JP2021132612A - Method for producing vanillin - Google Patents

Abstract

To provide a mutated aromatic carboxylic acid reductase (ACAR) useful for production of vanillin; and a method for producing vanillin using the same.SOLUTION: Vanillin is produced by using a mutated ACAR having mutation in an amino acid residue 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, I574 .SELECTED DRAWING: None

Description

The present invention relates to a method for producing vanillin.

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

Attempts have also been made to produce vanillin by bioengineering techniques. For example, a microorganism such as Corynebacterium glutamicum can be used to produce vanillin from a vanillin precursor such as vanillic acid.

Vanillin can produce 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 with 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.

An object of the present invention is to provide a mutant ACAR useful for the production of vanillin and a method for producing vanillin using the mutant ACAR.

The present inventors have found 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 the wild-type aromatic carboxylic acid reductase and having vanillin-producing activity.
Mutant aromatic carboxylic acid reductase: The particular mutation is a mutation at one or more 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, I574.
[2]
The mutant aromatic carboxylic acid reductase, wherein the particular mutation comprises a mutation in G386.
[3]
The mutant aromatic carboxylic acid reductase, wherein the particular mutation comprises one or more mutations selected from:
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, wherein the particular mutation comprises G386N.
[5]
The mutant aromatic carboxylic acid reductase, wherein the particular mutation comprises one of the following:
G386M / M271Y, G386M / S274N, G386M / I353F, G386M / I353L, G386N / M271Y, G386N / V312A, G386N / V312F, G386N / V312I, G386N / I353F, G386N / I353L, G386N / A384S, G386N A385V, G386N / V387I, G386N / I574A, G386N / I574M, G386N / I574T.
[6]
The mutant aromatic carboxylic acid reductase, wherein the wild aromatic carboxylic acid reductase is an aromatic carboxylic acid reductase of a bacterium belonging to the genus Gordonia.
[7]
The mutant aromatic carboxylic acid reductase, wherein the wild-type aromatic carboxylic acid reductase is one of the following proteins:
(A) A protein containing the amino acid sequence shown in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, a protein containing an amino acid sequence containing substitutions, deletions, insertions, and / or additions of 1 to 10 amino acid residues;
(C) A protein containing an amino acid sequence having 90% or more identity with respect to the amino acid sequence shown in SEQ ID NO: 2.
[8]
The mutant aromatic carboxylic acid reductase having an isovanillin by-product rate of 1.0% or less.
[9]
It ’s a method of making vanillin.
A method comprising converting vanillic acid to vanillin utilizing the mutant aromatic carboxylic acid reductase.
[10]
The method comprising culturing a microorganism having the mutant aromatic carboxylic acid reductase in a medium containing vanillic acid to produce and accumulate vanillin in the medium.
[11]
The method, wherein the conversion comprises causing the mutant aromatic carboxylic acid reductase to act on vanillic acid in the reaction solution to produce and accumulate vanillin in the reaction solution.
[12]
The method as described above, wherein the variant aromatic carboxylic acid reductase is utilized in the form of bacterial cells of a microorganism having the variant aromatic carboxylic acid reductase.
[13]
The method as described above, wherein the cells are used in the form of a culture solution of the microorganism, cells recovered from the culture solution, processed products thereof, or a combination thereof.
[14]
The method, wherein the microorganism is a bacterium or 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 Corynebacterium bacterium or an Escherichia bacterium.
[17]
The method as described above, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.
[18]
The method, wherein the microorganism has been modified to increase the activity of phosphopantetinyl transferase as compared to an unmodified strain.
[19]
The method, wherein the microorganism has been modified such that the activity of vanillate demethylase and / or alcohol dehydrogenase is reduced compared to an unmodified strain.
[20]
The method, further comprising recovering vanillin.
[21]
A gene encoding the mutant aromatic carboxylic acid reductase.
[22]
A vector carrying the gene.
[23]
A microorganism having the above gene.
[24]
The microorganism, which is a bacterium or yeast.
[25]
The microorganism, which is a coryneform bacterium or a bacterium of the family Enterobacteriaceae.
[26]
The microorganism, which is a bacterium of the genus Corynebacterium or a bacterium of the genus Escherichia.
[27]
The microorganism, which is Corynebacterium glutamicum or Escherichia coli.
[28]
Said microorganisms that have been modified to increase the activity of phosphopantetinyl transferase as compared to unmodified strains.
[29]
Said microorganisms that have been modified to reduce the activity of vanillate demethylase and / or alcohol dehydrogenase as compared to unmodified strains.

According to the present invention, it is possible to provide a mutant ACAR useful for the production of vanillin, and vanillin can be efficiently produced by using the mutant ACAR.

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

“Aromatic carboxylic acid reductase (ACAR)” may mean a protein that has the activity of catalyzing the reaction of reducing an aromatic carboxylic acid to produce the corresponding aromatic aldehyde (EC 1.2). .99.6 etc.). This activity is also called "ACAR activity". The gene encoding ACAR is also called "ACAR gene".

Specifically, "ACAR activity" may mean an activity that catalyzes a reaction that reduces an aromatic carboxylic acid in the presence of an electron donor and ATP to produce the corresponding aromatic aldehyde. Examples of electron donors include NADH and NADPH. Examples of electron donors include NADPH in particular.

Examples of the combination of the aromatic carboxylic acid and the corresponding aromatic aldehyde include a combination of vanillic acid and vanillin and a combination of isovanillic acid and isovanillin. The ACAR activity in which vanillic acid serves as a substrate, that is, the activity of catalyzing the reaction of reducing vanillic acid to produce vanillin is also referred to as "vanillin-producing activity". Specifically, "vanillin-producing activity" may mean an activity that catalyzes a reaction of reducing vanillic acid to produce vanillin in the presence of an electron donor and ATP. The ACAR activity in which isovanillin is used as a substrate, that is, the activity of catalyzing the reaction of reducing isovanillin to produce isovanillin is also referred to as "isovanillin-producing activity". Specifically, "isovanillin-producing activity" may mean an activity that catalyzes a reaction of reducing isovanillin acid to produce isovanillin in the presence of an electron donor and ATP.

ACAR activity (eg, vanillin-producing activity or isovanillin-producing activity) is, for example, ATP.
And can be measured by incubating the enzyme with a substrate (eg, vanillic acid or isovanillic acid) in the presence of NADPH and measuring the enzyme and substrate-dependent oxidation of NADPH (J. Biol. Chem. 2007, Vol. 282, No.1, modified the method described on p478-485). "Protein has ACAR activity" may mean that the protein has ACAR activity as measured under at least one suitable condition. The same applies to the activities of other proteins referred to in the present application.

An ACAR having a "specific mutation" is also referred to as a "mutant ACAR". The gene encoding the mutant ACAR is also referred to as a "mutant ACAR gene".

ACAR that does not have a "specific mutation" is also called "wild-type ACAR". The gene encoding wild-type ACAR is also referred to as "wild-type ACAR gene". The term "wild type" as used herein is a description for convenience to distinguish the "wild type" ACAR from the "mutant" ACAR, and can be obtained naturally as long as it does not have a "specific mutation". It is not limited to things. "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 not selected as a "specific mutation" as long as it does not have a mutation selected as a "specific mutation".

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

The wild-type ACAR will be described below.

The wild-type ACAR may or may not have ACAR activity such as vanillin-producing activity and isovanillin-producing activity as long as the corresponding mutant ACAR has vanillin-producing activity. Wild-type ACAR may usually have vanillin-producing activity and isovanillin-producing activity. Wild-type ACAR may have at least isovanillin-producing activity.

Examples of wild-type ACAR include ACAR of the genus Gordonia (WO2018 / 079705). Bacteria of the genus Gordonia include Gordonia effusa. The nucleotide sequence of the ACAR gene of 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. Unless otherwise specified, the expression "gene or protein has a base sequence or amino acid sequence" may mean that the gene or protein contains the base sequence or amino acid sequence, and the gene or protein has the base sequence or amino acid sequence. The case consisting of an amino acid sequence may also be included.

The wild-type ACAR gene may be a variant of the above-exemplified wild-type ACAR gene (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 1) as long as the encoding ACAR does not have a “specific mutation”. Similarly, wild-type ACAR may be a variant of the above-exemplified wild-type ACAR (eg, protein having the amino acid sequence set forth in SEQ ID NO: 2) as long as it does not have a "specific mutation". That is, the term "wild-type ACAR gene" may include variants thereof in addition to the above-exemplified wild-type ACAR gene (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 1). Similarly, the term "wild-type ACAR" may include variants thereof in addition to the above-exemplified wild-type ACAR (eg, proteins having the amino acid sequence set forth in SEQ ID NO: 2). The gene specified by the species of origin is not limited to the gene itself found in the species, but includes genes having the base sequence of the gene found in the species and variants thereof. In addition, the protein specified by the species of origin is not limited to the protein itself found in the species, but includes proteins having the amino acid sequence of the protein found in the species and variants thereof. These variants may or may not be found in the species. That is, for example, "ACAR of a bacterium of the genus Gordonia" is not limited to ACAR itself found in a bacterium of the genus Gordonia, but includes proteins having an amino acid sequence of ACAR found in a bacterium of the genus Gordonia and variants thereof. Examples of the variant include homologues and artificial variants of the above-exemplified genes and proteins.

The wild-type ACAR gene homolog or the wild-type ACAR homolog can be obtained from a public database by, for example, a BLAST search or a FASTA search using the nucleotide 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. Can be easily determined from. Further, the homologue of the wild-type ACAR gene can be obtained, for example, by PCR using chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequence of the wild-type ACAR gene exemplified above as primers. ..

The wild ACAR gene is one or several amino acids at one or several positions in the above amino acid sequence (eg, the amino acid sequence shown in SEQ ID NO: 2) unless the encoding ACAR has a "specific mutation". May encode a protein having an amino acid sequence substituted, deleted, inserted, and / or added. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. The above "1 or several" differs depending on the position and type of the amino acid residue in the protein structure, but specifically, for example, 1 to 50, 1 to 40, and 1 to 30. , 1-20, 1-10, 1-5, or 1-3.

Substitutions, deletions, insertions, or additions of one or several amino acids described above are conservative mutations that maintain the original function of the protein. A typical conservative mutation is a conservative substitution. Conservative substitutions are polar amino acids between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In some cases, between Gln and Asn, between Lys, Arg and His if it is a basic amino acid, and between Asp and Glu if it is an acidic amino acid, if it is an amino acid with a hydroxyl group. Is Ser
, Thr are mutations that replace each other. Substitutions that are considered conservative substitutions include, specifically, Ala to Ser or Thr substitutions, Arg to Gln, His or Lys substitutions, Asn to Glu, Gln.
, Lys, His or Asp substitution, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly , Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile , Met, Val or Phe replacement, Lys to Asn, Glu, Gln, His or Arg replacement, Met to Ile, Leu, Val or Phe replacement, Phe to Trp, Tyr, Met, Ile or Leu Substitution of, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu Replacement with. In addition, the above-mentioned amino acid substitutions, deletions, insertions, or additions may be caused by naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the organism from which the gene is derived. included.

