to produce protocatechuic acid. This activity is also referred to as "vanillate demethylase activity." A gene encoding vanillate demethylase is also referred to as "vanillate demethylase gene." Examples of vanillate demethylase include VanAB proteins encoded by vanAB genes (Current Microbiology, 2005, Vol.51, p59-65). The vanA gene and vanB gene encode subunit A and subunit B of vanillate demethylase, respectively. When reducing vanillate demethylase activity, for example, both vanAB genes may be disrupted, or only one of them may be disrupted. Examples of vanillate demethylase such as VanAB proteins include those of various organisms such as bacteria of the Enterobacteriaceae family and coryneform bacteria. Specific examples of vanillate demethylase include the VanAB proteins of C. glutamicum such as C. glutamicum ATCC 13032 and ATCC 13869 strains. The vanAB genes usually constitute the vanABK operon together with the vanK gene. Therefore, in order to reduce vanillate demethylase activity, the vanABK operon may be disrupted (e.g., deleted) en masse. In that case, the vanK gene may be introduced again into the host. For example, in the case of utilizing vanillic acid present outside the host cell, when the vanABK operon is disrupted (e.g., deleted) en masse, the vanK gene may be introduced again.

The host may be modified to reduce the activity of alcohol dehydrogenase (ADH).
"ADH" may refer to a protein having an activity of catalyzing a reaction in which an aldehyde is reduced to produce an alcohol in the presence of an electron donor (EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.71, etc.). This activity is also called "ADH activity." A gene encoding ADH is also called "ADH gene." Examples of electron donors include NADH and NADPH.

ADHs include those that have the activity of catalyzing the reaction of reducing vanillin to produce vanillyl alcohol in the presence of an electron donor. This activity is also called "vanillyl alcohol dehydrogenase activity." ADHs that have vanillyl alcohol dehydrogenase activity are also called "vanillyl alcohol dehydrogenase."

ADH includes the yqhD gene, the NCgl0324 gene, the NCgl0313 gene, the NCgl2709 gene, and the NCgl0
Examples of ADH include the YqhD protein, NCgl0324 protein, NCgl0313 protein, NCgl2709 protein, NCgl0219 protein, and NCgl2382 protein, which are encoded by the .219 gene and the NCgl2382 gene, respectively. Both the yqhD gene and the NCgl0324 gene encode vanillyl alcohol dehydrogenase. Examples of such ADH include those from various organisms such as bacteria of the Enterobacteriaceae family and coryneform bacteria. The yqhD gene can be found, for example, in bacteria of the Enterobacteriaceae family, such as E. coli. The NCgl0324 gene, the NCgl0313 gene, the NCgl2709 gene, the NCgl0219 gene, and the NCgl2382 gene can be found, for example, in coryneform bacteria, such as C. glutamicum. Specifically, the ADH may be the YqhD protein of E. coli such as E. coli K-12 MG1655 strain. Specifically, the ADH may be the NCgl0324 protein, NCgl0313 protein, NCgl2709 protein, NCgl0219 protein, and NCgl2382 protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The activity of one type of ADH may be reduced, or the activity of two or more types of ADH may be reduced. For example, the activity of one or more types of ADH among the NCgl0324 protein, NCgl2709 protein, and NCgl0313 protein may be reduced. In particular, the activity of at least the NCgl0324 protein may be reduced.

The genes and proteins used to modify the host may be, for example, genes and proteins having known nucleotide and amino acid sequences, respectively. The genes and proteins used to modify the host may also be conservative variants of genes and proteins having known nucleotide and amino acid sequences, respectively. "Conservative variant" may mean a variant that maintains the original function. Specifically, for example, the genes used to modify the host may be genes that encode proteins having an amino acid sequence in which one or several amino acids at one or several positions in the known amino acid sequence of the protein are substituted, deleted, inserted, and/or added, as long as the original function (i.e., enzyme activity, etc.) is maintained. The activity of each protein can be measured by, for example, the methods described in WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, or US2019-0161776A. For conservative variants of such genes and proteins, the descriptions regarding the ACAR gene and ACAR variants described above can be applied mutatis mutandis.

<2-2> Methods for Increasing Protein Activity Methods for increasing protein activity (including gene introduction methods) are described below.

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

The degree of increase in protein activity is not particularly limited, so long as the activity of the protein is increased compared to that of the unmodified strain. For example, the activity of the protein 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 the 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.

A modification that increases the activity of a protein can be achieved, for example, by increasing the expression of a gene that codes for the protein. "Gene expression is increased" may mean that the expression of the gene is increased compared to a non-modified strain such as a wild type or a parent strain. "Gene expression is increased" may specifically mean that the expression level of the gene per cell is increased compared to a non-modified strain. "Expression level of the gene per cell" may mean the average expression level of the gene per cell. "Gene expression is increased" 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. "Gene expression is increased" is also referred to as "gene expression is enhanced." Gene expression may be increased, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to a non-modified strain. Furthermore, "increasing gene expression" includes not only increasing the expression level of a target gene in a strain in which the target gene is originally expressed, but also expressing the target gene in a strain in which the target gene is not originally expressed. In other words, "increasing gene expression" may mean, for example, introducing the target gene into a strain that does not harbor the gene and expressing the gene.

Increasing gene expression can be achieved, for example, by increasing the copy number of the gene.

The copy number of a gene can be increased by introducing the gene into a host chromosome. The introduction of a gene into a chromosome can be achieved, for example, by using homologous recombination (Miller, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include a method using linear DNA such as the Red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U.S.A. 97:6640-6645 (2000)), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, a method using a suicide vector that does not have a replication origin that functions in the host, and a transduction method using a phage. 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 has multiple copies on a chromosome. Examples of sequences that exist in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of a transposon. Homologous recombination may also be performed by targeting an appropriate sequence on a chromosome, such as a gene that is not necessary for vanillin production. Genes can also be randomly introduced onto a chromosome using a transposon or Mini-Mu (JP Patent Publication 2-109985, US Pat. No. 5,882,888, EP805867B1).

Introduction of the target gene onto the chromosome can be confirmed by Southern hybridization using a probe with a sequence complementary to all or part of the gene, or by PCR using primers created based on the sequence of the gene.

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 is linked to a vector that functions in a host to construct an expression vector for the gene, and the host is transformed with the expression vector, thereby increasing 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 having the target gene as a template. A vector capable of autonomously replicating in the host cell can be used as the vector. The vector may be a multicopy vector. In addition, the vector may have a marker such as an antibiotic resistance gene in order to select a transformant. The vector may also have a promoter or a terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomously replicating in bacteria of the Enterobacteriaceae family, such as Escherichia coli, include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK-based vectors (Clontech), pKK233-2 (Clontech), pET-based vectors (Novagen), pQE-based vectors (Qiagen), pCold TF DNA (Takara Bio), pACYC-based vectors, and 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 Patent Publication No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP Patent Publication No. 2-72876, U.S. Patent No. 5,185,262); pCRY2 and pCRY 3 (JP Patent Publication No. 1-191686); pAJ655, pAJ611, and pAJ1844 (JP Patent Publication No. 58-192900); pCG1 (JP Patent Publication No. 57-134500); pCG2 (JP Patent Publication No. 58-35197); pCG4 and pCG11 (JP Patent Publication No. 57-183799); pPK4 (U.S. Pat. No. 6,090,597); pVK4 (JP Patent Publication No. 9-322774); pVK7 (JP Patent Publication No. 10-215883); pVK9 (WO2007/046389); pVS7 (WO2013/069634); and pVC7 (JP Patent Publication No. 9-070291). Furthermore, specific examples of vectors capable of autonomously replicating in coryneform bacteria include pVC7 variants such as pVC7H2 (WO2018/179834).

