WO2018199112A1 - Transgenic microorganism and use thereof - Google Patents

Abstract

The purpose of the present invention is to provide: a microorganism which enables the production of muconic acid with good economic efficiency and high yield using a syringyllignin-rich biomass as a raw material; and a method for producing muconic acid using the microorganism. The purpose can be achieved by: a transgenic microorganism of which a host microorganism is a microorganism belonging to the family Sphingomonad, having a protocatechuic acid degrading enzyme gene on the chromosome thereof and capable of utilizing a syringyllignin-derived aromatic compound and in which the protocatechuic acid degrading enzyme gene on the chromosome is deleted, inserted catA gene can be expressed and inserted aroY gene or aroY gene and inserted kpdB gene can also be expressed; a method for producing muconic acid using the transgenic microorganism; and others.

Description

Transformed microorganism and use thereof Cross-reference of related applications

This application claims the priority of Japanese Patent Application No. 2017-86595 filed on Apr. 25, 2017, the entire description of which is incorporated herein by reference.

The present invention relates to a transformed microorganism capable of producing muconic acid or protocatechuic acid, and a method for producing muconic acid or protocatechuic acid using the transformed microorganism. In particular, the present invention is a method for growing and muconic acid using a biomass containing an aromatic compound derived from syringyl lignin, an aromatic compound derived from guaiacyl lignin and / or an aromatic compound derived from p-hydroxyphenyl lignin as a carbon source, or The present invention relates to a transformed microorganism capable of producing protocatechuic acid.

Lignin is an amorphous polymer that exists as a component of plant vascular cell walls. It is a complex condensation of phenylpropane-based structural units, and the main feature of its chemical structure is the inclusion of methoxy groups. ing. Lignin has a function of sticking woody plant cells to each other and strengthening the tissue, and is present in about 18 to 36% in wood and about 15 to 25% in herbs. Therefore, in order to effectively use wood, various attempts have been made to decompose lignin and obtain useful compounds.

On the other hand, cis, cis-muconic acid (hereinafter sometimes simply referred to as muconic acid) is a highly reactive compound due to two double bonds and carboxy groups in the molecule. Various muconic acid derivatives starting from muconic acid are known, and examples include lactone, sulfone, polyamide, polyester, thioester, and addition polymer. Such muconic acid derivatives are known to have various uses, and can be used as, for example, surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents and the like.

As described above, muconic acid is used for various purposes, and if muconic acid can be produced from lignin, resource regeneration is achieved, which is very useful. Therefore, a method for producing muconic acid from lignin or a substance derived from lignin has been attempted. In particular, bioconversion using microorganisms has been studied as such a method.

For example, the following Non-Patent Document 1 (., Which is incorporated as disclosed herein entire disclosure of the document), the Pseudomonas putida and (Pseudomonas putida) as a host microorganism, pCAH gene and pcaG gene on chromosome (hereinafter combined And the catR gene, catB gene, catC gene and catA gene are disrupted, and the inserted catA gene and aroY gene, or the catA gene, aroY gene and ecdB gene are expressed. It is described that a transformed microorganism was produced, the transformed microorganism was grown with glucose, and then muconic acid was produced with p-coumaric acid. Further, in the following Non-Patent Document 2 (the entire description of which is incorporated herein by reference), the Sphingobium species SYK-6 strain is grown using vanillic acid and syringic acid as a carbon source. It is described that it can.

Certainly, the transformed microorganism described in Non-Patent Document 1 metabolizes aromatic compounds derived from p-hydroxyphenyl lignin such as p-coumaric acid and aromatic compounds derived from guaiacyl lignin such as ferulic acid. be able to. However, the transformed microorganism described in Non-Patent Document 1 cannot metabolize aromatic compounds derived from syringyl lignin, such as syringic acid. Therefore, using the transformed microorganism described in Non-Patent Document 1, there is a problem that muconic acid cannot be substantially produced using biomass containing a large amount of syringyl lignin as a raw material.

In addition, since the transformed microorganism described in Non-Patent Document 1 requires expensive glucose as a carbon source for growth, the method for producing muconic acid using the transformed microorganism described in Non-Patent Document 1 is economical. There is a problem that is bad. Furthermore, since two or more types of carbon sources are required, such as glucose for cell growth and p-coumaric acid as a substrate for muconic acid production, considering the total amount of these carbon sources, The method for producing muconic acid using the transformed microorganism described in Non-Patent Document 1 has a problem that the yield of muconic acid is poor.

On the other hand, according to Sphingobiium species SYK-6 described in Non-Patent Document 2, muconic acid cannot be produced using vanillic acid and syringic acid as a carbon source.

Accordingly, the present invention can use a biomass containing a large amount of syringyl lignin as a raw material, has good economic efficiency, and can produce muconic acid with a high yield, and mucon using the microorganism. An object of the present invention is to provide a method for producing an acid.

In an attempt to solve the above-mentioned problems, the present inventors have intensively studied a microorganism capable of producing muconic acid from biomass containing a large amount of syringyl lignin.

FIG. 1 shows the metabolic pathway of the transformed microorganism described in Non-Patent Document 1. As shown in FIG. 1, the transformed microorganism described in Non-Patent Document 1 is Pseudomonas putida in which the host microorganism undergoes degradation through the protocatechuic acid • 3,4-cleavage pathway. From this, when trying to efficiently produce muconic acid by the transformed microorganism described in Non-Patent Document 1, the pcaHG gene and catB gene on the chromosome of the host microorganism are deleted, and the inserted aroY gene is expressed. There must be. This is the first time that an aromatic compound derived from p-hydroxyphenyl lignin such as p-coumaric acid or an aromatic compound derived from guaiacyl lignin such as ferulic acid converges to protocatechuic acid and is converted to muconic acid. Will be able to.

However, as shown in FIG. 1, the transformed microorganism described in Non-Patent Document 1 can metabolize aromatic compounds derived from syringyl lignin such as syringic acid into protocatechuic acid and muconic acid. Furthermore, they cannot be propagated using these. In addition, since the transformed microorganism described in Non-Patent Document 1 lacks the pcaHG gene on the chromosome, a substrate other than lignin-derived aromatic compounds such as glucose is required for the growth of the microorganism. To do.

