# Patent

## Title
Escherichia coli capable of producing 3-amino-4-hydroxybenzoic acid

## Abstract

The present invention provides a method for conveniently and inexpensively producing an amino-hydroxybenzoic acid-type compound such as 3-amino-4-hydroxybenzoic acid by utilizing Escherichia coli that is a bacterium commonly used for a production process by biosynthesis. Specifically, the present invention provides an Escherichia coli having an ability to produce 3-amino-4-hydroxybenzoic acid, which is modified to reduce an activity of N-hydroxyarylamine O-acetyltransferase (NhoA); a method for producing a 3-amino-4-hydroxybenzoic acid-type compound using such Escherichia coli; and the like.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of, and claims priority under 35 U.S.C. §120 to, International Application No. PCT/JP2013/051778, filed Jan. 28, 2013, and claims priority therethrough under 35 U.S.C. §119 to Japanese Patent Application No. JP 2012-044452 filed on Feb. 29, 2012, the entireties of which are incorporated herein by reference. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: 2014-08-25T_US-520_Seq_List; File size: 45 KB; Date recorded: Aug. 25, 2014.


BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to an Escherichia coli bacterium having an ability to produce 3-amino-4-hydroxybenzoic acid and a method for producing an amino-hydroxybenzoic acid-type compound using the same.
Brief Description of the Related Art
Amino-hydroxybenzoic acid-type compounds are useful as intermediates in the manufacture of dyes, agricultural chemicals, pharmaceuticals, and other synthesized organic products, and as a monomer of a sophisticated and heat resistant polymer, polybenzoxazole. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is biosynthesized in two steps using the enzymes GriI and GriH. GriI catalyzes a carbon-carbon binding reaction between a C4 compound having an amino group and a C3 or C4 compound. GriH catalyzes cyclization of a C7 compound or cyclization of a C8 compound with decarboxylation. Dihydroxyacetone phosphate (DHAP) and aspartate semialdehyde (ASA) are used as substrates in the biosynthesis.




