Hydroquinone manufacturing method

Abstract

The object of the present invention is to provide a method for producing hydroquinone involving an enzyme that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604. [Solution] The above objective can be achieved by a method for producing hydroquinone, which includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 1-hydroxylase activity of 4-hydroxybenzoic acid derived from a microorganism.

Description

Technical Field

[0001] The present invention relates to a method for producing hydroquinone in which an enzyme having 4-hydroxybenzoate 1-hydroxylase activity that catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone is involved.

Background Art

[0002] Hydroquinone is a dihydric phenol having a structure in which the 1-position and the 4-position of the benzene ring are substituted with hydroxy groups (-OH). Hydroquinone is used as a cosmetic raw material as a kind of whitening ingredient.

[0003] Hydroquinone has a structure similar to tyrosine and can function as a tyrosine analog. Thus, hydroquinone can inhibit tyrosinase activity by antagonizing tyrosine and binding to tyrosinase. Since the biosynthesis of melanin is brought about by tyrosinase activity, if tyrosinase activity is inhibited by hydroquinone, the biosynthesis of melanin is suppressed. As a result, hydroquinone can exhibit a cosmetic improvement effect on the skin such as improvement of pigmentation such as freckles, age spots, and sunburn, and promotion of whitening.

[0004] Hydroquinone is mainly produced by chemical synthesis. However, the method by chemical synthesis has problems such as using substances harmful to the human body such as organic solvents and inorganic metal catalysts, difficulty in separating the product hydroquinone from raw materials and by-products, and inclusion of expensive substances not recognized as cosmetic ingredients in the raw materials. Therefore, it is required to produce hydroquinone biochemically or biologically safely and simply.

[0005] As a kind of hydroquinone-producing enzyme, 4-hydroxybenzoate 1-hydroxylase that catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone is known. As a microorganism expressing 4-hydroxybenzoate 1-hydroxylase, Candida parapsilosis ( White parapsilosis The CBS604 strain is known (see, for example, Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] MH Eppinket al, J Bacteriol., 1997 Nov; 179 (21): pages 6680-6687. [Non-Patent Document 2] Willem JH Van Berkel et al, FEMS Microbiology Letters, Volume 121, Issue 2, August 1994, Pages 207-215. [Overview of the project] [Problems that the invention aims to solve]

[0007] However, our researchers have found that the 4-hydroxybenzoic acid 1-hydroxylase present in Candida parapsis strain CBS604 has low catalytic activity, resulting in a slow conversion reaction from p-hydroxybenzoic acid to hydroquinone.

[0008] Furthermore, to date, there are very few known enzymes that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604, nor are there any known microorganisms that express such enzymes.

[0009] Therefore, the problem that the present invention aims to solve is to provide a method for producing hydroquinone involving an enzyme that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604. [Means for solving the problem]

[0010] The inventors of the present invention have diligently investigated new hydroquinone-producing enzymes in an attempt to solve the above problems. In doing so, they selected enzymes that are thought to function as hydroquinone-producing enzymes and further extracted genes encoding more than 40 proteins that are presumed to be hydroquinone-producing enzymes. Among these, some have hydroquinone-producing enzyme activity, while others do not. As a result, spasaspora ( Spathaspora ) Microorganisms of the genus Pycnoporus ( Pycnoporus ) genus microorganisms, Rhoderomyces ( Lodderomyces ) microorganisms and Deltaproteobacteria ( Deltaproteobacteria We discovered that microorganisms of the genus ) express proteins that possess 4-hydroxybenzoic acid 1-hydroxylase activity.

[0011] Then, when we created transformed microorganisms expressing the protein and evaluated their 4-hydroxybenzoate 1-hydroxylase activity, we were surprised to find that the 4-hydroxybenzoate 1-hydroxylase activity of proteins derived from microorganisms of the genera Spathaspora, Pycnoporus, Rhoderomyces, and Deltaproteobacteria was higher than that of the 4-hydroxybenzoate 1-hydroxylase possessed by Candida parapsis strain CBS604.

[0012] Based on the above findings, the inventors have finally succeeded in creating a hydroquinone production method involving an enzyme that can efficiently convert p-hydroxybenzoic acid to hydroquinone, thereby solving the problems of the present invention. The present invention is completed based on the first findings and success stories obtained by these inventors.