Accordingly, the present inventors have made intensive studies, which resulted in the focusing on sphingomyelin monad (Sphingomonad) family microorganism capable of metabolizing aromatic compounds from syringyl lignin such syringic acid. The present inventors then disrupted the protocatechuate-4,5-dioxygenase gene on the chromosome of the sphingomonad family microorganism, and then expressed the aroY gene, kpdB gene, catA gene, etc. inserted separately as foreign genes. Succeeded in creating transformed microorganisms. Surprisingly, the transformed microorganism produced by the present inventors grows using an aromatic compound derived from syringyl lignin such as syringic acid, that is, an aromatic compound derived from syringyl lignin. It has been found that muconic acid can be produced using an aromatic compound derived from guaiacyl lignin and p-hydroxyphenyl lignin. In this way, the present inventors can use the transformed microorganisms produced by the present inventors to use aromatic compounds derived from various types of lignin such as syringyl lignin, guaiacyl lignin, and p-hydroxyphenyl lignin. Succeeded in creating a method for producing muconic acid from

Surprisingly, the method using the transformed microorganisms produced by the present inventors requires the use of these lignin-derived aromatic compounds as carbon sources, but also uses glucose and lignin-derived aromatic compounds as substrates. It has been found that the yield of muconic acid is comparable or higher compared to the method using a transformed microorganism described in Non-Patent Document 1 or the like.

The present inventors apply a transformed microorganism capable of producing muconic acid to produce a transformed microorganism capable of producing protocatechuic acid, which is an intermediate of muconic acid, and further, using the transformed microorganism, We have succeeded in creating a method for producing protocatechuic acid from aromatic compounds derived from various types of lignin such as gill lignin, guaiacyl lignin and p-hydroxyphenyl lignin.

The lignin-derived aromatic compound can be obtained from biomass such as waste materials. In particular, hardwood is rich in syringyl lignin. Therefore, since the biomass derived from broad-leaved broad-leaved trees that are prosperous in Japan can be obtained at a very low cost, a method for producing muconic acid and protocatechuic acid using a transformed microorganism produced by the present inventors is described in Non-Patent Document 1. This is an economically advantageous method compared to a method using a transformed microorganism. The present invention has been completed based on these findings and successful examples.

Therefore, according to one aspect of the present invention, the following transformed microorganisms (1) to (6) are provided.
(1) the host microorganism has a protocatechuic acid degrading enzyme gene on a chromosome, and a Sphingomonas monad (Sphingomonad) family microorganism assimilates aromatic compounds from syringyl lignin,
The protocatechuate degrading enzyme gene on the chromosome is deleted,
Expressing the inserted catA gene, and
Expressing the inserted aroY gene or aroY gene and kpdB gene;
Transformed microorganisms.
(2) The transformed microorganism according to (1), wherein the inserted aroY gene, the kpdB gene, and the catA gene are under the control of the same promoter.
(3) The transformed microorganism according to any one of (1) to (2), wherein the transformed microorganism further expresses the inserted vanA gene and vanB gene.
(4) The transformed microorganism according to (3), wherein the inserted aroY gene, the kpdB gene, the catA gene, the vanA gene, and the vanB gene are under the control of the same promoter.
(5) The protocatechuic acid degrading enzyme gene is a gene selected from the group consisting of a ligA gene, a ligB gene, a pcaG gene, a pcaH gene and a prA gene, according to any one of (1) to (4) Transformed microorganisms.
(6) The transformed microorganism according to any one of (1) to (5), wherein the host microorganism is a Sphingobiium species SYK-6 strain.

According to another aspect of the present invention, there is provided the following method (7) for producing muconic acid.
(7) An aromatic compound derived from p-hydroxyphenyl lignin and / or an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from syringyl lignin according to any one of (1) to (6) A method for producing muconic acid, comprising the step of obtaining muconic acid by acting on the transformed microorganism described above.

According to another aspect of the present invention, the following transformed microorganism (8) is provided.
(8) the host microorganism is a sphingomonad family microorganism having a protocatechuate degrading enzyme gene on a chromosome and assimilating an aromatic compound derived from syringyl lignin;
The protocatechuate degrading enzyme gene on the chromosome is deleted,
Transformed microorganisms.

According to another one aspect | mode of this invention, the manufacturing method of the protocatechuic acid of the following (9) is provided.
(9) An aromatic compound derived from p-hydroxyphenyl lignin and / or an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from syringyl lignin are allowed to act on the transformed microorganism described in (8). The manufacturing method of protocatechuic acid including the process of obtaining protocatechuic acid by this.

According to the transformed microorganism of one embodiment of the present invention and the production method of one embodiment of the present invention, a biomass containing a large amount of syringyl lignin, which has been difficult or impossible by the method using a conventional microorganism, is used as a raw material at low cost and in a low yield. Muconic acid and protocatechuic acid can be produced while maintaining or improving the rate. Therefore, according to the transformed microorganism of one embodiment of the present invention and the production method of one embodiment of the present invention, as part of effective utilization of biomass containing various types of lignin and biomass relatively easily available in Japan, Production of muconic acid and protocatechuic acid on a scale can be expected.

FIG. 1 shows FIG. 1 is a schematic diagram of a metabolic pathway of Pseudomonas putida described in 1. FIG.

Hereinafter, the details of the transformed microorganism and the production method according to one aspect of the present invention will be described. However, the technical scope of the present invention is not limited only to the matters of this item, and the present invention achieves the object. As long as it does, various aspects can be taken.

(Overview of transformed microorganisms)
The transformed microorganism which is one embodiment of the present invention is a microorganism obtained by transforming a host microorganism so that a specific gene on the chromosome of the host microorganism is deleted. In addition, the transformed microorganism which is one embodiment of the present invention further transforms the host microorganism so as to express a specific gene involved in the muconic acid synthesis pathway from protocatechuic acid inserted as a foreign gene. There are two types of modes.

As used herein, “gene deletion” means that a gene does not function normally, such as a gene that is not normally transcribed or a protein that is to be produced by gene expression is not translated normally. It means that expression is prevented. Deletion of a gene can occur, for example, when the structure of the gene is changed due to destruction, deletion, substitution, insertion, or the like of all or part of the gene. However, gene deletion can also occur when gene expression is suppressed by means such as blocking the control region of a gene without causing a change in the structure of the gene.

As used herein, “gene expression” means that a protein encoded by a gene is produced in such a manner as to have an original structure or activity through transcription, translation, or the like. In addition, the term “overexpression of a gene” in the present specification means that a protein encoded by the gene is produced in excess of the amount originally expressed by the host microorganism by inserting the gene.