In JP 2004-283163-A, a method for producing 3,4-AHBA using Streptomyces griseus into which griI and griH were introduced is disclosed. The document also discloses that 3-acetylamino-4-hydroxybenzoic acid (3,4-AcAHBA), which is a byproduct of 3,4-AHBA, is formed and that 3,4-AcAHBA is deacetylated to form 3,4-AHBA. Use of a strong base such as sodium hydroxide and a strong acid such as hydrochloric acid is exemplified as a specific method of deacetylating 3,4-AcAHBA. However, this method has a problem that the strong base and the strong acid have to be used.
In International Publication WO2010/005099, it is disclosed that 3,4-AHBA is formed by the use of Corynebacterium glutamicum into which griI and griH were introduced.
In Suzuki et al., J. Biol. Chem., 281 (2006), 36944-36951, it is disclosed that 3,4-AHBA and 3,4-AcAHBA are formed by introducing griI and griH into Escherichia coli. 
In Suzuki et al., J. Bacteriol., 189 (2007), 2155-2159, it is disclosed that when an arylamine N-acetyltransferase gene (natA) is deleted in Streptomyces griseus, 3,4-AcAHBA is not formed in culture.
In Yamamura et al., Biochim. Biophys. Acta., 1475 (2000), 10-16, it is disclosed that an N-hydroxyarylamine O-acetyltransferase gene (nhoA) derived from Escherichia coli works to catalyze the acetylation of an aromatic amino group. Meanwhile, in Rude et al., J. Antibiot., vol. 59 (2006), p. 464, it is disclosed that Escherichia coli BAP1 strain forms an N-acetylated form (3,5-AcAHBA) as a byproduct of 3,5-AHBA (structural isomer of 3,4-AHBA). 3,5-AcAHBA is also produced as the byproduct in an nhoA gene-deleted strain (MAR1 strain) of Escherichia coli BAP1, and thus, the NhoA does not appear to be a major factor for N-acetylation of 3,5-AHBA.
SUMMARY OF THE INVENTION
It is an aspect of the present invention to provide a method for conveniently and inexpensively producing an aminohydroxybenzoic acid-type compound such as 3-amino-4-hydroxybenzoic acid by utilizing Escherichia coli. Escherichia coli is a bacterium commonly used for production processes by biosynthesis methods.
NhoA is involved in formation of 3,4-AcAHBA as a byproduct from 3,4-AHBA in Escherichia coli, and thus 3,4-AHBA, which is not acetylated, can be produced in large amounts by the use of a Escherichia coli bacterium modified to reduce an NhoA activity.
It is an aspect of the present invention to provide an Escherichia coli bacterium having an ability to produce 3-amino-4-hydroxybenzoic acid which has been modified to increase an activity of forming 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde, wherein the Escherichia coli bacterium has been modified to reduce an N-hydroxyarylamine O-acetyltransferase (NhoA) activity.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the NhoA activity is reduced by mutating or deleting an nhoA gene on the chromosome.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the ability to produce 3-amino-4-hydroxybenzoic acid is conferred by being transformed with a recombinant vector incorporating a DNA encoding a protein having an activity of forming 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the protein having the activity of forming 3-amino-4-hydroxybenzoic acid is GriI and GriH.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the GriI is a protein according to any one of the following (A) to (C) and the GriH is a protein according to any one of the following (D) to (F):
(A) a protein comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:14;
(B) a protein comprising an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in (A) above, and having an aldolase activity;
(C) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence represented in (A) above and having an aldolase activity;
(D) a protein comprising an amino acid sequence represented by SEQ ID NO:7 or SEQ ID NO:16;
(E) a protein comprising an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in (D) above, and having a 3-amino-4-hydroxybenzoic acid synthase activity; and
(F) a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in (D) above and having a 3-amino-4-hydroxybenzoic acid synthase activity.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the DNA encoding the protein having the activity of forming 3-amino-4-hydroxybenzoic acid comprises a griI gene and a griH gene.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the griI gene is a DNA according to any one of the following (a) to (c) and the griH gene is a DNA according to any one of the following (d) to (f):
(a) a DNA comprising a nucleotide sequence represented by SEQ ID NO:6 or SEQ ID NO:15;
(b) a DNA that hybridizes under a stringent condition with the nucleotide sequence complementary to the nucleotide sequence shown in (a) above and encodes a protein having an aldolase activity;
(c) a DNA having 70% or more identity to the nucleotide sequence shown in (a) above and having an aldolase activity;
(d) a DNA comprising a nucleotide sequence represented by SEQ ID NO:8 or SEQ ID NO:17;
(e) a DNA that hybridizes under a stringent condition with the nucleotide sequence complementary to the nucleotide sequence shown in (d) above and encodes a protein having a 3-amino-4-hydroxybenzoic acid synthase activity; and
(f) a DNA having 70% or more identity to the nucleotide sequence shown in (d) above and having a 3-amino-4-hydroxybenzoic acid synthase activity.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the griI gene and the griH gene are derived from an actinomycete.
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the griI gene and the griH gene are derived from the genus Streptomyces. 
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the griI gene and the griH gene are derived from Streptomyces griseus. 
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, wherein the griI gene and the griH gene are derived from Streptomyces murayamaensis. 
It is a further aspect of the present invention to provide the Escherichia coli bacterium as described above, having a gene encoding a mutated aspartokinase III in which feedback inhibition is canceled.
It is a further aspect of the present invention to provide a method for producing a 3-amino-4-hydrozybenzoic acid-type compound, comprising a step of culturing the Escherichia coli bacterium as described above.
It is a further aspect of the present invention to provide a method for producing a polymer containing a 3-amino-4-hydrozybenzoic acid-type compound as a component, comprising a step of polymerizing the 3-amino-4-hydrozybenzoic acid-type compound produced by the method as described above.
It is a further aspect of the present invention to provide the method as described above, wherein the polymer is a polybenzoxazole polymer.
According to the present invention, the aminohydroxybenzoic acid-type compound such as 3-amino-4-hydroxybenzoic acid can be produced conveniently and inexpensively.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1(a)-(c) shows the results of analysis by reverse phase column chromatography for (a) a cultured supernatant of Escherichia coli BW25113 strain, (b) a cultured supernatant of Escherichia coli BW25113 ΔnhoA strain, and (c) a standard preparation of 3,4-AHBA.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides an Escherichia coli bacterium that is able to produce 3-amino-4-hydroxybenzoic acid. Formation of an acetylated byproduct (3,4-AcAHBA) can be inhibited in the bacterium.
<1> Modification to Reduce Activity of N-Hydroxyarylamine O-Acetyltransferase (NhoA)
In the bacterium, the formation of the acetylated byproduct can be inhibited by modifying the bacterium to reduce the activity of N-hydroxyarylamine O-acetyltransferase (NhoA). The NhoA activity is an N-hydroxyarylamine O-acetyltransferase activity, and can mean an activity of forming 3-acetylamino-4-hydroxybenzoic acid (3,4-AcAHBA) from 3-amino-4-hydroxybenzoic acid (3,4-AHBA).
The phrase “modified to reduce the NhoA activity” can mean that the NhoA activity becomes lower than the specific activity in an unmodified strain, or a wild type Escherichia coli. The NhoA activity can be reduced to 50% or less, 30% or less, or 10% or less per microbial cell, as compared with that in the unmodified or wild-type strain. The activity can also be completely eliminated. It is only necessary that the NhoA activity is lower than that in the wild-type strain or the unmodified strain, but further accumulation of 3,4-AHBA is desirably enhanced compared with these strains. The phrase “modified to reduce the NhoA activity” can mean when the number of NhoA molecules per cell is decreased and also when the NhoA activity per molecule is reduced, and the like. Specifically, the modification to reduce the NhoA activity can be introduced by conventional mutagenesis or gene engineering treatment. Examples of the mutagenesis may include irradiation with X ray or ultraviolet ray and treatment with a mutagenic agent such as N-methyl-N′-nitro-N-nitrosoguanidine. Examples of such modification may include introducing a mutation into a nhoA gene (including an expression regulatory region) on a chromosome or deleting a part of or all of the nhoA gene so that the NhoA activity can be reduced or disappear compared with non-mutated strains. Examples of methods for mutating or deleting the gene may include modification of the expression regulatory region such as a promoter sequence and Shine-Dalgarno (SD) sequence, introduction of a miss-sense mutation, a nonsense mutation or a frameshift mutation into an open reading frame, as well as partial deletion of the gene (Qiu et al., J. Biol. Chem. 1997, 272 (13): 8611-7). The mutation or the deletion of the nhoA gene can be introduced into a microorganism by using a homologous recombination method in which a wild-type gene on a chromosome is replaced with a gene having a mutation or a deletion or by using a transposon or an IS factor. The homologous recombination method may include methods using a linear DNA, a temperature-sensitive plasmid and a non-replication plasmid. These methods are described in Datsenko et al., Proc. Natl. Acad. Sci. USA., 2000 Jun. 6; 97(12): 6640-5, U.S. Pat. No. 6,303,383, JP 05-007491-A, and the like.
Activity levels and a reduced activity of a target enzyme can be confirmed by measuring enzyme activity using a cell extract or a purified fraction obtained from a candidate microbial strain, and comparing the activity with that of a wild-type strain or an unmodified strain. For example, the NhoA activity can be measured by the method described in Biochim. Biophys. Acta., 1475 (2000), 10-16.
Escherichia coli modified to reduce the NhoA activity may include Escherichia coli inherently able to produce 3,4-AHBA, as well as Escherichia coli that is not inherently able to produce 3,4-AHBA but has been engineered to produce 3,4-AHBA. The ability to produce 3,4-AHBA can be engineered by a method described herein. Appropriate Escherichia coli strains can be used in the present invention, and examples thereof may include K12 strain (ATCC10798) or its substrains (e.g., BW25113 (CGSC7630), DH1 (ATCC33747), MG1655 (ATCC700926), W3110 (ATCC27325)), and B strain or its substrains (e.g., BL21 (ATCCBAA-1025), REL606 (CGSC12149)). Those that include the CGSC number can be obtained from The Coli Genetic Stock Center (cgsc.biology.yale.edu). Those that include with the ATCC number can be obtained from American Type Culture Collection (atcc.org).