In addition, the wild-type ACAR gene is, for example, 50% or more, 65% or more, 80% or more, 90% or more, 95% of the entire amino acid sequence, unless the encoding ACAR has a "specific mutation". As described above, it may be a gene encoding a protein having an amino acid sequence having 97% or more, or 99% or more identity.

In addition, the wild-type ACAR gene is a probe that can be prepared from the above base sequence (for example, the base sequence shown in SEQ ID NO: 1) as long as the encoded ACAR does not have a "specific mutation", for example, the whole or one of the above base sequences. It may be a complementary sequence to the part, and a gene that hybridizes under stringent conditions, such as DNA. The "stringent condition" may mean a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. To give an example, DNAs with high identity, for example, DNAs having 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC, which are conditions under which DNAs that hybridize and have lower identities do not hybridize with each other, or under normal Southern hybridization washing conditions. Conditions can be mentioned for washing once, preferably 2-3 times, at a salt concentration and temperature corresponding to 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS.

As described above, the probe used for 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, as a probe, a DNA fragment having a length of about 300 bp can be used. When a DNA fragment having a length of about 300 bp is used as the probe, the conditions for washing the hybridization include 50 ° C., 2 × SSC, and 0.1% SDS.

In addition, since the codon degeneracy differs depending on the host, the wild-type ACAR gene may be a codon in which an arbitrary codon is 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 reduction of the genetic code. For example, the wild-type ACAR gene may be modified to have the optimal codon depending on the codon usage frequency of the host used. Examples of the codon-optimized wild-type ACAR gene include an ACAR gene having the nucleotide sequence shown in SEQ ID NO: 177, which is codon-optimized according to the codon use of E. coli.

The "identity" between amino acid sequences is calculated by blastp using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Presence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). It means the identity between the amino acid sequences. In addition, "identity" between base sequences means the identity between base sequences calculated by blastn using the default setting Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear). do.

Hereinafter, the mutant ACAR will be described.

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 having a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2. In addition, the mutant ACAR has, for example, a "specific mutation" in the amino acid sequence shown in SEQ ID NO: 2, and one or several amino acid substitutions or deletions are made at locations other than the "specific mutation". It may be a protein having an amino acid sequence including insertion and / or addition and having vanillin-producing activity.

In other words, the mutant ACAR may be a protein having the same amino acid sequence as the wild-type ACAR, except that it has a "specific mutation". That is, the mutant ACAR may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 2, except that it has a “specific mutation”. In addition, the mutant ACAR is an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence shown in SEQ ID NO: 2, except that it has, for example, a “specific mutation”. It may be a protein having a vanillin-producing activity. Moreover, mutant ACAR, for example, except with a "specific mutation", to the amino acid sequence shown in SEQ ID NO: 2, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably It may be a protein having an amino acid sequence having 97% or more, particularly preferably 99% or more identity, 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. In addition, the mutant ACAR may be expressed, for example, in a form containing an additional sequence (that is, as a fusion protein with the additional sequence), and finally, a part or all of the additional sequence may be lost. "Mutant ACAR contains an addendum" or "mutant ACAR is a fusion protein with an addendum" means that the final mutant ACAR contains an addendum, unless otherwise noted. On the other hand, "mutant ACAR is expressed in a form containing an additional sequence" or "mutant ACAR contains an additional sequence at the time of expression" means that the mutant ACAR contains at least an additional sequence at the time of expression, unless otherwise specified. However, it does not necessarily mean that the finally obtained mutant ACAR contains an additional sequence. In other words, the mutant ACAR gene may contain a base sequence encoding an additional sequence in addition to the base sequence of the mutant ACAR gene as exemplified above. The same is true for wild-type ACAR and wild-type ACAR genes. The addition sequence is not particularly limited as long as the mutant ACAR has vanillin-producing activity. The additional sequence can be appropriately selected according to various conditions such as the purpose of use. Examples of the additional sequence include a peptide tag, a signal peptide (also referred to as a signal sequence), and a protease recognition sequence. The additional sequence may be linked, for example, to the N-terminus, C-terminus, or both of the mutant ACAR. 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.

Specifically, as peptide tags, His tag, FLAG tag, GST tag, Myc tag, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (Thioredoxin), GFP (green fluorescent protein), HRP (horseradish) Peroxidase), ALP (Alkaline Phosphatase), Fc region of antibody. Examples of His tags include 6xHis tags. Peptide tags can be used, for example, for the detection and purification of expressed mutant ACAR.

The signal peptide is not particularly limited as long as it functions in a host expressing the mutant ACAR. Examples of the signal peptide include a signal peptide recognized by the Sec secretory pathway and a signal peptide recognized by the Tat secretory pathway. Specific examples of the signal peptide recognized by the Sec system secretory pathway include the signal peptide of the cell surface protein of coryneform bacteria. Specific examples of the signal peptide of the cell surface protein of coryneform bacteria include the PS1 signal sequence of C. glutamicum, the PS2 (CspB) signal sequence (Special Table 6-502548), and the SlpA (CspA) signal sequence of C. stationis. (Japanese Patent Laid-Open No. 10-108675). Tat
Specific signal peptides recognized in the system secret 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 of Arthrobacter globiformis. The signal sequence is mentioned (WO2013 / 118544). The signal peptide can be used, for example, for the secretory production of mutant ACAR. When a mutant ACAR is secreted and produced using a signal peptide, the signal peptide is cleaved at the time of secretion, and the mutant ACAR having no signal peptide can be secreted outside the cells. That is, typically, the resulting mutant ACAR does not have to have a signal peptide.

Specific examples of the protease recognition sequence include a Factor Xa protease recognition sequence and a proTEV protease recognition sequence. The recognition sequence of the protease can be used, for example, to cleave the expressed mutant ACAR. Specifically, for example, when the mutant ACAR is expressed as a fusion protein with a peptide tag, the protease is expressed from the expressed mutant ACAR by introducing a protease recognition sequence into the link between the mutant ACAR and the peptide tag. It can be used to cleave the peptide tag to obtain a mutant ACAR without the peptide tag.

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

Hereinafter, the "specific mutation" will be described.

"Specific mutation" means a mutation useful for the production of vanillin. Specifically, the “specific mutation” may mean a mutation that imparts properties suitable for vanillin production to the wild-type ACAR when introduced into the wild-type ACAR. "Specific mutations" include mutations that improve the substrate specificity of ACAR for vanillic acid. Mutations that improve the substrate specificity of ACAR for vanillic acid include mutations that reduce the isovanillin by-product rate (iVNN by-product rate) of ACAR. That is, mutant ACAR may exhibit a lower iVNN by-product rate than the corresponding wild-type ACAR. The iVNN by-product rate of the mutant ACAR is, for example, 99% or less, 97% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% of the iVNN by-product rate of the corresponding wild-type ACAR. Below, 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, It may be 10% or less, 7% or less, 5% or less, 3% or less, 2% or less, or 1% or less. The iVNN by-product rate of mutant ACAR is, 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
It may be% or less, 0.2% or less, 0.1% or less, or 0. Mutations that result in these ranges of iVNN by-product rates include mutations that result in these ranges of iVNN by-product rates in the Examples. A decrease in the iVNN by-product rate of ACAR may be achieved, for example, by an increase in Km of ACAR for isovanillic acid, a decrease of Km of ACAR for vanillic acid, or a combination thereof. A decrease in the iVNN by-product rate of ACAR may be achieved, for example, by a decrease in the specific activity of ACAR's isovanillin-producing activity, an increase in the specific activity of ACAR's vanillin-producing activity, or a combination thereof.

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

"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.

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

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

In the above notation for identifying an amino acid residue, the number indicates the position in the amino acid sequence shown in SEQ ID NO: 2, and the character on the left side of the number indicates the amino acid residue at each position in the amino acid sequence shown in SEQ ID NO: 2 (that is, each position). Amino acid residues before modification) are shown respectively. 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, each of these amino acid residues represents "an amino acid residue corresponding to the amino acid residue in the amino acid sequence shown in SEQ ID NO: 2". That is, for example, "G386" in any wild-type ACAR indicates an 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 unmodified amino acid residue. Specific examples of the modified amino acid residues include K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), and G ( Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E ( Amino acid residues selected from Glu), N (Asn), and Q (Gln) other than the amino acid residues before modification. As the modified amino acid residue, one effective for vanillin production (for example, one that reduces the iVNN by-product rate of ACAR) may be selected.

Specific examples of "specific mutations" include the following mutations:
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, R) 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. In addition, "specific mutation" includes, for example, one or more mutations selected from these mutations and other mutations C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, etc. One or more amino acids selected from T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, G386, V387, S388, I389, Y467, I574 It may be a combination with a mutation in the residue.

Also, C28, C135, R241, G249, R252, W259, M265, V270, M271, S274, T275, Q278, Q310, V312, I353, S356, I376, E377, G378, Y379, S381, T382, A384, A385, Mutations in G386, V387, S388, I389, Y467, and I574 are, for example, C28A, C135 (A, V), R241 (K, Q), G249 (N, S, T), R252 (K, Q, respectively). , 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) It may be there.

Mutations in G386 include, in particular, G386N.

In the above notation for identifying a mutation, the meanings of the numbers and the letters to the left of them are the same as described above. In the above notation for identifying mutations, the letters to the right of the numbers indicate the modified amino acid residues 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 the A (Ala) residue. Further, for example, "G386 (C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y)" is the sequence number 2. The G (Gly) residue at position 386 in the amino acid sequence shown in is C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Indicates a mutation that is replaced by a Y residue.

In any wild-type ACAR, each of these mutations indicates "a mutation corresponding to that mutation in the amino acid sequence set forth in SEQ ID NO: 2". In any wild 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" means "a mutation in the amino acid sequence shown in SEQ ID NO: 2." It shall be read as "a mutation in which an amino acid residue corresponding to the amino acid residue at position X is replaced with a certain amino acid residue". That is, for example, in any wild-type ACAR, "G386N" means that 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. Indicates a mutation.

The combination of mutations is not particularly limited. Examples of the combination of mutations include combinations including mutations in G386. Specific examples of mutation combinations 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 A385V, G386N / V387I, G386N / I574A, G386N / I574M, G386N / I574T.

Combinations of mutations include, among others:
G386M / M271Y, G386M / S274N, G386M / I353F, G386M / I353L, G386N / M271Y, G386N / V312A, G386N / V312F, G386N / V312I, G386N / I353F, G386N / I353L, G386N / A384S, G386N V387I, G386N / I574A, G386N / I574M, G386N / I574T.

