JP2020022377A - High acid-resistant microorganism having glucaric acid-producing ability, and method for producing glucaric acid using the same - Google Patents

Abstract

To suppress the amount of neutralizer added during fermentation production, and improve the efficiency of purification, in fermentative production of glucaric acid.SOLUTION: Glucaric acid is produced by allowing highly acid-resistant microorganisms or their treated products to act on organic raw materials in an aqueous medium having a pH of 5 or less, the highly acid-resistant microorganisms or their treated products having myo-inositol-1-phosphate synthase activity, myo-inositol monophosphatase activity, myo-inositol oxygenase activity, and uronic acid dehydrogenase activity.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a highly acid-resistant microorganism having a glucaric acid-producing ability, and a method for producing glucaric acid using the same.

  Glucaric acid (tetrahydroxyadipic acid) is an acidic saccharide contained in apple, grapefruit, broccoli and the like. Outside Japan, it is used as a functional food material utilizing its β-glucuronidase inhibitory action, as an antioxidant, a chelating agent and the like. In 2004, the U.S. Department of Energy specified it as one of 12 types of biorefinery basic substances.

  Glucaric acid has wide versatility as an industrial intermediate material such as a polymer and a surfactant. In particular, biomass-derived basic compounds that can be derived into various useful chemicals, such as 2,5-furandicarboxylic acid (FDCA), which is attracting attention as a new polymer raw material, and a raw material for 2,5-furandicarboxylic acid diester It is expected as.

It has been known that glucaric acid can be synthesized by a nitric acid oxidation reaction of starch or a catalytic oxidation reaction in the presence of a basic bleaching agent.
In recent years, attempts have been made to biosynthesize glucaric acid from common organic raw materials such as glucose using bacteria such as Escherichia coli or yeast belonging to the genus Pichia (Patent Document 1, Non-Patent Document 1).

WO 2013/125509 pamphlet JP 2012-61006 A

Ye Liua, et al., Enzyme and Microbial Technology, 91 (2016) 8-16

  When glucaric acid is produced by microbial fermentation, the pH of the fermented solution decreases with the accumulation of glucaric acid. Generally, the growth of microorganisms and fermentation production capacity are maximized near neutrality, so a large amount of neutralizing agent is added to the fermentation liquor to maintain the pH at neutral, which causes an increase in cost . In fact, even in the methods described in Patent Literature 1 and Non-Patent Literature 1, the acidity of the fermentation liquor is maintained near neutral or pH 5.5 by the neutralizing agent. In addition, glucaric acid produced by fermentation near neutrality is dissolved in the fermentation broth in the form of salt, and in order to obtain free glucaric acid from this state, the property that the solubility of glucaric acid decreases under acidic conditions. It is necessary to perform acid crystallization for purification, and the cost increase due to the increase in the number of process steps and the cost increase due to the use of a neutralizing agent are recognized as issues in achieving industrial production using the fermentation method. ing.

  By the way, regarding lactic acid biosynthesis, a method of fermenting in a low pH environment (around pH 3) without neutralization using acid-resistant Saccharomyces yeast is disclosed (Patent Document 2). In such a method, although the purification load can be reduced, the fermentation results are lower than in the case where a neutralizing agent is added.

  In view of such a situation, an object of the present invention is to suppress the amount of a neutralizing agent added during fermentation production and improve purification efficiency in fermentative production of glucaric acid.

  The present inventors have conducted intensive studies in order to solve the above problems, and as a result, have found that glucaric acid can be fermentatively produced under strongly acidic conditions by using a highly acid-resistant microorganism imparted with glucaric acid-producing ability. . Then, the present inventors have found that Candida yeast has excellent high acid resistance performance, and efficient glucaric acid fermentation production is realized by a transformant having myo-inositol oxygenase activity and uronic acid dehydrogenase activity using the yeast as a host. And completed the present invention.

That is, according to the present invention, the following inventions are provided.
[1] A highly acid-resistant microorganism having myo-inositol-1-phosphate synthase activity, myo-inositol monophosphatase activity, myo-inositol oxygenase activity, and uronic acid dehydrogenase activity.
[2] The microorganism according to [1], wherein the highly acid-resistant microorganism is a yeast of the genus Candida.
[3] The microorganism according to [2], wherein the highly acid-resistant microorganism is Candida boidinii.
[4] The microorganism according to any one of [1] to [3], which expresses an enzyme having myo-inositol oxygenase activity and an enzyme having uronate dehydrogenase activity as a fusion protein.
[5] The microorganism according to any one of [1] to [4], which expresses an enzyme having myo-inositol oxygenase activity as a solubilized protein.
[6] A method for producing glucaric acid, comprising a step of allowing the microorganism according to any one of [1] to [5] or a processed product thereof to act on an organic material in an aqueous medium having a pH of 5 or less.
[7] The production method according to [6], wherein the organic raw material contains at least one selected from the group consisting of glucose, xylose, sucrose, starch, molasses, myo-inositol, glycerol, ribitol, and erythritol.
[8] The production method according to [6] or [7], wherein the aqueous medium has a pH of 3.5 or less.
[9] The production method according to any one of [6] to [8], wherein the aqueous medium has a pH of 2.5 or more.
[10] The production method according to any one of [6] to [9], which does not include a step of neutralizing the aqueous medium.
[11] The production method according to any one of [6] to [10], wherein the crystals are precipitated while generating glucaric acid in the aqueous medium.
[12] A step of producing glucaric acid by the method according to any one of [6] to [11], and a step of converting glucaric acid obtained in the above step as a raw material into 2,5-furandicarboxylic acid. And a method for producing 2,5-furandicarboxylic acid.
[13] The step of producing glucaric acid by the method according to any one of [6] to [11], and the step of converting glucaric acid obtained in the above step into a 2,5-furandicarboxylic acid diester as a raw material. A method for producing a 2,5-furandicarboxylic acid diester.

  According to the present invention, glucaric acid can be efficiently produced from common organic raw materials such as glucose and galactose while suppressing by-products.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the biosynthesis pathway of glucaric acid. It is a graph which shows growth of various yeasts under pH3 conditions. 7 is a graph showing the change over time in the amount of inositol accumulation in Example 2. It is a construction scheme of a plasmid for expressing MIOX-Udh fusion protein. It is a construction scheme of a plasmid for expressing His-tagged MIOX-Udh fusion protein. It is a construction scheme of a plasmid for superfolderGFP expression. 9 is a graph showing the change over time in the amount of accumulated glucaric acid in Example 4.

  Hereinafter, embodiments of the present invention will be described in detail.

<Microorganism of the present invention>
The microorganism of the present invention is a highly acid-resistant microorganism having myo-inositol-1-phosphate synthase activity, myo-inositol monophosphatase activity, myo-inositol oxygenase activity, and uronic acid dehydrogenase activity.

As used herein, the term "highly acid-resistant microorganism" refers to a microorganism having high growth ability and metabolic ability even under low pH conditions. More specifically, in a 200 mL Erlenmeyer flask, 20 mL of a YPD medium (1% YEAST EXTRACT, 2% HIPOLYPEPTON, 2% glucose) of pH 3 was added at 30 ° C. for 72 hours without depleting glucose. The OD ₆₆₀ at the time of rotating and shaking culture at a shaking speed of 180 rpm is 5 or more, preferably 10 or more, more preferably 40 or more, and still more preferably 50 or more as a relative value when 1 at 0 hour. Microorganism.
In the present invention, by imparting glucaric acid-producing ability to highly acid-resistant microorganisms, glucaric acid fermentation under low pH conditions becomes possible, and as a result, generation of by-products is suppressed and purification load is reduced. Efficient glucaric acid production is realized.