NhoA may include a protein that includes an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or 99% or more identity to an amino acid sequence represented by SEQ ID NO:2 and having the NhoA activity. Also the nhoA gene may include a nucleotide sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or 99% or more identity to a nucleotide sequence represented by SEQ ID NO:18 and encoding the protein having the NhoA activity.
Homology, that is, identity or similarity between the amino acid sequences or between the nucleotide sequences can be determined by using algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Programs termed BLASTP and BLASTN were developed based on this algorithm BLAST (see ncbi.nlm.nih.gov). Thus, the homology between the amino acid sequences and between the nucleotide sequences may be calculated using these programs using the default settings. Also, for example, a numerical value obtained by calculating as a percentage using a full length portion of a polypeptide encoded in ORF using software GENETYX Ver. 7.0.9 with a setting of Unit Size to Compare=2, which is available from Genetyx Corporation employing Lipman-Pearson method may be used as the homology between the amino acid sequences. The lowest value in the values derived from these calculations may be employed as the homology between the amino acid sequences and between the nucleotide sequences.
The nucleotide sequence of the nhoA gene is sometimes different depending on the strain of Escherichia coli. Examples of the protein encoded by the nhoA gene may include proteins having an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions at one or multiple positions in the amino acid sequence represented by SEQ ID NO:2 and having the NhoA activity. Here, the term “several” can mean that the amino acid can vary depending on locations or the amino acid residues in a three dimensional structure of a protein, but can mean 1 to 50, 1 to 20, 1 to 10, or 1 to 5. Such a substitution, deletion, insertion addition or the like includes those due to a naturally occurring mutation (mutant or variant) based on individual difference of a microorganism having the nhoA gene.
The nhoA gene may also be DNA that hybridizes with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:18 under stringent conditions and encodes the protein having the NhoA activity. Here, the term “stringent conditions” refers to when a so-called specific hybrid is formed while a non-specific hybrid is not formed. One example is when polynucleotides having high homology (e.g., identity or similarity), for example 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more homology hybridize while polynucleotides having lower homology than that do not hybridize. Specifically, such conditions may include hybridization in 6×SSC (sodium chloride/sodium citrate) at about 45° C. followed by one or two or more washings in 0.2×SSC and 0.1% SDS at 50 to 65° C.
<2> Modification to Increase Activity to Produce 3-amino-4-hydroxybenzoic Acid from Dihydroxyacetone Phosphate and Aspartate Semialdehyde
A bacterium may be modified to increase the activity to produce 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from dihydroxyacetone phosphate (DHAP) and aspartate semialdehyde (ASA). Such a modification can be accomplished by, for example, transforming the bacterium with a recombinant vector incorporating DNA encoding a protein having the activity to form 3,4-AHBA from DHAP and ASA. The protein having the activity to form 3,4-AHBA from DHAP and ASA is not particularly limited as long as the protein contributes to the formation of 3,4-AHBA from DHAP and ASA, and includes, for example, proteins having an enzyme activity to catalyze the formation of a carbon-carbon bond between DHAP and ASA (hereinafter sometimes abbreviated as an “aldolase activity”), and proteins having an enzyme activity to catalyze cyclization of a C7 compound obtained by forming the carbon-carbon bond between DHAP and ASA (hereinafter sometimes abbreviated as a 3-amino-4-hydroxybenzoic acid synthase activity). Hereinafter, both the above activities are sometimes referred to as the ability to biosynthesize 3,4-AHBA.
A gene encoding the protein having the enzyme activity to catalyze the formation of the carbon-carbon bond between DHAP and ASA may include a griI gene or a griI gene homolog (both the griI gene and the griI gene homolog are sometimes simply referred to as the griI gene) derived from Streptomyces griseus. The griI gene homolog refers to a gene that is derived from another microorganism, exhibits high homology to the above gene derived from Streptomyces griseus, and encodes a protein having the aldolase activity. Such a gene can be searched using BLAST. Example thereof may include an nspI gene derived from Streptomyces murayamaensis (SEQ ID NO:14 and 15), fructose-bisphosphate aldolase (Accession no. YP_483282) and fructose-bisphosphate aldolase (Accession no. YP_481172) derived from Frankia sp., fructose-bisphosphate aldolase derived from Streptomyces scabies (sanger.ac.uk/cgi-bin/blast/submitblast/s_scabies), fructose-bisphosphate aldolase (Accession no. Q39NQ9) derived from Burkholderia sp. 383, fructose-bisphosphate aldolase (Accession no. NP_247374) derived from Methanococcus jannaschii, and a dhn gene (Accession no. NC_000913) derived from Escherichia coli (Suzuki et al., Journal of Biological Chemistry vol. 281, NO. 48, pp. 36944-36951, supplementary data).
The gene encoding the protein having the enzyme activity to catalyze the cyclization of the C7 compound obtained by forming the carbon-carbon bond between DHAP and ASA may include a griH gene or a griH gene homolog (both the griH gene and the griH gene homolog are sometimes simply referred to as the griH gene) derived from Streptomyces griseus. The griH gene homolog refers to a gene that is derived from another microorganism, exhibits the high homology to the gene derived from Streptomyces griseus, and encodes the protein having the 3-amino-4-hydrosybenzoic acid synthase activity. Such a gene can be searched using BLAST. Examples thereof may include an nspH gene derived from Streptomyces murayamaensis (SEQ ID NO:16 and 17), 3-dehydroquinate synthase (Accession no. YP_483283) and 3-dehydroquinate synthase (Accession no. YP_481171) derived from Frankia sp., 3-dehydroquinate synthase (Accession no. YP_366552) and 3-dehydroquinate synthase (Accession no. YP_366553) derived from Burkholderia sp. 383, 3-dehydroquinate synthase derived from Streptomyces scabies (sanger.ac.uk/cgi-bin/blast/submitblast/s_scabies), and 3-dehydroquinate synthase (Accession no. NP_248244) derived from Methanococcus jannaschii (Suzuki et al, Journal of Biological Chemistry vol. 281, NO. 48, pp. 36944-36951, supplementary data).
GriI and GriH or the griI gene and the griH gene derived from any organism can be used in the present invention. For example, they may be derived from microorganisms such as the bacteria or actinomycetes described above. Examples of actinomycetes may include microorganisms belonging to genus Streptomyces. Examples of the microorganisms belonging to genus Streptomyces may include Streptomyces griseus, Streptomyces murayamaensis, Streptomyces lividans, and Streptomyces scabies. GriI and GriH, or the griI gene and the griH gene, may be derived from the same microorganism or different microorganisms.
The GriI homolog can include a protein having an amino acid sequence with 70% or more, 80% or more, 90% or more, 95% or more, or 98% or 99% or more identity to SEQ ID NO:5 or 14, a protein encoded by the above griI gene, and/or a protein having the aldolase activity. Examples thereof may include SEQ ID NOS:9, 11, 13, 15, 17, 19, and 21 in WO2010/005099. Also, the griI gene homolog can include a nucleotide sequence with 70% or more, 80% or more, 90% or more, 95% or more, or 98% or 99% or more identity to SEQ ID NO:6 or 15, and/or a griI gene and encoding the protein having aldolase activity. Examples thereof may include SEQ ID NOS:8, 10, 12, 14, 16, 18 and 20 in WO2010/005099.
The GriH homolog can include a protein having an amino acid sequence with 70% or more, 80% or more, 90% or more, 95% or more, or 98% or 99% or more identity to SEQ ID NO:7 or 16, a protein encoded by the above griH gene, and/or a protein having the 3-amino-4-hydroxybenzoic acid synthase activity. Examples thereof may include SEQ ID NOS:23, 25, 27, 29, 31, 33, and 35 in WO2010/005099. Also, the griH gene homolog can include a nucleotide sequence with 70% or more, 80% or more, 90% or more, 95% or more, or 98% or 99% or more identity to SEQ ID NO:8 or 17, and/or a griH gene encoding the protein having the 3-amino-4-hydroxybenzoiic acid synthase activity. Examples thereof may include SEQ ID NOS:22, 24, 26, 28, 30, 32, and 34 in WO2010/005099.
The person of ordinary skill in the art would readily be able to recognize position(s) in an amino acid sequence at which a mutation would have no effect on the activity of the protein, but a sequence alignment can be referenced regarding protein mutants. Specifically, those skilled in the art can (1) compare amino acid sequences of a plurality of homolog proteins, (2) determine relatively conserved regions and relatively not conserved regions, then (3) predict a region or regions that may or may not be functionally important from the relatively conserved regions and the relatively not conserved regions, respectively, and thus recognize correlativity of structures and functions. WO2010/005099 discloses the alignment of the amino acid sequence of the above griI gene homolog (FIGS. 1 and 2 in WO2010/005099), the alignment of the amino acid sequence of the above griH gene homolog (FIGS. 3 and 4 in WO2010/005099), and their consensus (common) sequence (SEQ ID NOS:36 and 37 in WO2010/005099). The above griI gene homolog includes a gene encoding an amino acid sequence represented by SEQ ID NO:36 in WO2010/005099, and the above griH gene homolog includes a gene encoding an amino acid sequence represented by SEQ ID NO:37 in WO2010/005099.
The homology (e.g., identity or similarity) between the amino acid sequences and between the nucleotide sequences can be determined as described above.