(Gene to be deleted or inserted)
For transformed microorganisms, the host microorganism has a protocatechuate degrading enzyme gene on the chromosome. The protocatechuic acid degrading enzyme gene is not particularly limited as long as it expresses an enzyme having an activity of degrading protocatechuic acid, and examples thereof include ligA gene, ligB gene, pcaG gene, pcaH gene, and prA gene. Among these genes, transformed microorganisms lack part or all of the protocatechuate degrading enzyme gene on the chromosome of the host microorganism.

The ligA gene and the ligB gene are genes that express the small and large subunits of protocatechuic acid and 4,5-dioxygenase, respectively, or genes that express protocatechuic acid and 4,5-dioxygenase consisting of a single polypeptide. If it is, it will not specifically limit, For example, the gene etc. which have the base sequence of sequence number 19 and 20, respectively are mentioned. Protocatechuic acid 4,5-dioxygenase, for example, possessed by Sphingobium species SYK-6, etc., produces 4-carboxy-2-hydroxymuconic acid-6-semialdehyde from protocatechuic acid It has an activity of catalyzing the reaction to be performed and requires Fe ²⁺ as a cofactor.

The pcaH gene and the pcaG gene are not particularly limited as long as they are genes that express the β subunit and α subunit of protocatechuate and 3,4-dioxygenase, respectively. For example, they have the nucleotide sequences of SEQ ID NOs: 35 and 36, respectively. Examples include genes. Protocatechuic acid • 3,4-dioxygenase is possessed by, for example, Pseudomonas putida KT2440 strain, and has an activity of catalyzing the reaction to produce 3-carboxymuconic acid from protocatechuic acid, and contains Fe ³⁺ Require as a cofactor.

The prA gene is not particularly limited as long as it is a gene that expresses protocatechuic acid-2,3-dioxygenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 37. Protocatechuic acid-2,3-dioxygenase is, for example, possessed by Paenibacillus species JJ-1b and the like, and produces 5-carboxy-2-hydroxymuconic acid-6-semialdehyde from protocatechuic acid. Has the activity of catalyzing

The transformed microorganism of one embodiment of the present invention (hereinafter referred to as transformed microorganism (1)) expresses the inserted catA gene. The transformed microorganism (1) further expresses the inserted aroY gene or aroY gene and kpdB gene. On the other hand, none of the catA gene, the aroY gene, and the kpdB gene is inserted into the transformed microorganism of another embodiment of the present invention (hereinafter referred to as transformed microorganism (2)). That is, the transformed microorganism (2) cannot substantially produce muconic acid from protocatechuic acid. In the present specification, when the transformed microorganisms (1) and (2) are collectively referred to, they are simply referred to as “transformed microorganisms”.

The catA gene is not particularly limited as long as it is a gene that expresses catechol 1,2-dioxygenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 21. Catechol 1,2-dioxygenase (EC 1.13.11.1) is also called 1,2-dihydroxybenzene 1,2-dioxygenase and the like. Catechol 1,2-dioxygenase is possessed by, for example, Pseudomonas putida KT2440 strain, has an activity of catalyzing the reaction of generating cis, cis-muconic acid from catechol, and co-factors Fe ³⁺ As request.

Intradiol dioxygenase domain is contained in the amino acid sequence of catechol 1,2-dioxygenase (accession no. Q88I35). The domain is [LIVMF] -xGx- [LIVM] -x (4)-[GS] -x (2)-[LIVMA] -x (4)-[LIVM]-[DE]-[LIVFMFYC ] -X (6) -Gx- [FY] (Prosite entry no. P00083), and Y in the sequence is related to the binding of Fe ³⁺ as a cofactor. L137 to Y165 in the amino acid sequence of catechol 1,2-dioxygenase correspond to the above domain.

The aroY gene is not particularly limited as long as it is a gene that expresses protocatechuic acid / decarboxylase, and examples thereof include a gene having the base sequence of SEQ ID NO: 22. Protocatechuic acid decarboxylase (EC 4.1.1.63) is also called 3,4-dihydroxybenzoate carboxy-lyase. Protocatechuic acid / decarboxylase is not particularly limited as long as it is an enzyme that catalyzes a reaction for producing catechol from protocatechuic acid. An enzyme having an activity of catalyzing a reaction for producing 3-methoxycatechol from 3-O-methylgallic acid or a reaction for producing pyrogallol from gallic acid may be used as protocatechuic acid / decarboxylase. In addition, vanillic acid decarboxylase and 4-hydroxybenzoic acid decarboxylase may also be used as protocatechuic acid / decarboxylase because protocatechuic acid may be decarboxylated. Protocatechuic acid decarboxylase is structurally classified into a protein group (UbiD superfamily) containing a UbiD domain (Domain architecture ID 10487953).

Examples of protocatechuic acid decarboxylase, Klebsiella pneumoniae subsp. Pneumoniae (Klebsiella pneumoniae subsp pneumoniae.) Protein from A170-40 strain (ATCC twenty-five thousand five hundred and ninety-seven strain) (accession no.AB479384; AB479384 protein) etc. Can be mentioned. As a protein having an amino acid sequence having high amino acid sequence with the amino acid sequence of AB479384 protein, protocaterate decarboxylase derived from Enterobacter cloacae MBRL1077 (accession no. AMJ70686; sequence identity 87.2%), E. coli. Examples include, but are not limited to, proteins registered as protocatechuate decarboxylase such as protocatechuate decarboxylase (accession no. CZU76022; sequence identity 85.7%) derived from cloacae e1026 strain. These enzymes are characterized as a series of enzymes that require Mn ²⁺ , prenylated flavin mononucleotide (prenyl-FMN) as cofactors.

The kpdB gene is not particularly limited as long as it is a gene that expresses flavin / prenyltransferase, and examples thereof include a gene that expresses 4-hydroxybenzoic acid / decarboxylase / subunit B. Specifically, SEQ ID NO: 23 And a gene having the nucleotide sequence of Among the UbiX family is Phenolic acid (hydroxylylic acid) decarboxylase subunit B, one of which is 4-hydroxybenzoic acid, decarboxylase, subunit B. In addition, among the phenolic acid decarboxylase, there are homo-oligomeric enzymes such as AroY and Fdc (ferric acid decarboxylase derived from yeast), and BCD such as 4-hydroxybenzoic acid / decarboxylase and vanillic acid / decarboxylase. There are hetero-oligomeric enzymes consisting of subunits.

Flavin prenyltransferase catalyzes the reaction of synthesizing prenyl-FMN by binding a dimethylallyl structure from dimethylallyl monophosphate (DMAP) to the flavin backbone of flavin mononucleotide (FMN).