[0013] Therefore, according to one aspect of the present invention, the following embodiments are provided. [1] A method for producing hydroquinone, The process includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from microorganisms, and The aforementioned microorganism is Spasaspora ( Spathaspora ) Microorganisms of the genus Pycnoporus ( Pycnoporus ) genus microorganisms, Rhoderomyces ( Lodderomyces ) microorganisms and Deltaproteobacteria ( Deltaproteobacteria It is at least one microorganism selected from the group consisting of microorganisms of the ) genus. The aforementioned method. [2] The enzyme according to item [1], wherein the enzyme has the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 80% or more sequence identity with any of the amino acid sequences of SEQ ID NOs. 2-5 (2) In any of the amino acid sequences of Sequence ID No. 2 to 5, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [3] A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, It contains as an active ingredient an enzyme having 1-hydroxylase activity of 4-hydroxybenzoic acid derived from microorganisms, and The aforementioned microorganism is Spasaspora ( Spathaspora ) Microorganisms of the genus Pycnoporus ( Pycnoporus ) genus microorganisms, Rhoderomyces ( Lodderomyces ) microorganisms and Deltaproteobacteria ( Deltaproteobacteria It is at least one microorganism selected from the group consisting of microorganisms of the ) genus. The aforementioned composition. [4] DNA fragments for transforming non-human host organisms, The gene for an enzyme possessing 1-hydroxylase activity of 4-hydroxybenzoic acid, derived from microorganisms, At least one gene different from the said gene and including, and The aforementioned microorganism is Spasaspora ( Spathaspora ) Microorganisms of the genus Pycnoporus ( Pycnoporus ) genus microorganisms, Rhoderomyces ( Lodderomyces ) a microorganism selected from the group consisting of microorganisms belonging to the genus Microorganism and Deltaproteobacteria Deltaproteobacteria ), the DNA fragment. [5] A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, A gene encoding an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from a microorganism has been introduced as a foreign gene, and The microorganism is Sparassis Spathaspora ), microorganisms belonging to the genus Piceoporus Pycnoporus ), microorganisms belonging to the genus Rhodomyces Lodderomyces ), and at least one microorganism selected from the group consisting of microorganisms belonging to the genus Deltaproteobacteria Deltaproteobacteria ), the transformant. [6] The transformant according to item [5], wherein the host organism is selected from the group consisting of microorganisms belonging to the genus Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingobium and Ralstonia. [7] The enzyme has the amino acid sequence of (1) or (2) below, and the composition, DNA fragment or transformant according to any one of items [3] to [6]. (1) An amino acid sequence having 80% or more sequence identity with any one of the amino acid sequences of SEQ ID NOs: 2 to 5 (2) An amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit of 100 amino acids in any one of the amino acid sequences of SEQ ID NOs: 2 to 5 [Advantages of the Invention]

[0014] According to the present invention, hydroquinone can be produced from p-hydroxybenzoic acid with greater efficiency compared to using the 4-hydroxybenzoic acid 1-hydroxylase present in Candida parapsis strain CBS604. Furthermore, since the present invention employs a biochemically and biologically safe and simple production method, it is expected that hydroquinone can be produced in large quantities on an industrial scale. [Modes for carrying out the invention]

[0015] The details of each aspect of the present invention will be described below, but the present invention is not limited to the matters described in this section and can take various forms insofar as it achieves the objective of the present invention.

[0016] In this specification, unless otherwise specified, each term is used in the sense commonly used by those skilled in the art in the fields of biochemistry, biotechnology, microbiology, etc., and should not be interpreted as having an unreasonably restrictive meaning. Furthermore, since the assumptions and theories made herein are based on the inventors' prior knowledge and experience, the present invention is not limited solely to such assumptions and theories.

[0017] "To include" means that elements other than those explicitly included can be added (synonymous with "at least include"), but it also encompasses "consisting of" and "essentially consisting of." In other words, "to include" can mean that it includes the explicitly included elements and any one or more of those elements, consists of the explicitly included elements, or essentially consists of the explicitly included elements. Examples of elements include components, processes, conditions, parameters, and other limitations. "To have" is synonymous with "to include." The terms "and / or" mean any one of the listed related items, any combination of two or more, or any combination of all of them. The "~" in a numerical range indicates a range that includes the numbers before and after it, as well as the range excluding one of the limit values ​​that include them. For example, "0%~100%" can be any of the following: 0% or more, 100% or less, or 0% or more and 100% or less. "Approximately" means a quantity within ±10% of the quantity that follows the term. For example, "approximately 100" means 100 ± 10%, i.e., 90 to 110. The number of digits in an integer value matches the number of significant figures. For example, 1 has 1 significant figure, and 10 has 2 significant figures. Similarly, the number of digits after the decimal point in a decimal value matches the number of significant figures. For example, 0.1 has 1 significant figure, and 0.10 has 2 significant figures.

[0018] "Foreign genes" refer to genes that are not naturally present on the chromosomal (genomic) DNA of the organism being introduced, and are also called heterogenes. In this specification, genome and chromosome are synonymous. "Genetic expression" means that proteins (gene-encoded proteins) that have an amino acid sequence encoded by part or all of the nucleotide sequence of a gene are produced through transcription, translation, etc., in order to acquire their original structure and activity. "Wild organism" refers to an organism that exists naturally and has not been genetically modified. "Transformed organism" refers to an organism that has been genetically modified. "Wild-type genes" refer to genes that are naturally present on the genomic DNA of wild-type organisms. "Wild-type proteins" refer to proteins encoded by wild-type genes. Proteins that have the activity to catalyze a specific reaction are called "enzymes."