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

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

The position of the amino acid residue referred to in each of the above mutations is a description for convenience to identify the amino acid residue to be modified, and it is not necessary to indicate the absolute position in the wild-type ACAR. That is, the position of the amino acid residue in each of the above mutations indicates a relative position based on the amino acid sequence shown in SEQ ID NO: 2, and is absolute due to deletion, insertion, addition, etc. of the amino acid residue. The position may move back and forth. For example, in the amino acid sequence shown in SEQ ID NO: 2, when one amino acid residue is deleted or inserted at a position closer to the N-terminal side than the X-position, the original amino acid residue at the X-position is each N-terminal. It is the X-1st or X + 1th amino acid residue counted from, but is regarded as "the amino acid residue corresponding to the X-position amino acid residue of the amino acid sequence shown in SEQ ID NO: 2". In addition, the amino acid residue before modification referred to in each of the above mutations is a description for convenience for identifying the amino acid residue to be modified, and does not need to be conserved in wild-type ACAR. That is, when the wild-type ACAR does not have the amino acid sequence shown in SEQ ID NO: 2, the unmodified amino acid residue mentioned in each of the above mutations may not be conserved. That is, in each of the above mutations, when the amino acid residue before modification referred to in each of the above mutations is not conserved, the amino acid residue is another amino acid residue (for example, after modification referred to in each of the above mutations). Mutations that are replaced with amino acid residues) may also be included. For example, "mutation in G386" is a mutation in which an amino acid residue corresponding to G386 is conserved (that is, it is a G (Gly) residue) and the amino acid residue is replaced with another amino acid residue. However, a mutation that replaces the amino acid residue with another amino acid residue when the amino acid residue corresponding to G386 is not conserved (that is, it is not a G (Gly) residue) may be included. 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 corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2" is determined by the amino acid sequence and SEQ ID NO of the arbitrary ACAR. It can be determined by aligning with the amino acid sequence shown in 2. Alignment can be performed using, for example, known gene analysis software. Specific software includes DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et. al., Journal of molecular biol
ogy, 198 (2), 327-37. 1987).

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

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

Hereinafter, the production of mutant ACAR using a host having the mutant ACAR gene will be described in detail.

<2-1> Host A host having a mutant ACAR gene can be obtained by introducing the mutant ACAR gene into an appropriate host. "Introducing a mutant ACAR gene into a host" may also include modifying an ACAR gene such as a wild-type ACAR gene possessed by the host to encode a mutant ACAR. "Having a mutant ACAR gene" is also referred to as "having a mutant ACAR".

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

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

Bacteria belonging to the family Enterobacteriaceae include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, and Photolabdas. Bacteria belonging to the genus (Photorhabdus), Providencia, Salmonella, Morganella and the like can be mentioned. Specifically, according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) Bacteria classified in the Department of Enterobacteriaceae can be used. The bacterium belonging to the genus Escherichia is not particularly limited, and examples thereof include bacteria classified into the genus Escherichia according to a classification known to microbiology experts. Examples of Escherichia bacteria include the books of 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). Bacteria of the genus Escherichia include, for example, Escherichia coli. Escherichia coli includes, for example, Escherichia coli K-12 strain such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia strain such as BL21 (DE3) strain. Kori B strains; and their derivatives. As an Enterobacter bacterium,
For example, Enterobacter agglomerans and Enterobacter aerogenes. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of the bacterium of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of the bacterium belonging to the genus Klebsiella include Klebsiella planticola.

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

Specific examples of the coryneform bacterium include the following species.
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium crenatum
Corynebacterium glutamicum
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum (Corynebacterium glutamicum)
Brevibacterium flavum (Corynebacterium glutamicum) (Brevibacterium)
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 the coryneform bacterium 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

Bacteria belonging to the genus Corynebacterium were previously classified into the genus Brevibacterium, but bacteria currently integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)) are also included. included. In addition, Corynebacterium stationis includes bacteria that were previously classified as Corynebacterium ammoniagenes, but have been reclassified into Corynebacterium stationis by 16S rRNA nucleotide sequence analysis, etc. (Int. J). . Syst. Evol. Microbiol., 60, 874-879 (2010)).

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

Yeasts include Saccharomyces cerevisiae and other Saccharomyces cerevisiae, Candida utilis and other Candida genus, Pichia pastoris and other Pichia genus, and Hansenula polymorpha (Hansenula).
Examples thereof include yeasts belonging to the genus Hansenula such as polymorpha) and the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe.

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

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

The gene can be modified by a known method. For example, a site-specific mutation method can be used to introduce a desired mutation into a target site of DNA. That is, for example, site-specific mutagenesis can modify the coding region of a gene such that the encoded protein comprises substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. .. As a site-specific mutation method, 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 methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 ( 1987)).

The method for introducing the mutant ACAR gene into the host is not particularly limited. The mutant ACAR gene may be retained in the host so that it can be expressed. The mutant ACAR gene can be introduced into the host in the same manner as the gene transfer described later in "Method for increasing protein activity".

In addition, when the host already has an ACAR gene such as a wild-type ACAR gene on the chromosome or the like, the host is modified so as to have the mutant ACAR gene by modifying the ACAR gene so as to encode the mutant ACAR gene. It can also be modified. Modification of the ACAR gene present on the chromosome or the like can be carried out by, for example, natural mutation, mutation treatment, or genetic engineering.

The host may have any property as long as it can produce the mutant ACAR.

The host may be modified, for example, to have properties useful for the production of vanillin. Specifically, the host may be modified to have properties useful for the production of vanillin from, for example, vanillic acid. Such modifications include vanillin productivity as described in WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 079684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, and US2019-0161776A. Modifications to grant or enhance. Such modifications include, specifically, enhanced activity of phosphopantetinylating enzyme, enhanced activity of vanillate uptake system, decreased activity of vanillate demethylase, alcohol dehydrogenase (ADH). )
There is a decrease in the activity of. Such modifications include, in particular, enhanced activity of the phosphopantetinylating enzyme, reduced activity of vanillate demethylase, and reduced activity of alcohol dehydrogenase. These modifications can be used alone or in combination as appropriate. The order of modification for constructing the host is not particularly limited.

The host may be modified to increase the activity of the phosphopantetinylating enzyme. ACAR can become an active enzyme by being phosphopantetinylated (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 phosphopantetinylation of proteins (also referred to as "phosphopantetinylation enzyme"). Examples of the phosphopantetheinyl transferase include phosphopantetheinyl transferase (PPT).

By "phosphopantetheinyl transferase (PPT)" may mean a protein having the activity of catalyzing the reaction of phosphopantethenylizing ACAR in the presence of a phosphopantetheinyl group donor. This activity is also called "PPT activity". The gene encoding PPT is also referred to as the "PPT gene". Examples of the phosphopantetinyl group donor include coenzyme A (CoA). As PPT, EntD encoded by the entD gene
Examples include proteins. Examples of PPTs such as EntD protein include those of various organisms. Specific examples of PPT include E. coli EntD protein such as E. coli K-12 MG1655 strain. The nucleotide sequence of the entD gene of the 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. Specifically, the PPTs are Nocardia brasiliensis PPT, Nocardia farcinica IFM10152 PPT (J. Biol. Chem. 2007, Vol. 282, No.1, pp.478-485), and C. glutamicum PPT (PPT). App. Env.
Microbiol. 2009, Vol.75, No.9, pp.2765-2774) can also be mentioned. Examples of C. glutamicum include the above-exemplified strains such as C. glutamicum ATCC 13032 strain and 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 a function of taking up vanillic acid from the outside of the cell into the intracellular. This activity is also referred to as "vanillic acid uptake activity". A gene encoding a vanillic acid uptake system is also referred to as a "vanillic acid uptake system gene". Examples of the vanillic acid uptake system include the VanK protein encoded by the vanK gene (MT Chaudhry, et al., Microbiology, 2007. 153: 857-865). Examples of the vanillic acid uptake system such as VanK protein include those of various organisms such as coryneform bacteria. As a vanillic acid uptake system, specifically, C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.
VanK protein is mentioned.

The host may be modified to reduce the activity of vanillate demethylase. "Vanillate demethylase" may mean a protein that has the activity of catalyzing the reaction of demethylating vanillic acid to produce protocatechuic acid. This activity is also referred to as "vanillate demethylase activity". The gene encoding vanillate demethylase is also referred to as "vanillate demethylase gene". Vanillate demethylase includes the VanAB protein encoded by the vanAB gene (Current Microbiology, 2005, Vol.51, p59-65). The vanA and vanB genes encode subunit A and subunit B of vanillate demethylase, respectively. When reducing the 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 protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of the vanillate demethylase include the VanAB protein of C. glutamicum such as the C. glutamicum ATCC 13032 strain and the ATCC 13869 strain. The vanAB gene usually constitutes the vanK gene and the vanABK operon. Therefore, the vanABK operons may be collectively destroyed (for example, deleted) in order to reduce the vanillate demethylase activity. In that case, the vanK gene may be introduced into the host again. For example, when vanillic acid existing outside the host cell is used and the vanABK operon is collectively destroyed (for example, deleted), the vanK gene may be introduced again.

The host may be modified to reduce the activity of alcohol dehydrogenase (ADH). "Alcohol dehydrogenase
"ADH)" may mean a protein that has the activity of catalyzing the reaction of reducing aldehydes to produce alcohols in the presence of electron donors (EC 1.1.1.1, EC 1.1.1.2, EC 1.1. 1.71 etc.). This activity is also called "ADH activity". The gene encoding ADH is also referred to as the "ADH gene". Examples of electron donors include NADH and NADPH.

ADHs include those having an activity of catalyzing a reaction of reducing vanillin to produce vanillyl alcohol in the presence of an electron donor. This activity is also particularly referred to as "vanillyl alcohol dehydrogenase activity". ADH having vanillyl alcohol dehydrogenase activity is also particularly referred to as "vanillyl alcohol dehydrogenase".

As ADH, yqhD gene, NCgl0324 gene, NCgl0313 gene, NCgl2709 gene, NCgl0
Examples thereof include YqhD protein, NCgl0324 protein, NCgl0313 protein, NCgl2709 protein, NCgl0219 protein, and NCgl2382 protein encoded by the 219 gene and the NCgl2382 gene, respectively. Both the yqhD gene and the NCgl0324 gene encode vanillyl alcohol dehydrogenase. Such ADH includes Enterobacteriaceae.
Bacteria of various organisms such as coryneform bacteria and coryneform bacteria can be mentioned. The yqhD gene can be found, for example, in bacteria of the Enterobacteriaceae family, such as E. coli. The NCgl0324 gene, NCgl0313 gene, NCgl2709 gene, NCgl0219 gene, and NCgl2382 gene can be found in coryneform bacteria such as C. glutamicum, for example. That is, as ADH, specifically, E. coli
Examples include the YqhD protein of E. coli such as K-12 MG1655 strain. Specific examples of ADH include 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 ADH may be reduced, or the activity of two or more ADHs may be reduced. For example, the activity of one or more of the NCgl0324, NCgl2709, and NCgl0313 proteins may be reduced. In particular, at least the activity of the NCgl0324 protein may be reduced.