The nucleotide sequence of the griI gene or the griH gene may be different depending on the species and/or microbial strain of the chosen microorganism. Thus, the griI gene and the griH gene are only necessary to be able to enhance the ability to produce 3,4-AHBA in Escherichia coli by expressing them in Escherichia coli, e.g., augmenting their expression. For example, a protein that includes one or several amino acid substitutions, deletions, insertions additions or the like at one or multiple positions in the amino acid sequence of the protein encoded by the griI gene (SEQ ID NO:5 or 14) and having the aldolase activity is desirable as the protein encoded by the griI gene. Examples thereof may include SEQ ID NOS:9, 11, 13, 15, 17, 19, and 21 in WO2010/005099. A protein that includes one or several amino acid substitutions, deletions, insertions additions or the like at one or multiple positions in the amino acid sequence of the protein encoded by the griH gene (SEQ ID NO:7 or 16) and having the 3-amino-4-hydroxybenzoic acid synthase activity is desirable as the protein encoded by the griH gene. Examples thereof may include SEQ ID NOS:23, 25, 27, 29. 31, 33, and 35 in WO2010/005099. Here, the term “several” can mean that the amino acid can vary depending on the particular amino acid or the location of the amino acid in the three dimensional structure of a protein, but can be 1 to 50, 1 to 20, 1 to 10 or 1 to 5. Such an amino acid substitution, deletion, insertion addition or the like includes those due to naturally occurring mutation (mutant or variant) based on individual difference or species difference of the microorganism having the griI gene or the griH gene. The substitution can be a conservative substitution that is a neutral substitution in which a function is not affected. The conservative substitution is as described above.
Furthermore, degeneracy of the griI gene and the griH gene varies depending on the chosen host. Thus, codons may be replaced with codons available in Escherichia coli. Likewise, the griI gene and the griH gene may be genes encoding proteins that are extended or truncated on an N terminal side and/or a C terminal side as long as the gene has a function to enhance the ability to produce 3,4-AHBA in Escherichia coli. For example, a length of extended or truncated residues can be 50 or less, 20 or less, 10 or less or 5 or less of amino acid residues. More specifically, the gene may be a gene encoding a protein in which 50 to 5 amino acid residues on the N terminal side or 50 to 5 amino acid residues on the C terminal side have been extended or truncated.
Such a gene that is homologous to the griI gene or the griH gene can be acquired by, for example, modifying the gene encoding an amino acid sequence by site-specific mutagenesis so that an amino acid residue at a particular position of the encoded protein can include the substitution, deletion, insertion or addition. Such a homologous gene can also be acquired by conventionally known mutation treatments as follows. A method of treating the griI gene or the griH gene with hydroxylamine and the like in vitro and a method of treating a microorganism carrying the gene with ultraviolet ray or a mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) generally used for the mutation treatment, error prone PCR (Cadwell, R. C. PCR Meth. Appl. 2, 28 (1992)), DNA shuffling (Stemmer, W. P. Nature 370, 389 (1994)), and StEP-PCR (Zhao, H. Nature Biotechnol. 16, 258 (1998)) are available as mutation treatments. Utilizing these treatments, a mutation can be artificially introduced into the griI gene or the griH gene by gene recombination to acquire a gene encoding an enzyme with high activity.
The griI gene can also be DNA that hybridizes with a nucleotide sequence complementary to a nucleotide sequence of the griI gene or its homolog gene (e.g., SEQ ID NO:6 or 15, or SEQ ID NO:8, 10, 12, 14, 16, 18 or 20 in WO2010/005099) under stringent conditions and encodes the protein having the aldolase activity. The griH gene can also be DNA that hybridizes with a nucleotide sequence complementary to a nucleotide sequence of the griH gene or its homolog gene (e.g., SEQ ID NO:8 or 17, or SEQ ID NO:22, 24, 26, 28, 30, 32 or 34 in WO2010/005099) under stringent conditions and encodes the protein having the 3-amino-4-hydroxybenzoic acid synthase activity. The “stringent conditions” is the same as described above.
The descriptions concerning the above gene homologs and the conservative substitution are applied to the other genes described herein in the same manner.
Whether the griI gene and griH gene and their homologs do or do not encode the protein that enhances the ability to produce 3,4-AHBA can be confirmed by introducing these genes into a bacterium and the like having a gene encoding aspartokinase that is mutated so that feedback inhibition is canceled and examining whether the activity of forming 3,4-AHBA is enhanced or not. In such a case, the effect can be verified more clearly by quantifying 3,4-AHBA using reverse phase chromatography according to, for example, Suzuki, et al.'s method [J. Bio. Chem., 281, 823-833 (2006)].
<3> Recombinant Vector
A recombinant vector that can be used in the present invention can be obtained by introducing a desired gene into an expression vector. For example, when both the griI and the griH are used, they may be on separate recombinant vectors for transformation, or may be linked via an appropriate spacer on the same recombinant vector, as long as they are present in a transformant in an expressible state. The griI gene and the griH gene may be derived from the same microorganism or different microorganisms. When the griI gene and the griH gene are derived from the same microorganism and located in close proximity on a chromosome, a DNA fragment including both griI and griH may be cut out and carried on a vector.
The recombinant vector used in the present invention generally has a promoter, the aforementioned DNA of the present invention (e.g., griI and griH) and regulatory regions (operator and terminator) necessary for expression of the genes in Escherichia coli at appropriate positions so that they are functional.
The expression vector that can be used as the recombinant vector is not particularly limited, but must be able to function in Escherichia coli, and may be self-replicating, such as a plasmid, or may be integrated into the chromosome of a bacterium. Specifically, examples of the expression vector may include pSTV (e.g., pSTV28), pUC (e.g., pUC18, pUC19), pBR (e.g., pBR322), pHSG (e.g., pHSG298, pHSG299, pHSG399, pHSG398), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW118, pMW119, pMW218, pMW219), pQE (e.g., pQE) and derivatives thereof.
The promoter that can be used in the present invention is not particularly limited, and a promoter generally used for production of a foreign protein in Escherichia coli can be used. Examples thereof may include potent promoters such as a T7 promoter, a lac promoter, a trp promoter, a trc promoter, a tac promoter, a PR promoter and PL promoter of a lambda phage, a T5 promoter, and the like.
<4> Transformant
The bacterium of the present invention is not particularly limited as long as it is an Escherichia coli that is modified to have the ability to produce 3-amino-4-hydroxybenzoic acid and to reduce the activity of N-hydroxyarylamine O-acetyltransferase (NhoA), and can be a transformant. The transformant of the present invention can be obtained by using with a recombinant vector into which DNA encoding a protein having the activity of forming 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde has been introduced.
The host Escherichia coli bacterium can be a strain that can efficiently supply dihydroxyacetone phosphate and aspartate semialdehyde, which are the substrates for biosynthesis of a 3-amino-4-hydroxybenzoic acid-type compound. Escherichia coli has aspartokinase III (AKIII) that is a non-coupled enzyme and works alone. AKIII in Escherichia coli in its native form is subject to feedback inhibition by lysine. The Escherichia coli of the present invention preferably has an AKIII gene having a mutation capable of canceling the feedback inhibition by lysine.
The mutation capable of canceling the feedback inhibition by an amino acid such as lysine has been reported for aspartokinase derived from various microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Serratia marcescens. For example, the mutation of glutamic acid to lysine at position 250 (E250K), the mutation of methionine to isoleucine at position 318 (M318I), the mutation of threonine to methionine at position 344 (T344M), the mutation of serine to leucine at position 345 (S345L), and the mutation of threonine to isoleucine at position 352 (T352I) have been reported as mutations capable of canceling the feedback inhibition by lysine in AKIII in Escherichia coli (see e.g., Kikuchi et al., FEMS Microbiology Letters 173, 211-215 (1999), and Falco et al., BioTechnology 13, 577-582 (1995)). Therefore, Escherichia coli having the AKIII gene in which such a mutation has been introduced can be used in the present invention. Several amino acid residues are different even in wild-type AKIII depending on Escherichia coli strain from which the AKIII is derived, and such an allelic mutant may be used. The position to be modified for canceling the feedback inhibition in the allelic mutant can be identified by performing the sequence alignment publicly known to those skilled in the art. The modification to cancel the feedback inhibition in AKIII can be accomplished by a method known to those skilled in the art, e.g., by obtaining a mutant strain having resistance to a lysine analog such as 2-aminoethyl cysteine or by introducing site specific mutation by gene replacement utilizing homologous recombination. Also, Escherichia coli having an augmented activity of mutated AKIII in which the feedback inhibition was canceled can be obtained by transforming Escherichia coli with a plasmid containing a mutated AKIII gene in which the feedback inhibition was canceled.
The expression of a pyruvate carboxylase gene may further be augmented in Escherichia coli having the mutated AKIII in which the feedback inhibition was canceled.
According to methods known in the art, Escherichia coli can be transformed with a recombinant vector incorporating DNA encoding a protein having the activity of forming 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde. For example, a protoplast method (Kiyoshi et al., Gene, 39, 281-286 (1985)), an electroporation method (Dunican et al., Bio/Technology, 7, 1067-1070 (1989)), and the like can be used. When transformation for canceling the feedback inhibition in AKIII is performed, either the transformation for conferring the activity of forming 3,4-AHBA or the transformation for canceling the feedback inhibition in AKIII may be performed in first.
<5> Methods for Producing 3-amino-4-hydroxybenzoic Acid-Type Compound and Polymer Including the Same
The present invention also provides a method for producing a 3-amino-4-hydroxybenzoic acid-type compound, which includes a step of culturing the bacterium of the present invention.
The 3-amino-4-hydroxybenzoic acid-type compound in the present invention includes 3-amino-4-hydroxybenzoic acid (hereinafter sometimes abbreviated as “3,4-AHBA”) having the following structure as well as a derivative and a salt thereof.