[0019] [Summary of the Invention] One aspect of the present invention is a method for producing hydroquinone. One embodiment of the present invention is a method for producing hydroquinone, which includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from a microorganism. The microorganism is Spasaspora ( Spathaspora) Microorganisms of the genus Pycnoporus ( Pycnoporus ) genus microorganisms, Rhoderomyces ( Lodderomyces ) microorganisms and / or Deltaproteobacteria ( Deltaproteobacteria These are microorganisms of the genus Spathaspora. In this specification, enzymes possessing 4-hydroxybenzoic acid 1-hydroxylase activity derived from microorganisms of the genus Spathaspora, Pycnoporus, Rhoderomyces, and Deltaproteobacteria may be referred to as SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H, respectively. Furthermore, genes encoding enzymes possessing 4-hydroxybenzoic acid 1-hydroxylase activity derived from microorganisms of the genus Spathaspora, Pycnoporus, Rhoderomyces, and Deltaproteobacteria may be referred to as the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene, respectively. Note that either "hydro" or "hydro" is used as the Japanese pronunciation of the English word "hydro".

[0020] Another aspect of the present invention is a hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid. A composition according to one embodiment of the present invention comprises SPj-S1H, PCi-S1H, LE-S1H and / or DBa-S1H as active ingredients.

[0021] SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H used in each aspect of the present invention may be isolated and purified enzymes, or enzyme-containing materials such as living organisms, processed organisms, or cultures of microorganisms from which each originates or transformants transformed to express SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H. Furthermore, SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H may be synthesized using a cell-free protein synthesis system with the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene, which are the genes encoding SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H.

[0022] Another aspect of the present invention is a DNA fragment for transforming a non-human host organism. A DNA fragment according to one aspect of the present invention comprises the SPj-S1H gene, the PCi-S1H gene, the LE-S1H gene and / or the DBa-S1H gene and at least one gene different from said gene.

[0023] Another aspect of the present invention is a transformant capable of producing hydroquinone from p-hydroxybenzoic acid. A transformant according to one embodiment of the present invention is obtained by transduction of the SPj-S1H gene, PCi-S1H gene, LE-S1H gene and / or DBa-S1H gene as an exogenous gene.

[0024] [Enzymes possessing 1-hydroxylase activity of 4-hydroxybenzoic acid: SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H] As shown in the following scheme (1), 4-hydroxybenzoic acid 1-hydroxylase has the activity to catalyze the reaction that converts p-hydroxybenzoic acid to hydroquinone (hereinafter also simply referred to as hydroxylase activity). After diligent investigation, it was found that when 4-hydroxybenzoic acid is reacted with SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H, the same reaction as in scheme (1) occurs. [ka]

[0025] As for 4-hydroxybenzoic acid 1-hydroxylases already known, there are Candida parapsis (as described in Non-Patent Documents 1 and 2) White parapsilosisThe CBS604 strain possesses 4-hydroxybenzoic acid 1-hydroxylase. SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H have hydroxylase activity, but their amino acid sequence identity with the 4-hydroxybenzoic acid 1-hydroxylase derived from Candida parapsis CBS604 is 32% to 71%. Therefore, SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H may be enzymes having hydroxylase activity and amino acid sequences that have 60% or more, preferably 65% ​​or more, more preferably 70% or more, even more preferably 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the wild-type enzymes (SEQ ID NOs. 2-5) of SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H.

[0026] There are no particular limitations on the method for determining the sequence identity of amino acids, but for example, it can be determined by using a program that aligns the amino acid sequences of two proteins using commonly known methods and calculates the degree of sequence agreement between the two.

[0027] For calculating the degree of synchrony between two amino acid sequences, for example, the Karlin and Altschul algorithm (Proc.Natl.Acad.Sci.USA 87:2264-2268, 1990; Proc.Natl.Acad.Sci.USA 90:5873-5877, 1993) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J.Mol.Biol.215:403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res.25:3389-3402, 1997). Those skilled in the art can use the above programs to search a database for sequences that show high sequence identity with a given sequence. These are available, for example, on the website of the U.S. National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0028] Examples of amino acid sequences with 60% or more sequence identity to the amino acid sequence of the wild-type enzyme include amino acid sequences in which one or more amino acids are deleted, substituted, or added compared to the amino acid sequence of the wild-type enzyme. The range of "several" is determined by the sequence identity of the amino acid sequence. For example, if 100 amino acids in the amino acid sequence are considered as one unit, then each unit may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21-30 amino acids, preferably 1-20, and more preferably 1-10. Furthermore, "amino acid deletion" means the absence or disappearance of an amino acid residue in the sequence, "amino acid substitution" means that an amino acid residue in the sequence is replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue is inserted into the sequence.

