Method for producing hydroquinone

Abstract

The purpose of the present invention is to provide a method for producing hydroquinone that involves an enzyme that can convert p-hydroxybenzoic acid into hydroquinone at higher efficiencies than the 4-hydroxybenzoate 1-hydroxylase possessed by Candida parapsilosis strain CBS604. This purpose is achieved, for example, by a method for producing hydroquinone, the method comprising a step for obtaining hydroquinone by reacting p-hydroxybenzoic acid with a 4-hydroxybenzoate 1-hydroxylase derived from Spathaspora passalidarum.

Description

Hydroquinone manufacturing method

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

[0002] Hydroquinone is a divalent phenol with a structure in which the 1st and 4th positions of the benzene ring are substituted with hydroxyl groups (-OH). Hydroquinone is used as a cosmetic ingredient and is a type of skin whitening agent.

[0003] Hydroquinone has a structure similar to tyrosine and can function as a tyrosine analog. This allows hydroquinone to inhibit tyrosinase activity by binding to tyrosinase in opposition to tyrosine. Since melanin biosynthesis is driven by tyrosinase activity, inhibiting tyrosinase activity by hydroquinone suppresses melanin biosynthesis. As a result, hydroquinone can exhibit cosmetic effects on the skin, such as improving pigmentation like age spots, freckles, and sunburn, and promoting skin whitening.

[0004] Hydroquinone is primarily produced through chemical synthesis. However, this method has several drawbacks, including the use of substances harmful to the human body, such as organic solvents and inorganic metal catalysts; the difficulty in separating the hydroquinone product from the raw materials and by-products; and the inclusion of expensive raw materials that are not approved as cosmetic ingredients. Therefore, there is a need for a safe and simple biochemical or biological method of producing hydroquinone.

[0005] One type of hydroquinone-producing enzyme is 4-hydroxybenzoic acid 1-hydroxylase, which catalyzes the reaction that converts p-hydroxybenzoic acid to hydroquinone. Candida parapsilosis CBS604 strain is known as a microorganism that expresses 4-hydroxybenzoic acid 1-hydroxylase (see, for example, Non-Patent Documents 1 and 2). Non-Patent Document 3 describes the amino acid sequence derived from Spasaspora passaridarum.

[0006] MH Eppinket al, J Bacteriol., 1997 Nov; 179 (21): pages 6680-6687. Willem JH Van Berkel et al, FEMS Microbiology Letters, Volume 121, Issue 2, August 1994, Pages 207-215. Accession No. XM_007376104.1, NCBI[online], December 29, 2023, https: / / www.ncbi.nlm.nih.gov / nuccore / XM_007376104.1

[0007] However, the inventors have found that the 4-hydroxybenzoic acid 1-hydroxylase present in Candida parapsis strain CBS604 has low catalytic activity, resulting in a very slow conversion reaction from p-hydroxybenzoic acid to hydroquinone. Non-patent document 1 only discloses the amino acid sequence, and does not disclose the reactivity of the enzyme encoded by that amino acid sequence.

[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.

[0010] In an attempt to solve the above problems, the inventors diligently investigated novel hydroquinone-producing enzymes. As a result, they discovered that the Spathaspora passalidarum strain NRRL Y-27907 expresses a protein having 4-hydroxybenzoic acid 1-hydroxylase activity.

[0011] Then, when we created transformed microorganisms expressing the protein and evaluated their 4-hydroxybenzoic acid 1-hydroxylase activity, we were surprised to find that the 4-hydroxybenzoic acid 1-hydroxylase activity of the protein was significantly higher than that of the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis CBS604 strain.