The gene and protein used to modify the host may be, for example, a gene and protein having a known base sequence and amino acid sequence, respectively. In addition, the gene and protein used to modify the host may be conservative variants of the gene and protein having a known base sequence and amino acid sequence, respectively. "Conservative variant" may mean a variant that retains its original function. Specifically, for example, a gene used to modify a host can be used at one or several positions in a known amino acid sequence of a protein as long as its original function (ie, enzymatic activity, etc.) is maintained. It may be a gene encoding a protein in which one or several amino acids have a substituted, deleted, inserted, and / or added amino acid sequence. The activity of each protein is measured, for example, by the method described in WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, or US2019-0161776A. can do. For conservative variants of such genes and proteins, the above description of ACAR genes and variants of ACAR can be applied mutatis mutandis.

<2-2> Method for increasing protein activity A method for increasing protein activity (including a method for introducing a gene) will be described below.

"Increased protein activity" may mean that the activity of the protein is increased as compared to the unmodified strain. "Increased protein activity" may specifically mean that the per-cell activity of the protein is increased relative to the unmodified strain. The "activity of a protein per cell" may mean the average value of the activity of the protein per cell. The term "unmodified strain" as used herein may mean a control strain that has not been modified to increase the activity of the target protein. Examples of the unmodified strain include a wild strain and a parent strain. Specific examples of the unmodified strain include a type strain of the species to which the host belongs. Further, as the unmodified strain, specifically, the strain exemplified in the description of the host can be mentioned. That is, in one embodiment, the activity of the protein may be increased relative to the reference strain (ie, the reference strain of the species to which the host belongs). Also, in another embodiment, the activity of the protein may be increased compared to the C. glutamicum ATCC 13869 strain. Also, in another embodiment, the activity of the protein may be increased compared to the C. glutamicum ATCC 13032 strain. Also, in another embodiment, the activity of the protein may be increased compared to the E. coli K-12 MG1655 strain. It should be noted that "increasing protein activity" is also referred to as "enhancing protein activity". "Increased protein activity" means, more specifically, that the number of molecules of the protein per cell is increased as compared to the unmodified strain, and / or per molecule of the protein. It may mean that the function is increasing. That is, the "activity" in the case of "increasing the activity of a protein" means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or the translation amount (protein amount) of the gene encoding the protein. You may. The “number of molecules of a protein per cell” may mean the average value of the number of molecules of the protein per cell. In addition, "increasing the activity of a protein" means not only increasing the activity of the protein in a strain that originally has the activity of the target protein, but also that the strain of the protein that originally does not have the activity of the target protein has the same protein. It also includes imparting activity. Further, as long as the activity of the protein is increased as a result, the activity of the target protein originally possessed by the host may be reduced or eliminated, and then the activity of the suitable target protein may be imparted.

The degree of increase in protein activity is not particularly limited as long as the protein activity is increased as compared with the unmodified strain. The activity of the protein may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain. When the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein. For example, the activity of the protein is measured. It may be produced to the extent possible.

Modifications that increase the activity of the protein can be achieved, for example, by increasing the expression of the gene encoding the protein. "The expression of a gene is increased" may mean that the expression of the gene is increased as compared with an unmodified strain such as a wild strain or a parent strain. Specifically, "the expression of a gene is increased" may mean that the expression level of the gene per cell is increased as compared with the unmodified strain. The “expression level of a gene per cell” may mean the average value of the expression level of the gene per cell. "Increased gene expression" means, more specifically, an increase in the amount of transcription (mRNA) of a gene and / or an increase in the amount of translation (amount of protein) of a gene. It's okay. It should be noted that "increased gene expression" is also referred to as "enhanced gene expression". The expression of the gene may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain. In addition, "increasing gene expression" means not only increasing the expression level of the gene in the strain in which the target gene is originally expressed, but also in the strain in which the target gene is not originally expressed. Expression of the same gene is also included. That is, "the expression of a gene is increased" may mean, for example, introducing the gene into a strain that does not carry the target gene and expressing the gene.

Increased gene expression can be achieved, for example, by increasing the number of copies of the gene.

An increase in the number of copies of a gene can be achieved by introducing the gene into the host chromosome. Gene transfer into chromosomes can be performed, for example, by utilizing homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene transfer methods that utilize homologous recombination include the Red-driven integration method (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645. (2000)), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugation-transmissible plasmid, a method using a suicidal vector having no replication origin that functions in the host, A transmission method using a phage can be mentioned. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, a large number of copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence having a large number of copies on the chromosome. Sequences in which a large number of copies are present on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. In addition, homologous recombination may be performed by targeting an appropriate sequence on the chromosome such as a gene unnecessary for vanillin production. The gene can also be randomly introduced onto the chromosome using a transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US5,882,888, EP805867B1).

To confirm that the target gene was introduced on the chromosome, Southern hybridization using a probe having a sequence complementary to all or part of the gene was used, or a primer prepared based on the sequence of the gene was used. It can be confirmed by PCR or the like.

The increase in the number of copies of a gene can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing a target gene can be linked to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the number of copies of the gene. can. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of the microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication in the cell of the host can be used. The vector may be a multicopy vector. Also, to select transformants, the vector may carry markers such as antibiotic resistance genes. The vector may also include a promoter or 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, a phagemid or the like. Specific examples of vectors capable of autonomous replication in enterobacteriaceae bacteria such as Escherichia coli, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA ( Takara Bio)
, PACYC-based vector, 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)); A plasmid having an improved drug resistance gene; pCRY30 (Japanese Patent Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-Open No. 2-72876, US Pat. No. 5,185,262) PCRY2 and pCRY3 (Japanese Patent Laid-Open No. 1-191686); pAJ655, pAJ611, and pAJ1844 (Japanese Patent Laid-Open No. 58-192900); pCG1 (Japanese Patent Laid-Open No. 57-134500); pCG2 (Japanese Patent Laid-Open No. 58-35197); pCG4 and pCG11 (Japanese Patent Laid-Open No. 57-183799); pPK4 (US Patent No. 6,090,597); pVK4 (Japanese Patent Laid-Open No. 9-322774); pVK7 (Japanese Patent Laid-Open No. 10-215883); pVK9 (WO2007 / 046389); pVS7 (WO2013 / 069634) ; PVC7 (Japanese Patent Laid-Open No. 9-070291) can be mentioned. Further, as a vector capable of autonomous replication in a coryneform bacterium, specifically, a variant of pVC7 such as pVC7H2 can be mentioned (WO2018 / 179834).

When introducing a gene, the gene may be expressed by the host. Specifically, the gene may be retained so as to be expressed under the control of a promoter that functions in the host. "Promoter functioning in a host" may mean a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a promoter unique to the gene to be introduced, or may be a promoter of another gene. As the promoter, for example, a stronger promoter as described herein may be used.

A terminator for transcription termination can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterogeneous terminator. The terminator may be a unique terminator of the gene to be introduced, or may be a terminator of another gene. As terminators, specifically, for example, T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trp.
A Terminator can be mentioned.

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

In addition, when introducing two or more genes, each gene may be retained in the host so as to be expressible. For example, each gene may be all retained on a single expression vector or all on a chromosome. In addition, each gene may be separately retained on a plurality of expression vectors, or may be separately retained on a single or a plurality of expression vectors and on a chromosome. In addition, an operon may be composed of two or more genes and introduced. When "introducing two or more genes", a gene encoding two or more proteins (for example, an enzyme) is introduced, or a single protein complex (for example, an enzyme complex) is introduced. There is a case where a gene encoding each of two or more subunits constituting the above is introduced.

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 host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, genomic DNA of an organism having the gene, a plasmid carrying the gene, or the like as a template. Further, the gene to be introduced may be totally synthesized, for example, based on the base sequence of the gene (Gene, 60 (1), 115-127 (1987)). The acquired gene can be used as it is or after being appropriately modified. That is, the variant can be obtained by modifying the gene. The gene can be modified by a known method. For example, a site-specific mutation method can be used to introduce a desired mutation into a target site of DNA. That is, for example, site-specific mutagenesis can modify the coding region of a gene such that the encoded protein comprises substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. .. As a site-specific mutation method, 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 methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 ( 1987)). Alternatively, the gene variant may be totally synthesized.

When a protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein is increased as a result. That is, for example, when increasing the activity of a protein by increasing the expression of a gene, the expression of all the genes encoding each of these subunits may be enhanced, or only a part of the expression may be enhanced. good. Usually, it is preferable to enhance the expression of all genes encoding those subunits. In addition, each subunit constituting the complex may be derived from one organism or two or more different organisms as long as the complex has the function of the target protein. That is, for example, a gene derived from the same organism that encodes a plurality of subunits may be introduced into the host, or a gene derived from a different organism may be introduced into the host.

In addition, an increase in gene expression can be achieved by improving the transcription efficiency of the gene. In addition, an increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvements in gene transcription efficiency and translation efficiency can be achieved, for example, by modifying the expression regulatory sequence. The "expression regulatory sequence" may be a general term for sites that affect gene expression. Examples of the expression regulatory sequence include a promoter and a Shine-Dalgarno (SD) sequence (ribosome binding site (ribosome binding site).
RBS)), and the spacer region between the RBS and the start codon. The expression regulatory sequence can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression regulatory sequences can be modified by, for example, a method using a temperature-sensitive vector or a Red-driven integration method (WO2005 / 010175).

Improvements in gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. A "stronger promoter" may mean a promoter in which transcription of a gene is improved over a wild-type promoter that originally exists. More potent promoters include, for example, the well-known highly expressed promoters T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and Pm1 derived from Bifidobacterium. Promoters include promoters, PR promoters, and PL promoters. More potent promoters that can be used in coryneform bacteria include, for example, the artificially redesigned P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)) in coryneform bacteria. The pta, aceA, aceB, adh, amyE promoters that can be induced with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, tuf (EF-Tu) promoters (Journal of), which are strong promoters with high expression levels in coryneform bacteria. 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 Examples include promoters and F1 promoters (WO2018 / 179834). Further, as a stronger promoter, a highly active form of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be enhanced by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00/18935). Examples of the highly active promoter include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for assessing promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

Improvements in gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as the ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. A "stronger SD sequence" may mean an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. More potent SD sequences include, for example, the RBS of gene 10 derived from phage T7 (Olins PO et al, Gene,
1988, 73, 227-235). In addition, the substitution, insertion, or deletion of several nucleotides in the spacer region between the RBS and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), contributes to mRNA stability and translation efficiency. It is known to have a great effect, and the translation efficiency of genes can be improved by modifying these.