[0029] Specific examples of "deletion, substitution, and addition of amino acids" include cases where an amino acid is replaced by another chemically similar amino acid. For example, this could include substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the relevant field for each amino acid. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0030] SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H may have a tag peptide to facilitate separation and purification. Examples of such tag peptides include His tags, preferably 6×His, which consists of six histidine molecules linked together.

[0031] When attempting to obtain recombinant enzymes SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H using genetic engineering technology, they may exist as intracellular enzymes depending on the host organism used, making enzyme isolation and purification difficult. Therefore, when attempting to obtain recombinant SPj-S1H, recombinant Pci-S1H, recombinant LE-S1H, and recombinant DBa-S1H, it is preferable to modify these recombinant enzymes to become extracellular enzymes. However, if the recombinant enzyme is expressed as an extracellular enzyme in the transformant, extracellular enzyme modification is not necessary.

[0032] Methods for extracellular enzymatic modification of recombinant enzymes include, but are not limited to, adding secretory signals such as peptides or proteins that function to cause the recombinant enzyme to be expressed as an extracellular enzyme to the C-terminus and / or N-terminus of the recombinant enzyme.

[0033] Specific embodiments of SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H are those having an amino acid sequence that has 80% or more sequence identity with the amino acid sequences of SEQ ID NOs. 2-5. Another specific embodiment of SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H is that having an amino acid sequence in which, in the amino acid sequences of SEQ ID NOs. 2-5, one to ten amino acids are deleted, substituted, and / or added per unit consisting of 100 amino acids.

[0034] [Genes of enzymes possessing 4-hydroxybenzoic acid 1-hydroxylase gene activity: SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene] The SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene each contain nucleotide sequences that encode the amino acid sequences of SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H, respectively, and express SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H. These nucleotide sequences in the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene may be coding sequences (CDS) or open reading frames (ORF), but are preferably ORF.

[0035] The SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene may consist of nucleotide sequences encoding SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H, respectively, and may also include transcriptional regulatory sequences such as promoters and terminators, and non-coding sequences such as introns. A gene is not merely sequence information, but a DNA molecule (fragment) made up of linked nucleotides.

[0036] The SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H genes may contain nucleotide sequences optimized for codons, secondary structure, GC content, etc., by utilizing the fact that there are several types of codons corresponding to a single amino acid. Preferably, the codon-modified nucleotide sequence is one which has been modified to be easily expressed in a host organism.

[0037] The wild-type genes for SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H each have the nucleotide sequences of SEQ ID NOs.6-9. As a specific example of the SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H genes, the wild-type genes are derived from the Coryne genus ( Corynebacterium Examples include genes with nucleotide sequences of sequence numbers 10-13 that have been codon-optimized for microorganisms.

[0038] By introducing the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene as exogenous genes into a host organism, a host organism that was originally unable to express SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H can be transformed to express SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H as recombinant enzymes. The expression of the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene may be autonomously carried out by the transcriptional regulatory sequences of the genes, or by the transcriptional regulatory mechanisms of the host organism, but it is preferable that it be autonomously carried out by the transcriptional regulatory sequences of the genes.

[0039] For the SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H genes to be expressed autonomously within a host organism, it is preferable that they contain a promoter, ORF, and terminator. The promoter and terminator can be appropriately selected according to the host organism.

[0040] [DNA fragment] A DNA fragment according to one aspect of the present invention includes, in addition to a first gene, which is one of the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene, at least one second gene that is different from the first gene. However, for example, if the first gene is the SPj-S1H gene, the second gene may be the PCi-S1H gene, the LE-S1H gene, and / or the DBa-S1H gene. Furthermore, if the first gene is an artificially designed and synthesized gene that is codon-optimized for the host, the second gene may not be included. In the DNA fragment, the copy number of the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and DBa-S1H gene may be any of the above, and may be one copy or two or more copies.

[0041] The second gene may be a selection marker gene, for example, to facilitate the selection of transformants. Examples of selection marker genes include drug resistance genes such as kanamycin resistance genes and ampicillin resistance genes, but other selection marker genes such as nutritional requirement marker genes may also be used. The second gene may not have a protein-coding nucleotide sequence and may consist of, for example, a transcriptional regulatory sequence, a non-coding sequence, etc.

[0042] The DNA fragment may also be a DNA construct such as a plasmid or vector. Specific examples of such DNA constructs are the SPj-S1H expression DNA construct, PCi-S1H expression DNA construct, LE-S1H expression DNA construct, and DBa-S1H expression DNA construct described in the examples below.