When a gene is introduced, the gene may be expressed by the host. Specifically, the gene may be 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 specific to the gene to be introduced or a promoter of another gene. For example, a stronger promoter as described in this specification may be used as the promoter.

A terminator for terminating transcription can be placed downstream of the gene. There are no particular limitations on the terminator, so long as it functions in the host. The terminator may be a terminator derived from the host or a terminator derived from a heterologous species. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene. Specific examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

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.

When introducing two or more genes, each gene only needs to be retained in the host in an expressible manner. For example, all of the genes may be retained on a single expression vector, or all of the genes may be retained on a chromosome. The genes may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Two or more genes may be introduced as an operon. Examples of "introducing two or more genes" include introducing genes that each code for two or more proteins (e.g., enzymes) and introducing genes that each code for two or more subunits that make up a single protein complex (e.g., an enzyme complex).

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 by PCR, for example, using primers designed based on the base sequence of the gene and using the genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. The gene to be introduced can also be totally synthesized based on the base sequence of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as is or after appropriate modification. That is, by modifying the gene, its variant can be obtained. Gene modification can be performed by a known method. For example, a desired mutation can be introduced into a target site of DNA by site-directed mutagenesis. That is, for example, a coding region of a gene can be modified by site-directed mutagenesis so that the encoded protein contains substitution, deletion, insertion, and/or addition of amino acid residues at a specific site. Site-specific mutagenesis methods include PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, gene variants may be totally synthesized.

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 activity of the protein is increased as a result. That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of all or only some of the genes encoding the subunits may be enhanced. Usually, it is preferable to enhance the expression of all of the genes encoding the subunits. In addition, each subunit constituting the complex may be derived from one 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 the host, or genes derived from different organisms may be introduced into the host.

In addition, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. In addition, the increase in gene expression can be achieved by improving the translation efficiency of the gene. The improvement in gene transcription efficiency and translation efficiency can be achieved, for example, by modifying the expression regulatory sequence. "Expression regulatory sequence" may be a general term for a site that affects gene expression. Examples of expression regulatory sequences include promoters, Shine-Dalgarno (SD) sequences (also called 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 gene 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).

The efficiency of gene transcription can be improved, for example, by replacing the promoter of a gene on a chromosome with a stronger promoter. A "stronger promoter" may mean a promoter that improves gene transcription more than the wild-type promoter that is originally present. Examples of stronger promoters include the well-known high-expression promoters T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter derived from Bifidobacterium, PR promoter, and PL promoter. Examples of stronger promoters that can be used in coryneform bacteria include the artificially designed P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679(2000)), 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 Dec;71(12):8587-96.), P2 promoter (WO2018/079684), P3 promoter (WO2018/079684), lac promoter, tac promoter, trc promoter, and F1 promoter (WO2018/179834). In addition, as a stronger promoter, a highly active version of a conventional promoter may be obtained by using various reporter genes. For example, promoter activity can be increased by making the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 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 Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

The improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence. The term "stronger SD sequence" may mean an SD sequence that improves the translation of mRNA more than the wild-type SD sequence that is originally present. An example of a stronger SD sequence is the RBS of gene 10 derived from phage T7 (Olins PO et al., Gene,
1988, 73, 227-235). Furthermore, it is known that substitution, insertion, or deletion of several nucleotides in the spacer region between the RBS and the initiation codon, particularly in the sequence immediately upstream of the initiation codon (5'-UTR), greatly affects the stability and translation efficiency of mRNA, and modification of these regions can also improve the translation efficiency of genes.

The improvement of gene translation efficiency can also be achieved, for example, by modifying codons. 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 an optimal codon depending on the codon usage frequency of the host to be used. Codon replacement 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 replaced codon may be totally synthesized. The frequency of codon usage in various organisms is disclosed in the "Codon Usage Database"(http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).

Increasing gene expression can also be achieved by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

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

Modifications that increase the activity of a protein can also be achieved, for example, by 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 a metabolite, the activity of the protein can be increased by mutating a gene or protein in a host so that feedback inhibition is desensitized. Unless otherwise specified, "desensitization of feedback inhibition" may include cases in which feedback inhibition is completely released and cases in which feedback inhibition is reduced. In addition, "feedback inhibition being desensitized" (i.e. feedback inhibition being reduced or released) is also referred to as "resistance to feedback inhibition". Proteins with enhanced specific activity can be obtained, for example, by searching for various organisms. Also, highly active proteins may be obtained by introducing mutations into existing proteins. The mutations introduced may be, for example, substitution, deletion, insertion, and/or addition of one or several amino acids at one or several positions of the protein. The introduction of mutations can be performed, for example, by the site-directed mutagenesis method described above. Mutations may also be introduced by, for example, a mutation treatment. Examples of mutation treatments 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 may also be induced in vitro by directly treating DNA with hydroxylamine. Enhancement of specific activity may be used alone or in any combination with the above-mentioned methods for enhancing gene expression.

The method of transformation is not particularly limited, and a conventionally known method can be used. For example, a method of treating recipient cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), or a method of preparing competent cells from cells in the growth stage and introducing DNA, as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, 1977. Gene 1: 153-167), can be used. Alternatively, a method known for Bacillus subtilis, actinomycetes, and yeasts, in which the cells of the DNA recipient are transformed into protoplasts or spheroplasts that easily incorporate recombinant DNA, and then the recombinant DNA is introduced into the DNA recipient (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) can also be applied. Alternatively, an electric pulse method (JP Patent Publication 2-207791) reported for coryneform bacteria can also be used.

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

Increased protein activity can also be confirmed by confirming increased expression of the gene that codes for the protein. Increased gene expression can be confirmed by confirming increased transcription levels of the gene or increased amount of protein expressed from the gene.

The increase 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 such as a wild-type strain or a parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, and the like (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., the number of molecules per cell) may be increased, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to that of a non-modified strain.

The increase in the amount of the protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein (e.g., the number of molecules per cell) may be increased, for example, by 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain.

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

<2-3> Methods for reducing protein activity Methods for reducing protein activity are described below.