In the derivative of 3-amino-4-hydroxybenzoic acid, (1) at least one of the carboxyl group at position 1, the amino group at position 3, and/or the hydroxyl group at position 4 is derivatized, and/or (2) the carboxyl group at position 1, the amino group at position 3, and the hydroxyl group at position 4 are kept and a hydrogen atom on at least one carbon atom at positions 2, 5 and/or 6 is substituted with another atom or group. Examples of the other atom or group in (2) above may include halogen atoms (e.g., a fluorine atom, a bromine atom, a chlorine atom, an iodine atom), alkyl groups (e.g., alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, and hexyl), a hydroxyl group, an alkyloxy group (the alkyl moiety is the same as described above), an amino group, a mono- or di-alkylamino group (the alkyl moiety is the same as described above), a cyano group, a nitro group, a sulfonyl group, and a carboxyl group. Specifically, examples of the derivative in (1) above may include derivatives in which the carboxyl group at position 1 is derivatized (e.g., 3-amino-4-hydroxybenzaldehyde in which the carboxyl group in 3-amino-4-hydroxybenzoic acid is aldehydated), derivatives in which the amino group at position 3 is derivatized with the group such as the above alkyl group (e.g., 3-alkylamino derivatives), and derivatives in which the hydroxyl group at position 4 is derivatized with the group such as the above alkyl group (e.g., 4-mono- or di-alkylamino derivatives).
Examples of salts include basic salts such as alkali metal (e.g., sodium, potassium, lithium) salts and alkali earth metal (e.g., calcium, magnesium) salts of carboxylic acid, and acid addition salts such as hydrochloride salts, sulfate salts, lead nitrate and phosphate salts.
The 3-amino-4-hydroxybenzoic acid-type compound can be produced by culturing the bacterium of the present invention and recovering the 3-amino-4-hydroxybenzoic acid-type compound produced in the medium.
The medium for culturing the bacterium of the present invention is not particularly limited as long as Escherichia coli is able to grow, and the bacterium can be cultured according to methods publicly known in the art. For example, the bacterium can be cultured in an ordinary medium containing a carbon source, a nitrogen source, and inorganic ions. Organic trace nutrients such as vitamins and amino acids may be added if necessary in order to obtain higher proliferation. A cultivation temperature is generally 25 to 42° C., and it is desirable to control pH to 5 to 8. A cultivation time period is generally 20 to 90 hours.
The cultivation of the bacterium of the present invention can be performed under conditions that control oxygen supply. Specifically, it is desirable to keep oxygen at 2.0 ppm or less when bacterial growth enters the logarithmic growth phase.
A recovery method used in steps of recovering and purifying the 3-amino-4-hydroxybenzoic acid-type compound from the culture medium can be appropriately selected from publicly known methods. For example, 3-amino-4-hydroxybenzoic acid-type compounds can be recovered from a culture medium supernatant obtained by removing microbial cells by centrifugation or membrane filtration after adjusting the pH of the culture medium to an acidic pH at which the solubility of the 3-amino-4-hydroxybenzoic acid-type compound is high. The recovery method of 3-amino-4-hydroxybenzoic acid from the culture medium supernatant can include purification by a porous adsorbent, crystallization and precipitation.
The porous adsorbent can be a porous solid adsorbent having a large surface area, and specifically includes hydrophilic adsorbents typified by silica gel, alumina, zeolite, bauxite, magnesia, activated white earth, acrylic synthetic adsorbents, and the like, and hydrophobic adsorbents typified by vegetable charcoal, bone charcoal, activated charcoal and aromatic synthetic adsorbents. Any adsorbent can be used without particular limitation as long as the purity of the 3-amino-4-hydroxybenzoic acid-type compound can be enhanced by adsorbing the impurities. In this regard, however, the impurities adsorbed by the porous adsorbent may contain abundant aromatic compounds mainly produced in the process of biochemical synthesis. Thus, the hydrophobic adsorbent typified by the activated charcoal and the aromatic synthetic adsorbent to which these compounds easily adsorb is suitably used in the present invention. These hydrophobic adsorbents may be used alone or in combination of two or more.
When the activated charcoal is used, its raw material is not particularly limited, and may include, but is not particularly limited to, plant raw materials such as wood powder and palm shell, coal/petroleum-based raw materials such as smokeless coal, petroleum pitch and cokes, synthetic resin-based raw materials such as acrylic resins, phenol resins, epoxy resins and polyester resins. The activated charcoal can be in the form of a powder, grain, and/or fibrous, and secondary processed articles such as filters and cartridges, and other forms that easily handled may be appropriately selected.
Meanwhile, when an aromatic synthetic adsorbent is used, the raw material thereof is not particularly limited, and for example, porous resins that can be used include 1) unsubstituted aromatic resins, 2) aromatic resins having a hydrophobic substituent(s), and 3) aromatic resins obtained by giving a special treatment to the unsubstituted aromatic resins. Specific compounds may include, for example, styrene- and divinylbenzene-based resins.
As mentioned above, an objective of contacting the 3-amino-4-hydroxybenzoic acid-type compound in the culture medium with the porous adsorbent is to adsorb the impurities to the porous adsorbent and to improve the purity of the 3-amino-4-hydroxybenzoic acid-type compound. However, 3-amino-4-hydroxybenzoic acid, which is an objective product, is sometimes adsorbed together in no small part with the impurities to the porous adsorbent. Thus, it is also possible to isolate and recover the 3-amino-4-hydroxybenzoic acid-type compound by contacting the 3-amino-4-hydroxybenzoic acid-type compound in the culture medium to the porous adsorbent, then contacting the porous adsorbent with a polar organic solvent to detach and dissolve the 3-amino-4-hydroxybenzoic acid-type compound in the polar organic solvent. The polar organic solvent used in the present invention refers to the organic solvent composed of polar molecules having a high dielectric constant, and can be used without particular limitation as long as the 3-amino-4-hydroxybenzoic acid-type compound can be detached from the porous adsorbent and the 3-amino-4-hydroxybenzoic acid-type compound can be dissolved in the polar organic solvent. The polar organic solvent may be used alone or in combination with two or more at a desired combination ratio.
Crystallization or the precipitation refers to a manipulation to produce a crystal or a precipitate, respectively, by evaporating the solvent in which an objective substance is dissolved to concentrate, or lowering the temperature, or keeping the concentration higher than a saturation solubility by adding a poor solvent to the solvent in which an objective substance is dissolved, and is not particularly limited including conventionally and publicly known methods. The produced crystal or precipitate can be separated by precipitation, filtration, centrifugation or the like.
The present invention also provides a method of producing a polymer that includes a 3-amino-4-hydroxybenzoic acid-type compound as a component. The production method of a polymer includes a step of polymerizing a 3-amino-4-hydroxybenzoic acid-type compound as at least one constituent of the polymer.
For example, the 3-amino-4-hydroxybenzoic acid-type compound that is purified from the culture medium of the bacterium of the present invention by using the porous adsorbent or by crystallization, precipitation or the like is polymerized by condensation polymerization in a non-oxidizing solvent acid such as methanesulfonic acid or polyphosphoric acid at high temperature (e.g., WO91/01304). In the production method of a polymer according to the present invention, the 3-amino-4-hydroxybenzoic acid-type compound may be polymerized with other constituents of a polymer. Examples of the other constituents include terephthalic acid and bisphenol A, or terephthalic acid and p-phenylenediamine. Known polymerizing method can be used as described in the following: U.S. Pat. Nos. 5,142,021, 5,219,981 and 5,422,416, and Kricheldorf et. al., (1992) Makromol. Chem., 193, 2467-2476, and Marcos-Fernandez et. al., (2001) Polymer, 42, 7933-7941. Examples of the polymer that includes a 3-amino-4-hydroxybenzoic acid-type compound which is produced by the method of the present invention include polybenzoxazole polymer, polyester and polyamide.
EXAMPLES
Embodiments of the present invention are further described with reference to the following non-limiting examples.
Example 1
Search for an Enzyme that Catalyzes Conversion of 3,4-AHBA in Escherichia coli 
(1) Search for the 3,4-AHBA Conversion Enzyme Based on Genomic Information of Escherichia coli 
It has been reported that arylamine N-acetyltransferase (convertible term: NatA; NCBI accession ID:BAF46971.1) catalyzes an N-acetylation reaction of 3,4-AHBA in Streptomyces griseus IFO13350 strain, and the amino acid sequence of NatA has been reported [Suzuki et. al., (2007) J. Bacteriol., 189, 2155-2159].
The amino acid sequence of NatA is shown in SEQ ID NO:1. In order to search for an enzyme having the same function as that of NatA, the genomic information of Escherichia coli K-12 strain was searched to find sequences with homology to the sequence of NatA. As a result of this search utilizing the published database (EcoCyc, ecocyc.org, Keseler et al., (2005) Nucleic Acids Res., 33, 334-337) and using BLASTP, it was found that N-hydroxyarylamine O-acetyltransferase (convertible term: NhoA, EC: 2.3.1.118, NCBI accession ID: NP_415980.1) in Escherichia coli K-12 strain exhibited 49% homology to NatA derived from Streptomyces griseus IFO13350 strain. The amino acid sequence of NhoA is shown in SEQ ID NO:2.
(2) Analysis of 3,4-AHBA Conversion Ability of nhoA Gene-Deleted Mutant Strain and Wild-Type Strain in Escherichia coli 
Escherichia coli BW25113 ΔnhoA (same strain: JW1458, Keio Collection) was used as a strain having a deletion of a gene which codes NhoA (convertible term: nhoA, GenBank accession No.: NC_000913.2, GI: 947251). Escherichia coli BW25113 ΔnhoA strain was obtained by deleting the nhoA gene in Escherichia coli BW25113 strain (Haldimann et. al., (2001) J. Bacteriol., 183, 6384-6393) (CGSC7630) (Baba et. al., (2006) Mol. Syst. Biol., 2, 2006-2008). Escherichia coli BW25113 ΔnhoA strain is available from National Institute of Genetics (nig.ac.jp/). Escherichia coli BW25113 strain is available from The Coli Genetic Stock Center (cgsc.biology.yale.edu/).
The ability to convert 3,4-AHBA in Escherichia coli BW25113 ΔnhoA strain and BW25113 strain was calculated according to the following procedure. Microbial cells of each strain were uniformly applied onto an LB plate, and cultured at 37° C. for 24 hours. One loopful of the microbial cells from the resulting plate was inoculated in 4 mL of MS glucose/3,4-AHBA medium in a test tube, and cultured at 30° C. for 30 hours on a reciprocal shaking cultivation apparatus. A composition of the MS glucose/3,4-AHBA medium is as described in the following Table 1.