Improvements in 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 the rare codon present in the gene with a synonymous codon that is used more frequently. That is, the gene to be introduced may be modified to have an optimum codon according to the codon usage frequency of the host used, for example. Codon substitution can be performed, for example, by a site-specific mutation method that introduces the desired mutation into the target site of DNA. In addition, the gene fragment in which the codon has been replaced may be totally synthesized. The frequency of codon use in various organisms is described in the "Codon Use Database"(http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). It is disclosed in.

Elevated 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 method for increasing gene expression may be used alone or in any combination.

Modifications that increase the activity of the 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 metabolites, the activity of the protein can be increased by mutating the gene or protein in the host so that the feedback inhibition is desensitized. Unless otherwise specified, the "desensitization of feedback inhibition" may include a case where the feedback inhibition is completely canceled and a case where the feedback inhibition is reduced. Further, "the feedback inhibition is desensitized" (that is, the feedback inhibition is reduced or canceled) is also referred to as "tolerance to the feedback inhibition". Proteins with enhanced specific activity can be obtained by searching for, for example, various organisms. In addition, a highly active form may be obtained by introducing a mutation into a conventional protein. The mutation introduced may be, for example, one or several amino acids substituted, deleted, inserted, and / or added at one or several positions of the protein. The mutation can be introduced by, for example, the site-specific mutation method as described above. Further, the introduction of the mutation may be carried out by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And other treatments with a mutagen. Alternatively, the DNA may be treated directly with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in combination with the above-mentioned method for enhancing gene expression.

The transformation method 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. Methods of preparing competent cells from proliferating cells and introducing DNA (Duncan, CH, Wilson, GA and Young, FE), as reported in 1970, 53, 159-162) and Bacillus subtilis. , 1977. Gene 1: 153-167) can be used. Alternatively, a cell of a DNA-receptive bacterium, as known for Bacillus subtilis, actinomycetes, and yeast.
Easily Incorporating Recombinant DNA A method of introducing recombinant DNA into a DNA receptor bacterium in the state of protoplast or spheroplast (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, the electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

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

The increase in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has increased. The increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased and that the amount of protein expressed from the gene has increased.

Confirmation that the transcription amount of the gene is increased can be confirmed by comparing the amount of mRNA transcribed from the gene with an unmodified 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, etc. (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 (for example, the number of molecules per cell) is 1.2 times or more, 1.5 times that of the unmodified strain, for example.
It may increase more than twice, more than twice, or more than three times.

Confirmation of elevated protein levels can be confirmed by Western blotting using antibodies (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring). Harbor (USA), 2001
). The amount of protein (eg, number of molecules per cell) may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain.

The above-mentioned method for increasing the activity of a protein can be used to enhance the activity of an arbitrary protein or the expression of an arbitrary gene.

<2-3> Method for reducing protein activity A method for reducing protein activity will be described below.

"Reduced activity of a protein" may mean that the activity of the protein is reduced as compared to an unmodified strain. "Reduced protein activity" may specifically mean that the per-cell activity of the protein is reduced as compared to the unmodified strain. The "activity of a protein per cell" may mean the average value of the activity of the protein per cell. The term "unmodified strain" as used herein may mean a control strain that has not been modified to reduce the activity of the target protein. Examples of the unmodified strain include a wild strain and a parent strain. Specific examples of the unmodified strain include a type strain of the species to which the host belongs. Further, as the unmodified strain, specifically, the strain exemplified in the description of the host can be mentioned. That is, in one embodiment, the activity of the protein may be reduced compared to the reference strain (ie, the reference strain of the species to which the host belongs). Also, in another embodiment, the activity of the protein is C. glutamicum ATCC.
May be reduced compared to 13869 strains. Also, in another embodiment, the activity of the protein is C.I.
May be reduced compared to glutamicum ATCC 13032 strain. Also, in another embodiment, protein activity may be reduced compared to E. coli K-12 MG1655 strain. The "decrease in protein activity" may include the case where the activity of the protein is completely eliminated. More specifically, "reduced protein activity" means that the number of molecules of the protein per cell is reduced as compared with the unmodified strain, and / or per molecule of the protein. It may mean that the function is deteriorated. That is, the "activity" in the case of "reducing the activity of a protein" means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or the translation amount (protein amount) of the gene encoding the protein. You may. The “number of molecules of a protein per cell” may mean the average value of the number of molecules of the protein per cell. The fact that "the number of molecules of a protein per cell is reduced" may include the case where the protein does not exist at all. In addition, "the function per molecule of the protein is reduced" may include the case where the function per molecule of the protein is completely lost. The degree of decrease in protein activity is not particularly limited as long as the protein activity is decreased as compared with the unmodified strain. The activity of the protein may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Modifications that reduce the activity of the protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "The expression of a gene is reduced" may mean that the expression of the gene is reduced as compared with an unmodified strain such as a wild strain or a parent strain. Specifically, "reduced gene expression" may mean that the per-cell expression level of the gene is reduced as compared with the unmodified strain. The “expression level of a gene per cell” may mean the average value of the expression level of the gene per cell. "Reduced gene expression" means, more specifically, a decrease in the transcription amount (mRNA amount) of a gene and / or a decrease in the translation amount (protein amount) of a gene. It's okay. "Reduced expression of a gene" may include the case where the gene is not expressed at all. It should be noted that "decreased gene expression" is also referred to as "weakened gene expression". Gene expression may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Decreased gene expression is the modification of expression regulatory sequences such as the gene promoter, Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)), and the spacer region between the RBS and the start codon. Can be achieved by. When the expression regulatory sequence is modified, the expression regulatory sequence is modified by 1 or more bases, 2 or more bases, or 3 or more bases. A decrease in gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. A "weaker promoter" may mean a promoter in which transcription of a gene is weaker than the native wild-type promoter. Weaker promoters include, for example, inducible promoters. That is, an inducible promoter can function as a weaker promoter under non-inducible conditions (eg, in the absence of an inducer). Weaker promoters include, for example, the P4 promoter (WO2018 / 079684) and the P8 promoter (WO2018 / 099684). In addition, a part or all of the expression regulatory sequence may be deleted. In addition, a decrease in gene expression can also be achieved by, for example, manipulating factors involved in expression control. Examples of factors involved in expression control include small molecules (inducing substances, inhibitors, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) involved in transcription and translation control. 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, gene expression can be reduced by replacing codons in the coding region of a gene with synonymous codons that are used less frequently in the host. Also, for example, gene disruption as described herein can reduce gene expression itself.

In addition, modifications that reduce the activity of the protein can be achieved, for example, by disrupting the gene encoding the protein. "Gene disruption" may mean that the gene is modified so that it does not produce a normally functioning protein. "Do not produce a normally functioning protein" means that no protein is produced from the gene, or that the gene produces a protein whose per-molecule function (eg, activity or property) is reduced or eliminated. May also be included.

Gene disruption can be achieved, for example, by deleting (deficient) a gene on the chromosome. "Gene deletion" may mean deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. As long as reduced protein activity can be achieved, the regions to be deleted are the N-terminal region (ie, the region encoding the N-terminal side of the protein), the internal region, and the C-terminal region (ie, the region encoding the C-terminal side of the protein). It may be any region such as. In general, the longer the region to be deleted, the more reliable the gene can be inactivated. The region to be deleted is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the total length of the coding region of the gene. Alternatively, it may be a region having a length of 95% or more. Further, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match. A reading frame mismatch can result in a frameshift downstream of the region to be deleted.

Gene disruption can be, for example, by introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, by introducing a stop codon (nonsense mutation), or by adding or deleting one or two bases (a nonsense mutation). It can also be achieved by introducing a 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)).

Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the base sequence to be inserted, the more reliable the gene can be inactivated. Further, it is preferable that the reading frames do not match in the arrangement before and after the insertion site. A frameshift can occur downstream of the insertion site due to a mismatch in the leading frames. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include a marker gene such as an antibiotic resistance gene and a gene useful for the production of vanillin.

Gene disruption may be carried out, in particular, so that the amino acid sequence of the encoded protein is deleted. In other words, modifications that reduce the activity of a protein are specifically modified by deleting the amino acid sequence of the protein (a part or all of a region of the amino acid sequence), specifically, one of the amino acid sequences (one of the amino acid sequences). This can be achieved by modifying the gene to encode a protein lacking part or all regions. In addition, "deletion of amino acid sequence of protein" may mean deletion of a part or all region of amino acid sequence of protein. Further, "deletion of the amino acid sequence of a protein" may mean that the original amino acid sequence does not exist in the protein, and may include the case where the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region changed to another amino acid sequence by a frame shift may be regarded as a deleted region. Amino acid sequence deletions typically reduce the overall length of the protein, but the overall length of the protein may not change or may be extended. For example, by deleting a part or all of the coding region of a gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the encoded protein. Further, for example, by introducing a stop codon into the coding region of a gene, the region encoded by the region downstream from the introduction site can be deleted in the amino acid sequence of the encoded protein. Further, for example, a frameshift in a gene coding region can cause a region encoded by the frameshift site to be deleted. Regarding the position and length of the region to be deleted in the deletion of the amino acid sequence, the description of the position and length of the region to be deleted in the deletion of the gene can be applied mutatis mutandis.

Modifying a gene on a chromosome as described above is, for example, producing a disrupted gene modified so as not to produce a normally functioning protein, and transforming the host with a recombinant DNA containing the disrupted gene. This can be achieved by substituting the wild gene on the chromosome with the disruptive gene by causing homologous recombination between the disruptive gene and the wild gene on the chromosome. At that time, if the recombinant DNA contains a marker gene according to a trait such as auxotrophy of the host, it is easy to operate. Destructive genes include genes lacking part or all of the coding region of a gene, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, transposons and marker genes. Examples include the inserted gene. Even if the protein encoded by the 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. Gene disruption by gene substitution using such homologous recombination has already been established, and a method called "Red-driven integration" (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)),
Linear chain such as a method combining the Red driven integration method and a λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175). There are a method using a morphological DNA, a method using a plasmid containing a temperature-sensitive origin of replication, a method using a plasmid capable of conjugation, and a method using a suicide vector having no origin of replication that functions in the host (US Patent No. 6303383).
No., Japanese Patent Application Laid-Open No. 05-007491).

In addition, modifications that reduce the activity of the protein may be performed, for example, by mutation treatment. Mutation treatment includes X-ray irradiation, ultraviolet irradiation, and N-methyl-N'.
Treatment with mutant agents such as -nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methylmethanesulfonate (MMS) can be mentioned.