"The activity of a protein is reduced" may mean that the activity of the protein is reduced compared to that of a non-modified strain. "The activity of a protein is reduced" may specifically mean that the activity of the protein per cell is reduced compared to that of a non-modified strain. "The activity of a protein per cell" may mean the average activity of the protein per cell. "Non-modified strain" here may mean a control strain that has not been modified to reduce the activity of the target protein. Examples of non-modified strains include wild-type strains and parent strains. Examples of non-modified strains include type strains of the species to which the host belongs. Examples of non-modified 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 decreased compared to that of the E. coli K-12 MG1655 strain. The term "decreased activity of a protein" may also include a case where the activity of the protein is completely lost. More specifically, the term "decreased activity of a protein" may mean that the number of molecules of the protein per cell is decreased and/or the function of the protein per molecule is decreased compared to a non-modified strain. That is, the "activity" in the term "decreased activity of a protein" is not limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of a gene encoding the protein. The term "number of molecules of a protein per cell" may mean the average number of molecules of the protein per cell. The term "decreased number of molecules of a protein per cell" may also include a case where the protein is completely absent. The term "decreased function of a protein per molecule" may also include a case where the function of the protein per molecule is completely lost. The degree of decrease in the activity of a protein is not particularly limited as long as the activity of the protein is decreased compared to a non-modified strain. The activity of the protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the unmodified strain.

Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of a gene encoding the protein. "Reduced gene expression" may mean that the expression of the gene is reduced compared to a non-modified strain such as a wild type or a parent strain. "Reduced gene expression" may specifically mean that the expression level of the gene per cell is reduced compared to a non-modified 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. "Reduced gene expression" is also referred to as "weakened 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 the non-modified strain.

The reduction in gene expression may be due to, for example, a reduction in transcription efficiency, a reduction in translation efficiency, or a combination thereof. The reduction in gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter, a Shine-Dalgarno (SD) sequence (also called a ribosome binding site (RBS)), or a 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. The reduction in gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" may mean a promoter that weakens the transcription of a gene compared to the wild-type promoter that is originally present. An example of a weaker promoter is an inducible promoter. That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (for example, in the absence of an inducer). Examples of weaker promoters include the P4 promoter (WO2018/079684) and the P8 promoter (WO2018/079684). In addition, a part or all of the expression regulatory sequence may be deleted. In addition, the reduction in 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. In addition, the reduction in gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces the expression of the gene. For example, the expression of the gene can be reduced by replacing the codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, the expression of the gene itself can be reduced by disrupting the gene as described herein.

Modifications that reduce the activity of a protein can be achieved, for example, by disrupting the gene that codes for the protein. "The gene is disrupted" may mean that the gene is modified so that it does not produce a protein that functions normally. "Does not produce a protein that functions normally" may also include cases where no protein is produced from the gene, or cases where the gene produces a protein with reduced or lost function (e.g., activity or property) per molecule.

The destruction of a gene can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Deletion of a gene" may mean the deletion of a part or the entire region of the coding region of the gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. As long as the activity of the protein can be reduced, the region to be deleted may be any region, such as the N-terminal region (i.e., the region coding the N-terminal side of the protein), the internal region, or the C-terminal region (i.e., the region coding the C-terminal side of the protein). 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, a region with a length of 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. In addition, it is preferable that the sequences before and after the region to be deleted do not match in reading frame. A mismatch in reading frame may cause a frame shift downstream of the region to be deleted.

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 or 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)).

Disruption of a gene can also be achieved, for example, by inserting another base sequence into the coding region of the gene on a chromosome. The insertion site may be in any region of the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. It is also preferable that the sequences before and after the insertion site do not match in reading frame. A mismatch in reading frame can cause a frameshift downstream of the insertion site. There is no particular limit to the other base sequence as long as it reduces or eliminates the activity of the encoded protein, but examples of such sequences include marker genes such as antibiotic resistance genes and genes useful for producing vanillin.

The gene may be destroyed so that the amino acid sequence of the encoded protein is deleted (missing). In other words, a modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein (a part or all of the region of the amino acid sequence), specifically by modifying the gene so that the gene encodes a protein from which the amino acid sequence (a part or all of the region of the amino acid sequence) is deleted. The term "deletion of the amino acid sequence of a protein" may mean the deletion of a part or all of the region of the amino acid sequence of the protein. The term "deletion of the amino acid sequence of a protein" may mean that the original amino acid sequence is no longer present in the protein, and may also include cases in which the original amino acid sequence is changed to a different amino acid sequence. That is, for example, a region that has been changed to a different amino acid sequence by a frameshift may be considered to be a deleted region. Although the deletion of an amino acid sequence typically shortens the full length of the protein, the full length of the protein may not change or may be extended. For example, the deletion of a part or all of the region of the coding region of a gene can delete the region coded by the deleted region in the amino acid sequence of the encoded protein. Also, for example, by introducing a stop codon into the coding region of a gene, the region coded by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. Also, for example, by causing a frameshift in the coding region of a gene, the region coded by the frameshift site can be deleted. The position and length of the region deleted in the deletion of an amino acid sequence can be explained mutatis mutandis with the explanation of the position and length of the region deleted in the deletion of a gene.

The above modification of a gene on a chromosome can be achieved, for example, by preparing a disrupted gene modified so as not to produce a protein that functions normally, transforming a host with recombinant DNA containing the disrupted gene, and causing 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 if it contains a marker gene according to the host's characteristics such as nutritional requirements. Examples of disrupted genes include genes in which a part or all of the coding region of a gene has been deleted, genes in which a missense mutation has been introduced, genes in which a nonsense mutation has been introduced, genes in which a frameshift mutation has been introduced, and genes in which a transposon or a marker gene has been inserted. Even if a protein encoded by a disrupted gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Such gene disruption by gene replacement using homologous recombination has already been established, and includes a method using linear DNA such as "Red-driven integration" (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), a method combining the Red-driven integration method with an excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005/010175), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, and a method using a suicide vector that does not have a replication origin that functions in the host (U.S. Patent No. 6,303,383, JP-A-05-007491).

Furthermore, a modification that reduces the activity of a protein may be performed, for example, by a mutation treatment. Examples of the mutation treatment include irradiation with X-rays, irradiation with ultraviolet light, and N-methyl-N'
Examples of such mutagenic agents include treatment with mutagens such as N-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

When a protein functions as a complex consisting of multiple subunits, all or only some of the subunits may be modified, so long as the activity of the protein is reduced as a result. That is, for example, all or only some of the genes encoding each of the subunits may be destroyed, etc. Furthermore, when a protein has multiple isozymes, the activity of all or only some of the isozymes may be reduced, so long as the activity of the protein is reduced as a result. That is, for example, all or only some of the genes encoding each of the isozymes may be destroyed, etc.

The above methods for reducing protein activity may be used alone or in any combination.

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

A decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene that codes for 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.

The reduced amount of gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Methods for evaluating the amount of mRNA 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 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.

The reduction in the amount of the protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein (e.g., the 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 a non-modified strain.

Depending on the method used for the disruption, gene disruption can be confirmed by determining the base sequence, restriction enzyme map, or full length of part or all of the gene.

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

<2-4> Culturing the host The mutant ACAR can be expressed by culturing the host carrying the mutant ACAR gene.