[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] Accordingly, according to each aspect of the present invention, the following embodiments are provided: [I-1] A method for producing hydroquinone, comprising the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum. [I-2] The method according to item [1], wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum has the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which, in the amino acid sequence of Sequence ID No. 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [I-3] A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, comprising an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum as an active ingredient. [I-4] A DNA fragment for transforming a host organism other than a human, comprising a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum and at least one gene different from said gene. [I-5] A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, wherein a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum is introduced as an exogenous gene. [I-6] The transformant according to item [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.[I-7] The enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum is a composition, DNA fragment or transformant according to any one of items [3] to [6], having the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which, in the amino acid sequence of Sequence ID No. 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [II-1] A method for producing hydroquinone, comprising the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum, wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum has the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which, in the amino acid sequence of Sequence ID No. 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [II-2] A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, comprising an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum as an active ingredient, wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum has the amino acid sequence of (1) or (2) below.(1) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which, in the amino acid sequence of Sequence ID No. 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [II-3] A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, wherein the transformant is transfected by introducing a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum as an exogenous gene, and the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum has the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which, in the amino acid sequence of Sequence ID No. 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [II-4] The transformant described in item [II-3], 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. [III-1] An enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum, having the amino acid sequence of (1) or (2) below. (1) An amino acid sequence having 80% or more but less than 100% sequence identity with the amino acid sequence of Sequence ID No. 2. (2) An amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids in the amino acid sequence of Sequence ID No. 2. [III-2] An enzyme described in item [III-1] having at least one property selected from the group consisting of (A) to (E) below. (A) Vmax value: 13 μmol / min / mg (B) Km value: 70 μM (C) kcat value: 11 s. －１ (D) Optimal temperature: 37°C to 40°C (E) Optimal pH: around 7

[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.

[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] "Includes" means that elements other than those explicitly included can be added (synonymous with "at least include"), but it also includes "consists of" and "essentially consists of." That is, "includes" can mean including the explicitly included elements and any one or more of those elements, consisting of the explicitly included elements, or essentially consisting of the explicitly included elements. Examples of elements include components, processes, conditions, parameters, and other limitations. "Has" is synonymous with "includes." The terms "and / or" mean any one of the multiple related items listed, any combination of two or more, or all of them. The "~" in a numerical range means a range that includes the numbers before and after it, and also includes the range excluding one of the limit values ​​in which they are included. For example, "0% to 100%" could be 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 one significant figure, and 10 has two 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 one significant figure, and 0.10 has two significant figures.

[0018] "Foreign gene" refers to a gene that is not naturally occurring on the chromosome (genomic) DNA of the organism being introduced (host organism), and is also called a heterogene. Foreign genes include genes that are inserted at a different location than their naturally occurring position, even if they are naturally present on the chromosome DNA. In this specification, genome and chromosome are synonymous. "Gene expression" means the production of proteins (gene-encoded proteins) having the amino acid sequence encoded by part or all of the nucleotide sequence of a gene, through transcription, translation, etc., so that they possess their original structure and activity. "Gene overexpression" means that, as a result of gene introduction, the host organism produces a protein encoded by that gene in an amount exceeding its naturally expressed level. "Wild-type" refers to naturally occurring organisms that have not been artificially genetically modified. "Transformed organism" refers to an organism that has been artificially genetically modified. "Wild-type gene" refers to a gene naturally present on the genomic DNA of a wild-type organism. "Wild-type protein" refers to a protein encoded by a wild-type gene. 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 Spathaspora passalidarum. In this specification, the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum may be called SP-S1H, and the gene encoding the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum may be called the SP-S1H gene. In addition, either "Hydro" or "Hydro" is used as the Japanese reading of the English word "hydro".

[0020] Another aspect of the present invention is a hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid. One embodiment of the present invention comprises SP-S1H as an active ingredient.

[0021] SP-S1H used in each aspect of the present invention may be an isolated and purified enzyme, or it may be an enzyme-containing material such as a living organism, a biologically processed product, or a biological culture of a transformant transformed to express Spasaspora passaridarum or SP-S1H. Furthermore, SP-S1H may be synthesized using a cell-free protein synthesis system with the SP-S1H gene, which is the gene encoding SP-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 embodiment of the present invention comprises the SP-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. The transformant according to one embodiment of the present invention is obtained by transduction of the SP-S1H gene as an exogenous gene.