Since 4-hydroxybenzoic acid / decarboxylase / subunit B is an enzyme classified into the flavoprotein, UbiX / Pad1 family, the amino acid sequence thereof is, for example, E. coli . amino acid sequence sequence identity with (accession no.P0AG03) of coli K-12 strain derived from Flavin prenyltransferase (UbiX) is 50%; S. cerevisiae S288c derived amino acid sequence of Flavin prenyltransferase (Pad1) of (accession no.P33751) and sequence identity is 39.8%; B. It has 54.5% sequence identity with the amino acid sequence (accession no. P94404) of Phenolic acid decarboxylase subunit B (BcdB) derived from subtilis 168 strain. According to White MD et al. (Nature, 522: 502-506, 2015; the entire description is incorporated herein by reference), S37 and R123, which are involved in FMN binding in the amino acid sequence of UbiX, Y153 and R169 involved in the binding were identified, and the amino acid residues involved in the binding of FMN and DMAP are also conserved in the amino acid sequences of proteins classified into the UbiX / Pad1 family such as KpdB.

With respect to the transformed microorganism (1), the host microorganism has the ability to grow (utilize) an aromatic compound derived from syringyl lignin such as syringic acid and syringaldehyde as a carbon source. In order for a host microorganism to have the ability to grow using an aromatic compound having a syringyl nucleus such as syringic acid or syringaldehyde as a sole carbon source, the host microorganism has a syringate / demethylase gene (eg desA on the chromosome). ), 3-O-methylgallic acid · 3,4-dioxygenase gene (eg desZ), 3-O-methylgallic acid · demethylase gene (eg vanAB, ligM), 2-pyrone-4,6-dicarboxylic acid · Hydrolase gene (eg ligI), gallic acid / dioxygenase gene (eg desB), 4-oxalomesaconic acid / tautomerase (eg ligU), 4-oxalomesaconic acid / hydratase gene (eg ligJ), 4-carboxy-4-hydroxy- 2-oxoadipic acid, al Hydrolase gene (e.g. ligK), such as oxaloacetate decarboxylase gene (e.g. ligK), it is preferable to have a gene that produces an enzyme for metabolism of syringic acid to pyruvate.

Among the above genes, for example, the desA gene is not particularly limited as long as it is a gene that expresses syringate O-demethylase, and examples thereof include a gene having the base sequence of SEQ ID NO: 38. The syringic acid demethylase is possessed by, for example, Sphingobium sp. Strain SYK-6 and has an activity of catalyzing a reaction for producing 3-O-methylgallic acid from syringic acid.

In addition, VanAB can convert syringic acid into 3-O-methylgallic acid, and thus can also be used as syringic acid / demethylase.

The host microorganism preferably has a gene expressing vanillate demethylase in addition to the protocatechuate degrading enzyme gene on the chromosome. When the host microorganism does not have a gene expressing vanillate demethylase on the chromosome, it is preferable to insert a gene expressing vanillate demethylase into the transformed microorganism (1). A gene that expresses vanillate demethylase is not particularly limited, and examples thereof include a ligM gene, a vanA gene, and a vanB gene.

The ligM gene is not particularly limited as long as it is a gene that expresses tetrahydrofolate-dependent vanillic acid / 3-O-methylgallic acid / O-demethylase (tetrahydrofolate-dependent vanillate / 3-O-methylgallate O-demethylase). And a gene having the base sequence of SEQ ID NO: 24.

The vanA gene is not particularly limited as long as it is a gene that expresses a vanillate / demethylase / oxygenase component (vanillate demethylase oxygenase component), and examples thereof include a gene having the base sequence of SEQ ID NO: 25.

Examples of the vanillic acid / demethylase / oxygenase component (EC 1.14.13.82) include VanA (accession no. Q88GI6) derived from Pseudomonas putida KT2440. The vanillic acid / demethylase / oxygenase component cleaves the methyl ether bond of vanillic acid using an NADH or NADPH-derived electron supplied via Oxidoductase component and an oxygen atom supplied from molecular oxygen, Protocatechuic acid, formaldehyde and water are produced.

The vanillic acid / demethylase / oxygenase component has Rieske [2Fe-2S] iron-sulfur domain (W7-V107, PROSITE entry no. PS51296) in its amino acid sequence, and C and H (C47, H49) in the domain , C66, H69) are related to the Fe—S bond.

The vanB gene is not particularly limited as long as it is a gene that expresses a vanillate / demethylase / oxidoreductase component (vanillate demethylase oxidoreductase component), and examples thereof include a gene having the base sequence of SEQ ID NO: 26.

Examples of the vanillic acid / demethylase / oxidoreductase component (EC 1.14.13.82) include VanB (accession no. Q88GI5) derived from Pseudomonas putida KT2440. The vanillic acid / demethylase / oxidoreductase component is known as one of the oxidoreductases that extract electrons from NADH or NADPH and transfer them to an oxygenase (oxygenase). The vanillic acid / demethylase / oxidoreductase component transmits NADH or NADPH-derived electrons to VanA which is a vanillic acid / demethylase / oxygenase component.

The vanillic acid / demethylase / oxidoreductase component has 2Fe-2S ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and C in the amino acid sequence 5 (C26) , C270, C273, and C303) are involved in the Fe—S bond. In addition, vanillic acid / demethylase / oxidoreductase components include NAD-binding domain (L109-D201, Pfammentry nono. PF00175) and Ferredoxinreductasetype FAD-BindingPrindoMindPrindoMinder PS 51384).

For the transformed microorganism, the host microorganism preferably has a desA gene and a ligM gene on the chromosome. When the host microorganism does not have the desA gene and the ligM gene on the chromosome, it is preferable to insert the desA gene and the ligM gene into the transformed microorganism.

The gene to be inserted may not be completely the same as the gene originally possessed by the organism having the gene (that is, the wild type gene), and at least the protein that is expressed by the wild type gene (that is, the wild type protein), As long as it is a gene that expresses a protein having similar enzymatic properties, it may be a DNA having a base sequence that hybridizes with a base sequence complementary to the base sequence of the wild-type gene under stringent conditions. Good.

The term “base sequence that hybridizes under stringent conditions” in the present specification refers to a colony hybridization method, plaque hybridization method, Southern blot hybridization method using a DNA having a base sequence of a wild-type gene as a probe. It means the base sequence of DNA obtained by using.

The term “stringent conditions” in the present specification is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal. The hybridization system used, the type of probe, and the sequence It depends on the length. Such conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration. If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration.