The medium used is not particularly limited as long as the host can grow and a functional mutant ACAR can be expressed. For example, a normal 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 necessary. The types and concentrations of medium components may be appropriately set depending on various conditions such as the type of host.

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. In addition, plant-derived raw materials can be preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. There are no particular limitations on the form in which plant-derived raw materials are used, and they can be used in any form, such as raw products, squeezed juice, crushed products, and purified products. In addition, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from, for example, 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. Since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in the plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses. Xylose may be supplied by conversion of hexoses such as glucose, 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.

The concentration of the carbon source in the medium is not particularly limited as long as the host can grow and a functional mutant ACAR is expressed. The concentration of the carbon source in the medium may be as high as possible, for example, within a range in which the production of mutant ACAR is not inhibited. 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%. In addition, the carbon source may be additionally supplied to the medium as appropriate. For example, the carbon source may be additionally supplied to the medium in response to a decrease or depletion of the carbon source as the culture progresses. As long as mutant ACAR is ultimately produced, the carbon source may be temporarily depleted, but it may be preferable to carry out the culture so that the carbon source is not depleted or the state in which the carbon source is depleted is not continued.

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 hydrolysates; ammonia; and urea. Ammonia gas and aqueous ammonia, which are used to adjust the pH, may also be used as the 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.

Specific examples of phosphate sources 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.

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.

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

When using auxotrophic mutants that require nutrients such as amino acids for growth, it is preferable to supplement the medium with such required nutrients.

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

The culture 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 be the same or different. The mutant ACAR may be expressed at least 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 liquid with an OD660 of 4 to 100 may be added at 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, to the medium for main culture at the start of the culture.

Cultivation can be performed 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 performed separately into a seed culture and a main culture, the cultivation form 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 performed by batch culture, or the seed culture may be performed by batch culture and the main culture may be performed by fed-batch culture or continuous culture.

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

The culture 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 more, and preferably 1.5 ppm or more. The oxygen concentration may be specifically controlled, for example, to 1 to 50% of the saturated oxygen concentration, and preferably about 5%. The culture can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. 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°C to 37°C. The culture period may be, for example, 10 hours to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the host activity is lost.

By culturing the host in this manner, a culture containing mutant ACAR can be obtained. The mutant ACAR can accumulate, for example, within the host's bacterial cells. "Bacterial cells" may be interpreted as "cells" as appropriate depending on the type of host. Depending on the design of the host and/or mutant ACAR gene used, it may be possible to accumulate mutant ACAR in the periplasm or to secrete and produce mutant ACAR outside the bacterial cells.

The mutant ACAR may be used for a desired purpose such as the production of vanillin while remaining contained in the culture (specifically, the medium or the bacterial cells), or may be purified from the culture (specifically, the medium or the bacterial cells) and used for a desired purpose such as the production of vanillin. Purification can be performed to a desired extent. That is, the mutant ACAR may be a purified mutant ACAR or a fraction containing the mutant ACAR. In other words, the mutant ACAR 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 the form of a combination thereof. Such a fraction is not particularly limited, so long as the mutant ACAR is contained in such a fraction so that it can act on its substrate. Such fractions include cultures of hosts having a mutant ACAR gene (i.e., hosts having mutant ACAR), cells recovered from the cultures, culture supernatants recovered from the cultures, processed products thereof (e.g., processed products of cells such as cell disruption products, cell lysates, cell extracts, and others described below), partially purified products thereof (i.e., crude products), and combinations thereof. Note that "purified mutant ACAR" may also include crude products. These fractions can be used alone or in appropriate combinations.

The mutant ACAR may be used for a desired purpose such as the production of vanillin, particularly in the form contained in the bacterial cells. The bacterial cells may be used for a desired purpose such as the production of vanillin while still contained in the culture (specifically, the medium), or may be recovered from the culture (specifically, the medium) and used for a desired purpose such as the production of vanillin. The bacterial cells may be subjected to an appropriate treatment before being used for a desired purpose such as the production of vanillin. That is, examples of the bacterial cells include a culture of a host having a mutant ACAR gene (i.e., a host having a mutant ACAR), 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 mutant ACAR gene (i.e., a host having a mutant ACAR), 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.

The method for recovering the bacterial cells from the culture solution is not particularly limited, and for example, a known method can be used. Examples of such methods include 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 collected cells can be resuspended in an appropriate medium as appropriate. Media that can be used for washing or suspending include, for example, aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

Examples of treatments for the 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 an organic solvent. These treatments can be used alone or in appropriate combination.

The mutant ACAR may also be diluted or concentrated as appropriate and used for the desired purpose, such as the production of vanillin.

The mutant ACAR may be produced alone or together with other proteins, for example, with a phosphopantetheinylating enzyme such as PPT.
For example, a mutant ACAR gene and a PPT gene can be produced simultaneously by expressing these genes in a host carrying the mutant ACAR gene and a PPT gene.

In addition, by culturing a host having a mutant ACAR gene, the host can be used as a mutant ACAR for a desired purpose, such as the production of vanillin.

<3> Use of mutant ACAR The use of mutant ACAR is not particularly limited. Mutant ACAR can be used, for example, to produce vanillin. Vanillin can be produced, for example, from a carbon source or a vanillin precursor. Examples of vanillin precursors include vanillic acid. That is, vanillin can be produced, in particular, from vanillic acid. The production of vanillin using a mutant ACAR can be carried out in a manner similar to the production of vanillin using ACAR (e.g., production of vanillin using a microorganism having ACAR) described in, for example, WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, or US2019-0161776A, except that a mutant ACAR is used as the ACAR.

The mutant ACAR can be suitably used, for example, for producing vanillin in the presence of isovanillic acid. The mutant ACAR can be suitably used, in particular, for producing vanillin from vanillic acid in the presence of isovanillic acid. In other words, by using the mutant ACAR, vanillin can be efficiently produced while reducing the by-production of isovanillin from isovanillic acid.

Below, we will explain an example of a method for producing vanillin from vanillic acid using mutant ACAR.

The present invention provides a method for producing vanillin, comprising the step of producing vanillin from vanillic acid using a mutant ACAR. This step is also referred to as a "vanillin production step." Specifically, the vanillin production step may be a step of converting vanillic acid into vanillin using a mutant ACAR.

Vanillic acid may be used in its free form, as its salt, or as a mixture thereof. In other words, unless otherwise specified, "vanillic acid" may mean vanillic acid in its free form, its salt, or a mixture thereof. Examples of the salt include 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.