[0024] [Enzyme with 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum (SP-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). Specifically, p-hydroxybenzoic acid, NAD(P)H and H ＋ and O ２ Therefore, hydroquinone and NAD(P) ＋ and H ２ O and CO ２It catalyzes the reaction that produces (see https: / / www.brenda-enzymes.org / enzyme.php?ecno=1.14.13.64). After diligent investigation, it was found that when salicylate hydroxylase from Spathaspora passaridarum is treated with 4-hydroxybenzoic acid, the same reaction as in scheme (1) occurs. In other words, it was found that salicylate hydroxylase from Spathaspora passaridarum is an enzyme that possesses 4-hydroxybenzoic acid 1-hydroxylase activity.

[0025] A known example of 4-hydroxybenzoic acid 1-hydroxylase is the 4-hydroxybenzoic acid 1-hydroxylase found in Candida parapsilosis strain CBS604, as described in Non-Patent Documents 1 and 2. SP-S1H possesses hydroxylase activity, yet its amino acid sequence identity with that of Candida parapsilosis CBS604 is 62%. Therefore, SP-S1H may be an enzyme having hydroxylase activity and an amino acid sequence that has 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 of the amino acid sequence identity with the wild-type enzyme of SP-S1H (SEQ ID NO: 2). The upper limit may be 100% or less, or less than 100%, specifically 99% or less, 98% or less, 97% or less, 95% or less, 90% or less, etc. However, SP-S1H with less than 100% amino acid sequence identity with the wild-type enzyme is not a wild-type enzyme.

[0026] There are no particular limitations on the method for determining 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] As a program for calculating the degree of agreement between two amino acid sequences, for example, the algorithm by Karlin and Altschul (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 National Center for Biotechnology Information in the United States (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 to 30 amino acids, preferably 1 to 20, and more preferably 1 to 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 added to the sequence by insertion.

[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] SP-S1H may have a tag peptide that facilitates 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 SP-S1H using genetic engineering technology, depending on the host organism used, it may exist as an intracellular enzyme, making enzyme isolation and purification difficult. Therefore, when attempting to obtain recombinant SP-S1H, it is preferable to modify the recombinant SP-S1H to become an extracellular enzyme. However, if recombinant SP-S1H is expressed as an extracellular enzyme in the transformant, extracellular enzyme modification is not necessary.

[0032] Methods for extracellular enzymatic modification of recombinant SP-S1H include, but are not limited to, adding secretory signals such as peptides or proteins that function to cause recombinant SP-S1H to be expressed as an extracellular enzyme to the C-terminal and / or N-terminal side of recombinant SP-S1H. Even if recombinant SP-S1H has a different amino acid sequence from the wild-type protein, its enzyme activity may be comparable to that of the wild-type protein, for example, with a Vmax value of 13 μmol / min / mg, a Km value of 70 μM, and a kcat value of 11s. －１ The optimal temperature is 37°C to 40°C, and / or the optimal pH is around 7. These parameters can be measured by the method described in the examples below.

[0033] A specific embodiment of SP-S1H is an SP-S1H having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. Another specific embodiment of SP-S1H is an SP-S1H having an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids.

[0034] [Genesis of an enzyme with 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum (SP-S1H gene)] The SP-S1H gene has a nucleotide sequence that codes for the amino acid sequence of SP-S1H and expresses SP-S1H. These nucleotide sequences in the SP-S1H gene may be a coding sequence (CDS) or an open reading frame (ORF), but are preferably ORFs.

[0035] The SP-S1H gene may consist of a nucleotide sequence that codes for SP-S1H, or it 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 SP-S1H gene may include a nucleotide sequence optimized for codons, secondary structure, GC content, etc., by utilizing several types of codons corresponding to one amino acid. The nucleotide sequence subjected to codon modification is preferably, for example, a nucleotide sequence subjected to codon modification so as to be easily expressed in a host organism.

[0037] The wild-type gene of the SP-S1H gene has the nucleotide sequence of SEQ ID NO: 3. Specific embodiments of the SP-S1H gene include a gene having the nucleotide sequence of SEQ ID NO: 4 in which the wild-type gene is codon-optimized for Corynebacterium microorganisms.