TABLE 1







Table 1. MS glucose/3,4-AHBA medium










Components
Final concentration (g/L)














Glucose
40



(NH4)2SO4
24



3,4-AHBA
2



KH2PO4
1



MgSO4•7H2O
1



FeSO4•7H2O
0.01



MnSO4•7H2O
0.0082



Yeast Extract
2



CaCO3
50










The pH value of the medium was adjusted to 7.0 with KOH, and the medium was autoclaved at 120° C. for 20 minutes. Glucose and MgSO4.7H2O were mixed and sterilized separately. CaCO3 was added after dry heat sterilization. 3,4-AHBA was dissolved at 20 g/L in distilled water, then adjusted to pH 7.0 with KOH, sterilized through a filter, and subsequently added at a final concentration of 2.0 g/L.
After completion of the cultivation, an optical density (OD) value of the culture medium was measured using a spectrophotometer (HITACHI U-2900) at 600 nm. 3,4-AHBA in a culture supernatant was separated using reverse phase column chromatography, and its concentration was quantified (Suzuki et. al., (2006) J. Biol. Chem., 281, 36944-36951). The OD value at 600 nm, the concentration of 3,4-AHBA in the culture supernatant, and a conversion rate of 3,4-AHBA obtained from each microbial strain are shown in Table 2. The conversion rate of 3,4-AHBA was calculated using the following formula:

Conversion rate of 3,4-AHBA (%)]=100×{2 (g/L)−[Concentration of 3,4-AHBA in culture medium supernatant after completion of cultivation (g/L)]}/2 (g/L)








TABLE 2







Table 2. Effect of nhoA deletion on ability to convert 3,4-AHBA












Concentration of
Conversion rate


Strain name
OD (600 nm)
3,4-AHBA (g/L)
of 3,4-AHBA (%)





BW25113
34.3 ± 0.4
0.60 ± 0.10
70


BW25113ΔnhoA
36.7 ± 1.0
1.80 ± 0.10
10









As a result, the concentration of 3,4-AHBA in the culture supernatant after the cultivation of Escherichia coli BW25113 strain was decreased by 30% of the initial concentration. On the other hand, the decrease of the 3,4-AHBA concentration in the culture supernatant after the cultivation in Escherichia coli BW25113 ΔnhoA strain was around 10%. Therefore, it was strongly suggested that NhoA catalyzed the conversion reaction of 3,4-AHBA.
Charts of the reverse phase column chromatography for the culture supernatant of Escherichia coli BW25113 strain, the culture supernatant of Escherichia coli BW25113 ΔnhoA strain, and a 3,4-AHBA standard preparation (supplied from Tokyo Chemical Industry CO., Ltd., Cat. No.: A0859) are shown in FIG. 1. These results suggest that 3,4-AHBA was converted to a compound detected at a retention time (R.T.) of 9.5 minutes in Escherichia coli BW25113 strain.
(3) Identification of Molecular Weight of Product Converted from 3,4-AHBA (R.T., 9.5 Minutes)
The molecular weight of the product converted from 3,4-AHBA (R.T., 9.5 minutes) described in Example 1(2) and present in the culture supernatant after the cultivation of BW25113 strain was identified by LC/MS. The analysis conditions are as follows.
Column: Inertsil ODS-3 2 μm 2.1×75 mm (supplied from GL Science)
Mobile phase: A=0.1% formic acid/H2O
B=0.1% formic acid/acetonitrile
Gradient program: 0 minute: A/B=100/0

    
    
        3 minutes: A/B=100/0
        23 minutes: A/B=20/80
        25 minutes: A/B=20/80

Flow rate: 0.2 mL/minute

Column temperature: room temperature (25° C.)

Detection wavelength: 254 nm (PDA)

MS ionization mode: ESI

Analysis machine model: Agilent Infinity1290 (LC)

Agilent Quadrupole LC/MS 6130 (MS)

    
    