As vanillic acid, a commercially available product may be used, or one obtained by appropriate production may be used. The method for producing vanillic acid is not particularly limited, and for example, a known method may be used. Vanillic acid may be produced, for example, by a chemical synthesis method, an enzymatic method, a bioconversion method, a fermentation method, an extraction method, or a combination thereof. That is, for example, vanillic acid may be produced from a vanillic acid precursor using an enzyme (also called a "vanillic acid-producing enzyme") that catalyzes the conversion reaction from a vanillic acid precursor to vanillic acid. Also, for example, vanillic acid may be produced from a carbon source or a vanillic acid precursor using a microorganism capable of producing vanillic acid. "Microorganisms capable of producing vanillic acid" may mean microorganisms capable of producing vanillic acid. "Microorganisms capable of producing vanillic acid" may specifically mean microorganisms capable of producing vanillic acid from a carbon source and/or a vanillic acid precursor. Microorganisms capable of producing vanillic acid and the production of vanillic acid using the same are described, for example, in WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, or US2019-0161776A. Vanillic acid can be produced, for example, by an enzymatic method using O-methyltransferase (OMT) from protocatechuic acid as a precursor or a bioconversion method using a microorganism having OMT (J. Am. CHm. Soc., 1998, Vol.120). Vanillic acid can be produced, for example, by a bioconversion method using ferulic acid as a precursor and Pseudomonas sp. AV ₁₀ strain (J. App. Microbiol., 2013, Vol.116, p903-910). Vanillic acid may be produced as described above. Vanillic acid may be produced, in particular, by using a microorganism capable of producing vanillic acid. The method for producing vanillin may further include a step of producing vanillic acid. The method for producing vanillin may specifically include a step of producing vanillic acid as described above. The method for producing vanillin may particularly include a step of producing vanillic acid by using a microorganism capable of producing vanillic acid. The produced vanillic acid can be used for the production of vanillin as it is, or after being subjected to treatment such as concentration, dilution, drying, dissolution, fractionation, sterilization, extraction, purification, etc., as appropriate. That is, as vanillic acid, for example, a purified product purified to a desired degree may be used, or a material containing vanillic acid may be used. Vanillic acid may be used, in particular, in the form of a material containing vanillic acid. The vanillic acid-containing material is not particularly limited as long as the mutant ACAR (e.g., a microorganism having a mutant ACAR) can utilize vanillic acid. Specific examples of the vanillic acid-containing material include a culture or reaction solution containing vanillic acid, a supernatant separated from the culture or reaction solution, and processed products such as concentrates (e.g., concentrates), dilutions (e.g., dilutions), and dried products thereof. The vanillic acid-containing material may contain, for example, isovanillic acid in addition to vanillic acid. For example, when vanillic acid is produced using OMT or a microorganism having OMT with protocatechuic acid as a precursor, isovanillic acid may be produced as a by-product depending on the substrate specificity of OMT.

The mutant ACAR may be used in the production of vanillin in any of the forms described above. That is, examples of the use of the mutant ACAR include use in any of the forms described above. The mutant ACAR may be used, for example, in a form contained in a host cell, such as a microbial cell. The mutant ACAR may also be used, for example, in a form not contained in a host cell, such as a purified enzyme. The mutant ACAR may be used, in particular, in the form of a microorganism having the mutant ACAR. That is, examples of the use of the mutant ACAR include use of a microorganism having the mutant ACAR. Examples of the use of a microorganism having the mutant ACAR include culturing a microorganism having the mutant ACAR and using the cells of a microorganism having the mutant ACAR. That is, a microorganism having the mutant ACAR may be used as a mutant ACAR, for example, by culturing the microorganism. Also, a microorganism having the mutant ACAR may be used, for example, as a mutant ACAR.

In one embodiment, the vanillin production process can be carried out by culturing a microorganism having a mutant ACAR. This embodiment is also referred to as the "first embodiment of the vanillin production process". That is, the vanillin production process may be, for example, a process of culturing a microorganism having a mutant ACAR in a medium containing vanillic acid and converting vanillic acid into vanillin. Specifically, the vanillin production process may be a process of culturing a microorganism having a mutant ACAR in a medium containing vanillic acid and producing and accumulating vanillin in the medium.

The medium used is not particularly limited as long as it contains vanillic acid, the microorganism having mutant ACAR can grow, and vanillin is produced. The culture conditions are not particularly limited as long as the microorganism having mutant ACAR can grow and vanillin is produced. For the culture in the first embodiment of the method for producing vanillin, the description of the culture of the host in the production of mutant ACAR described above (e.g., the description of the medium and culture conditions) can be applied mutatis mutandis, except that in this embodiment, the medium contains vanillic acid and vanillin is produced.

Vanillic acid may be contained in the medium throughout the entire culture period, or may be contained in the medium only during a part of the culture period. In other words, "culturing a microorganism having a mutant ACAR in a medium containing vanillic acid" does not require that vanillic acid be contained in the medium throughout the entire culture period. For example, vanillic acid may or may not be contained in the medium from the start of culture. If vanillic acid is not contained in the medium at the start of culture, vanillic acid is added to the medium after the start of culture. The timing of addition can be appropriately set depending on various conditions such as culture time. For example, vanillic acid may be added to the medium after the microorganism having a mutant ACAR has grown sufficiently. In either case, vanillic acid may be added to the medium as appropriate. For example, vanillic acid may be added to the medium in response to a decrease or depletion of vanillic acid due to the production of vanillin. There are no particular limitations on the means for adding vanillic acid to the medium. For example, vanillic acid can be added to the medium by feeding a feed medium containing vanillic acid to the medium. In addition, for example, a microorganism having a mutant ACAR and a microorganism having vanillic acid production ability can be co-cultured to allow the microorganism having vanillic acid production ability to produce vanillic acid in the medium, thereby adding vanillic acid to the medium. The "component" in the case of "adding a certain component to the medium" may also include a component that is produced or regenerated in the medium. These means of addition may be used alone or in appropriate combination. The vanillic acid concentration in the medium is not particularly limited as long as the microorganism having the mutant ACAR can use vanillic acid as a raw material for vanillin. The vanillic acid concentration in the medium may be, for example, 0.1 g/L or more, 1 g/L or more, 2 g/L or more, 5 g/L or more, 10 g/L or more, or 15 g/L or more, or 200 g/L or less, 100 g/L or less, 50 g/L or less, or 20 g/L or less, or a combination thereof, calculated in terms of the weight of the free form. Vanillic acid may or may not be contained in the medium at the concentration exemplified above throughout the entire culture period. Vanillic acid may be contained in the medium at the concentration exemplified above, for example, at the start of culture, or may be added to the medium after the start of culture so that the concentration becomes the concentration exemplified above. When culture is performed separately into a seed culture and a main culture, vanillin only needs to be produced during at least the main culture period. Therefore, vanillic acid only needs to be contained in the medium during at least the main culture period, i.e., the entire period of the main culture period or a part of the main culture period. In other words, vanillic acid may or may not be contained in the medium during the seed culture period. In such cases, descriptions about culture (e.g., "culture period (period of culture)" and "start of culture") can be read as referring to the main culture.

In another embodiment, the vanillin production process can be carried out by utilizing a mutant ACAR (e.g., a microorganism cell having a mutant ACAR). This embodiment is also referred to as the "second embodiment of the method for producing vanillin". That is, the vanillin production process may be, for example, a process of converting vanillic acid in a reaction solution into vanillin by utilizing a mutant ACAR (e.g., a microorganism cell having a mutant ACAR). Specifically, the vanillin production process may be a process of allowing a mutant ACAR (e.g., a microorganism cell having a mutant ACAR) to act on vanillic acid in a reaction solution to produce and accumulate vanillin in the reaction solution. The vanillin production process in the second embodiment of the method for producing vanillin is also referred to as the "conversion reaction". The conversion reaction may be carried out, in particular, by utilizing a microorganism cell having a mutant ACAR.