As a specific example of stringent conditions, for example, a DNA probe is used as a probe, and hybridization is 5 × SSC, 1.0% (w / v), a blocking reagent for nucleic acid hybridization (Roche Diagnostics) , 0.1% (w / v) N-lauroyl sarcosine, 0.02% (w / v) SDS, overnight (about 8 to 16 hours). Washing is performed using 0.1 to 0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS, twice for 15 minutes. Do. The temperature for performing hybridization and washing is 65 ° C or higher, preferably 68 ° C or higher.

In addition, as the DNA having a base sequence that hybridizes under stringent conditions, for example, using a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter on which the DNA fragment is immobilized, After hybridization at 40 ° C. to 75 ° C. in the presence of DNA obtained by hybridization under the above stringent conditions or 0.5 M to 2.0 M NaCl, preferably 0.7 to 1 After hybridization at 65 ° C in the presence of 0.0 M NaCl, 0.1 to 1 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 65 ° C. DNA that can be identified by washing the filter with The Probe preparation and hybridization methods are described in Molecular Cloning: A laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter these documents may be referred to as reference technical documents. The entire description of these documents is incorporated herein by reference. ) And the like. In addition to the conditions such as the salt concentration and temperature of the buffer, those skilled in the art will consider other conditions such as probe concentration, probe length, reaction time, etc. Conditions for obtaining a DNA having a base sequence that hybridizes under stringent conditions with a complementary base sequence can be appropriately set.

Examples of DNA containing a base sequence that hybridizes under stringent conditions include DNA having a certain sequence identity with a base sequence of a DNA having a base sequence of a wild-type gene used as a probe. 80% or more, preferably 85% or more, more preferably 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, more preferably 99.5% or more of DNA having sequence identity. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

The base sequence that hybridizes with the base sequence complementary to the base sequence of the wild type gene under stringent conditions is, for example, 1 to several, preferably 1 to 50, more preferably, in the base sequence of the wild type gene. 1 to 30, more preferably 1 to 20, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base deletions, substitutions, additions, etc. The base sequence which has. Here, “base deletion” means that there is a deletion or disappearance in the base in the sequence, and “base replacement” means that the base in the sequence is replaced with another base, “Addition of a base” means that a new base is added to be inserted.

The protein encoded by the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild-type gene under stringent conditions is 1 to several in the amino acid sequence of the protein encoded by the base sequence of the wild-type gene. Although it is likely to be a protein having an amino acid sequence having deletion, substitution, addition, etc. of individual amino acids, it has the same enzyme activity as the protein encoded by the base sequence of the wild-type gene.

A protein having the same or similar enzymatic properties as the wild-type protein has an amino acid sequence having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild-type protein It may consist of. Here, the range of “1 to several” in the “deletion, substitution and addition of one to several amino acids” of the amino acid sequence is not particularly limited, but for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 About 1, more preferably about 1, 2, 3, 4 or 5. In addition, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, and “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue. “Addition of amino acid” means that a new amino acid residue is added to the sequence.

A specific embodiment of “deletion, substitution, addition of 1 to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid. For example, a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid, a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the basic amino acid having a positive charge include arginine, histidine, and lysine. Examples of acidic amino acids having a negative charge include aspartic acid and glutamic acid.

Examples of amino acid sequences having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild type protein include amino acid sequences having a certain sequence identity with the amino acid sequence of the wild type protein. For example, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% with the amino acid sequence of the wild-type protein As mentioned above, amino acid sequences having sequence identity of 97% or more, 98% or more, or 99% or more, and more preferably 99.5% or more can be mentioned. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

(Means for calculating sequence identity)
The method for determining the sequence identity of the base sequence or amino acid sequence is not particularly limited. For example, by using a commonly known method, the amino acid sequence of the wild type gene or the wild type protein expressed by the wild type gene and the target base It is obtained by aligning sequences and amino acid sequences and using a program for calculating the coincidence ratio between the sequences.

As a program for calculating the coincidence rate between two base sequences and amino acid sequences, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993; the entire description of this document is incorporated herein by reference), and a BLAST program using this algorithm has been developed by Altschul et al. Mol. Biol. 215: 403-410, 1990; the entire description of this document is hereby incorporated by reference.) Furthermore, Gapped BLAST, which is a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997; the entire description of this document is incorporated herein by reference). . Therefore, a person skilled in the art can search, for example, a sequence showing high sequence identity from a database using a program described above. These can be used, for example, on the website of the US National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Each of the above methods can be generally used for searching a sequence showing sequence identity from a database. As a means for determining the sequence identity of individual sequences, Genetyx network version version 12.0. 1 (Genetics) homology analysis can also be used. This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985; the entire description of this document is incorporated herein by reference). When analyzing the sequence identity of a base sequence, a region encoding a protein (CDS or ORF) is used if possible.

(The origin of the gene to be inserted)
The gene to be inserted is derived from a microorganism or the like carrying the gene to be inserted. Examples of the organism derived from the gene to be inserted include microorganisms that can produce muconic acid from protocatechuic acid, microorganisms that can grow by assimilating protocatechuic acid, and the like.

Specific examples of organisms from which genes are inserted include sphingo such as Sphingobium species, Sphingomonas species, Novosphingobium aromaticibolans, Alter erythrobacter species, and erythrobacter for ligM gene and desA gene. Microorganisms such as monads, Arthrobacter castelli, Arthrobacter species such as Arthrobacter sp., Microbacteriaceae such as Leifsonia sp. Micrococcidae microorganisms such as Gangotriensis, Citrococcus sp., Tersicoccus sp.・ Microbacteriaceae microorganisms such as Spices, etc .; For the catA gene, Pseudomonas petitda, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas rennekei and Acinetobacter calcoaceticus, Acinetobacter radioresistence microorganisms Rhodococcus microorganisms such as Opacas, Rhodococcus pyridiniboras, Rhodococcus rhodochrous, etc .; for aroY and kpdB genes, Klebsiella microorganisms such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasi pneumoniae, and Enterobacter cloacaeros enthusiata Enterobacter microorganisms, Cediment bacter hydroxybenzoicus, etc .; for the vanA gene and vanB gene, Pseudomonas microorganisms such as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas renonovans, Pseudomonas aeruginosa, Comamonas testosteroni, Comamonas thiooxy Examples include, but are not limited to, microorganisms of the genus Comamonas such as dance, microorganisms of the genus Acetobacter such as Acetobacter patsulianus, Acetobacter aceti, Acetobacter tropicalis, and the like.