As a result of the analysis, the m/z value of the converted product (R.T., 9.5 minutes) was 195.1, which is consistent with a calculated m/z value of an acetylated product of 3,4-AHBA (195.1). Hereinafter, this converted product (R.T., 9.5 minutes) is referred to as 3,4-AcAHBA.
Example 2
Construction of Bacteria Producing 3,4-AHBA by Introducing 3,4-AHBA Synthetase Gene Group Derived from Streptomyces griseus and Evaluation of Amounts of Accumulated 3,4-AHBA and 3,4-AcAHBA
Construction of Plasmid pSTV28-Ptac-Ttrp for Expression
Subsequently, the effect of nhoA deletion on accumulation of 3,4-AHBA was verified in a 3,4-AHBA producing bacteria. First, the expression plasmid pSTV28-Ptac-Ttrp was constructed for the purpose of producing 3,4-AHBA.
To begin with, a DNA fragment was chemically synthesized that includes a tac promoter (convertible term: Ptac) region (deBoer, et al., (1983) Proc. Natl. Acad. Sci. U.S.A., 80, 21-25) and a terminator (convertible term: Ttrp) region of a tryptophan operon derived from Escherichia coli (Wu et al., (1978) Proc. Natl. Acad. Sci. U.S.A., 75, 5442-5446), and also having a KpnI site at the 5′ terminus and a BamHI site at the 3′ terminus (the nucleotide sequence is shown as SEQ ID NO:3). The resulting DNA fragment was digested with KpnI and BamHI to obtain a DNA fragment containing Ptac and Ttrp. The purified DNA fragment and pSTV28 (supplied from Takara Bio Inc.) digested with KpnI and BamHI were ligated by a ligation reaction using DNA ligase. The resulting plasmid was designated as pSTV28-Ptac-Ttrp (the nucleotide sequence is shown as SEQ ID NO:4). An objective gene can be expressed and amplified by cloning the objective gene downstream of Ptac in this plasmid.
(2) Chemical Synthesis of griI Gene and griH Gene (griIH Gene) Corresponding to Codon Usage in Escherichia coli 
It has been reported that synthesis of 3,4-AHBA is catalyzed by a 3,4-AHBA synthetase group consisting of aldolase (convertible term: SGR_4249, GriI) and 3,4-AHBA synthase (convertible term: SGR_4248, GriH) in Streptomyces griseus IFO13350 strain, and sequences of the genes encoding these enzymes have been reported (Suzuki et. al., (2006) J. Biol. Chem., 281, 36944-36951). GriI is encoded by the griI gene (GenBank accession no. AB259663.1, nucleotides: 13956 to 14780; GI: 117676060). The amino acid sequence of the GriI protein and a nucleotide sequence of the griI gene are shown as SEQ ID NO:5 and SEQ ID NO:6, respectively. GriH is encoded by the griH gene (GenBank accession no. AB259663.1, nucleotides: 12690 to 13880; GI: 117676059). The amino acid sequence of the GriH protein and the nucleotide sequence of the griH gene are shown as SEQ ID NO:7 and SEQ ID NO:8, respectively.
In order to efficiently express the griI gene and the griH gene in Escherichia coli, a sequence was designed so as to correspond to the codon usage in Escherichia coli by changing the sequences of the griI gene and the griH gene and to express as an operon, and this sequence was designated as EcGriIH. A DNA fragment was chemically synthesized by adding an EcoRI restriction enzyme recognition sequence to the 5′ terminus and a HindIII restriction enzyme recognition sequence to the 3′ terminus of EcGriIH (the sequence is shown as SEQ ID NO:9). EcGriIH, both termini of which the restriction enzyme recognition sequence had been added to, was digested with EcoRI and HindIII, and then cloned into pUC57 (supplied from GenScript) which had been digested with the same enzymes. The resulting vector was designated as pUC57-EcGri. The full length sequence of pUC57-EcGri is shown as SEQ ID NO:10.
(3) Construction of Plasmid for Expressing the griI Gene and griH Gene (griIH Gene)
An expression plasmid for expressing the griI gene and the griH gene in Escherichia coli was constructed using the following procedure. PCR was performed with pUC57-EcGri as a template using a synthesized oligonucleotide represented by SEQ ID NO:11 and further a synthesized oligonucleotide represented by SEQ ID NO:12 as primers and using PrimeStar GXL polymerase (supplied from Takara Bio Inc.). A reaction solution was prepared according to a composition attached to the kit, and a reaction at 98° C. for 10 seconds, 55° C. for 15 seconds and 68° C. for 150 seconds was performed in 30 cycles. As a result, a PCR product of about 2.1 kbp containing the EcGriIH gene fragment was obtained. Subsequently, the purified EcGriIH gene fragment and pSTV28-Ptac-Ttrp digested with SmaI were ligated using In-Fusion HD Cloning Kit (supplied from Clontech). The resulting plasmid for expressing the griIH gene was designated as pSTV28-EcGri. A full length sequence of pSTV28-EcGri is shown as SEQ ID NO:13.
(4) Construction of 3,4-AHBA Producing Bacteria
Competent cells of Escherichia coli BW25113 strain and BW25113 ΔnhoA strain were prepared, subsequently pSTV28-EcGri was introduced thereto by electroporation, then the cells were uniformly applied onto an LB plate containing 30 mg/L of chloramphenicol, and cultured at 37° C. for 24 hours. Transformants that were resistant to chloramphenicol were obtained from the resulting plate. pSTV28-EcGri was introduced into the BW25113 strain, and the resulting strain was designated as BW25113/pSTV28-EcGri strain, and pSTV28-EcGri was introduced into Escherichia coli BW25113 ΔnhoA strain, and the resulting strain was designated as BW25113ΔnhoA/pSTV28-EcGri strain.
(5) Evaluation of Cultivation for Producing 3,4-AHBA
Microbial cells of BW25113ΔnhoA/pSTV28-EcGri strain and BW25113/pSTV28-EcGri strain were uniformly applied onto an LB plate containing 30 mg/L of chloramphenicol, and cultured at 37° C. for 24 hours. One loopful of the microbial cells from the resulting plate was inoculated in 4 mL of MS glucose/Asp medium containing 30 mg/L of chloramphenicol and 0.1 mM isopropyl-β-thiogalactopyranoside in a test tube, and cultured at 30° C. for 48 hours on the reciprocal shaking cultivation apparatus. A composition of the MS glucose/Asp medium is as described in the following Table 3.







TABLE 3







Table 3. MS glucose/Asp medium










Components
Final concentration (g/L)














Glucose
40



(NH4)2SO4
24



Aspartic acid
5



KH2PO4
1



MgSO4•7H2O
1



FeSO4•7H2O
0.01



MnSO4•7H2O
0.0082



Yeast Extract
2



CaCO3
50










The pH value of the medium was adjusted to 7.0 with KOH, and the medium was autoclaved at 120° C. for 20 minutes. Glucose and MgSO4.7H2O were mixed and sterilized separately. CaCO3 was added after dry heat sterilization.
After the cultivation, the OD value of the culture medium at 600 nm was measured using the spectrophotometer (HITACHI U-2900). 3,4-AHBA and 3,4-AcAHBA were separated using the reverse phase column chromatography, and their concentrations were quantified (Suzuki et. al., (2006) J. Biol. Chem., 281, 36944-36951). The OD values at 600 nm in the culture medium and the concentrations of 3,4-AHBA and 3,4-AcAHBA in the cultured supernatant are shown in Table 4. In BW25113ΔnhoA/pSTV28-EcGri strain, no 3,4-AcAHBA was detected and the concentration of 3,4-AHBA was much higher than that in the control strain BW25113/pSTV28-EcGri.







TABLE 4







Table 4. Effect of nhoA deletion on amount


of accumulated 3,4-AHBA and 3,4-AcAHBA












Concentration
Concentration



OD
of 3,4-AHBA
of 3,4-AcAHBA


strain name
(600 nm)
(g/L)
(g/L)





BW25113/
30.2 ± 0.2
0.11 ± 0.02
0.26 ± 0.02


pSTV28-EcGri


BW25113ΔnhoA/
30.5 ± 0.3
0.30 ± 0.03
N.D.


pSTV28-EcGri





N.D.: not detected






Each value is a mean value in the cultivation in triplicate.