The microorganism of the present invention can be prepared by genetic engineering using a host microorganism.
The host microorganism is not particularly limited as long as it is highly acid-resistant. For example, yeast, Escherichia coli, coryneform bacteria, Bacillus bacteria, Lactobacillus bacteria, Actinobacillus bacteria, Pseudomonas ( Pseudomonas) bacteria, filamentous fungi and the like.

  Among them, yeast is more preferable, and examples thereof include yeast of the genus Candida, yeast of the genus Kluyveromyces, yeast of the genus Pichia, and yeast of the genus Saccharomyces. Further, among these, yeasts of the genus Candida are preferred because they are more excellent in acid resistance, and examples thereof include Candida boidinii, Candida parapsilosis, Candida glabrata and the like. Furthermore, Candida boidiny is particularly preferred because the genome sequence has been analyzed in detail and a genetic recombination technique has been established.

  The microorganism may be a wild-type strain, a mutant strain obtained by ordinary mutation treatment such as UV irradiation or NTG treatment, or a recombinant strain derived by a genetic technique such as cell fusion or gene recombination. May be used.

A reaction route when glucose is used as a starting material or an intermediate will be described with reference to FIG.
The reaction may be started with glucose as a starting material, or may be started with glucose produced by a suitable reaction from other starting materials. Glucose-6-phosphate converted from glucose by the metabolic pathway that microorganisms have in general is converted to myo-inositol-1-phosphate by myo-inositol-1-phosphate synthase activity. Next, using myo-inositol-1-phosphate as a substrate, myo-inositol is generated by myo-inositol monophosphatase activity. Next, myo-inositol is converted to glucuronic acid by myo-inositol oxygenase activity, and glucaric acid is generated from glucuronic acid by uronic acid dehydrogenase activity.

Therefore, the microorganism of the present invention must have myo-inositol-1-phosphate synthase activity, myo-inositol monophosphatase activity, myo-inositol oxygenase activity, and uronic acid dehydrogenase activity, as described above. When the host microorganism does not contain proteins having these enzyme activities, a foreign gene may be introduced so as to express these proteins. Here, “not endogenous” refers to a case where there is no gene encoding the protein having the enzyme activity, a case where the protein encoded by the gene is not substantially expressed, and the like. In addition, even when the protein is endogenous in the host microorganism, modification may be performed by introducing a foreign gene or by recombination so as to enhance the protein as compared with an unmodified strain.
It should be noted that yeast has a protein having myo-inositol-1-phosphate synthase activity and a protein having myo-inositol monophosphatase activity.

  Examples of the method for enhancing the target enzyme activity include, for example, a method of treating a parent strain with a mutagen, a method of increasing the copy number of a gene encoding a protein having the activity, a method of modifying the gene or the promoter of the gene, Examples include a method of randomly inserting an arbitrary gene capable of enhancing the expression of the gene into the genome. Further, a plurality of these methods may be combined.

Hereinafter, a specific method for producing a strain modified so as to enhance the target enzyme activity will be described. For the strain having the enhanced enzyme activity, the parent strain is treated with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonic acid (EMS) which is commonly used for mutagenesis. However, it can be obtained by selecting a strain having the increased activity.
In addition, a strain in which the desired enzyme activity is enhanced can also be obtained by modifying a gene encoding a protein having the activity. Specifically, it can be achieved by increasing the copy number of the gene, and increasing the copy number can be achieved by transforming with a vector containing the gene, or by transferring the gene on a chromosome by a technique such as homologous recombination. This can be achieved by, for example, introduction and multicopying on a chromosome.

Further, the strain in which the desired enzyme activity is enhanced can be obtained by introducing a mutation into a gene encoding a protein having the activity on a chromosome or a plasmid vector, thereby reducing the activity per molecule of the protein encoded by the gene. It can also be achieved by increasing it.
Further, the strain in which the expression of the gene is enhanced, by introducing a mutation into the promoter of the gene on a chromosome or a plasmid vector, by increasing the expression of the gene by replacing it with a stronger promoter, etc. Can also be achieved.

The expression vector of the protein having the desired enzyme activity may be inserted so that the gene encoding the protein can be expressed in a known expression vector. By transforming with this expression vector, a strain modified so as to enhance the desired enzyme activity can be obtained. Alternatively, a strain modified so that the desired enzyme activity is enhanced can also be obtained by integrating the DNA encoding the protein into the chromosomal DNA of the host microorganism such that it can be expressed by homologous recombination or the like. In addition, transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When the gene is introduced on a chromosome or a plasmid, a suitable promoter is incorporated 5'-upstream of the gene, more preferably a terminator is incorporated 3'-downstream. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in a microorganism used as a host, and may be the promoter and terminator of the gene itself, or may be other promoters. And a terminator. The vectors, promoters, terminators, and the like that can be used in these various microorganisms are described in detail, for example, in “Basic Microbiology Course 8 Genetic Engineering / Kyoritsu Publishing”.

In the present invention, “myo-inositol-1-phosphate synthase (INO1) activity” refers to an activity of catalyzing a reaction of converting glucose-6-phosphate to myo-inositol-1-phosphate. Whether or not the enzyme has this enzyme activity is determined by a usual assay method as to whether or not the protein to be measured can produce myo-inositol-1-phosphate using glucose-6-phosphate as a substrate. Thus, the determination can be made. For example, an enzyme to be measured is allowed to act on glucose-6-phosphate, and the amount of myo-inositol-1-phosphate converted from glucose-6-phosphate is measured to determine the enzyme activity. Can be confirmed. The resulting myo-inositol-1-phosphate is treated with myo-inositol-1-phosphate with sodium periodate to generate inorganic phosphorus, and the resulting inorganic phosphorus is subjected to a conventional method such as a molybdenum blue colorimetric method. It can be measured by colorimetric determination (determined from the absorption intensity at 620 nm) according to (J. Adhikari et al., Plant Physiol. (1987) 85 611-614).
The gene encoding the protein having the INO1 activity is not particularly limited. For example, the gene encoding the known GenBank Accession Nos. AB032073, AF052625, AF071033, AF078915, AF120146, AF207640, AF284065, BC111160, L23520, U32511 and the like. In particular, a myo-inositol-1-phosphate synthase gene having a coding region nucleotide sequence represented by SEQ ID NO: 1 can be suitably used.
In addition, genes derived from other microorganisms, animals and plants can also be used. In this case, it is preferable to use a nucleotide sequence optimized for the codon usage of the host microorganism. A gene encoding a protein having INO1 activity based on homology with the DNA sequence shown in SEQ ID NO: 1 and the like, isolated from chromosomes of microorganisms, animals and plants, and determined for its nucleotide sequence can be used. After the nucleotide sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In order to enhance INO1 activity, a plurality of types of genes derived from different microorganisms, animals and plants may be used.

In the present invention, "myo-inositol monophosphatase (INM) activity" refers to an activity of catalyzing a reaction of dephosphorylating gumio-inositol-1-phosphate to convert it to myo-inositol. Whether or not the enzyme has this enzyme activity is determined by measuring whether or not the protein to be measured can produce myo-inositol using myo-inositol-1-phosphate as a substrate by a usual assay method. Is possible. For example, an enzyme to be measured is allowed to act on myo-inositol-1-phosphate, and the amount of myo-inositol converted from myo-inositol-1-phosphate is measured using a differential refractive index (RI) detector. The enzymatic activity can be confirmed by directly detecting the peak derived from myo-inositol by HPLC used.
The gene encoding a protein having INM activity is not particularly limited, and the gene (suhB gene) derived from many known organisms can be used. For example, GenBank Accession Nos. ZP_04619988, YP_001451
848 and the like. In particular, the use of the Escherichia coli-derived suhB gene (SEQ ID NO: 3: AAC75586 (MG1655)) is convenient when Escherichia coli is used as a host cell.
In addition, genes derived from other microorganisms, animals and plants can also be used. In this case, it is preferable to use a nucleotide sequence optimized for the codon usage of the host microorganism. A gene encoding a protein having INM activity based on homology with the DNA sequence shown in SEQ ID NO: 3 or the like from a chromosome of a microorganism, animal, plant or the like, and a base sequence thereof can be used. After the nucleotide sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. In order to enhance INM activity, a plurality of types of genes derived from different microorganisms, animals and plants may be used.