As described above, the organism from which the inserted gene is derived is not particularly limited, but the gene expressed in the transformed microorganism is not inactivated by the growth conditions of the host microorganism, or is transformed by inserting the gene so as to exhibit activity. A host microorganism to be converted or a microorganism having similar growth conditions to the host microorganism is preferable.

(Cloning of genes using genetic engineering techniques)
The gene to be deleted and inserted can be inserted into various known vectors. Furthermore, this vector can be introduced into a suitable known host microorganism to produce a transformant (transformed microorganism) having the gene deleted or inserted. The gene to be deleted is preferably one in which the whole or a part of the wild-type gene is altered in its structure due to destruction, deletion, substitution, insertion or the like. The inserted gene is preferably a gene that expresses the same or similar protein as the wild-type gene.

Those skilled in the art can obtain methods for obtaining genes to be deleted and inserted, methods for obtaining information on the base sequences of these genes and amino acid sequences of proteins expressed by these genes, methods for producing various vectors, methods for producing transformed microorganisms, etc. Can be selected as appropriate. Moreover, in this specification, a transformation and a transformant include a transduction and a transductant, respectively. An example of cloning of the gene to be deleted and inserted will be described later without limitation.

For example, chromosomal DNA or mRNA can be extracted from an organism derived from a wild-type gene associated with a gene to be deleted or inserted or various microorganisms by a conventional method, for example, a method described in a reference technical document. CDNA can be synthesized using the extracted mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA thus obtained.

For example, the gene to be inserted can be obtained by cloning using a chromosomal DNA or cDNA of a derived organism having a wild-type gene related to the gene as a template. The wild-type gene-derived organisms are as described above, and specific examples include Sphingobium sp. SYK-6, Pseudomonas putida KT 2440, and Klebsiella pneumoniae sub-species, depending on the type of gene. Examples include, but are not limited to, Pneumonier A170-40. For example, Pseudomonas putida KT2440 strain is cultured, water is removed from the obtained bacterial cells, and it is physically ground using a mortar while cooling in liquid nitrogen to obtain fine powdered bacterial cell pieces. A chromosomal DNA fraction is extracted from the cell fragments by a conventional method. For the chromosomal DNA extraction operation, a commercially available chromosomal DNA extraction kit such as DNeasy Blood & TissueKit (Qiagen) can be used. In this specification, chromosomal DNA and genomic DNA are synonymous.

Next, the DNA is amplified by performing a polymerase chain reaction (PCR) using a chromosomal DNA as a template and a synthetic primer complementary to the 5 'end sequence and the 3' end sequence. The primer is not particularly limited as long as it can amplify a DNA fragment containing a gene to be inserted. Examples thereof include primers represented by SEQ ID NOs: 11 and 12, which are designed with reference to the genome sequence of Pseudomonas putida KT2440 strain as an amplifying catA gene. In addition, when such a primer is used, the target gene full length can be amplified. As another method, DNA containing a target gene fragment is amplified by screening a target gene clone from a shotgun library, or by appropriate PCR such as Inverse PCR, Nested PCR, 5′RACE method, or 3′RACE method. These can be ligated to obtain DNA containing the full-length target gene.

In addition, the method for obtaining a gene to be deleted or inserted is not particularly limited as described above, and it is possible to construct a gene using, for example, a chemical synthesis method without using a genetic engineering technique.

Confirmation of the base sequence in the amplification product amplified by PCR or the chemically synthesized gene can be performed, for example, as follows. First, a DNA whose sequence is to be confirmed is inserted into an appropriate vector according to a normal method to produce a recombinant DNA. For cloning into vectors, known or commercially available kits such as In-Fusion HD Cloning Kit (Takara Bio), TA Cloning Kit (Invitrogen), etc .; pAK405 (Andreas Kaczmarczzyk et al., Applied and Environmental Micro10, Bio201, 78 ) 3774-3777; the entire description of this document is incorporated herein by reference.), PMCL200 (see Gene, vol. 162, p157-158, 1995; the entire description of this document is incorporated herein by reference) PJB866 (Plasmid, vol. 38, p35-51, 1997; the entire description of which is incorporated herein by reference), pUC118 (taka Bio), pQE30 (Qiagen), pUC4K (Gene, vol. 19, p259-268, 1982; the entire description of this document is incorporated herein by reference), pEX18Amp (Gene, vol. 212, See p77-86, 1998; the entire description of this document is incorporated herein by reference.), pPS858 (see Gene, vol. 212, p77-86, 1998; the full description of this document is hereby incorporated by reference) ), Known or commercially available plasmid vectors such as pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio); known or commercially available bacteriophage vectors such as λEMBL3 (Stratagene) Etc. can be used.

When a large amount of the constructed recombinant DNA is to be obtained, the recombinant DNA is introduced into, for example, Escherichia coli , preferably E. coli JM109 (Takara Bio) or E. coli DH5α (Takara Bio). Then, the recombinant DNA contained in the obtained transformant can be purified using QIAGEN Plasmid Mini Kit (Qiagen) or the like.

For the determination of the base sequence of each gene inserted into the recombinant DNA, see the dideoxy method (Methods in Enzymology, 101, 20-78, 1983, etc.); the entire description of this document is incorporated herein by reference. ) Etc. The sequence analysis apparatus used for determining the base sequence is not particularly limited, and examples thereof include Li-COR MODEL 4200L Sequencer (Aloka), 370 DNA Sequence System (PerkinElmer), and CEQ2000XL DNA Analysis System (Beckman). Can be mentioned. Based on the determined base sequence, the amino acid sequence of the translated protein can be known.

(Construction of recombinant vectors containing genes)
Recombinant vector (recombinant DNA) containing a gene to be deleted or inserted is obtained by linking PCR amplification products containing the gene to be deleted or inserted and various vectors in such a manner that the gene can be deleted or expressed. Can be constructed. In the case of a deleted gene, the recombinant vector is deleted because the gene in the recombinant vector is replaced with the gene in the host microorganism by homologous recombination by introducing the recombinant vector into the host microorganism. It is preferable to include regions upstream and downstream of the gene.

As a non-limiting example, a method for producing a recombinant vector containing a gene to be inserted is, for example, excising a DNA fragment containing any of the genes to be inserted with an appropriate restriction enzyme and cleaving the DNA fragment with an appropriate restriction enzyme. The plasmid vector thus constructed can be constructed by ligation using a commercially available recombinant vector production kit such as In-Fusion HD Cloning Kit (Takara Bio Inc.). Alternatively, a DNA fragment containing a gene having a sequence homologous to a plasmid vector added to both ends and a plasmid-derived DNA fragment amplified by inverse PCR are commercially available, such as In-Fusion HD Cloning Kit (Takara Bio Inc.). It can be obtained by ligation using a recombinant vector preparation kit.