Example 3
Construction of Bacteria Producing 3,4-AHBA by Introducing 3,4-AHBA Synthetase Gene Group Derived from Streptomyces murayamaensis and Evaluation of Amounts of Accumulated 3,4-AHBA and 3,4-AcAHBA
(1) Chemical Synthesis of nspI Gene and nspH Gene Corresponding to Codon Usage in Escherichia coli 
It has been reported that the synthesis of 3,4-AHBA is catalyzed by a 3,4-AHBA synthetase group consisting of aldolase (convertible term: NspI; NCBI accession ID: BAJ08171.1) and 3,4-AHBA synthase (convertible term: NspH; NCBI accession ID: BAJ08172.1) in Streptomyces murayamaensis (Furusaki et. al., (1972) Isr. J. Chem., 10, 173-187), and sequences of the genes encoding these enzymes have been reported (Noguchi et. al., (2010) Nat. Chem. Biol., 6, 641-643). NspI is encoded by an nspI gene (GenBank accession no. AB530136, nucleotides 8730 to 9584; GI: 296784943). An amino acid sequence of the NspI protein and a nucleotide sequence of the nspI gene are shown as SEQ ID NO:14 and SEQ ID NO:15, respectively. NspH is encoded by an nspH gene (GenBank accession no. AB530136, nucleotides 9599 to 10702; GI: 296784944). An amino acid sequence of the NspH protein and a nucleotide sequence of the nspH gene are shown as SEQ ID NO:16 and SEQ ID NO:17, respectively.
In order to efficiently express the nspI gene and the nspH gene in Escherichia coli, a sequence was designed so as to correspond to the codon usage in Escherichia coli by changing the sequences of the nspI gene and the nspH gene and to express as an operon, and this sequence was designated as EcNspIH. A DNA fragment was chemically synthesized by adding an EcoRI restriction enzyme recognition sequence to the 5′ terminus and a HindIII restriction enzyme recognition sequence to the 3′ terminus of EcNspIH. EcNspIH, both termini of which the restriction enzyme recognition sequence had been added to, was digested with EcoRI and HindIII, and then cloned into pUC57 (supplied from GenScript) digested with the same restriction enzymes. The resulting vector was designated as pUC57-EcNsp.
(2) Construction of Plasmid for Expressing nspI Gene and nspH Gene (nspIH Gene)
An expression plasmid for expressing the nspI gene and the nspH gene in Escherichia coli was constructed using the following procedure. PCR was performed with pUC57-EcNsp as a template using synthesized oligonucleotides as primers and using PrimeStar GXL polymerase (supplied from Takara Bio Inc.). A reaction solution was prepared according to a composition attached to the kit, and a reaction at 98° C. for 10 seconds, 55° C. for 15 seconds and 68° C. for 150 seconds was performed in 30 cycles. As a result, a PCR product of about 2.1 kbp containing the EcNspIH gene fragment was obtained. Subsequently, the purified EcNspIH gene fragment and pSTV28-Ptac-Ttrp digested with SmaI (described in Example 2) were ligated using In-Fusion HD Cloning Kit (supplied from Clontech). The resulting plasmid for expressing the nspIH gene was designated as pSTV28-EcNsp.
(3) Construction of 3,4-AHBA Producing Bacteria
Competent cells of Escherichia coli BW25113 strain and BW25113 ΔnhoA strain were prepared, subsequently pSTV28-EcNsp was introduced thereto by electroporation, then the cells were uniformly applied onto an LB plate containing 30 mg/L of chloramphenicol, and cultured at 37° C. for 24 hours. Transformants that were resistant to chloramphenicol were obtained from the resulting plate. pSTV28-EcNsp was introduced into the BW25113 strain, and the resulting strain was designated as BW25113/pSTV28-EcNsp strain, and pSTV28-EcNsp was introduced into Escherichia coli BW25113 ΔnhoA strain, and the resulting strain was designated as BW25113ΔnhoA/pSTV28-EcNsp strain.
(4) Evaluation of Cultivation for Producing 3,4-AHBA
Microbial cells of BW25113/pSTV28-EcNsp strain and BW25113ΔnhoA/pSTV28-EcNsp strain were uniformly applied onto an LB plate containing 30 mg/L of chloramphenicol, and cultured at 37° C. for 24 hours. One loopful of the microbial cells from the resulting plate was inoculated in 4 mL of MS glucose/Asp medium containing 30 mg/L of chloramphenicol and 0.1 mM isopropyl-β-thiogalactopyranoside (see Table 3) in a test tube, and cultured at 30° C. for 48 hours on the reciprocal shaking cultivation apparatus.
After the cultivation, the OD value of the culture medium at 600 nm was measured using the spectrophotometer (HITACHI U-2900). 3,4-AHBA and 3,4-AcAHBA were separated using the reverse phase column chromatography, and their concentrations were quantified (Suzuki et. al., (2006) J. Biol. Chem., 281, 36944-36951). The OD values at 600 nm in the culture medium and the concentrations of 3,4-AHBA and 3,4-AcAHBA in the cultured supernatant were shown in Table 5. In BW25113ΔnhoA/pSTV28-EcNsp strain, no 3,4-AcAHBA was detected and the concentration of 3,4-AHBA was much higher than that in the control strain BW25113/pSTV28-EcNsp.







TABLE 5







Table 5. Effect of nhoA deletion on amount


of accumulated 3,4-AHBA and 3,4-AcAHBA












Concentration
Concentration



OD
of 3,4-AHBA
of 3,4-AcAHBA


Strain name
(600 nm)
(g/L)
(g/L)





BW25113/
30.2 ± 0.5
0.17 ± 0.01
0.28 ± 0.03


pSTV28-EcNsp


BW25113ΔnhoA/
31.5 ± 2.7
0.47 ± 0.02
N.D.


pSTV28-EcNsp





N.D.: not detected






Each value is a mean value in the cultivation in triplicate.

INDUSTRIAL APPLICABILITY
According to the present invention, it is possible to conveniently and inexpensively produce an amino-hydroxybenzoic acid-type compound that is useful as an intermediate in the manufacture of dyes, agricultural chemicals, pharmaceuticals and other organic synthesized articles and as a monomer for polybenzoxazole. Thus, for example, polybenzoxazole (PBO) is obtained by polymerizing 3-amino-4-hydroxybenzoic acid obtained by the present invention, thereby enabling to inexpensively provide PBO fibers and PBO films having high strength, high elastic modulus and high resistance to heat. It is also possible to produce a 3-amino-4-hydroxybenzoic acid-type compound that can be used as a raw material in various biosyntheses. Thus, the method of the present invention is a process with low environmental load and an environmentally-friendly production method.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing a 3-amino-4-hydroxybenzoic acid or a salt thereof, comprising a step of culturing an Escherichia coli bacterium having an ability to produce 3-amino-4-hydroxybenzoic acid which has been genetically modified to express DNA which encodes an enzyme to increase the activity of an enzyme, wherein said enzyme is capable of catalyzing formation of 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde as compared to a non-modified Escherichia coli bacterium, and wherein said enzyme is GriI and GriH; and wherein the Escherichia coli bacterium also has been modified to inactivate an N-hydroxyarylamine O-acetyltransferase (NhoA) as compared to a non-modified Escherichia coli bacterium by mutating or deleting an nhoA gene on the chromosome of the Escherichia coli bacterium.

2. The method according to claim 1, further comprising a step of polymerizing the 3-amino-4-hydroxybenzoic acid.

3. The method according to claim 2, wherein the polymer is a polybenzoxazole polymer.

4. The method according to claim 1, wherein the GriI is a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO: 14;
(B) a protein comprising an amino acid sequence having 1 to 10 amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in (A) above, and having an aldolase activity; and
(C) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence represented in (A) above and having an aldolase activity;

and the GriH is a protein selected from the group consisting of:
(D) a protein comprising the amino acid sequence represented by SEQ ID NO:7 or SEQ ID NO: 16;
(E) a protein comprising an amino acid sequence having 1 to 10 amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in (D) above, and having a 3-amino-4-hydroxybenzoic acid synthase activity; and
(F) a protein comprising an amino acid sequence having 95% or more identity to the amino acid sequence shown in (D) above and having a 3-amino-4-hydroxybenzoic acid synthase activity.

5. The method according to claim 1, wherein said DNA comprises a griI gene and a griH gene.

6. The method according to claim 5, wherein the griI gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:6 or SEQ ID NO: 15;
(b) a DNA that hybridizes under a stringent condition with the nucleotide sequence complementary to the nucleotide sequence shown in (a) above and encodes a protein having an aldolase activity; and
(c) a DNA having 95% or more identity to the nucleotide sequence shown in (a) above and encoding a protein having an aldolase activity;

and the griH gene is selected from the group consisting of:
(d) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8 or SEQ ID NO: 17;
(e) a DNA that hybridizes under a stringent condition with the nucleotide sequence complementary to the nucleotide sequence shown in (d) above and encodes a protein having a 3-amino-4-hydroxybenzoic acid synthase activity; and
(f) a DNA having 95% or more identity to the nucleotide sequence shown in (d) above and encoding a protein having a 3-amino-4-hydroxybenzoic acid synthase activity;
wherein said stringent condition of (b) and (e) above comprises hybridization in 6×SSC at about 45° C. followed by washing once, twice, or more times in 0.2×SSC and 0.1% SDS at 50 to 65° C.

7. The method according to claim 6, wherein the griI gene and the griH gene are from an actinomycete.

8. The method according to claim 6, wherein the griI gene and the griH gene are from the genus Streptomyces.

9. The method according to claim 6, wherein the griI gene and the griH gene are from Streptomyces griseus.

10. The method according to claim 6, wherein the griI gene and the griH gene are from Streptomyces murayamaensis.

11. The method according to claim 1, having wherein said Escherichia coli bacterium also has a gene encoding a mutated aspartokinase III in which feedback inhibition is canceled.