In the present invention, “myo-inositol oxygenase (MIOX) activity” refers to an activity of catalyzing a reaction of oxidizing myo-inositol to convert to glucuronic acid. Whether or not the enzyme has this enzyme activity can be determined by measuring whether or not the protein to be measured can generate glucuronic acid using myo-inositol as a substrate by a usual assay method. For example, an enzyme to be measured is allowed to act on myo-inositol, and the amount of glucuronic acid converted from myo-inositol is directly detected by HPLC using a UV (210 nm) detector. , The enzyme activity can be confirmed.
The gene encoding the protein having the MIOX activity is not particularly limited, but may be a gene derived from many known organisms. For example, GenBank ACCESSION No. AY732258, NM101319, NM00111065, NM001030266, NM214102, AY0644416, NM001247664, XM630762, NM1455771, NM017584, NM001131282 and the like. In particular, a mouse miox gene having the nucleotide sequence of the coding region represented by SEQ ID NO: 5 can be suitably used.
In addition, genes derived from other microorganisms, animals and plants can also be used. In this case, it is preferable to use a nucleotide sequence optimized for the codon usage of the host microorganism. For example, when Candida boidiny is used as a host, SEQ ID NO: 7 obtained by optimizing SEQ ID NO: 5 to codon usage of Candida boidiny can be used. Further, a gene encoding a protein having MIOX activity based on homology with the DNA sequence shown in SEQ ID NO: 5 may be isolated from a chromosome of a microorganism, animal, plant, or the like, and a base sequence thereof may be used. it can. After the nucleotide sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. For enhancing MIOX activity, a plurality of types of genes derived from different microorganisms, animals and plants may be used.

In the present invention, “uronate dehydrogenase (Udh) activity” refers to an activity of catalyzing a reaction of converting glucuronic acid to glucaric acid. Whether or not the enzyme has this enzyme activity can be determined by measuring whether or not the protein to be measured can generate glucaric acid using glucuronic acid as a substrate by a usual assay method. For example, an enzyme to be measured is allowed to act on glucuronic acid, and the amount of glucaric acid converted from glucuronic acid is directly detected by HPLC using a UV (210 nm) detector to detect the peak derived from glucaric acid. By doing so, the enzyme activity can be confirmed.
The gene encoding a protein having Udh activity is not particularly limited, and may be a gene derived from many known organisms. For example, GenBank ACCESSION No. BK00642, EU377538 and the like. In particular, the Udh gene of Pseudomonas syringae having the nucleotide sequence of the coding region represented by SEQ ID NO: 9 can be suitably used.
In addition, genes derived from other microorganisms, animals and plants can also be used. In this case, it is preferable to use a nucleotide sequence optimized for the codon usage of the host microorganism. For example, when Candida boidiny is used as a host, SEQ ID NO: 11 obtained by optimizing SEQ ID NO: 9 to the codon usage of Candida boidiny can be used. In addition, a gene encoding a protein having Udh activity based on homology with the DNA sequence shown in SEQ ID NO: 9 may be isolated from chromosomes of microorganisms, animals and plants, and those whose nucleotide sequences have been determined may be used. it can. After the nucleotide sequence is determined, a gene synthesized according to the sequence can be used. These can be obtained by amplifying a region containing a promoter and an ORF portion by a hybridization method or a PCR method. For enhancing the Udh activity, a plurality of types of genes derived from different microorganisms, animals and plants may be used.

In the microorganism of the present invention, the enzyme having MIOX activity and the enzyme having Udh activity are preferably expressed as a fusion protein.
This is because the efficiency of the conversion of myo-inositol to glucaric acid via glucuronic acid in the reaction is increased by the two enzymes participating in the glucaric acid production reaction as fusion proteins. In addition, by making the two enzymes into a fusion protein, the solubility of the enzyme having MIOX activity can be improved, and the enzyme reaction can be promoted as described later.
The fusion protein may be one obtained by linking the above two enzymes with a suitable linker, for example, an embodiment in which the two enzymes are linked by about 1 to 10 amino acids. Such a fusion protein may be designed and constructed by using the above-mentioned expression vector by a well-known technique, introduced into a host microorganism, and expressed.

In the microorganism of the present invention, the enzyme having MIOX activity is preferably expressed as a solubilized protein.
In the glucaric acid production reaction, the conversion of myo-inositol to glucuronic acid is presumed to be rate-determining, but the enzyme reaction can be promoted by solubilizing an enzyme having MIOX activity.
As a method for solubilizing the target protein, there is a method of expressing the target protein by fusing a specific fusion protein or peptide, and various such fusion proteins and peptides are known (Kato et al., Biophysics, 48 ( 3) 185-189 (2008)).

  As described above, a microorganism having a glucaric acid-producing ability can be produced by performing a modification that imparts or enhances each enzyme activity. In the present invention, “glucaric acid-producing ability” means that when a microorganism is cultured in a medium, the microorganism can generate and accumulate glucaric acid in the medium.

The method for producing glucaric acid of the present invention includes a step of allowing the microorganism of the present invention or a processed product thereof to act on an organic raw material in an acidic aqueous medium (hereinafter, referred to as a “fermentation step”). The method for producing glucaric acid of the present invention may further include a step of collecting the generated glucaric acid (hereinafter, referred to as a “recovering step”).
In general, in glucaric acid fermentation, the pH of the fermentation solution decreases with the accumulation of glucaric acid, and the growth of microorganisms and the fermentation productivity tend to decrease. On the other hand, since the microorganism of the present invention is excellent in acid resistance as described above, the growth and fermentation productivity of the microorganism are not reduced even in an acidic aqueous medium, so that glucaric acid can be efficiently produced. Therefore, in the production method of the present invention, it is possible to reduce the amount of a neutralizing agent to be added to suppress a decrease in pH due to fermentation production. Furthermore, glucaric acid is usually produced and precipitated in the form of an acid by fermentatively producing glucaric acid under acidic conditions, and the purification step of acid crystallization performed in the production of glucaric acid can be omitted. Generation of by-products can be suppressed. As a result, the load on the entire manufacturing process can be reduced, and the cost can be reduced.

  In the method for producing glucaric acid of the present invention, a processed product of the microorganism of the present invention can also be used. Examples of processed microorganisms include immobilized microorganisms in which microorganisms are immobilized with acrylamide, carrageenan, etc., crushed microorganisms, crushed microorganisms, centrifuged supernatants, or fractions obtained by partially purifying the supernatants with ammonium sulfate treatment or the like. And the like. Usually, these processed products include various enzymes involved in glucaric acid biosynthesis possessed by the microorganism of the present invention.

In using the microorganism of the present invention in the method for producing glucaric acid of the present invention, those that have been slope-cultured on a solid medium such as an agar medium may be used directly. Those cultured in a liquid medium may be used. That is, by performing seed culture or main culture described below, the fermentation step can be performed after the microorganism of the present invention has been grown in advance.
The seed culture or main culture described below and the fermentation step described later can be performed simultaneously without distinction. Glucaric acid can also be produced by reacting an organic raw material while growing a seed-cultured or main-cultured microorganism in a reaction solution.