[0043] [Transformed body] A transformant according to one aspect of the present invention is obtained by transduction of the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene as exogenous genes. The transformant has the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene introduced as exogenous genes and expresses the introduced genes.

[0044] Transformants are created by introducing the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene as exogenous genes into a host organism.

[0045] The host organism is not particularly limited as long as it can be introduced with the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene as an exogenous gene, and can express the introduced SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene. Examples include microorganisms, insect cells and insect bodies, plant cells and plant bodies, animal cells and animal bodies, etc. However, the host organism is not a human. Examples of host organisms include microorganisms of the genera Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Sphingovium, Novosphingovium, Ralstonia, Burkholderia, Alkaligenes, and Arthrobacter. In particular, microorganisms such as Pseudomonas, Burkholderia, Alkaligenes, Sphingovium, Rhodococcus, and Arthrobacter, which include strains that degrade p-hydroxybenzoic acid, are preferred. Preferably, the host organism is a Corynebacterium, which has a proven track record as a host organism for genetic modification and does not assimilate the product hydroquinone. In the following, the present invention will be described in detail assuming that the host organism is a microorganism.

[0046] The host microorganism may be a naturally occurring wild-type organism or a transformed microorganism obtained by introducing mutations into a wild-type organism. For example, if the host microorganism has a gene for an enzyme that has hydroquinone-degrading activity, it is preferable to delete the hydroquinone-degrading enzyme gene in the host microorganism or to inactivate the expression of the hydroquinone-degrading enzyme gene. Transformed microorganisms may also be obtained by introducing the SPj-S1H gene, PCi-S1H gene, LE-S1H gene and / or DBa-S1H gene into a wild-type organism, and then deleting or inactivating the hydroquinone-degrading enzyme gene.

[0047] The vectors, culture media, and procedures used to produce transformed microorganisms can be found in the examples described later. For example, methods for transforming host microorganisms by introducing DNA fragments include electroporation, protoplasts, and calcium ion methods, but electroporation is preferred.

[0048] The method for introducing the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene into a host microorganism is not particularly limited. Examples include introducing a DNA construct, such as a plasmid vector incorporating the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene, into the host microorganism so that it autonomously proliferates and expresses the gene, or incorporating the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene into the genomic DNA of the host microorganism by utilizing homologous recombination. However, the method of incorporating the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene into the genomic DNA of the host microorganism is preferred because there is a probability that the gene will be distributed during cell division.

[0049] Other molecular biological, biotechnological, and biochemical methods used to produce transformed microorganisms can be found in literature such as Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997.

[0050] One specific embodiment of the transformed microorganism is a transformed microorganism (1) in which the host microorganism is Corynebacterium glutamicum, and the SPj-S1H gene, PCi-S1H gene, LE-S1H gene, and / or DBa-S1H gene have been introduced as foreign genes and the transformed microorganism expresses the introduced genes. By using the transformed microorganism (1), SPj-S1H, PCi-S1H, LE-S1H, and / or DBa-S1H can be obtained, and furthermore, the cells, cell treatment products, and cell cultures of the transformed microorganism (1) can be used as SPj-S1H-containing products, PCi-S1H-containing products, LE-S1H-containing products, and / or DBa-S1H-containing products. Specific examples of the transformed microorganism (1) are described in the examples below as SPj-S1H-expressing transformed Corynebacterium microorganisms, PCi-S1H-expressing transformed Corynebacterium microorganisms, LE-S1H-expressing transformed Corynebacterium microorganisms, and DBa-S1H-expressing transformed Corynebacterium microorganisms.

[0051] [Method for producing hydroquinone] A method for producing hydroquinone according to one aspect of the present invention includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with SPj-S1H, PCi-S1H, LE-S1H and / or DBa-S1H.

[0052] A method for reacting p-hydroxybenzoic acid with SPj-S1H, PCi-S1H, LE-S1H, and / or DBa-S1H is any method in which p-hydroxybenzoic acid comes into contact with SPj-S1H, PCi-S1H, LE-S1H, and / or DBa-S1H so that hydroquinone can be produced and / or accumulated by SPj-S1H, PCi-S1H, LE-S1H, and / or DBa-S1H. For example, a method in which p-hydroxybenzoic acid reacts with SPj-S1H, PCi-S1H, LE-S1H, and / or DBa-S1H under conditions of standing, stirring, or shaking, with a pH of 5 to 9, a temperature of 20°C to 45°C, a duration of several minutes to several tens of hours.