[0038] By introducing the SP-S1H gene into a host organism as a foreign gene, a host organism that could not originally express SP-S1H can be transformed to express SP-S1H as a recombinant enzyme. The expression of the SP-S1H gene may be carried out autonomously by the transcriptional control sequence possessed by the gene, or may be carried out using the transcriptional control mechanism of the host organism, but is preferably carried out autonomously by the transcriptional control sequence possessed by the gene.

[0039] In order for the SP-S1H gene to be expressed autonomously in a host organism, it preferably includes a promoter, an ORF, and a terminator. The promoter and the terminator can be appropriately selected according to the host organism.

[0040] [DNA Fragment] The DNA fragment of one embodiment of the present invention includes at least one gene different from the SP-S1H gene in addition to the SP-S1H gene. In the DNA fragment, the copy number of the SP-S1H gene may be any, and may be 1 copy or 2 or more copies.

[0041] The gene different from the SP-S1H gene can be any gene that codes for a protein different from SP-S1H, and may, for example, be a selection marker gene 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 may also be other selection marker genes such as nutritional requirement marker genes. The gene different from the SP-S1H gene may not have a protein-coding nucleotide sequence and may consist of, for example, a transcriptional regulatory sequence or a non-coding sequence.

[0042] The DNA fragment may also be a DNA construct such as a plasmid or vector. A specific example of such a DNA construct is the SP-S1H expression DNA construct described in the examples below.

[0043] [Transformant] A transformant according to one aspect of the present invention is obtained by transduction of the SP-S1H gene as an exogenous gene. The transformant has the SP-S1H gene introduced as an exogenous gene and expresses or overexpresses the introduced gene.

[0044] Transformed organisms are created by introducing the SP-S1H gene as an exogenous gene into a host organism.

[0045] The host organism is not particularly limited as long as it is an organism that can be introduced with the SP-S1H gene as an exogenous gene and can express the introduced SP-S1H gene, for example, 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. A transformed microorganism may also be obtained by introducing the SP-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 SP-S1H gene into a host microorganism is not particularly limited. Examples include introducing a DNA construct, such as a plasmid vector incorporating the SP-S1H gene, into the host microorganism so that it autonomously proliferates and expresses the gene, or incorporating the SP-S1H gene into the host microorganism's genomic DNA by utilizing homologous recombination. However, the method of incorporating the SP-S1H gene into the host microorganism's genomic DNA 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, the SP-S1H gene is introduced as an exogenous gene, and the transformed microorganism (1) expresses the introduced gene. By using the transformed microorganism (1), SP-S1H can be obtained, and furthermore, the cells, cell treatment products, and cell cultures of the transformed microorganism (1) can be used as SP-S1H-containing products. A specific example of the transformed microorganism (1) is the SP-S1H-expressing transformed Corynebacterium microorganism described in the examples below.

[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 SP-S1H.

[0052] The method for reacting p-hydroxybenzoic acid with SP-S1H is any method in which p-hydroxybenzoic acid and SP-S1H come into contact and hydroquinone is produced and / or accumulated by SP-S1H. For example, a method in which p-hydroxybenzoic acid reacts with SP-S1H under conditions of pH 5 to 9, temperature 20°C to 45°C, duration of several minutes to several tens of hours, and with standing, stirring, or shaking.

[0053] When transformed microorganisms are used as SP-S1H-containing materials, 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] Hydroquinone is generally 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.

[0059] [1. Isolation of Hydroquinone-Producing Enzymes] Hydroquinone-producing enzymes have enzymatic activity that catalyzes the reaction that converts p-hydroxybenzoic acid (pHBA) to hydroquinone (HQ), and 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 a novel hydroquinone-producing enzyme and obtained the SP-S1H protein, which has the amino acid sequence of SEQ ID NO: 2, as a candidate enzyme. The gene encoding SP-S1H is present in the genome of Spathaspora passalidarum NRRL Y-27907 strain and consists of the nucleotide sequence of SEQ ID NO: 3. The GenBank accession number version of SP-S1H is XP_007376166.1.