(Seed culture)
Seed culture is performed to prepare the microorganism of the present invention to be used for main culture. The medium used for seed culture may be a usual medium used for culturing microorganisms, but is preferably a medium containing a nitrogen source, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as the microorganism of the present invention can assimilate and grow, and specifically, ammonium salts, nitrates, urea, hydrolyzed soybeans, hydrolyzed casein, peptone And various organic and inorganic nitrogen compounds such as yeast extract, meat extract, corn steep liquor and the like. As the inorganic salt, various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc are used. In addition, factors that promote the growth of vitamins such as biotin, thiamine, pantothenic acid, inositol, nicotinic acid, nucleotides and amino acids are added as necessary.

  In the seed culture, a carbon source may be added to the medium as needed. The carbon source used for the seed culture is not particularly limited as long as the microorganism can assimilate and grow, and is usually a carbohydrate such as glucose, fructose, galactose, lactose, xylose, arabinose, sucrose, starch, or cellulose; Glycerol, mannitol, inositol, fermentable carbohydrates such as polyalcohols such as ribitol are used, among which glucose, fructose, galactose, lactose, sucrose, xylose, or arabinose is preferable, particularly glucose, galactose, sucrose or lactose is preferred. preferable. These carbon sources may be added alone or in combination.

  The seed culture can be performed at a general optimum growth temperature, but is preferably performed at a temperature at which the growth rate of the highly acid-resistant microorganism is the highest. The specific culture temperature is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. In particular, in the case of Candida yeast, the temperature is usually 20 ° C to 45 ° C, preferably 25 ° C to 35 ° C.

  Seed culture can be carried out at a general optimum growth pH, but is preferably carried out at a pH at which the growth rate of the highly acid-resistant microorganism is the highest. The specific culture pH is usually from pH 2 to 10, and preferably from pH 5 to 8. In particular, in the case of Candida yeast, the pH is usually 2 to 10, preferably 2.1 to 8, and more preferably 2.5 to 6.

The culture time of the seed culture is not particularly limited as long as a certain amount of microorganisms can be obtained, but is usually from 6 hours to 96 hours. In seed culture, it is preferable to supply oxygen by aeration or stirring.

  The microorganism after the seed culture can be used for the main culture described below, but the seed culture may be omitted, or the one obtained by slant culture on a solid medium such as an agar medium may be directly used for the main culture. If necessary, seed culture may be repeated several times.

(Main culture)
The main culture is performed to prepare the microorganism of the present invention to be used in the glucaric acid production reaction described below, and its main purpose is to increase the amount of the microorganism. When the above-described seed culture is performed, main culture is performed using a microorganism obtained by the seed culture.

  The medium used for the main culture may be a usual medium used for culturing microorganisms, but is preferably a medium containing a nitrogen source, an inorganic salt, and the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and proliferated by the present microorganism. Specifically, ammonium salts, nitrates, urea, soybean hydrolysates, casein hydrolysates, peptone, yeast Various organic and inorganic nitrogen compounds such as extract, meat extract, corn steep liquor and the like can be mentioned. As the inorganic salt, various phosphates, sulfates, metal salts such as magnesium, potassium, sodium, calcium, manganese, iron, zinc, and copper are used. In addition, factors that promote the growth of vitamins such as biotin, thiamine, pantothenic acid, inositol, nicotinic acid, nucleotides and amino acids are added as necessary. In order to suppress foaming during culturing, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

  In the main culture, it is preferable to add a carbon source to the medium. The carbon source used in the main culture is not particularly limited as long as the microorganism can assimilate and proliferate, but usually, carbohydrates such as glucose, fructose, galactose, lactose, xylose, arabinose, sucrose, starch, and cellulose; Glycerol, mannitol, inositol, fermentable carbohydrates such as polyalcohols such as ribitol are used, among which glucose, fructose, galactose, lactose, sucrose, xylose, or arabinose is preferable, particularly glucose, galactose, sucrose or lactose is preferred. preferable. These carbon sources may be added alone or in combination.

Also, a starch saccharified solution, molasses or the like containing the above-mentioned fermentable sugar is used, and the above-mentioned fermentable sugar is preferably a sugar solution extracted from a plant such as sugar cane, sugar beet or sugar maple.
These carbon sources may be added alone or in combination.

  The concentration of the carbon source used is not particularly limited, but it is advantageous to add it within a range that does not inhibit the growth of microorganisms, and it is usually 0.1 to 10% (W / V), preferably 0 to 10% with respect to the culture solution. It can be used within a range of 0.5 to 5% (W / V). Further, a carbon source may be additionally added in accordance with the decrease of the carbon source accompanying the growth.

  In addition, the main culture is preferably performed at a general optimum growth temperature. The specific culture temperature is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. In particular, in the case of Candida yeast, the temperature is usually 20 ° C to 45 ° C, preferably 25 ° C to 35 ° C.

Further, it is preferable that the main culture is performed at a general optimum pH for growth. The specific culture pH is usually from pH 2 to 10, and preferably from pH 5 to 8. Particularly, in the case of Candida yeast, the pH is usually 2 to 10, preferably pH 2.1 to 8, and more preferably pH 2.5 to 6.
However, when the main culture is performed simultaneously with the fermentation step, the main culture is performed at pH 5 or lower, and preferably at pH 3.5 or lower.

  The culture time of the main culture is not particularly limited as long as a certain amount of microorganisms can be obtained, but is usually 6 hours or more and 96 hours or less. In the main culture, it is preferable to supply oxygen by aeration or stirring.

  The microorganism after the main culture can be used for a glucaric acid production reaction described below, but the culture solution may be used directly, or may be used after the microorganism is recovered by centrifugation, membrane separation, or the like.

(Fermentation process)
In the fermentation step, glucaric acid is produced by allowing the above-described microorganism having glucaric acid-producing ability or a processed product thereof to act on an organic raw material in an acidic aqueous medium. The reaction occurring in this fermentation step is hereinafter referred to as “glucaric acid production reaction”.

  Here, the acidic aqueous medium refers to an aqueous solution for performing a glucaric acid production reaction in the fermentation step, and is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like, as described later. A glucaric acid production reaction can be performed by reacting the microorganism of the present invention or a processed product thereof with an organic raw material in the aqueous medium. In this specification, the acidic aqueous medium means all liquids having a pH of 5 or less and contained in a reaction vessel.

  As the aqueous medium, for example, a medium for culturing microorganisms or a buffer such as a phosphate buffer may be used, but the reaction solution is an aqueous solution containing a nitrogen source or an inorganic salt. Is preferred. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source capable of assimilating glucaric acid by the microorganism of the present invention, and specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein Various organic and inorganic nitrogen compounds such as decomposition products, peptone, yeast extract, meat extract, corn steep liquor and the like can be mentioned. As the inorganic salt, various phosphates, sulfates, metal salts such as magnesium, potassium, sodium, calcium, manganese, iron, zinc, and copper are used. In addition, factors that promote the growth of vitamins such as biotin, thiamine, pantothenic acid, inositol, nicotinic acid, nucleotides and amino acids are added as necessary. In order to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the reaction solution.

The organic raw material used in the method for producing glucaric acid of the present invention is not particularly limited as long as the microorganism of the present invention can assimilate and produce glucaric acid, and a so-called general saccharide can be used.
Specifically, a monosaccharide having 3 carbon atoms such as glyceraldehyde (triose); a monosaccharide having 4 carbon atoms such as erythrose, threose and erythrulose (tetroose); ribose, lyxose, xylose, arabinose, deoxyribose, xylulose, C5 monosaccharide such as ribulose (pentose); C6 monosaccharide such as allose, talose, growth, glucose, altrose, mannose, galactose, idose, fucose, fucrose, rhamnose, psicose, fructose, sorbose, tagatose, etc. A monosaccharide having 7 carbon atoms (heptose) such as sedoheptulose; disaccharides such as sucrose, lactose, maltose, trehanose, turanose and cellobiose; trisaccharides such as raffinose, melesitose and maltotriose; Kutoorigo sugars, galactooligosaccharides, oligosaccharides such Man'naorigo sugar; starch, dextrin, cellulose, hemicellulose, glucans, polysaccharides pentosan like; glycerol, mannitol, inositol, polyalcohols such as ribitol, and the like.