When a protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein is reduced as a result. That is, for example, all of the genes encoding these subunits may be destroyed, or only a part of them may be destroyed. In addition, when a plurality of isozymes are present in a protein, all the activities of those isozymes may be reduced or only a part of the activities may be reduced as long as the activity of the protein is reduced as a result. That is, for example, all of the genes encoding these isozymes may be destroyed, or only a part of them may be destroyed.

The above-mentioned method for reducing the activity of a protein may be used alone or in any combination.

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

The decrease in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased and that the amount of protein expressed from the gene has decreased.

Confirmation that the transcription amount of the gene has decreased can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the unmodified 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 is, for example,
It may be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Confirmation of reduced protein levels can be made by Western blotting using antibodies (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring). Harbor (USA), 2001
). The amount of protein (eg, 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 the unmodified strain.

It can be confirmed that the gene is disrupted by determining the base sequence of a part or all of the gene, the restriction enzyme map, the total length, etc., depending on the means used for the disruption.

The above-mentioned method for reducing the activity of a protein can be used to reduce the activity of an arbitrary protein or the expression of an arbitrary gene.

<2-4> Culturing of a host By culturing a host having a mutant ACAR gene, the mutant ACAR can be expressed.

The medium used is not particularly limited as long as the host can proliferate and express a functional mutant ACAR. As the medium, for example, a normal medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain a medium component such as a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and various other organic components and inorganic components, if necessary. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of host.

Specific examples of the carbon source include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste sugar honey, starch hydrolyzate, and biomass hydrolyzate, acetic acid, citric acid, and succinic acid. , Organic acids such as fructose, alcohols such as ethanol, glycerol and crude glycerol, and fatty acids. As the carbon source, a plant-derived raw material can be preferably used. Plants include, for example, corn, rice, wheat, soybeans, sugar cane, beets and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing them, and decomposition products of those plant organs. The form of use of the plant-derived raw material is not particularly limited, and for example, any form such as an unprocessed product, a squeezed juice, a crushed product, and a refined product can be used. Further, 5-carbon sugar such as xylose, 6-carbon sugar such as glucose, or a mixture thereof can be obtained from, for example, plant biomass and used. Specifically, these saccharides 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, hemicellulose in plant biomass may be hydrolyzed in advance to liberate 5-carbon sugar, and then cellulose may be hydrolyzed to produce 6-carbon sugar. good. Further, xylose may be supplied, for example, by having the host possess a conversion pathway from 6-carbon sugar such as glucose to xylose and converting from 6-carbon sugar. 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 proliferate and express a functional mutant ACAR. The concentration of the carbon source in the medium may be as high as possible, for example, as long as 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, a carbon source may be additionally supplied to the medium as appropriate. For example, additional carbon sources may be supplied to the medium in response to the decrease or depletion of carbon sources as the culture progresses. The carbon source may be temporarily depleted as long as the mutant ACAR is finally produced, but the culture is carried out so that the carbon source is not depleted or the carbon source is not depleted continuously. May be preferable.

Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract and soybean protein decomposition products, ammonia and urea. Ammonia gas or aqueous ammonia used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphate. As the phosphoric acid source, one kind of phosphoric acid source may be used, or two or more kinds of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source 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.

As various other organic components and inorganic components, specifically, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine. Vitamins such as acids, nicotinic acid amides and vitamin B12; amino acids; nucleic acids; organic components such as peptone, casamino acid, yeast extract and soybean proteolytic products containing these can be mentioned. 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.

In addition, when an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, it is preferable to supplement the medium with such required nutrients.

The culture conditions are not particularly limited as long as the host can proliferate and a functional mutant ACAR is expressed. Culturing can be carried out under normal conditions used for culturing microorganisms such as bacteria and yeast, for example. The culture conditions may be appropriately set according to various conditions such as the type of host. Moreover, if necessary, the expression of the mutant ACAR gene can be induced.

Culturing can be carried out using a liquid medium. At the time of culturing, for example, the host cultured in a solid medium such as an agar medium may be directly inoculated into the liquid medium, or the host is seed-cultured in the liquid medium and inoculated into the liquid medium for main culture. You may. That is, the culture may be divided into a seed culture and a main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same. The mutant ACAR may be expressed at least in the main culture. The amount of host contained in the medium at the start of culturing is not particularly limited. For example, a seed culture solution having an OD660 = 4 to 100 may be added at the start of the culture in an amount of 0.1% by mass to 100% by mass, preferably 1% by mass to 50% by mass, based on the medium for main culture.

Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The medium at the start of culturing is also referred to as "initial medium". Further, a medium supplied to a culture system (for example, a fermenter) in fed-batch culture or continuous culture is also referred to as "fed-batch medium". In addition, supplying a fed-batch medium to a culture system in fed-batch culture or continuous culture is also referred to as "fed-batch". When the culture is divided into a seed culture and a main culture, the culture forms of the seed culture and the main culture may or may not be the same. For example, both seed culture and main culture may be carried out in batch culture, seed culture may be carried out in batch culture, and main culture may be carried out in fed-batch culture or continuous culture.

In the present invention, various components such as a carbon source may be contained in the initial medium, the fed-batch medium, or both. That is, in the process of culturing, various components such as a carbon source may be additionally supplied to the medium alone or in any combination. All of these components may be supplied once or multiple times, or may be supplied continuously. The type of component contained in the initial medium may or may not be the same as the type of component contained in the fed-batch medium. Further, 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 fed-batch medium. In addition, two or more fed-batch media having different types and / or concentrations of the components contained may be used. For example, when multiple feedings are performed intermittently, the type and / or concentration of the components contained in each fed-batch medium may or may not be the same.

The culture can be carried out, for example, under aerobic conditions. The "aerobic condition" may mean that the dissolved oxygen concentration in the medium is 0.33 ppm or more, and preferably 1.5 ppm or more. Specifically, the oxygen concentration may be controlled to, for example, 1 to 50%, preferably about 5% of the saturated oxygen concentration. The culture can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, pH 3-10, preferably pH 4.0-9.5. During culturing, the pH of the medium can be adjusted as needed. The pH of the medium is various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. Can be adjusted using. 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. Culturing may continue, for example, until the carbon source in the medium is consumed or until the host becomes inactive.

By culturing the host in this way, a culture containing the mutant ACAR is obtained. Mutant ACAR can accumulate, for example, in the host cells. The term "cell" may be appropriately read as "cell" depending on the type of host. Depending on the host used and / or the design of the mutant ACAR gene, it may be possible to accumulate the mutant ACAR in the periplasm or secrete and produce the mutant ACAR outside the cells.

The mutant ACAR may be used for desired purposes such as the production of vanillin while still contained in the culture (specifically, the medium or cells), and the culture (specifically, the medium or cells). It may be purified from and used for a desired application such as the production of vanillin. Purification can be carried out to the desired extent. That is, examples of the mutant ACAR include purified mutant ACAR and fractions containing the mutant ACAR. In other words, the mutant ACAR may be utilized in the form of purified enzymes, in the form of such fractions (ie, in the form contained in such fractions), and of those It may be used in the form of a combination. Such a fraction is not particularly limited as long as the mutant ACAR is contained so as to act on the substrate. Such fractions include cultures of hosts carrying the mutant ACAR gene (ie, hosts carrying mutant ACAR), cells recovered from the culture, culture supernatants recovered from the culture, and their treatment. Substances (for example, crushed cells, lysates of cells, extracts of cells, and other processed products of cells, which will be described later), partially purified products thereof (that is, crude purified products), and combinations thereof. Can be mentioned. The "purified mutant ACAR" may include a crude product. These fractions can be used alone or in combination as appropriate.

The mutant ACAR may be used, in particular, for desired applications such as the production of vanillin in the form contained in the cells. The cells may be used for desired purposes such as production of vanillin while being contained in the culture (specifically, the medium), and may be recovered from the culture (specifically, the medium) to produce vanillin. It may be used for a desired purpose such as. In addition, the cells may be appropriately treated and then used for a desired purpose such as production of vanillin. That is, examples of the bacterial cells include cultures of a host having a mutant ACAR gene (that is, a host having a mutant ACAR), bacterial cells recovered from the culture, and processed products thereof. In other words, the cells are utilized in the form of a culture of a host carrying the mutant ACAR gene (ie, a host having the mutant ACAR), cells recovered from the culture, treatments thereof, or a combination thereof. It's okay. Examples of the treated product include those obtained by subjecting the bacterial cells (for example, the bacterial cells contained in the culture or the bacterial cells recovered from the culture) to the treatment. The cells of these embodiments can be used alone or in combination as appropriate.

The method for recovering the cells from the culture solution is not particularly limited, and for example, a known method can be used. Such techniques include, for example, natural sedimentation, centrifugation and filtration. Alternatively, a flocculant may be used. These methods can be used alone or in combination as appropriate. The recovered cells can be appropriately washed using an appropriate medium.
In addition, the recovered cells can be appropriately resuspended using an appropriate medium. Examples of the medium that can be used for washing or suspension include an aqueous medium (aqueous solvent) such as water or an aqueous buffer solution.

Examples of the treatment of the cells include an immobilization treatment on a carrier such as acrylamide and carrageenan, a freeze-thaw treatment, and a treatment for increasing the permeability of the membrane. The permeability of the membrane can be enhanced by utilizing, for example, a surfactant or an organic solvent. These treatments can be used alone or in combination as appropriate.

In addition, the mutant ACAR may be appropriately diluted or concentrated to be used for desired purposes such as production of vanillin.

The mutant ACAR may be produced alone or in combination with other proteins. The mutant ACAR may be produced together with, for example, a phosphopantetinylating enzyme such as PPT.
For example, by expressing those genes in a host having a mutant ACAR gene and a PPT gene, the mutant ACAR and PPT can be produced together.

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

<3> Use of mutant ACAR The use of mutant ACAR is not particularly limited. Mutant ACAR can be used, for example, in the production of vanillin. Vanillin can be produced, for example, from a carbon source or vanillin precursor. Vanillin precursors include vanillic acid. That is, vanillin can be produced, in particular, from vanillic acid. Production of vanillin using mutant ACAR is, for example, WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018, except that mutant ACAR is used as ACAR. It can be carried out in the same manner as the production of vanillin using ACAR (for example, production of vanillin using a microorganism having ACAR) described in / 079705, US2018-0334693A, or US2019-0161776A.

The mutant ACAR can be suitably used for vanillin production in the presence of, for example, isovanillinic acid. Mutant ACAR is particularly suitably available for vanillin production from vanillic acid in the presence of isovalinic acid. That is, by using the mutant ACAR, vanillin can be efficiently produced while reducing the by-product of isovanillin from isovanillinic acid.

Hereinafter, a method for producing vanillin from vanillic acid using a mutant ACAR will be illustrated.