Host cells, such as bacterial cells of a microorganism having a mutant ACAR, may maintain metabolic activity. "Maintaining metabolic activity" may mean that the host cells have the ability to utilize a carbon source to generate or regenerate substances useful for the production of vanillin. Such substances include ATP, electron donors such as NADH and NADP, and phosphopantetheinylation enzymes such as PPT. The host cells may or may not have the ability to grow.

The conversion reaction can be carried out in a suitable reaction solution. Specifically, the conversion reaction can be carried out by allowing mutant ACAR (e.g., cells of a microorganism having mutant ACAR) and vanillic acid to coexist in a suitable reaction solution. The conversion reaction can be carried out in a batch or column format. In the case of a batch format, for example, the conversion reaction can be carried out by mixing mutant ACAR (e.g., cells of a microorganism having mutant ACAR) and vanillic acid in a reaction solution in a reaction vessel. The conversion reaction can be carried out by standing, or by stirring or shaking. In the case of a column format, for example, the conversion reaction can be carried out by passing a reaction solution containing vanillic acid through a column filled with immobilized enzymes such as immobilized cells. Examples of the reaction solution include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

The reaction solution may contain, in addition to vanillic acid, components other than vanillic acid as necessary. Examples of components other than vanillic acid include ATP, electron donors such as NADH and NADPH, phosphopantetheinylating enzymes such as PPT, metal ions, buffers, surfactants, organic solvents, carbon sources, phosphate sources, and various other medium components. That is, for example, a medium containing vanillic acid may be used as the reaction solution. That is, the description of the medium in the first embodiment of the method for producing vanillin can be applied mutatis mutandis to the reaction solution in the second embodiment of the method for producing vanillin. The type and concentration of the components contained in the reaction solution may be appropriately set depending on various conditions such as the properties and mode of use of the mutant ACAR.

The conditions of the conversion reaction (dissolved oxygen concentration, pH of the reaction solution, reaction temperature, reaction time, concentration of various components, etc.) are not particularly limited as long as vanillin is produced. The conversion reaction can be carried out, for example, under normal conditions used for substance conversion using enzymes (for example, substance conversion using purified enzymes or substance conversion using microbial cells such as resting cells). The conditions of the conversion reaction may be set appropriately depending on various conditions such as the properties and usage mode of the mutant ACAR. The conversion reaction may be carried out, for example, under aerobic conditions. "Aerobic conditions" may mean conditions under which the dissolved oxygen concentration in the reaction solution is 0.33 ppm or more, or 1.5 ppm or more. Specifically, the oxygen concentration may be controlled, for example, to about 1 to 50% or 5% of the saturated oxygen concentration. The pH of the reaction solution may be, for example, usually 6.0 to 10.0, or 6.5 to 9.0. The reaction temperature may be, for example, usually 15 to 50°C, 15 to 45°C, or 20 to 40°C. The reaction time may be, for example, 5 minutes to 200 hours. In the case of the 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, for example, normal conditions used for culturing microorganisms such as bacteria and yeast. In this case, when host cells such as cells of a microorganism having a mutant ACAR are used, the host cells may or may not grow. That is, for the conversion reaction in the second embodiment of the method for producing vanillin, the description of the culture in the first embodiment of the method for producing vanillin can be applied mutatis mutandis, except that in this embodiment, the host cells may or may not grow. In such a case, the culture conditions for obtaining the host cells and the conditions for the conversion reaction may or may not be the same. The concentration of vanillic acid in the reaction solution, calculated as the weight of the free form, may be, for example, 0.1 g/L or more, 1 g/L or more, 2 g/L or more, 5 g/L or more, 10 g/L or more, or 15 g/L or more, or 200 g/L or less, 100 g/L or less, 50 g/L or less, or 20 g/L or less, or a combination thereof. The concentration of host cells in the reaction solution, calculated as the optical density (OD) at 600 nm, may be, for example, 1 or more, 300 or less, or a combination thereof.

During the conversion reaction, mutant ACAR (e.g., cells of a microorganism having mutant ACAR), vanillic acid, and other components may be added to the reaction solution alone or in any combination. For example, vanillic acid may be added to the reaction solution in response to a decrease or depletion of vanillic acid accompanying the production of vanillin. These components may be added once or multiple times, or may be added continuously.

There are no particular limitations on the means for adding various components such as vanillic acid to the reaction solution. All of these components can be added to the reaction solution by directly adding them to the reaction solution. In addition, for example, a microorganism having a mutant ACAR and a microorganism capable of producing vanillic acid can be co-cultured to cause the microorganism capable of producing vanillic acid to produce vanillic acid in the reaction solution, thereby adding vanillic acid to the reaction solution. In addition, for example, all of the components such as ATP and electron donors may be produced or regenerated in the reaction solution, may be produced or regenerated within the host cells, or may be produced or regenerated by intercellular coupling. For example, when metabolic activity is maintained in the host cells, components such as ATP, electron donors, and methyl group donors can be produced or regenerated in the host cells using a carbon source. Examples of methods for generating or regenerating ATP include a method in which ATP is supplied from a carbon source using Corynebacterium bacteria (Hori, H et al., Appl. Microbiol. Biotechnol. 48(6): 693-698 (1997)), a method in which ATP is regenerated using yeast cells and glucose (Yamamoto, S et al., Biosci. Biotechnol. Biochem. 69(4): 784-789 (2005)), a method in which ATP is regenerated using phosphoenolpyruvate and pyruvate kinase (C. Aug'e and Ch. Gautheron, Tetrahedron Lett. 29: 789-790 (1988)), and a method in which ATP is regenerated using polyphosphate and polyphosphate kinase (Murata, K et al., Agric. Biol. Chem. 52(6): 1471-1477 (1988)). When "adding a component to a reaction solution," the "component" may also include those that are generated or regenerated in the reaction solution.

In addition, the reaction conditions may be uniform from the start to the end of the conversion reaction, or may change during the conversion reaction. "The reaction conditions change during the conversion reaction" is not limited to the reaction conditions changing over time, but may also include the reaction conditions changing over space. "The reaction conditions change over space" may mean, for example, that when a conversion reaction is carried out in a column, reaction conditions such as reaction temperature and enzyme density (e.g., bacterial cell density) differ depending on the position on the flow path.

By carrying out the vanillin production process in this manner, a culture solution (specifically, a medium) or reaction solution containing vanillin is obtained.

The production of vanillin can be confirmed by known techniques used for the detection or identification of compounds. Such techniques include, for example, HPLC, UPLC, LC/MS, GC/MS, and NMR. These techniques can be used alone or in appropriate combination. These techniques can also be used to determine the concentrations of various components present in the medium or reaction solution.