[0053] When transformed microorganisms are used as SPj-S1H-containing, PCi-S1H-containing, LE-S1H-containing, and / or DBa-S1H-containing products, hydroquinone can be produced by culturing the transformed microorganisms under various culture conditions suitable for the transformed microorganisms, using a culture medium containing p-hydroxybenzoic acid and suitable for the growth of the transformed microorganisms. The culture method can be any method suitable for culturing the host microorganism. For example, if the host microorganism is a Corynebacterium, solid culture or liquid culture methods performed under aeration conditions can be used. Alternatively, the recovered transformed microorganisms may be suspended in a liquid medium containing p-hydroxybenzoic acid in the presence of flavin mononucleotide as a coenzyme and NADH as an electron donor, and incubated at a temperature of 20°C to 45°C for several minutes to several tens of hours.

[0054] The method for obtaining hydroquinone from the culture after the completion of cultivation is not particularly limited. For example, the reaction mixture can be subjected to conventional solid-liquid separation treatments such as filtration and centrifugation to separate the solids from the reaction mixture, and hydroquinone can be extracted from the recovered reaction mixture by solid-phase extraction using a column or solvent extraction using a hydroquinone-soluble solvent. The extraction solvent is not particularly limited as long as it is capable of dissolving hydroquinone, and examples include ethyl acetate and diethyl ether. Hydroquinone may also be obtained as a fractionated solution using a chromatograph such as a liquid chromatograph.

[0055] Qualitative or quantitative analysis of hydroquinone may be performed by HPLC as described in the examples below.

[0056] In the method for producing hydroquinone, various steps and operations can be added before, after, or during the above-described steps, as long as the objective of the present invention can be achieved.

[0057] [Uses of hydroquinone] Generally, hydroquinone is used as a cosmetic ingredient for its expected cosmetic effects on the skin, such as improving pigmentation like age spots, freckles, and sunburn, and promoting skin whitening. Hydroquinone obtained by hydroquinone manufacturing methods can also be used as a cosmetic ingredient as a type of skin whitening agent. Industrial uses include photographic developers, dyes, organic synthesis raw materials, pharmaceutical intermediates, antioxidants, and polymerization inhibitors. Hydroquinone can also be used as a raw material for these industrial materials.

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and can take various forms as long as it can solve the problems of the present invention. [Examples]

[0059] [1. Isolation of hydroquinone-producing enzymes] Hydroquinone-producing enzymes possess enzymatic activity that catalyzes the reaction that converts p-hydroxybenzoic acid (pHBA) to hydroquinone (HQ). Some of these are known as 4-hydroxybenzoate 1-hydroxylase or 4-hydroxybenzoate 1-monooxygenase. The EC number (Enzyme Commission number) for 4-hydroxybenzoate 1-hydroxylase is EC1.14.13.64.

[0060] The inventors searched for novel hydroquinone-producing enzymes and obtained more than 40 candidate proteins. They identified the nucleotide sequences encoding these candidate proteins and used them in microorganisms of the genus Corynebacter. Corynebacterium glutamicA nucleotide sequence optimized for the codon of ) was obtained. Based on the nucleotide sequence thus obtained, a DNA construct for transformation and a transformant were obtained as described later. Then, the 4-hydroxybenzoic acid 1-hydroxylase activity was evaluated using the wet bacterial cells of the obtained transformant. As a result, it was found that out of more than 40 candidate proteins, about 20 candidate proteins possessed 4-hydroxybenzoic acid 1-hydroxylase activity. Of these, proteins with amino acid sequences of SEQ ID NOs. 2 to 5 showed relatively high 4-hydroxybenzoic acid 1-hydroxylase activity.

[0061] Proteins with the amino acid sequence of Sequence ID No. 2 are from Spathaspora species. Spathaspora Derived from strain sp.)JA1, it was named SPj-S1H. SPj-S1H is presumed to be 3-hydroxybenzoate 6-hydroxylase. The gene encoding SPj-S1H is present on the genome of Spathaspora species JA1 strain and consists of the nucleotide sequence of SEQ ID NO: 6. The GenBank accession number version of SPj-S1H is RLV89829.1.

[0062] The protein having the amino acid sequence of SEQ ID NO: 3 is Pycnoporus cinnabarinus ( Pycnoporus cinnabar It was named PCi-S1H, derived from [the original source]. PCi-S1H is presumed to be a FAD-dependent monooxygenase. The gene encoding PCi-S1H is located on the genome of Pycnoporus cinavarinus and consists of the nucleotide sequence of SEQ ID NO: 7. The GenBank accession number version of PCi-S1H is CDO72572.1.

[0063] The protein having the amino acid sequence of SEQ ID NO: 4 is Rhoderomyces elongisporus ( Lodderomyces long-sporeIt was named LE-S1H, derived from ). LE-S1H is presumed to be a FAD-binding domain-containing protein. The gene encoding DBa-S1H is located on the genome of Rhoderomyces elongisporus and consists of the nucleotide sequence of SEQ ID NO: 8. The GenBank accession number version of LE-S1H is XP_001527239.2.