Among the saccharides described above, at least one selected from the group consisting of glucose, xylose, sucrose, starch, molasses, myo-inositol, glycerol, ribitol, and erythritol is more preferable. These are because raw materials are easily available and inexpensive.
The organic raw material used in the method for producing glucaric acid of the present invention may contain one kind of saccharide alone or two or more kinds of saccharides.

  The organic raw material used in the method for producing glucaric acid of the present invention is not particularly limited as long as it contains the saccharide. For example, one or more saccharides dissolved in water to form an aqueous solution, And a plant obtained by decomposing a plant or a part thereof containing the carbohydrate as a constituent, or a plant obtained by extracting a carbohydrate from a plant or a part thereof containing one or more kinds of the carbohydrate as a constituent. Can be used. Specific examples include a lignocellulose decomposition raw material, a sucrose-containing raw material, and a starch decomposition raw material as described below.

The organic raw material used in the method for producing glucaric acid of the present invention may be used by diluting it with water or the like to reduce the concentration of carbohydrate, or may be used by concentrating it to increase the concentration of carbohydrate. Good.
The concentration of the saccharide contained in the organic raw material in the method for producing glucaric acid of the present invention is not particularly limited because it greatly varies depending on the origin of the organic raw material, the type of the saccharide contained, and the like. In consideration of the productivity of the chemical conversion process, it is usually at least 0.1% by mass, preferably at least 2% by mass, and usually at most 80% by mass, preferably at most 70% by mass. However, when two or more saccharides are contained, the total concentration is shown.

Preferred organic raw materials include lignocellulose decomposition raw materials.
Lignocellulose is an organic substance composed of cellulose, a structural polysaccharide, hemicellulose, and lignin, which is a polymer of an aromatic compound. Lignocellulose is usually not edible, and is usually discarded or incinerated. Therefore, lignocellulose is preferable because it can be supplied stably and resources can be effectively used.
Raw materials for decomposing lignocellulose include herbaceous biomass such as bagasse, corn stover, straw, rice straw, switchgrass, napiergrass, elianthus, sasa, and pampas grass, and woody biomass such as waste wood, sawdust, bark, and used paper. Etc. can be suitably used. Among them, bagasse, corn stover, and straw are preferred.

  The method of obtaining an organic raw material from the above-described lignocellulose decomposition raw material is not particularly limited, for example, after performing pretreatment as necessary on lignocellulose, an enzyme, an acid, subcritical water, supercritical water, or the like. Examples of the method include hydrolysis and thermal decomposition.

Preferred organic raw materials include sucrose-containing raw materials.
Sucrose is included in plants that can accumulate sucrose in cells, and such plants are hereinafter referred to as "plants containing sucrose". Examples of plants containing sucrose include those used as raw materials for sugars such as sugar cane, sugar beet, sugar maple, sugar beet, and sorghum. Among them, sugar cane and sugar beet are preferable.

  The method for obtaining an organic raw material from a plant containing sucrose is not particularly limited, and examples thereof include a method in which the plant is crushed and then squeezed or leached. In the method for producing glucaric acid of the present invention, sucrose-containing plant juice (for example, cane juice in the case of sugarcane), crude sugar, molasses, and the like thus obtained can also be used as the organic raw material.

Preferred organic raw materials include starch decomposition raw materials.
In addition, starch is included in plants capable of accumulating starch in cells, and such plants are hereinafter referred to as "plants containing starch". Examples of plants containing starch include cassava, corn, potato, wheat, sweet potato, sago palm, rice, kudzu, katakuri, mung bean, bracken, ouyuri, and the like. Among them, cassava, corn, potato, and wheat are preferable.

  The method for obtaining an organic raw material from a plant containing starch is not particularly limited, and examples thereof include a method of hydrolyzing starch extracted from the plant.

  The use concentration of the organic raw material is not particularly limited, but it is advantageous and preferable from the viewpoint of productivity if it is as high as possible within a range that does not inhibit the production of glucaric acid. The concentration of the organic raw material contained in the aqueous medium is the concentration of the saccharide contained therein, and is usually 5% (W / V) or more, preferably 10% (W / V) or more based on the aqueous medium. On the other hand, it is usually 30% (W / V) or less, preferably 20% (W / V) or less. Further, the organic raw material may be additionally added in accordance with the decrease of the organic raw material accompanying the progress of the glucaric acid production reaction.

The acidic aqueous medium during the glucaric acid production reaction has a pH of 5 or less, and preferably has a pH of 3.5 or less. Further, it is preferably pH 1 or more, more preferably pH 1.5 or more, and still more preferably pH 2.5 or more. By fermenting under such conditions, glucaric acid can be efficiently produced and recovered without lowering the growth of microorganisms and the fermentation productivity as described above.
In addition, it is preferable to always maintain pH 5 or less during the glucaric acid production reaction. Usually, the acidity of the aqueous medium does not reach pH 5 or more due to the generated glucaric acid.

  The pH of the aqueous medium can be adjusted by adding an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, an organic acid such as acetic acid, a mixture thereof, or supplying carbon dioxide gas.

  The amount of the microorganism used for the glucaric acid production reaction is not particularly limited, but is usually 1 g / L or more, preferably 10 g / L or more, more preferably 20 g / L or more as a wet weight, while usually 700 g / L. Or less, preferably 500 g / L or less, more preferably 400 g / L or less.

  The time for the glucaric acid production reaction is not particularly limited, but is usually 1 hour or more, preferably 3 hours or more, while it is usually 300 hours or less, preferably 200 hours or less.

The glucaric acid production reaction can be performed at the same temperature as the optimal growth temperature of the microorganism to be used. The specific culture temperature is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C. In particular, in the case of Candida yeast, the temperature is usually 20 ° C to 45 ° C, preferably 25 ° C to 35 ° C.
It is not necessary to always maintain the above temperature range during the glucaric acid production reaction, but it is desirable to keep the temperature range within 50% or more, preferably 80% or more of the total reaction time.

  The glucaric acid production reaction may be carried out under an anaerobic atmosphere without supplying air and supplying no oxygen, but it is preferable to carry out ventilation and stirring. Specifically, the oxygen transfer rate is usually at least 0 mmol / L / h, preferably at least 10 mmol / L / h, more preferably at least 20 mmol / L / h, while usually at most 200 mmol / L / h, preferably Is 150 mmol / L / h or less, more preferably 100 mmol / L / h or less.

  The method for producing glucaric acid in the method for producing glucaric acid of the present invention is not particularly limited, but can be applied to any of a batch reaction, a semi-batch reaction, and a continuous reaction.

  The present invention is also excellent in that the risk of contamination by various bacteria is reduced because the aqueous medium is acidic.

(Recovery process)
In the method for producing glucaric acid of the present invention, glucaric acid is generated by the above-described glucaric acid production reaction and can be accumulated in the reaction solution. The accumulated glucaric acid is recovered from the aqueous medium according to a conventional method. Specifically, the recovery step can be performed, for example, by removing microbial solids by centrifugation, filtration, or the like and recrystallizing the same, whereby high-purity glucaric acid can be recovered. In the present invention, since the aqueous medium is acidic, it is excellent in that it is not necessary to newly add an acid during purification and that it can be recovered in the form of glucaric acid.
The obtained glucaric acid can be used as a raw material for 2,5-furandicarboxylic acid or 2,5-furandicarboxylic acid diester, which is attracting attention as a new polymer raw material, and can be derived into various other useful chemicals. is there.