A recombinant vector containing a gene to be deleted or inserted includes at least a gene to be deleted or inserted and a gene (base sequence) derived from a plasmid vector. Examples of the recombinant vector include a recombinant vector containing a catA gene and an aroY gene; a recombinant vector containing a catA gene, an aroY gene and a kpdB gene. The recombinant vector can contain a vanA gene and a vanB gene in addition to the catA gene, aroY gene and kpdB gene. In addition, the recombinant vector may contain a gene other than the genes described above as long as it does not interfere with the solution of the problem of the present invention.

The recombinant vector preferably includes a heterologous gene or heterologous nucleic acid sequence. The heterologous gene is not particularly limited as long as it is not naturally occurring in the host microorganism (for example, a synthetic gene that does not depend on the nucleic acid sequence derived from the host microorganism, or an organism in which the inserted gene and the derived organism are different). Examples include genes derived from organisms such as other microorganisms different from host microorganisms, plants, animals, and viruses. Specific examples of the heterologous gene when the host microorganism is a Pseudomonas microorganism include, but are not limited to, a DNA fragment derived from pUC118, such as a lactose promoter region (Plac).

(Method for producing transformed microorganism)
A method for producing a transformed microorganism is not particularly limited, and examples thereof include a method of inserting into a host microorganism in such a manner that gene deletion or insertion is realized according to a conventional method. Specifically, a DNA construct in which any of the inserted genes is inserted between an expression-inducing promoter and a terminator is prepared, and then a host microorganism is transformed with the DNA construct. A converted microorganism is obtained. Alternatively, a transformed microorganism that lacks the gene can be obtained by preparing a DNA construct that includes the gene to be deleted and regions upstream and downstream of the gene, and then transforming the host microorganism with the DNA construct. In the present specification, recombinant vectors prepared for transforming host microorganisms are collectively referred to as DNA constructs.

The method for introducing the DNA construct into the host microorganism is not particularly limited. For example, the DNA construct is introduced into the host microorganism so that the introduced DNA construct autonomously proliferates to express the gene, as known to those skilled in the art. Method: Examples include a method of directly inserting a DNA construct into the chromosome of a host microorganism by utilizing homologous recombination.

In a method using homologous recombination as a method for introducing a DNA construct containing a gene to be inserted into a host microorganism, the DNA construct is ligated between sequences upstream and downstream of the recombination site on the chromosome. Can be inserted into the genome of the host microorganism.

The vector-host system used for the production of the transformed microorganism is not particularly limited as long as it is a system in which the inserted gene can be expressed or a gene on the chromosome can be deleted in the host microorganism. For example, pJB866- Sphingomonad family microorganism system, pKT230 (Gene, vol. 16, p237-247, 1981; the entire description of this document is incorporated herein by reference)-a sphingomonad family microorganism system.

The DNA construct containing the gene to be inserted does not introduce into the chromosome of the host microorganism, and autonomously amplifies and expresses the gene to be inserted, but expresses the gene to be inserted in the form introduced into the chromosome of the host microorganism. Or either.

The DNA construct may include a marker gene to allow selection of transformed cells. The marker gene is not particularly limited, and examples thereof include drug resistance genes for drugs such as kanamycin, tetracycline, ampicillin, and carbenicillin. The marker gene may be included in the middle of the deleted gene or so as to replace the deleted gene.

Depending on the type of gene, the DNA construct containing the gene to be inserted is not limited to a promoter and terminator that allow the gene to be expressed in the host microorganism, and other regulatory sequences (for example, cis involved in transcription control such as an operator). An array, etc.).

Examples of one embodiment of the DNA construct include, but are not limited to, the pTS082 plasmid vector, the pTS084 plasmid vector, and the pTS079 plasmid vector described in the Examples described later.

As a method for transformation into a sphingomonad family microorganism, a method known to those skilled in the art can be appropriately selected, and for example, electroporation (electroporation) method, junction transfer method and the like can be performed.

As a medium for selecting and growing transformed microorganisms, an appropriate medium is used according to the host microorganism and marker gene to be used. For example, when the Sphingobium sp. Strain SYK-6 is used as the host microorganism and the kanamycin and tetracycline resistance genes are used as the marker genes, selection and growth of the transformed microorganism can be achieved by, for example, transforming the transformed microorganism into these drugs. It can be carried out by culturing in an LB medium containing

Confirmation that a transformed microorganism has been produced can be achieved by, for example, transforming the transformed microorganism under conditions where only the transformed microorganism lacking the gene can survive or under conditions where only the transformed microorganism expressing the inserted gene can survive. It can be achieved by culturing and the like. In addition, by culturing the transformed microorganism and then confirming that the amount of muconic acid in the culture obtained after culturing is larger than the amount of muconic acid in the culture of the host microorganism cultured under the same conditions, etc. It can be confirmed that a transformed microorganism has been produced.

Confirmation that a transformed microorganism has been produced can be obtained by extracting chromosomal DNA from the transformed microorganism, performing PCR using this as a template, and generating a PCR product that can be amplified when transformation occurs. You may carry out by confirming a characteristic, a base sequence, etc.

For example, PCR is performed with a combination of a forward primer for the promoter base sequence of the gene to be deleted or inserted and a reverse primer for the base sequence of the marker gene to confirm that a product of the expected length is generated.

When performing transformation by homologous recombination, perform PCR with a combination of a forward primer located upstream from the upstream homologous region used and a reverse primer located downstream from the downstream homologous region used, It is preferable to confirm that a product of the expected length is produced when homologous recombination occurs.

(Host microorganism)
The host microorganism is not particularly limited as long as it is a sphingomonad family microorganism having a protocatechuate-degrading enzyme gene on a chromosome and assimilating an aromatic compound derived from syringyl lignin, for example, protocatechuate degradation It is a sphingomonad family microorganism having an enzyme gene and growing using syringic acid and syringaldehyde. Such a sphingomonad family microorganism is preferably a sphingomonado family microorganism capable of degrading protocatechuic acid, and sphingomonado that assimilates syringic acid or syringaldehyde even if the protocatechuate degrading enzyme is deleted. More preferred are family microorganisms, and sphingomonad family microorganisms having ligA gene and ligB gene on the chromosome are more preferred. Specific preferred embodiments of the host microorganism include Sphingobium sp. SYK-6 strain, Sphingobium sp. 66-54 strain, Sphingomonas hengshuiensis WHSC-8 strain, Novosphingobium strain Novosphingium sp. A sphingomonad family microorganism such as PP1Y strain, Novosphingobium sp. AAP93 strain, more preferably ligA gene and ligB gene on the chromosome, and syringic acid and syringaldehyde Sphingobium species to be utilized SYK-6.