The produced vanillin can be appropriately recovered. That is, the method for producing vanillin may further include a step of recovering vanillin. This step is also called a "recovery step". The recovery step may be a step of recovering vanillin from a culture solution (specifically, a medium) or a reaction solution. The recovery of vanillin can be carried out 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, a extraction method, a distillation method, and a crystallization method. Specifically, vanillin 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.

If vanillin precipitates in the medium or reaction solution, it can be recovered, for example, by centrifugation or filtration. The vanillin precipitated in the medium or reaction solution may be isolated together with the vanillin dissolved in the medium or reaction solution after crystallization.

The recovered vanillin may contain other components in addition to vanillin, such as mutant ACAR (e.g., cells of a microorganism having mutant ACAR), medium components, reaction solution components, water, and metabolic by-products of the microorganism. The purity of the recovered vanillin 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.

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

<1> Construction of expression plasmids and expression strains for wild-type ACAR and mutant ACAR <1-1> Construction of expression plasmid pET-28a-GeACAR_entD for wild-type ACAR The expression plasmid pET-28a-Ge_ACAR-entD for wild-type ACAR was constructed as follows. This plasmid allows coexpression of wild-type ACAR derived from Gordonia effusa (Ge_ACAR; SEQ ID NO: 2) and PPT derived from E. coli (EntD; SEQ ID NO: 4).

PCR was performed using the plasmid pVK9::Ptuf*-Ge_ACAR-entD (WO2018/079705) as a template and the synthetic DNAs of SEQ ID NOs: 5 and 6 as primers to obtain a PCR product containing the Ge_ACAR gene (codon-optimized according to the codon usage of E. coli) and the ORF sequence of the entD gene. Next, this PCR product was inserted into a pET-28a(+) vector (Novagen) treated with NdeI and EcoRI using In Fusion HD cloning lit (Clontech). This DNA was used to transform competent cells of E. coli JM109 (Takara Bio), which were then spread on LB agar medium containing 50 μg/mL of kanamycin and cultured overnight. The colonies that appeared were then picked up, single colonies were isolated, and transformants were obtained. Plasmids were extracted from the obtained transformants, and the one into which the desired PCR product had been inserted was named pET-28a-GeACAR_entD.

<1-2> Construction of wild-type ACAR expression strain E. coli pET-28a-GeACAR_entD/BL21(DE3) Plasmid pET-28a-GeACAR_entD was introduced into E. coli BL21(DE3) strain (Novagen) by electric pulse method. The cells were spread on LB agar medium containing 50 μg/mL kanamycin and cultured at 37°C. The grown strain was purified on the same agar medium and named pET-28a-GeACAR_entD/BL21(DE3) strain. The strain was inoculated into a test tube containing 2 mL of LB medium containing 50 μg/mL kanamycin and cultured with shaking at 37°C for about 18 hours. 0.8 mL of the obtained culture solution was mixed with 0.8 mL of 40% glycerol solution and stored at -80°C.

<1-3> Construction of expression plasmid and expression strain for mutant ACAR Using the plasmid pET-28a(+)-GeACAR-entD as a template, the plasmid with the mutation introduced was amplified by PCR using the primers shown in Table 1. PCR was performed using KAPA(TM) HiFi HotStart ReadyMix according to the manual. The PCR conditions were (95°C, 2 min) → [(98°C, 20 sec) → (60°C, 15 sec) → (72°C, 9.5 min) → (95°C, 2 min)] × 18 times → (72°C, 5 min). To digest the template plasmid, 0.8 μL of the restriction enzyme DpnI was added to the PCR product, stirred, and incubated at 37°C for 3 hr. E. coli JM109 competent cells were transformed with the reaction solution and plated on LB agar medium containing 25 μg/mL Kanamycin. The resulting transformant colonies were inoculated into 3 mL of LB medium containing 25 μg/mL Kanamycin and cultured (120 rpm, 37°C, overnight). Plasmids were extracted from 2 mL of the culture using a QIAprep Spin Miniprep Kit. The plasmid sequences were determined by Eurofins Genomics, and the plasmids with the correct mutations were used as expression plasmids for mutant ACAR. These plasmids enable the co-expression of mutant ACAR and PPT. E. coli BL21(DE3) competent cells were transformed with these plasmids to obtain the mutant ACAR expression strains shown in Table 1.

Similarly, other mutations (M271G, M271N, M271R, M271S, M271T, V312C, V312M, V312P, V312Q) were amplified using appropriately designed primers with the plasmid pET-28a(+)-GeACAR-entD as a template.
We constructed expression plasmids for mutant ACARs carrying the codons V312Y, A384C, A384F, A384G, A384H, A384I, A384K, A384L, A384N, A384Q, A384S, A384T, A385D, A385E, A385K, A385R, I574C, or I574P, and obtained strains expressing these mutant ACARs. The codon changes due to these mutations are shown in Table 2.

Similarly, the expression plasmids of mutant ACAR of G386N or G386M constructed above were used as templates to introduce double mutations (G386M/M271Y, G386M/S274N, G386M/I353F, G386M/I353L, G386N/M271Y, G386N/V312A, G386N/V312F, G3 An expression plasmid for mutant ACAR carrying the double mutations G386N/V312I, G386N/I353F, G386N/I353L, G386N/A384S, G386N/A385M, G386N/A385V, G386N/V387I, G386N/I574A, G386N/I574M, or G386N/I574T was constructed, and an expression strain for mutant ACAR carrying the double mutations was obtained. The codon changes due to I353F, A385M, and A385V are shown in Table 2.

<2> Analysis of substrate specificity of wild-type ACAR and mutant ACAR <2-1> Preparation of crude enzyme solution of wild-type ACAR and mutant ACAR A colony of each mutant ACAR expression strain was inoculated into 2 mL of LB medium containing 25 μg/mL Kanamycin and precultured (200 rpm, 37°C, overnight). 160 μL of the preculture solution was inoculated into 8 mL of LB medium containing 25 μg/mL Kanamycin placed in a 50 mL Falcon tube. The Falcon tube was tightly capped and cultured until OD ₆₆₀ = 1.0 (200 rpm, 37°C). IPTG was added to a final concentration of 0.1 mM to induce expression of mutant ACAR, and the culture was continued (200 rpm, 16°C, 18 to 20 hr). The culture medium was dispensed in 2 mL portions into 2 mL Violamo microtubes and centrifuged (6,800 g, 4°C, 3 min) to collect the bacteria. The obtained bacteria were suspended in 300 μL of 1×PBS, and 300 μL of each portion was placed in a homogenizer tube and ultrasonically disrupted (HIGH mode, 10 min, 30 sec, 30 sec interval for each disruption). The homogenate was centrifuged (15,000 g, 4°C, 15 min), and the supernatant was used as a crude enzyme solution of mutant ACAR. Similarly, wild-type ACAR expression strain E. coli pET-28a-GeACAR_entD/BL21(DE3) was cultured to obtain a crude enzyme solution of wild-type ACAR. The protein concentration of the crude enzyme solution was measured by the Bradford method using Quick Start(TM) Bradford Protein Assay (Bio-Rad). The standard solution for protein concentration measurement was the Quick Start BSA Standard Set (Bio-Rad).