[0064] Proteins with the amino acid sequence of Sequence ID No. 5 are found in Deltaproteobacteria bacterium. Deltaproteobacteria bacteria It was named DBa-S1H, derived from [the original source]. DBa-S1H is presumed to be an FAD-binding domain-containing protein. The gene encoding DBa-S1H is located on the genome of Deltaproteobacteria bacterium and consists of the nucleotide sequence of sequence number 9. The GenBank accession number version of DBa-S1H is MBI3799240.1.

[0065] [2. Construction of the DNA construct] The nucleotide sequence encoding the SPj-S1H protein having the amino acid sequence of Sequence ID No. 2 was identified by a Corynebacterium ( Corynebacterium glutamic The nucleotide sequence of SEQ ID NO: 10 was obtained by optimizing for the codon of ). pMKsf vector containing the kanamycin resistance gene ( Corynebacterium glutamic A DNA construct for SPj-S1H expression was constructed by inserting the nucleotide sequence of SEQ ID NO: 10 into the multi-cloning site of the ATCC31808-derived shuttle vector pCG1 (US4617267A). Similarly, DNA constructs for PCi-S1H expression, LE-S1H expression, and DBa-S1H expression were obtained.

[0066] [3. The creation of transformed microorganisms] Corynebacteria distributed by the National Institute of Technology and Evaluation (NITE) Corynebacterium glutamic ATCC13032 (NBRC12168) was transformed by homologous recombination using electroporation and heat shock to obtain CT10 (ΔcglIM-IR-IIR, ΔpobA, ΔpcaHG, ΔgenH, Δpcaben), a transformed Coryne microorganism lacking the cgl gene, pobA gene, pcaHG gene, genH gene, and pcaben gene.

[0067] CT10 was subjected to transduction treatment using electroporation and heat shock with a DNA construct for SPj-S1H expression. After transduction treatment, CT10 was cultured on LB agar medium containing kanamycin at 32°C for 20-24 hours to select SPj-S1H-expressing Coryne microorganisms. Similarly, PCi-S1H-expressing Coryne microorganisms, LE-S1H-expressing Coryne microorganisms, and DBa-S1H-expressing Coryne microorganisms were selected.

[0068] On the other hand, Candida parapsis described in Non-Patent Documents 1 and 2 ( White parapsilosis The amino acid sequence (SEQ ID NO: 1) of 4-hydroxybenzoic acid 1-hydroxylase from strain CBS604 was obtained from GenBank with accession number version XP_036663424.1. This 4-hydroxybenzoic acid 1-hydroxylase was named CP-4HB1H. Following the same procedure as above, CP-4HB1H-expressing transformed Corynebacterium microorganisms were selected through codon optimization, DNA construct construction, and transformation.

[0069] [4. Evaluation of enzyme activity] The obtained SPj-S1H-expressing transformed Coryne microorganisms were inoculated into 2 ml of a growth medium prepared by mixing CGXIIa medium and LB medium in a 1:1 ratio. Subsequently, the inoculated growth medium was inoculated with 25 μg / mL of kanamycin and incubated overnight at 32°C with shaking at 250 rpm.

[0070] The composition of CGXIIa medium (pH 6.6~6.8) is 83.3 mM urea, 151.4 mM ammonium sulfate, 10 mM dipotassium hydrogen phosphate, 10 mM potassium dihydrogen phosphate, 10% glucose, 250 mg / L magnesium sulfate, 10 mg / L calcium chloride, 200 μg / L biotin, 0.1 mM ferrous sulfate, 10 mg / L manganese sulfate monohydrate, 1 mg / L zinc sulfate heptahydrate, 0.2 mg / L copper sulfate, and 0.02 mg / L nickel chloride hexahydrate.

[0071] The composition of LB medium (pH 7.0) is 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0072] The obtained culture solution was subjected to centrifugation (13,000 rpm, 2 minutes), and the cell was recovered by removing the supernatant. The obtained wet cell was added to CGXIIa medium containing 10 mM pHBA and thoroughly suspended, and subjected to the HQ conversion reaction at 37°C overnight.

[0073] The solution after the HQ conversion reaction was subjected to centrifugation (13,000 rpm, 2 minutes), and the supernatant was collected as the reaction solution. The HQ concentration of the obtained reaction solution was measured by HPLC analysis.