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

Vanillic acid may be used as a free form, as a salt, or as a mixture thereof. That is, "vanillic acid" may mean free vanillic acid, or a salt thereof, or a mixture thereof, unless otherwise specified. Examples of the salt include an ammonium salt, a sodium salt, and a potassium salt. As the salt, one kind of salt may be used, or two kinds or more kinds of salts may be used in combination.

As the vanillic acid, a commercially available product may be used, or one obtained by appropriately producing the vanillic acid may be used. The method for producing vanillic acid is not particularly limited, and for example, a known method can be used. Vanillic acid can be produced, for example, by a chemical synthesis method, an enzymatic method, a biological conversion method, a fermentation method, an extraction method, or a combination thereof. That is, for example, vanillic acid can be produced from a vanillic acid precursor by utilizing an enzyme (also referred to as "vanillic acid-producing enzyme") that catalyzes the conversion reaction of the vanillic acid precursor to vanillic acid. Further, for example, vanillic acid can be produced from a carbon source or a vanillic acid precursor by utilizing a microorganism capable of producing vanillic acid. The “microorganism capable of producing vanillic acid” may mean a microorganism capable of producing vanillic acid. The "microorganism capable of producing vanillic acid" may specifically mean a microorganism capable of producing vanillic acid from a carbon source and / or a vanillic acid precursor. Regarding microorganisms capable of producing vanillic acid and the production of vanillic acid using them, for example, WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018 It is described in -0334693A or US2019-0161776A. Vanillic acid is, for example, using protocatechuic acid as a precursor and O
-Enzymatic method using O-methyltransferase (OMT) or OMT
It can be produced by a biological conversion method using a microorganism having (J. Am. CHm. Soc., 1998, Vol.120). In addition, vanillic acid can be produced, for example, _{by a biological conversion method using Pseudomonas sp. AV 10} strain using ferulic acid as a precursor (J. App. Microbiol.,,
2013, Vol.116, p903-910). Vanillic acid may be produced as described above. Vanillic acid may be produced in particular by utilizing a microorganism capable of producing vanillic acid. The method for producing vanillin may further include a step of producing vanillic acid. Specifically, the method for producing vanillin may include a step of producing vanillic acid as described above. The method for producing vanillin may particularly include a step of producing vanillic acid using a microorganism capable of producing vanillic acid. The produced vanillic acid can be used as it is or after being appropriately subjected to treatments such as concentration, dilution, drying, dissolution, fractionation, sterilization, extraction and purification, and then used for the production of vanillin. That is, as the vanillic acid, for example, a refined 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 material containing vanillic acid is not particularly limited as long as the mutant ACAR (for example, a microorganism having the mutant ACAR) can utilize vanillic acid. Specific examples of the material containing vanillic acid include a culture solution or reaction solution containing vanillic acid, a supernatant separated from the culture solution or reaction solution, a concentrate thereof (for example, a concentrate), and a diluted product (for example, a concentrate). For example, a treated product such as a diluted solution) and a dried product can be mentioned. The material containing vanillic acid may contain isovanillic acid in addition to vanillic acid, for example. For example, when vanillic acid is produced by using OMT or a microorganism having the same as a precursor of protocatechuic acid, isovanillic acid can be produced as a by-product depending on the substrate specificity of OMT.

The mutant ACAR may be utilized in the production of vanillin in any form as described above. That is, as the use of the mutant ACAR, the use in any form as described above can be mentioned. The mutant ACAR may be used, for example, in a form contained in a host cell such as a microbial cell. Further, the mutant ACAR may be used in a form not contained in the host cell such as a purified enzyme. Mutant ACAR may be utilized, in particular, in the form of microorganisms carrying mutant ACAR. That is, as the utilization of the mutant ACAR, in particular, the utilization of a microorganism having the mutant ACAR can be mentioned. Examples of the utilization of the microorganism having the mutant ACAR include the culture of the microorganism having the mutant ACAR and the utilization of the bacterial cells of the microorganism having the mutant ACAR. That is, a microorganism having a mutant ACAR may be utilized as a mutant ACAR by culturing the microorganism, for example. Further, for a microorganism having a mutant ACAR, for example, the cells thereof may be used as the mutant ACAR.

In one aspect, the vanillin production process can be performed by culturing a microorganism having a mutant ACAR. This aspect is also referred to as "the first aspect of the method for producing vanillin". That is, the vanillin production step may be, for example, a step of culturing a microorganism having a mutant ACAR in a medium containing vanillic acid to convert vanillic acid into vanillin. Specifically, the vanillin production step may be a step of culturing a microorganism having a mutant ACAR in a medium containing vanillic acid to generate and accumulate vanillin in the medium.

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

Vanillic acid may be contained in the medium for the entire period of the culture, or may be contained in the medium only for a part of the period of the culture. That is, "culturing a microorganism having a mutant ACAR in a medium containing vanillic acid" does not require that vanillic acid is contained in the medium during the entire culture period. For example, vanillic acid may or may not be contained in the medium from the beginning of the culture. If vanillic acid is not contained in the medium at the start of the culture, vanillic acid is added to the medium after the start of the culture. The timing of addition can be appropriately set according to various conditions such as culture time. For example, vanillic acid may be added to the medium after the microorganism having the mutant ACAR has fully grown. 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 the decrease or depletion of vanillic acid associated with the production of vanillin. The means for adding vanillic acid to the medium is not particularly limited. For example, vanillic acid can be added to the medium by feeding a fed-batch medium containing vanillic acid into the medium. Further, for example, by co-culturing a microorganism having a mutant ACAR and a microorganism having a vanillic acid-producing ability, vanillic acid is generated in the medium by the microorganism having a vanillic acid-producing ability, and thus vanillic acid is added to the medium. You can also do it. The "component" in the case of "adding a certain component to the medium" may also include those produced or regenerated in the medium. These adding means may be used alone or in combination as appropriate. The concentration of vanillic acid 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 is, 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 It may be 15 g / L or more, 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. Vanillic acid may or may not be contained in the medium at the concentrations illustrated above for the entire duration of the culture. Vanillic acid may be contained in the medium at the above-exemplified concentration at the start of culturing, or may be added to the medium at the above-exemplified concentration after the start of culturing. When the culture is divided into a seed culture and a main culture, vanillin may be produced at least during the main culture period. Therefore, vanillic acid may be contained in the medium at least during the main culture, that is, during the entire period of the main culture or a part of the main culture. That is, vanillic acid may or may not be contained in the medium during the seed culture period. In such a case, the description about the culture (for example, "culture period (culture period)" or "culture start") can be read as that for the main culture.

In another embodiment, the vanillin production process can be performed by utilizing a mutant ACAR (eg, a microbial cell of a microorganism having the mutant ACAR). This aspect is also referred to as "a second aspect of the method for producing vanillin". That is, the vanillin production step may be, for example, a step of converting vanillic acid in the reaction solution into vanillin using mutant ACAR (for example, bacterial cells of a microorganism having mutant ACAR). Specifically, the vanillin production step is a step in which a mutant ACAR (for example, a bacterial cell of a microorganism having a mutant ACAR) is allowed to act on vanillic acid in the reaction solution to generate and accumulate vanillin in the reaction solution. You may. The vanillin production step in the second aspect of the vanillin production method is also referred to as a "conversion reaction". The conversion reaction may be carried out, in particular, by utilizing the bacterial cells of the microorganism having the mutant ACAR.

A host cell such as a bacterial cell of a microorganism having a mutant ACAR may maintain its metabolic activity. "Maintaining metabolic activity" may mean that the host cell has the ability to assimilate the carbon source to produce or regenerate substances useful for the production of vanillin. Examples of such substances include electron donors such as ATP, NADH and NADP, and phosphopantetinylating enzymes such as PPT. The host cell 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 coexisting mutant ACAR (for example, cells of a microorganism having mutant ACAR) and vanillic acid in an appropriate reaction solution. The conversion reaction may be carried out in a batch manner or in a columnar manner. In the case of the batch type, for example, the conversion reaction can be carried out by mixing the mutant ACAR (for example, the cells of a microorganism having the mutant ACAR) and vanillic acid in the reaction solution in the reaction vessel. The conversion reaction may be carried out by standing still, or may be carried out by stirring or shaking. In the case of the column type, for example, the conversion reaction can be carried out by passing a reaction solution containing vanillic acid through a column packed with an immobilized enzyme such as an immobilized cell. Examples of the reaction solution include an aqueous medium (aqueous solvent) such as water and an aqueous buffer solution.

The reaction solution may contain components other than vanillic acid, if necessary, in addition to vanillic acid. Ingredients other than vaniphosphate include electron donors such as ATP, NADH and NADPH, phosphopantetinylating enzymes such as PPT, metal ions, buffers, surfactants, organic solvents, carbon sources, phosphoric acid sources, etc. Various medium components can be mentioned. That is, for example, a medium containing vanillic acid may be used as the reaction solution. That is, with respect to the reaction solution in the second aspect of the vanillin production method, the description of the medium in the first aspect of the vanillin production method can be applied mutatis mutandis. The type and concentration of the components contained in the reaction solution may be appropriately set according to various conditions such as the properties of the mutant ACAR and the mode of use.

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 under the usual conditions used for, for example, substance conversion using an enzyme (for example, substance conversion using a purified enzyme or substance conversion using a microbial cell such as a quiescent cell). The conditions of the conversion reaction may be appropriately set according to various conditions such as the properties of the mutant ACAR and the mode of use. The conversion reaction may be carried out under aerobic conditions, for example. The “aerobic condition” may mean a condition in which the dissolved oxygen concentration in the reaction solution is 0.33 ppm or more, or 1.5 ppm or more. Specifically, for example, the oxygen concentration may be controlled to about 1 to 50% or about 5% with respect to 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 15-50 ° C, 15-45 ° C, or 20-40 ° 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, a rate such that the reaction time is within the range of the reaction time exemplified above. Further, the conversion reaction can also be carried out under culture conditions such as normal conditions used for culturing microorganisms such as bacteria and yeast. At that time, when a host cell such as a bacterial cell of a microorganism having a mutant ACAR is used, the host cell may or may not grow. That is, the conversion reaction in the second aspect of the vanillin production method describes the culture in the first aspect of the vanillin production method, except that the host cells may or may not grow in the same embodiment. Can be applied mutatis mutandis. 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 is, 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 in terms of the weight of the free form. , Or 15 g / L or more, 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. good. The concentration of the host cells in the reaction solution may be, for example, 1 or more, 300 or less, or a combination thereof in terms of optical density (OD) at 600 nm.

In the course of the conversion reaction, mutant ACAR (eg, cells of microorganisms carrying 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 according to the 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.