[0061] [2. Construction of the DNA construct for SP-S1H expression] The SP-S1H protein having the amino acid sequence of SEQ ID NO: 2 was optimized for the codons of Corynebacterium glutamicum to obtain the nucleotide sequence of SEQ ID NO: 4. The DNA construct for SP-S1H expression was constructed by inserting the nucleotide sequence of SEQ ID NO: 4 into the multi-cloning site of a pMKsf vector containing a kanamycin resistance gene (modified shuttle vector pCG1 (US4617267A) derived from Corynebacterium glutamicum ATCC31808).

[0062] [3. Preparation of SP-S1H-expressing transformed microorganisms] Corynebacterium glutamicum ATCC13032 (NBRC12168) obtained from the National Institute of Technology and Evaluation (NITE) was transformed by homologous recombination using electroporation and heat shock to obtain CT10 (ΔcglIM-IR-IIR, ΔpobA, ΔpcaHG, ΔgenH, Δpcaben), a transformed Corynebacterium microorganism lacking the cgl gene, pobA gene, pcaHG gene, genH gene, and pcaben gene.

[0063] CT10 was subjected to transduction treatment using electroporation and heat shock with a DNA construct for SP-S1H expression. After transduction treatment, CT10 was cultured on LB agar medium containing kanamycin at 32°C for 20 to 24 hours to select SP-S1H-expressing transformed Coryne microorganisms.

[0064] On the other hand, the amino acid sequence (SEQ ID NO: 1) of 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsilosis strain CBS604, described in Non-Patent Documents 1 and 2, was obtained from GenBank with accession number version XP_036663424.1. This 4-hydroxybenzoic acid 1-hydroxylase was named CP-4HB1H. In the same manner as above, CP-4HB1H-expressing transformed Coryne microorganisms were selected through codon optimization, DNA construct construction, and transformation. The amino acid sequence identity between SP-S1H and CP-4HB1H was 62%.

[0065] [4. Activity Evaluation of SP-S1H] The obtained SP-S1H-expressing transformed Coryne microorganisms and CP-4HB1H-expressing transformed Coryne microorganisms were inoculated into 2 ml of growth medium prepared by mixing CGXIIa medium and LB medium in a 1:1 ratio. Next, the growth medium after inoculation was inoculated with 25 μg / mL of kanamycin and incubated overnight at 32°C and 250 rpm with shaking.

[0066] 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.

[0067] The composition of LB medium (pH 7.0) is 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0068] The obtained culture medium 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.

[0069] 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.

[0070] 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.

[0071] [5. Evaluation Results] 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 concentration in the reaction solution obtained using SP-S1H-expressing transformed Coryne microorganisms was 0.90 g / L.

[0072] Therefore, if the HQ-converting enzyme activity of CP-4HB1H is taken as 100%, the HQ-converting enzyme activity of SP-S1H was 215%. In this way, we were able to obtain a hydroquinone-producing enzyme with very high enzymatic activity that catalyzes the reaction that converts pHBA to HQ.

[0073] [6. Production of His-tagged SP-S1H] Escherichia coli BL21 (DE3) strain was transformed using an expression vector (pET-28a(+)) encoding the SP-S1H gene (SEQ ID NO: 5) optimized for Escherichia coli codons. The expression vector was designed to have six histidine residues added to its N-terminus.

[0074] The obtained transformant was selected by culturing it in LB medium containing kanamycin (final concentration 50 μg / mL). A single colony of the selected transformant was inoculated into 2 mL of the above medium and subjected to overnight preculture at 37 °C under shaking conditions of 250 rpm.

[0075] The preculture broth was inoculated into 100 ml of LB medium so that the absorbance OD ６００ｎｍ would be 0.1. The inoculated medium was subjected to shaking culture at 37 °C at 120 rpm. When the absorbance OD ６００ｎｍ of the culture broth reached 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture broth so that the final concentration would be 0.1 mM. By setting the temperature of the culture broth to 20 °C, induction of traits was performed for the transformant. The culture was continued overnight at 20 °C.