[0074] HPLC analysis was performed using a degasser "DGU-20A", liquid chromatograph "LC-20AR", autosampler "SIL-20AC", column oven "CTO-20AC", and detector "SPD-M20A" (all manufactured by Shimadzu Corporation). An Inertsil ODS-3 5μm column (diameter 4.6 mm, length 250 mm, particle size 5 μm, manufactured by GL Science) was used and maintained at 30°C. A gradient elution mode was used (solvent A: 0.1% (v / v) formic acid, solvent B: 100% (v / v) acetonitrile). After equilibration with solvent A, the proportion of solvent B was maintained at 30% for 2 minutes from the start of the analysis, then increased to 50% over 2 minutes, and then decreased to 30% over 1 minute. The mobile phase flow rate was set to 1.0 mL / min, the measurement wavelength for HQ was 290 nm, and the measurement wavelength for pHBA was 255 nm.

[0075] Similarly, HQ concentrations were measured using PCi-S1H-expressing transformed Coryneceae microorganisms, LE-S1H-expressing transformed Coryneceae microorganisms, DBa-S1H-expressing transformed Coryneceae microorganisms, and CP-4HB1H-expressing transformed Coryneceae microorganisms.

[0076] [5. Evaluation Results] Table 1 shows the results of enzyme activity and amino acid sequence identity (amino acid sequence homology) based on CP-4HB1H and CP-4HB1H-expressing transformed Coryne microorganisms. For reference, Table 1 also shows the relative activity and amino acid sequence identity of enzymes (Ake-S1H, SS-S1H, and AAr-S1H) obtained in the same manner from other microorganisms.

[0077] [Table 1]

[0078] When the HQ concentration was measured, the HQ concentration in the reaction solution obtained using CP-4HB1H-expressing transformed Coryne microorganisms was 0.42 g / L, while the HQ concentrations in the reaction solutions obtained using SPj-S1H-expressing transformed Coryne microorganisms, PCi-S1H-expressing transformed Coryne microorganisms, LE-S1H-expressing transformed Coryne microorganisms, and DBa-S1H-expressing transformed Coryne microorganisms were 0.58 g / L, 0.57 g / L, 0.46 g / L, and 0.45 g / L, respectively.

[0079] Therefore, when the HQ-converting enzyme activity of CP-4HB1H is set to 100%, the HQ-converting enzyme activities of SPj-S1H, PCi-S1H, LE-S1H, and DBa-S1H were 138%, 136%, 110%, and 107%, respectively, as shown in Table 1. In this way, we were able to obtain hydroquinone-producing enzymes with high enzymatic activity that catalyze the reaction to convert pHBA to HQ.

[0080] [6. Sequence Listing] The sequences listed in the sequence listing are shown in Tables 2A and 2B below.

[0081] [Table 2A]

[0082] [Table 2B] [Industrial applicability]

[0083] By utilizing the transformed microorganism and production method according to one aspect of the present invention, beneficial hydroquinones with physiological activity can be produced on an industrial scale from p-hydroxybenzoic acid through a biochemically or biologically safe and simple production method.

Claims

1. A method for producing hydroquinone, The process includes a step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from microorganisms, and The microorganism is at least one microorganism selected from the group consisting of microorganisms of the genera Spathaspora, Pycnoporus, Lodderomyces, and Deltaproteobacteria. The aforementioned method.

2. The method according to claim 1, wherein the enzyme has the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 80% or more sequence identity with any of the amino acid sequences of SEQ ID NOs. 2 to 5. (2) Amino acid sequences in any of Sequence IDs 2 to 5 in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids

3. A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, It contains as an active ingredient an enzyme having 1-hydroxylase activity of 4-hydroxybenzoic acid derived from microorganisms, and The microorganism is at least one microorganism selected from the group consisting of microorganisms of the genera Spathaspora, Pycnoporus, Lodderomyces, and Deltaproteobacteria. The aforementioned composition.

4. DNA fragments for transforming non-human host organisms, The gene for an enzyme possessing 1-hydroxylase activity of 4-hydroxybenzoic acid, derived from microorganisms, At least one gene different from the said gene and including, and The aforementioned microorganisms are selected from the group consisting of microorganisms of the genera Spathaspora, Pycnoporus, Lodderomyces, and Deltaproteobacteria. The aforementioned DNA fragment.

5. A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, It is formed by transducing a gene for an enzyme having 1-hydroxylase activity of 4-hydroxybenzoic acid derived from a microorganism as an exogenous gene, and The microorganism is at least one microorganism selected from the group consisting of microorganisms of the genera Spathaspora, Pycnoporus, Lodderomyces, and Deltaproteobacteria. The aforementioned transformed body.

6. The transformant according to claim 5, wherein the host organism is selected from the group consisting of microorganisms of the genera Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingovium, and Ralstonia.

7. The enzyme has the amino acid sequence of (1) or (2) below, and is a composition, DNA fragment or transformant according to any one of claims 3 to 6. (1) An amino acid sequence having 80% or more sequence identity with any of the amino acid sequences of SEQ ID NOs. 2 to 5. (2) Amino acid sequences in any of Sequence IDs 2 to 5 in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids

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