<2-2> Enzyme reaction using wild-type ACAR and mutant ACAR 500 μL of the reaction buffer shown in Table 3 was dispensed into a 1.5 mL Eppendorf tube. The reaction buffer was incubated for 10 min in a heat block or water bath at 30°C. A crude enzyme solution of wild-type ACAR or mutant ACAR in an amount equivalent to 50 μg of protein was added to the reaction buffer, and incubation was continued. 60 min after the addition, 50 μL of 10% TFA was added to the mixture and mixed to stop the enzyme reaction. The mixture was centrifuged (15,000 g, 15 min, 4°C), and vanillin and isovanillin in the supernatant were quantified. Vanillin and isovanillin were quantified by UHPLC analysis under the conditions shown in Table 4. The ratio of the amount of isovanillin to the amount of vanillin was calculated from the amounts of vanillin and isovanillin, and this was taken as the isovanillin by-production rate (iVNN by-production rate).

The results are shown in Tables 5 and 6. A mutant ACAR was found that had a reduced rate of iVNN by-production compared to the wild-type ACAR (i.e., improved substrate specificity for vanillic acid).

<Explanation of sequence listing>
SEQ ID NO: 1: Nucleotide sequence of the ACAR gene of Gordonia effusa SEQ ID NO: 2: Amino acid sequence of the ACAR protein of Gordonia effusa SEQ ID NO: 3: Nucleotide sequence of the entD gene of Escherichia coli MG1655 SEQ ID NO: 4: Amino acid sequence of the EntD protein of Escherichia coli MG1655 SEQ ID NOs: 5 to 176: Primers SEQ ID NO: 177: Nucleotide sequence of the ACAR gene of Gordonia effusa codon-optimized according to the codon usage of E. coli

Claims (29)

A mutant aromatic carboxylic acid reductase having a specific mutation in the amino acid sequence of a wild-type aromatic carboxylic acid reductase and having vanillin-producing activity,
The mutant aromatic carboxylic acid reductase, wherein the specific mutation is a mutation in one or more amino acid residues selected from the following:
C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, I574. The mutant aromatic carboxylic acid reductase according to claim 1, wherein the specific mutation includes a mutation at G386. The mutant aromatic carboxylic acid reductase according to claim 1 or 2, wherein the specific mutation comprises one or more mutations selected from the following:
C28A, C135 (A, V), R241 (K, Q), G249 (N, S, T), R252 (K, Q, T), W259 (F, Y), M265 (I, V), V270 (A, I), M271 (A, G, H, I, L, N, Q, R, S, T, V, Y), S274 (A, N), T275S, Q278D, Q310L, V312 (A, C, D, F, G, I, L, M, P, Q, S, T, Y), I353 (L, V), S356 (A, T), I376 (F, L, V), E377D, G378D, Y379 (F, L), S381 (A, T), T382S, A384 (C, F, G, H, I, K, L, N, Q, S, T), A385 (D, E, K, P, R), G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y), V387I, S388 (M, T), I389V, Y467 (F, H, W), I574 (A, C, F, G, L, L, M, N, P, T, V). The mutant aromatic carboxylic acid reductase according to any one of claims 1 to 3, wherein the specific mutation comprises G386N. The mutant aromatic carboxylic acid reductase according to any one of claims 1 to 4, wherein the specific mutation comprises any one of the following mutations:
G386M/M271Y, G386M/S274N, G386M/I353F, G386M/I353L, G386N/M271Y, G386N/V312A, G386N/V312F, G386N/V312I, G386N/I353F, G386N/I353L, G386N/A384S, G386N/A385M, G386N/A385V, G386N/V387I, G386N/I574A, G386N/I574M, G386N/I574T. The mutant aromatic carboxylic acid reductase according to any one of claims 1 to 5, wherein the wild-type aromatic carboxylic acid reductase is an aromatic carboxylic acid reductase from a bacterium belonging to the genus Gordonia. The mutant aromatic carboxylic acid reductase according to any one of claims 1 to 6, wherein the wild-type aromatic carboxylic acid reductase is any one of the following proteins:
(a) a protein comprising the amino acid sequence set forth in SEQ ID NO:2;
(b) a protein comprising the amino acid sequence shown in SEQ ID NO:2, which contains a substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues;
(c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:2. The mutant aromatic carboxylic acid reductase according to any one of claims 1 to 7, wherein the by-production rate of isovanillin is 1.0% or less. A method for producing vanillin, comprising the steps of:
A method for converting vanillic acid to vanillin by utilizing the mutant aromatic carboxylic acid reductase of any one of claims 1 to 8. The method according to claim 9, wherein the conversion comprises culturing a microorganism having the mutant aromatic carboxylic acid reductase in a medium containing vanillic acid, and producing and accumulating vanillin in the medium. The method according to claim 9, wherein the conversion comprises allowing the mutant aromatic carboxylic acid reductase to act on vanillic acid in a reaction solution to produce and accumulate vanillin in the reaction solution. The method according to claim 11, wherein the mutant aromatic carboxylic acid reductase is used in the form of a bacterial cell of a microorganism having the mutant aromatic carboxylic acid reductase. The method according to claim 12, wherein the bacterial cells are used in the form of a culture medium of the microorganism, bacterial cells recovered from the culture medium, a processed product thereof, or a combination thereof. The method of claim 12 or 13, wherein the microorganism is a bacterium or a yeast. The method according to any one of claims 12 to 14, wherein the microorganism is a coryneform bacterium or a bacterium of the Enterobacteriaceae family. The method according to any one of claims 12 to 15, wherein the microorganism is a bacterium of the genus Corynebacterium or Escherichia. The method according to any one of claims 12 to 16, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli. The method according to any one of claims 12 to 17, wherein the microorganism is modified so that the activity of phosphopantetheinyl transferase is increased compared to a non-modified strain. The method according to any one of claims 12 to 18, wherein the microorganism has been modified so that the activity of vanillic acid demethylase and/or alcohol dehydrogenase is reduced compared to a non-modified strain. The method according to any one of claims 9 to 19, further comprising recovering the vanillin. A gene encoding the mutant aromatic carboxylic acid reductase according to any one of claims 1 to 8. A vector carrying the gene according to claim 21. A microorganism having the gene according to claim 21. The microorganism according to claim 23, which is a bacterium or yeast. The microorganism according to claim 23 or 24, which is a coryneform bacterium or a bacterium of the Enterobacteriaceae family. The microorganism according to any one of claims 23 to 25, which is a bacterium of the genus Corynebacterium or Escherichia. The microorganism according to any one of claims 23 to 26, which is Corynebacterium glutamicum or Escherichia coli. The microorganism according to any one of claims 23 to 27, which has been modified so that the activity of phosphopantetheinyl transferase is increased compared to a non-modified strain. The microorganism according to any one of claims 23 to 28, which has been modified so that the activity of vanillic acid demethylase and/or alcohol dehydrogenase is reduced compared to a non-modified strain.