The means for adding various components such as vanillic acid to the reaction solution is not particularly limited. All of these components can be added to the reaction solution by adding them directly to the reaction solution. Further, for example, by co-culturing a microorganism having a mutant ACAR and a microorganism having a vanillic acid-producing ability, vanillic acid is generated in the reaction solution by the microorganism having a vanillic acid-producing ability, thereby producing vanillic acid in the reaction solution. It can also be added to. In addition, for example, components such as ATP and electron donors are
Both may be produced or regenerated in the reaction solution, may be produced or regenerated in the host cell, or may be produced or regenerated by intercellular conjugation. For example, when the metabolic activity is maintained in the host cell, a carbon source can be used to generate or regenerate components such as ATP, an electron donor, and a methyl group donor in the host cell. As a method for producing or regenerating ATP, for example, a method of supplying ATP from a carbon source using a bacterium belonging to the genus Corynebacterium (Hori, H et al., Appl. Microbiol. Biotechnol. 48 (6): 693-698 (1997)), Method of regenerating ATP using yeast cells and glucose (Yamamoto, S et al., Biosci. Biotechnol. Biochem. 69 (4): 784-789 (2005)), Phosphorus Method of Regenerating ATP Using Enol Pyruvate and Pyruvate Kinase (C. Aug'e and Ch. Gautheron, Tetrahedron Lett. 29: 789-790 (1988)), Using Polyphosphate and Polyphosphate Kinase Methods for regenerating ATP (Murata, K et al., Agric. Biol. Chem. 52 (6): 1471-1477 (1988)) can be mentioned. The "component" in the case of "adding a certain component to the reaction solution" may also include those produced or regenerated in the reaction solution.

Further, the reaction conditions may be uniform from the start to the end of the conversion reaction, or may be changed in the process of the conversion reaction. The fact that "the reaction conditions change in the process of the conversion reaction" is not limited to the temporal changes of the reaction conditions, but may also include the spatial changes of the reaction conditions. "The reaction conditions change spatially" means that, for example, when a conversion reaction is carried out by a column type, the reaction conditions such as the reaction temperature and the enzyme density (for example, cell density) depend on the position on the flow path. It may mean that they are different.

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

The production of vanillin can be confirmed by known techniques used to detect or identify compounds. Such techniques include, for example, HPLC, UPLC, LC / MS, GC / MS, NMR. These methods can be used alone or in combination as appropriate. These techniques can also be used to determine the concentration 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 process is also referred to as a "recovery process". The recovery step may be a step of recovering vanillin from the culture medium (specifically, the medium) or the reaction solution. Vanillin can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, an 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 combination as appropriate.

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

In addition to vanillin, the recovered vanillin includes, for example, mutant ACAR (for example, bacterial cells of microorganisms having mutant ACAR), medium components, reaction solution components, water, and other components such as metabolic by-products of microorganisms. May include. The purity of the recovered vanillin is, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w). It may be / w) or more, or 95% (w / w) or more.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

<1> Construction of wild-type ACAR and mutant ACAR expression plasmids and expression strains <1-1> Construction of wild-type ACAR expression plasmid pET-28a-GeACAR_entD Wild-type ACAR expression plasmid pET-28a-Ge_ACAR-entD It was constructed by the following procedure. The plasmid allows co-expression of wild-type ACAR (Ge_ACAR; SEQ ID NO: 2) derived from Gordonia effusa and PPT (EntD; SEQ ID NO: 4) derived from E. coli.

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

<1-2> Construction of wild-type ACAR expression strain E. coli pET-28a-GeACAR_entD / BL21 (DE3) The plasmid pET-28a-GeACAR_entD was subjected to the electric pulse method to the E. coli BL21 (DE3) strain (Novagen). Introduced to the company). The cells were applied on an LB agar medium containing 50 μg / mL of 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 of kanamycin, and shake-cultured at 37 ° C. for about 18 hours. 0.8 mL of the obtained culture solution was mixed with 0.8 mL of a 40% glycerol solution and stored at -80 ° C.

<1-3> Construction of expression plasmid and expression strain of mutant ACAR A plasmid into which the mutation was introduced by the PCR method using the plasmid pET-28a (+)-GeACAR-entD as a template and the primers shown in Table 1. Was amplified. PCR was performed using KAPA (TM) HiFi HotStart ReadyMix according to the manual. The PCR conditions are (95 ° C, 2 min) → [(98 ° C, 20 sec) → (60 ° C, 15 sec) → (72 ° C, 9.5 min) → (95 ° C, 2 min)] × 18 times → ( 72
℃, 5 min). 0.8 μL of restriction enzyme DpnI in the PCR product to digest the template plasmid
Add, stir and incubate 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 obtained transformant colonies were inoculated into 3 mL of LB medium containing 25 μg / mL Kanamycin and cultured (120 rpm, 37 ° C., overnight). 2 mL of the culture solution was taken and the plasmid was extracted using the QIAprep Spin Miniprep Kit. The nucleotide sequence of the plasmid was determined by requesting Eurofin Genomics, Inc., and the plasmid into which the correct mutation was introduced was used as the expression plasmid for mutant ACAR. These plasmids allow co-expression of mutant ACAR and PPT. E. coli BL21 (DE3) competent cells were transformed with these plasmids to obtain mutant ACAR expression strains shown in Table 1.

Similarly, using the plasmid pET-28a (+)-GeACAR-entD as a template and using appropriately designed primers, other mutations (M271G, M271N, M271R, M271S, M271T, V312C, V312M, V312P, V312Q)
, V312Y, A384C, A384F, A384G, A384H, A384I, A384K, A384L, A384N, A384Q, A384S, A384T, A385D, A385E, A385K, A385R, I574C, or I574P) Strains expressing these mutant ACARs were obtained. Table 2 shows the codon changes caused by these mutations.

Similarly, using the G386N or G386M mutant ACAR expression plasmid constructed above as a template, and using the primers shown in Table 1 or appropriately designed primers, double 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, or G386N / I574T) Was constructed to obtain a mutant ACAR expression strain having a double mutation. Table 2 shows the codon changes due to I353F, A385M, and A385V.

<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 25 μg / mL of colonies of each mutant ACAR-expressing strain The cells were inoculated into 2 mL of LB medium containing Kanamycin and precultured (200 rpm, 37 ° C, overnight). 160 μL of preculture was inoculated into 8 mL of LB medium containing 25 μg / mL kanamycin placed in a 50 mL falcon tube. Falcon tubes were closed tightly _{and cultured until OD 660} = 1.0 (200 rpm, 37 ° C). IPTG was added to a final concentration of 0.1 mM to induce the expression of mutant ACAR, and the culture was continued (200 rpm, 16 ° C., 18 to 20 hr). 2 mL of the culture solution was dispensed into 2 mL violamo microtubes and collected by centrifugation (6,800 g, 4 ° C, 3 min). The obtained cells were suspended in 300 μL of 1 × PBS, and 300 μL of the cells was placed in a crushing tube and ultrasonically crushed (HIGH mode, 10 min, 30 sec interval every 30 sec crushing). The crushed material was centrifuged (15,000 g, 4 ° C, 15 min), and the supernatant was used as a crude enzyme solution of mutant ACAR. Similarly, a 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. Quick Start (TM) Bradford for protein concentration of crude enzyme solution
It was measured by the Bradford method using the Protein Assay (Bio-Rad). The Quick Start BSA Standard Set (Bio-Rad) was used as the standard solution for protein concentration measurement.

<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 corresponding to 50 μg of protein was added to the reaction buffer, and the incubation was continued. 60 min after the addition, 50 μL of 10% TFA was added to the mixed solution and mixed to stop the enzymatic reaction. The mixture was centrifuged (15,000 g, 15 min, 4 ° C.) and vanillin and isovanillin in the supernatant were quantified. The quantification of vanillin and isovanillin was performed by UHPLC analysis under the conditions shown in Table 4. From the amounts of vanillin and isovanillin, the ratio of the amount of isovanillin to the amount of vanillin was calculated and used as the isovanillin by-product rate (iVNN by-product rate).

The results are shown in Tables 5 and 6. Mutant ACARs were found with reduced iVNN by-product rates (ie, improved substrate specificity for vanillic acid) compared to wild-type ACARs.

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

Claims (29)

A mutant aromatic carboxylic acid reductase having a specific mutation in the amino acid sequence of the wild-type aromatic carboxylic acid reductase and having vanillin-producing activity.
Mutant aromatic carboxylic acid reductase: The particular mutation is a mutation at one or more 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, I574. The mutant aromatic carboxylic acid reductase according to claim 1, wherein the specific mutation comprises a mutation in 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, R) 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 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 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 aromatic carboxylic acid reductase is an aromatic carboxylic acid reductase of 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 of the following proteins:
(A) A protein containing the amino acid sequence shown in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, a protein containing an amino acid sequence containing substitutions, deletions, insertions, and / or additions of 1 to 10 amino acid residues;
(C) A protein containing an amino acid sequence having 90% or more identity with respect 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 isovanillin by-product rate is 1.0% or less. It ’s a method of making vanillin.
A method comprising converting vanillic acid to vanillin using the mutant aromatic carboxylic acid reductase according to any one of claims 1-8. The method of claim 9, wherein the conversion comprises culturing the microorganism having the mutant aromatic carboxylic acid reductase in a medium containing vanillic acid to produce and accumulate vanillin in the medium. The method according to claim 9, wherein the conversion comprises causing the mutant aromatic carboxylic acid reductase to act on vanillic acid in the reaction solution to generate and accumulate vanillin in the reaction solution. 11. The method of claim 11, wherein the variant aromatic carboxylic acid reductase is utilized in the form of bacterial cells of a microorganism having the variant aromatic carboxylic acid reductase. The method according to claim 12, wherein the cells are used in the form of a culture solution of the microorganism, cells recovered from the culture solution, processed products thereof, or a combination thereof. The method of claim 12 or 13, wherein the microorganism is a bacterium or yeast. The method according to any one of claims 12 to 14, wherein the microorganism is a coryneform bacterium or a bacterium of the family Enterobacteriaceae. The method according to any one of claims 12 to 15, wherein the microorganism is a bacterium belonging to the genus Corynebacterium or a bacterium belonging to the genus 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 phosphopantetinyl transferase is increased as compared with the unmodified strain. The method of any one of claims 12-18, wherein the microorganism has been modified to reduce the activity of vanillate demethylase and / or alcohol dehydrogenase as compared to an unmodified strain. The method of any one of claims 9-19, further comprising recovering 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 family Enterobacteriaceae. The microorganism according to any one of claims 23 to 25, which is a bacterium belonging to the genus Corynebacterium or a bacterium belonging to the genus 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, wherein the activity of the phosphopantetinyl transferase is modified to increase as compared with the unmodified strain. The microorganism according to any one of claims 23 to 28, wherein the activity of vanillate demethylase and / or alcohol dehydrogenase is modified to be reduced as compared with the unmodified strain.