[0076] After completion of the culture, the cells were subjected to centrifugation at 4,000 g for 10 minutes at 4 °C to recover the cells. 0.15 g of wet cells were suspended in 5 ml of 10 mM phosphate buffer (pH 7.4, containing 2 mM PMSF; Phenylmethylsulfonyl fluoride). 10% (w / v) Tween 20 was added to the obtained suspension so that the final concentration would be 2% (w / v). After subjecting the obtained mixture to inversion mixing treatment, it was allowed to stand on ice for 30 minutes. Next, the ice-cooled mixture was subjected to three freeze-thaw treatments (a treatment of rapidly freezing the mixture in liquid nitrogen and then thawing it at room temperature). The obtained extract was subjected to centrifugation at 15,000 rpm for 10 minutes at 4 °C to recover the supernatant. The recovered supernatant was used as the water-soluble fraction.

[0077] The water-soluble fraction was passed through a Ni-NTA affinity column (His trap FF 1 ml) to purify the His-tag fusion protein. The column was pre-equilibrated with an equilibration / binding buffer (20 mM NaH ２ PO ４ (pH 7.4), 500 mM NaCl and 20 mM imidazole). After passing the water-soluble fraction through the column, the column was thoroughly washed by passing the same buffer through it to remove non-specifically bound proteins. The target protein was eluted with an elution buffer (20 mM NaH ２ PO４ Elution occurred with 500 mM NaCl and 500 mM imidazole (pH 7.4).

[0078] Figure 1 shows the results of SDS-PAGE treatment of the eluted fractions. As shown in Figure 1, a single band was observed at approximately 50.3 kDa in eluted fractions Elution 1 and Elution 2. SP-S1H consists of 447 amino acids and has a molecular weight of 50.3 kDa. The eluted fractions in which the single band was observed were desalted with Amicon Ultra (10 K, 0.5 ml) and replaced with a storage buffer containing 50 mM Tris-HCl (pH 7.5) and 10% (w / v) glycerol. The resulting purified protein was stored at -20°C or -80°C.

[0079] [7. Enzyme activity evaluation of His-tagged SP-S1H] The activity of His-tagged SP-S1H in reducing p-hydroxybenzoic acid (pHBA) to hydroquinone (HQ) was measured using the following method.

[0080] The His-tagged SP-S1H obtained in step 6 above was dissolved in Tris-HCl buffer (50 mM, pH 7.5) to prepare the test sample (final enzyme concentration 10 nM). pHBA (final concentration 0.05-10 mM) was added to the test sample as a substrate, and NADH (final concentration 0.2 mM) and FAD (final concentration 10 μM) were added as coenzymes. The final volume of the reaction solution was 1 mL, and the measurement was performed at a temperature of 37°C.

[0081] The reaction was tracked by measuring the change in absorbance at a wavelength of 340 nm over time using a UV-Vis spectrophotometer. The reaction was initiated by adding an enzyme, and the rate of decrease of NADH in the reaction solution was calculated from the initial linear region (approximately 30 to 120 seconds after initiation). The molar extinction coefficient of NADH was 6.22 mM. －１ ・cm －１ The consumption rate of NADH (μmol / min) was determined using [a specific method / tool]. From the obtained initial velocity value, the specific activity (μmol / min / mg) was calculated.

[0082] The initial velocity was measured with a variable substrate concentration, and the obtained data was analyzed using a nonlinear least squares method based on the Michaelis-Menten equation to determine the Km, Vmax, and kcat values ​​for the substrate.

[0083] The amount of HQ, the reaction product, was quantified by HPLC analysis. After a predetermined time, the reaction solution (500 μL) was collected, and the reaction was stopped by adding acetonitrile + 0.1% (v / v) formic acid (500 μL). The resulting solution was subjected to centrifugation at room temperature (15,000 × g, 5 minutes), and the recovered supernatant was subjected to HPLC analysis. The HPLC analysis was performed using the method described in 4 above.