<Method for producing 2,5-furandicarboxylic acid>
After producing glucaric acid by the above-mentioned method, 2,5-furandicarboxylic acid can be produced from the obtained glucaric acid according to a conventional method. Specific examples include a method of cyclizing and dehydrating in the presence of an acid catalyst, and a method of hydrolyzing a 2,5-furandicarboxylic acid diester described below.
The acid catalyst to be used is not particularly limited as long as this reaction proceeds, but sulfonic acid compounds such as paratoluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, sulfuric acid, phosphoric acid, and hydrobromic acid And inorganic acids such as hydrochloric acid, and heteropoly acids such as phosphotungstic acid and silicotungstic acid. From the viewpoint of reactivity, paratoluenesulfonic acid and hydrobromic acid are preferred.
The reaction temperature is usually preferably 100 ° C. or higher, more preferably 120 ° C. or higher.
A known technique can be used for the production of furandicarboxylic acid by hydrolysis from the furandicarboxylic acid diester.

  2,5-furandicarboxylic acid is a raw material for polyesters and polyamides, and can be converted to diols for raw materials for polycarbonates and polyesters by the reduction of carboxylic acids. Therefore, its manufacturing technology has attracted attention. Further, the polymer using 2,5-furandicarboxylic acid has a feature of being excellent in gas barrier properties.

<Method for producing 2,5-furandicarboxylic acid diester>
After producing glucaric acid by the above-mentioned method, 2,5-furandicarboxylic acid diester can be produced from the obtained glucaric acid as a raw material according to a conventional method. Specifically, for example, a method of cyclizing and dehydrating in the presence of a solvent and an acid catalyst, a method of dehydrating with 2,5-furandicarboxylic acid and an alcohol, a method of converting into a corresponding acid chloride, and then reacting with an alcohol are given. No.

The acid catalyst to be cyclized and dehydrated in the presence of a solvent and an acid catalyst is not particularly limited as long as the reaction proceeds, but sulfonic acid compounds such as paratoluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and camphorsulfonic acid, Inorganic acids such as sulfuric acid, phosphoric acid, hydrobromic acid and hydrochloric acid; and heteropoly acids such as phosphotungstic acid and silicotungstic acid. From the viewpoint of reactivity, paratoluenesulfonic acid, phosphoric acid, and sulfuric acid are preferred.
In order to directly produce an ester, the reaction is preferably performed while separating water. As a solvent capable of separating oil and water, an alcohol having 4 or more carbon atoms is preferable, and an alcohol having 8 or less carbon atoms is preferable in terms of post-reaction treatment. From the viewpoint of solvent recovery, a single solvent is preferred, but two or more solvents may be used in combination at any ratio.
When 2,5-furandicarboxylic acid is converted into an ester after synthesis, methyl ester and ethyl ester are preferable from the viewpoint of polymerization reactivity. Known methods can be used for the production of furandicarboxylic diesters by a method of dehydrating with 2,5-furandicarboxylic acid and alcohol, or a method of converting the acid into the corresponding acid chloride and then reacting with alcohol.

2,5-furandicarboxylate diester can be used as a raw material for polyesters and polyamides, and can be converted to diols used as raw materials for polycarbonates and polyesters by reducing ester sites, and can replace petroleum-based synthetic resins with biomass-based ones. Therefore, its manufacturing technology has attracted attention.
Further, polymers using these 2,5-furandicarboxylate diesters have a feature of being excellent in gas barrier properties.

  Hereinafter, the present invention will be described in more detail with reference to specific experimental examples, but the present invention is not limited to the following embodiments.

<Example 1> Evaluation test of acid resistance of various yeasts An evaluation example of the acid resistance performance of each yeast strain described in Table 1 is shown below. Inoculate one platinum loop from the freeze stock of each yeast strain into YPD agar medium (1% Yeast Extract, 2% HIPOLYPEPTON, 2% glucose, 2% agar) and incubate at 30 ° C for 3 days at 30 ° C. The cells grown in the above were inoculated into 3 mL of a YPD medium prepared in a test tube, and subjected to vortex shaking at 30 ° C. and 180 rpm for 24 hours. The obtained culture solution was inoculated to 20 mL of a YPD medium of pH 3 (pH adjusted by adding HCl) prepared in a 200 mL Erlenmeyer flask so that OD ₆₆₀ = 1.0, and glucose was added at 30 ° C. and 180 rpm for 72 hours at 180 rpm. Rotating shaking culture was carried out while appropriately adding so as not to cause depletion.
FIG. 2 shows the time-dependent changes in the cell growth. All of the evaluated yeast strains were confirmed to have a growth ability under acidic conditions of pH = 3. Among them, it was confirmed that the growth ability of Candida yeast was particularly high, indicating that they had extremely high acid resistance performance. Was. Moreover, Saccharomyces cerevisiae S288C and Pichia pastris showed the second highest acid resistance performance after Candida yeast, and the growth ability decreased in the order of Kluyveromyces yeast and Saccharomyces cerevisiae KA311A.

<Example 2> Preparation of Candida boidinii having high inositol-producing ability (1) Breeding of inositol-producing Candida boidinii An inositol-producing strain was obtained by a mutagenesis method using a chemical mutagen based on Candida boidinii MCB1. I got it. The cells of Candida boidinii MCB1 were treated with ethyl methanesulfonic acid (EMS), diluted appropriately, applied to a YPD agar medium, and cultured at 30 ° C. for 3 days in a static place. On the other hand, the cells obtained by culturing the inositol-requiring yeast Saccharomyces cerevisiae ATCC34893 in 5 mL of YPD medium (1% Yeast Extract, 2% HIPOLYPEPTON, 2% glucose) at 30 ° C. for 1 day, collect the cells once, and use physiological saline. After washing, an appropriate amount was applied to an agar medium (agar 1.5%) having the composition shown in Table 2.

Mutant-treated Candida boidinii MCB1 colonies grown on the above YPD agar medium were replicated on an agar medium of Table 2 coated with inositol-requiring yeast Saccharomyces cerevisiae ATCC34893 and cultured at 30 ° C. for 2 days. A colony in which growth of Saccharomyces cerevisiae ATCC34893 was confirmed around the replicated colony was isolated, and Candida boidinii
MCB2 was obtained.

(2) Flask culture of inositol-producing Candida boidinii MCB2
Candida boidinii MCB2 was pre-cultured in 3 mL of YPD medium at 30 ° C. for 24 hours, and then inoculated in a 200 mL Erlenmeyer flask containing 30 mL of MIS2 medium having the composition shown in Table 3 so that OD ₆₆₀ = 1, at 30 ° C. The cells were cultured at 160 rpm for 136 hours. The supernatant obtained by centrifugation (12000 rpm, 1 minute) from 1 mL of the obtained culture solution was passed through a 0.45 μm cosmospin filter (manufactured by Nippon Millipore Co., Ltd.), and the filtrate was used as an analysis sample.

The analysis was performed using HPLC (Shimadzu Corporation). The column used was a COSMOSIL Sugar-D 5 μm 4.6 × 250 mm Column (Nacalai Tesque). It was eluted isocratically with a 75% acetonitrile aqueous solution (flow rate 1 mL / min, temperature 30 ° C.) as a mobile phase, and detected by a differential refractive index detector (RID) (Shimadzu Corporation).
FIG. 3 shows the time course of the inositol accumulation obtained as a result of the analysis. The inositol accumulation amount at the end of the culture was 3.9 g / L.

(3) Breeding of Candida boidinii with high inositol productivity Using the inositol-producing Candida boidinii MCB2 obtained by the above-mentioned method as the original strain, the inositol productivity was further improved by a mutagenesis method using a chemical mutagen. Mutants were bred. Specifically, the inositol high-producing strain was appropriately diluted with a mutated cell in accordance with a standard method, applied to a YPD agar medium, and cultured at 30 ° C. for 3 days to isolate colonies that had grown, After pre-culturing at 30 ° C. for 24 hours in 3 mL of YPD medium, 0.75 mL was inoculated into a 200 mL Erlenmeyer flask containing 30 mL of MIS2 medium having the composition shown in Table 3, and cultured at 160 ° C. for 160 hours at 30 ° C. And the amount of accumulated inositol was determined by quantification. MCand2 was treated with 4-nitroquinoline-1-oxide (4-NQO) to obtain Candida boidinii MCB6. Further, MCB7 was obtained by NTG processing, MCB13 was obtained by twice 4-NQO processing, and then MCB23 was obtained by UV processing. Table 4 shows the results of flask culture of Candida boidinii MCB23. The amount of inositol accumulated at the end of the cultivation was 4.4 g / L for MCB2, whereas it was 8.8 g / L for MCB23, which was twice as large.
On June 18, 2018, the MCB23 strain was set up at the Patent and Microorganisms Depositary of the National Institute of Technology and Evaluation (Postal Code: 292-0818, Address: 2-5-8, Kazusa Kamatari, Kisarazu-shi, Chiba) ), A domestic deposit has been made (accession number: NITE P-02745).

<Example 3> Preparation of Candida boidinii capable of producing glucaric acid (1) Construction of plasmid for expressing MIOX and Udh fusion protein In a previous study using methanol-assimilating yeast Pichia pastoris as a host, the C-terminal of Udh and MIOX were used. N-terminal and Gly-Gly-Gly-Gly- Ser five consisting of the amino acid polypeptides G ₄ to fuse expressed by connecting the S linker improves the solubility and stability of MIOX, resulting glucaric acid production Has been reported (Y. Liu et al., Enzyme and Microbial Technology 91 (2016) 8-16).
Expecting a similar effect in Candida boidinii, a plasmid for expressing a fusion protein in which Udh and MIOX were linked by a G ₄ S linker was constructed. The expression promoter used was a glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene promoter that is constantly expressed.

From the pNOTeI (Sakai, Y., et al. High-level secretion of fungal glucoamylase using the Candida boidinii gene expression sysytem. Biochim. Biophys. Acta 1308: 81-87 (1996).) to the alcohol oxidase gene (AODI) promoter region It was excised by restriction enzyme treatment with NotI / EcoRI, and replaced with a TDH3 promoter fragment to prepare pTDH3eI. The TDH3 promoter fragment was obtained by amplification by PCR using Fw_pTDH3_EcoRI (SEQ ID NO: 13) and Rv_pTDH3_NotI (SEQ ID NO: 14) as primers and a plasmid pTDH3-Venus containing a TDH3 promoter region as a template. PCR amplification products were confirmed by 1% agarose (Agarose-RE: Nacalai Tesque, Inc.) gel electrophoresis and visualization by ethidium bromide staining to detect a fragment of about 1 kb. Recovery of the desired fragment from the gel was performed using ^{Wizard (R) SV Gel and PCR} Clean-UP System ( manufactured by Promega). Gene fragments treated with restriction enzymes with NotI / EcoRI were used.
The PCR reaction solution had a composition of 10 to 50 ng of template DNA, 25 μL of PrimeSTAR Max Premix, 10 pmol of each primer, and 50 μL of sterile water. As a reaction temperature condition, a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 1 minute / kbp was repeated 30 times using TaKaRa PCR Thermal Cylcer Dice Touch (model TP350). However, the heat retention at 94 ° C. in the first cycle was 2 minutes.

Subsequently, about 6800 bp of a gene fragment amplified by PCR using pTDH3eI as a template and Fw_pTDH3eI_AODt (SEQ ID NO: 15) and Rv_pTDH3eI_TDH3p (SEQ ID NO: 16) as a primer, and a mouse-derived MI obtained from the National Center for Biotechnology Information (NCBI)
The OX gene sequence was optimized to the codon frequency of Candida boidnii and the pUC57 plasmid vector containing the synthesized MIOX gene (SEQ ID NO: 7) was used as a template, and Fw_MIOX_pTDH3eI (SEQ ID NO: 17) and Rv_MIOX_pTDH3eI (SEQ ID NO: 18) were used as primers. Escherichia coli (DH5α strain) was transformed with a plasmid DNA obtained by ligating a gene fragment of about 880 bp obtained by PCR amplification using an In-Fusion HD Cloning Kit (TaKaRa). The recombinant E. coli thus obtained was spread on an LB agar medium containing 100 μg / mL ampicillin. Clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 100 μg / mL ampicillin, and plasmids were obtained from the resulting cells using the GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNA was designated as pTDH3eI-MIOX.
The PCR reaction solution had a composition of 10 to 50 ng of template DNA, 25 μL of PrimeSTAR Max Premix, 10 pmol of each primer, and 50 μL of total volume with sterilized water. As the reaction temperature conditions, a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 1 minute / kbp was repeated 30 times using TaKaRa PCR Thermal Cylcer Dice Touch (model TP350). However, the heat retention at 94 ° C. in the first cycle was set to 2 minutes.

  Furthermore, about 6800 bp of a gene fragment obtained by PCR amplification using the above-constructed pTDH3eI-MIOX as a template and Fw_pTDH3eIMIOX_MIOX (SEQ ID NO: 19) and Rv_pTDH3eIMIOX_TDH3pro (SEQ ID NO: 20) as primers, and from the National Center for Biotechnology Information (NCBI) The obtained Use gene of Pseudomonas syringae was optimized to the codon frequency of Candida boidnii, and the pUC57 plasmid vector containing the synthesized Udh gene (SEQ ID NO: 11) was used as a template and PCR was performed using Fw_Udh_TDH3p (SEQ ID NO: 21) and Rv_Udh_MIOX (SEQ ID NO: 22) as primers. The amplified gene fragment of about 830 bp was ligated using an In-Fusion HD Cloning Kit (TaKaRa) to prepare a plasmid pTDH3eI-Udh-MIOX for expressing a MIOX-Udh fusion protein (FIG. 4).

  Similarly, a plasmid for expressing a fusion protein in which a 6 × His tag was added to the C-terminal side of MIOX was prepared. After preparing pTDH3eI-MIOX-6xHis using the primer Rv_C_His_pNoteI_MIOX (SEQ ID NO: 23) instead of the above primer Rv_MIOX_pTDH3eI, pTDH3eI-Udh-MIOX-6xHis was prepared in the same manner as described above (FIG. 5). .

(2) Preparation of glucaric acid-producing Candida boidnii Candida boidnii for glucaric acid production was obtained by introducing the MIOX-Udh fusion protein expression cassette onto the chromosomal DNA of the uracil-requiring mutant MCB39 derived from the inositol-producing Candida boidnii MCB23 strain described above. Produced. The plasmids pTDH3eI-Udh-MIOX and pTDH3eI-Udh-MIOX-6xHis for expression of the MIOX-Udh fusion protein produced by the above method were each treated with a restriction enzyme with SpeI to make them linear, and a Fast Yeast Transformation Kit (TaKaRa) Was used to transform Candida boidnii MCB39 strain. The obtained recombinant yeast was subjected to SD agar medium (0.67% Yeast Nitrogen Base w / o Amino Acids) Difco containing Yeast Synthetic Drop-out Medium Supplements without uracil (SIGMA-ALDRICH), 2% glucose, 2% Agar). The clone that formed a colony on this medium was named MCB229.
The MCB229 strain was obtained on June 18, 2018, by the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (postal code: 292-0818, address: 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba). ), A domestic deposit has been made (accession number: NITE P-02746).