[0084] Figure 2 shows the S-V plot and the 1 / S-1 / V plot for substrate concentrations between 0.05 mM and 1 mM. From the graphs in Figure 2, the Vmax value of His-tagged SP-S1H is 13 μmol / min / mg, the Km value is 70 μM, and the kcat value is 11 s －１ It was found that...

[0085] [8. Optimal Temperature Evaluation of His-Tag Fusion SP-S1H] The optimal temperature for His-Tag Fusion SP-S1H was evaluated using the reaction system described in section 7 above, with the reaction temperature set to 25°C to 70°C.

[0086] Specifically, enzyme (10 nM) was added to a reaction mixture containing pHBA (0.5 mM), NADH (0.20 mM), and FAD (10 μM) in 50 mM Tris-HCl (pH 7.5) buffer, and the reaction was carried out at a temperature of 37°C. The absorbance at 340 nm was measured, and the relative activity was calculated by comparing the initial velocity values ​​obtained under each temperature condition.

[0087] Figure 3 shows the activity measurement results for His-tagged SP-S1H at different temperatures. As shown in Figure 3, the optimal temperature for His-tagged SP-S1H was found to be 37°C to 40°C.

[0088] [9. Optimal pH Evaluation of His-Tag Fusion SP-S1H] The optimal pH of His-Tag Fusion SP-S1H was evaluated by appropriately changing the buffer solution so that the pH value was between 4.0 and 9.0, using the reaction conditions described in section 7 above.

[0089] Acetate buffer (50 mM) was used when the pH value was in the range of 4.0 to 6.0, and Tris-HCl buffer (50 mM) was used when the pH value was in the range of 7.0 to 9.0. An enzyme (10 nM) was added to a reaction solution containing pHBA (0.5 mM), NADH (0.20 mM), and FAD (10 μM) in the buffer, and the reaction was carried out at 37°C.

[0090] A 500 μL reaction solution was collected after a predetermined time (within the range where the linearity of the initial reaction is maintained, usually 30 to 120 seconds), and the reaction was stopped by adding acetonitrile + 0.1% (v / v) formic acid (500 μL). The resulting solution was subjected to centrifugation at room temperature (15,000 × g, 5 minutes). The recovered supernatant was subjected to HPLC analysis, and the HQ concentration was quantified by HPLC. The relative activity was calculated from the HQ concentration and compared with the maximum activity set to 100%.

[0091] Figure 4 shows the activity measurement results for His-tagged SP-S1H at different pH levels. As shown in Figure 4, the optimal pH for His-tagged SP-S1H was found to be around 7.

[0092] [10. Sequence Listing] The sequences listed in the sequence listing are as shown in Table 1 below.

[0093]

[0094] 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.

[0095] Figure 1 is a gel photograph obtained by subjecting the eluted fraction containing His-tagged SP-S1H to SDS-PAGE, as described in the examples below. Figure 2 is a diagram showing the S-V plot and 1 / S-1 / V plot for His-tagged SP-S1H, as described in the examples below. Figure 3 shows the activity measurement results for His-tagged SP-S1H at different temperatures, as described in the examples below. Figure 4 shows the activity measurement results for His-tagged SP-S1H at different pH levels, as described in the examples below. Cross-reference of related applications

[0096] This application claims priority to Japanese Patent Application No. 2024-221622, filed on 18 December 2024, the entirety of which is incorporated herein by reference. Furthermore, the entirety of all documents referenced in the detailed description of the invention of this application, including Non-Patent Documents 1 to 3, is incorporated herein by reference.

Claims

1. A method for producing hydroquinone, comprising the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum.

2. The method according to claim 1, wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum has the amino acid sequence of (1) or (2) below: (1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (2) An amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 2, 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, comprising an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum as an active ingredient.

4. A DNA fragment for transforming a non-human host organism, comprising a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum, and at least one gene different from said gene.

5. A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, wherein the transformant is transfected by introducing a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum as an exogenous gene.

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 having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum has the amino acid sequence of (1) or (2) below, as described in any one of claims 3 to 6, the 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 the amino acid sequence of SEQ ID NO: 2; (2) an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 2, 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids.

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