(3) Acquisition of a random insertion mutant strain using a plasmid for superfolderGFP expression A random insertion mutation was introduced into the genome of the above-mentioned MCB229 strain, and an attempt was made to obtain a further high glucaric acid-producing strain. The plasmid for mutagenesis retains the Zeocin resistance gene as a marker gene for transformation, and a fluorescent protein superfolder GFP (JD Pedelacq et al., Nat. Biotechnology, 24 ( 2006) 79-88) The gene was designed as follows based on pTHD3eI-MIOX.

  First, a gene fragment amplified by PCR using pTHD3eI-MIOX as a template and Fw_MIOX_inverse (SEQ ID NO: 24) and Rv_TDH3p_inverse (SEQ ID NO: 25) as a primer, and a plasmid containing a gene encoding superfolderGFP (SEQ ID NO: 26) as a template, Fw_sGFP ( The plasmid pTDH3eI-sfGFP for superfolderGFP expression was prepared by ligating a gene fragment amplified by PCR using SEQ ID NO: 27) and Rv_sGFP (SEQ ID NO: 28) as primers using an In-Fusion HD Cloning Kit (Takara). did.

  Subsequently, the transformation marker gene of pTDH3eI-sfGFP was replaced from the ura3 gene to a Zeocin resistance gene. Specifically, a gene fragment amplified by PCR using pTDH3eI-sfGFP as a template and Fw_pTDH3eIMIOX-Amp (SEQ ID NO: 29) and Rv_pTDH3eIMIOX_AODt (SEQ ID NO: 30) as primers, and pREMI-Zc (JD Pedelacq et al., Nat. Biotechnology, 24 (2006) 79-88) as a template, and a gene fragment amplified by PCR using Fw_pREMIZc (SEQ ID NO: 31) and Rv_pREMIZc (SEQ ID NO: 32) as primers, and In-Fusion HD Cloning Kit (Takara) (Takara) ) To prepare pTDH3eI-sfGFP-Zc (FIG. 6).

  pTHD3eI-sfGFP-Zc was linearized by HindIII digestion and transformed into C. boidinii MCB229 strain using Fast Yeast Transformation Kit (Takara). The obtained recombinant yeast was spread on a YPD medium containing Zeocin 50 μg / mL. The insertion of the gene into the genome was confirmed by confirming the expression of the superfolderGFP protein in the strain obtained in this manner by fluorescence microscopy.

(4) Evaluation of glucaric acid production in mutants with plasmid insertion for superfolderGFP expression
For five strains of superfolderGFP having high fluorescence intensity (AHC006-10, 11, 13, 15, and 16; AHC006-16 was named MCB230), the cells were precultured in 5 mL of YPD medium at 30 ° C for 24 hours, and the results were shown in Table 1. The cells were inoculated to 5 mL of MIS2 medium having the composition shown in No. 3 so that OD ₆₆₀ = 0.1, and cultured at 30 ° C. and 180 rpm for 76 hours.

Analysis was performed using the ^{Applied Biosystems 4000 QTRAP (R) LC} / MS / MS System. The column used was SeQuant ZIC-HILIC 100 × 2.1 mm 3.5 μm 100 ° (Merck Millipore). As a mobile phase, a 20 mM ammonium formate aqueous solution (pH 7.5) (Solvent A) and 20 mM ammonium formate containing acetonitrile (pH 7.5) (Solvent B) were used, and the mixing ratio of Solvent B was changed from 0% (0 min) to 0% (2 min). → 20% (20 minutes) → 100% (24 minutes) → 100% (26 minutes) by increasing over time (flow rate 0.15 mL / min, temperature 40 ° C). Glucaric acid was detected and quantified by monitoring the peak of the mass-to-charge ratio (m / z = 209). As a result, the glucaric acid yield in the MCB230 strain was 200 mg / L in the culture at the 5 mL scale, which was about three times that of the MCB229 strain.
The MCB230 strain was acquired on June 18, 2018, by the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (zip code: 292-0818, address: 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba) ), A domestic deposit has been made (accession number: NITE P-02747).

<Example 4> Evaluation of 1 L jar fermenter culture of glucaric acid-producing C. boidinii MCB230 C. boidinii MCB230 prepared above was seed-cultured in 3 mL of YPD medium at 30 ° C for 8 hours, and then OD ₆₆₀ = 0 in 100 mL of YPD medium. 1 and then main-cultured at 30 ° C. and 160 rpm for 24 hours. The obtained cells were inoculated into a 1 L jar fermenter (Able) containing 400 mL of MIS3 medium having the composition shown in Table 5 so that OD ₆₆₀ = 0.5, and the culture temperature was 30 ° C., the stirring rotation speed was 800 rpm, The cells were cultured for 136 hours under the conditions of controlling the aeration rate at 1 vvm and pH = 3 or under non-neutralizing conditions (around pH = 2). 3.5 as neutralizer
% Ammonia water was used.

  The analysis was performed using HPLC (Shimadzu Corporation). The column used was a COSMOSIL HILIC 5 μm 4.6 × 250 mm Column (Nacalai Tesque). Glucaric acid was eluted isocratically using acetonitrile / 10 mM sodium phosphate buffer (pH 7.0) = 50/50 (flow rate 1 mL / min, temperature 30 ° C.) as a mobile phase and monitoring UV absorption at 210 nm. Was detected and quantified. FIG. 7 shows the change over time in the amount of accumulated glucaric acid obtained as a result of the analysis. The amount of glucaric acid accumulated at the end of the culture was 1.5 g / L under the condition of control pH = 3, and 0.4 g / L under the non-neutralizing condition.

  According to the present invention, glucaric acid can be produced efficiently and at low cost from common organic raw materials such as glucose and galactose while suppressing by-products, and thus has high industrial applicability.

Claims (13)

  A highly acid-resistant microorganism having myo-inositol-1-phosphate synthase activity, myo-inositol monophosphatase activity, myo-inositol oxygenase activity, and uronic acid dehydrogenase activity.   The microorganism according to claim 1, wherein the highly acid-resistant microorganism is a yeast of the genus Candida.   The microorganism according to claim 2, wherein the highly acid-resistant microorganism is Candida boidinii.   The microorganism according to any one of claims 1 to 3, which expresses an enzyme having myo-inositol oxygenase activity and an enzyme having uronic acid dehydrogenase activity as a fusion protein.   The microorganism according to any one of claims 1 to 4, which expresses an enzyme having myo-inositol oxygenase activity as a solubilized protein.   A method for producing glucaric acid, comprising a step of causing the microorganism or the processed product thereof according to any one of claims 1 to 5 to act on an organic raw material in an aqueous medium having a pH of 5 or less.   The method according to claim 6, wherein the organic raw material contains at least one selected from the group consisting of glucose, xylose, sucrose, starch, molasses, myo-inositol, glycerol, ribitol, and erythritol.   The method according to claim 6, wherein the aqueous medium has a pH of 3.5 or less.   The method according to any one of claims 6 to 8, wherein the aqueous medium has a pH of 2.5 or more.   The method according to any one of claims 6 to 9, wherein the method does not include a step of neutralizing the aqueous medium.   The production method according to any one of claims 6 to 10, wherein the crystals are precipitated while generating glucaric acid in the aqueous medium.   A method for producing glucaric acid by the method according to any one of claims 6 to 11, and a step of converting glucaric acid obtained in the step into 2,5-furandicarboxylic acid as a raw material, A method for producing 5-furandicarboxylic acid.   A method for producing glucaric acid by the method according to any one of claims 6 to 11, and a step of converting the glucaric acid obtained in the step into a 2,5-furandicarboxylic acid diester as a raw material, , 5-furandicarboxylic acid diester production method.

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