(Specific examples of genes to be deleted or inserted)
Specific examples of the ligA gene, the ligB gene, and the ligM gene as the genes to be deleted or inserted are the ligA gene, the ligB gene, and the ligM gene possessed by the Sphingobium species SYK-6 strain. Nos. 19 to 20 and 24. Specific examples of the catA gene, vanA gene, and vanB gene are the catA gene, vanA gene, and vanB gene possessed by Pseudomonas putida KT2440, and the base sequences thereof are those described in SEQ ID NOs: 21 and 25 to 26, respectively. . A specific example of the aroY gene is the aroY gene possessed by the Klebsiella pneumoniae subspecies pneumoniae A170-40 strain, and the nucleotide sequence is as set forth in SEQ ID NO: 22. A specific example of the kpdB gene is the kpdB gene possessed by Klebsiella pneumoniae, subspices pneumoniae NBRC14940, and the nucleotide sequence thereof is shown in SEQ ID NO: 23. The amino acid sequences of proteins expressed by these genes are those described in SEQ ID NOs: 27 to 34, respectively.

A method for obtaining a gene to be deleted or inserted from a microorganism other than Sphingobium sp., Pseudomonas putida, Klebsiella pneumoniae is not particularly limited. For example, the ligA gene, ligB possessed by Sphingobium sp. Nucleotide sequences of the gene and ligM gene (SEQ ID NOs: 19 to 20 and 24); Pseudomonas putida, nucleotide sequences of the catA gene, vanA gene and vanB gene (SEQ ID NOs: 21 and 25 to 26) possessed by the KT2440 strain; Klebsiella pneumoniae Subspecies pneumoniae A170-40 aroY gene nucleotide sequence (SEQ ID NO: 22); Klebsiella pneumoniae subspecies pneumoniae NBRC14940 strain kpd By performing a BLAST homology search on genomic DNA sequences of other microorganisms based on the base sequence of the gene (SEQ ID NO: 23), and identifying a gene having a base sequence with high sequence identity to the above base sequence Obtainable. Further, based on the total protein of other microorganisms, a protein having an amino acid sequence having a high sequence identity with the amino acid sequence (SEQ ID NO: 27 to 34) of the protein expressed by the gene is specified, and a gene that expresses the protein is identified. It can be obtained by specifying. The reason why the obtained gene corresponds to a gene to be deleted or inserted is that the derived organism is transformed as a host microorganism by the obtained gene, and the production amount of muconic acid is enhanced compared to the host microorganism. I can confirm.

Sphingobiium species SYK-6, Sphingobium species 66-54, Sphingomonas Hengshuiensis WHSC-8, Novosphingobium species PP1Y and Novosfingobium species AAP93 Since the genome structures are similar, there is a probability that they can be transformed into each other by inserting the genes they have. For example, the genes obtained from Sphingobium sp. Strain SYK-6 are used as host microorganisms, Sphingobium sp. Sp. 66-54 strain, Sphingomonas Hengshuiensis WHSC-8 strain, Novosphingobium sp. Strain and Novosphingobium sp. AAP93 strain can be transformed. It is also possible to introduce a gene obtained from Sphingobium sp. Strain SYK-6 into a Pseudomonas microorganism, Klebsiella microorganism, Enterobacter microorganism, Escherichia coli or the like as a host microorganism for expression. The gene to be inserted may be a gene whose codon, secondary structure, GC content and the like are optimized for expression in the host microorganism.

(One embodiment of transformed microorganism)
One specific aspect of the transformed microorganism (1) is that the host microorganism is a sphingobium species SYK-6 strain, a sphingobium species 66-54 strain, a sphingomonas hengshuiensis WHSC-8 strain, a nobosphingo A sphingomonad family microorganism selected from the group consisting of Bium sp. PP1Y strain and Novosphingobium sp. AAP93 strain, wherein at least one gene selected from the group consisting of ligA gene and ligB gene on the chromosome is present A transformed sphingomonad family microorganism that is deleted, expresses the inserted catA gene, and expresses the inserted aroY gene or aroY gene and kpdB gene. Another specific embodiment of the transformed microorganism (1) is a transformed sphingomonad microorganism that expresses the inserted vanA gene and vanB gene in the transformed sphingomonad family microorganism. In addition, when these transformed sphingomonad family microorganisms have a ligM gene on a chromosome, they may express the ligM gene.

In such a transformed microorganism (1), the ligA gene and / or ligB gene on the chromosome is deleted, the inserted catA gene is expressed, and the inserted aroY gene, aroY gene and kpdB gene are By taking at least the mode of expression, aromatic compounds derived from guaiacyl lignin such as vanillic acid and p-hydroxyphenyl such as 4-hydroxybenzoic acid, which are impossible with the transformed microorganism described in Non-Patent Document 1, Growth and muconic acid production are possible using lignin-derived aromatic compounds and syringyl lignin-derived aromatic compounds such as syringic acid as a carbon source. As described in the examples described later, a more preferable embodiment of the transformed microorganism is a transformed microorganism that expresses further inserted vanA gene and vanB gene.

Specific examples of the transformed microorganism (1) include SME257 / pTS084 strain described in Examples described later, but are not limited thereto.

One specific aspect of the transformed microorganism (2) is that the host microorganism is Sphingobiium species SYK-6, Sphingobiium species 66-54, Sphingomonas Hengshuiensis WHSC-8, Novosphingo Bium sp., A sphingomonad family microorganism selected from the group consisting of PP1Y strain and Novosphingobium sp. Strain AAP93, wherein at least one gene selected from the group consisting of ligA gene and ligB gene on the chromosome is present The transformed sphingomonad family microorganism which has been deleted.

Such a transformed microorganism (2) has at least an embodiment in which the ligA gene and / or the ligB gene on the chromosome is deleted, so that an aromatic compound derived from guaiacyl lignin such as vanillic acid or 4-hydroxy Growth and production of protocatechuic acid are possible using an aromatic compound derived from p-hydroxyphenyl lignin such as benzoic acid and an aromatic compound derived from syringyl lignin such as syringic acid as a carbon source.

Specific examples of the transformed microorganism (2) include the SME257 strain described in Examples described later, but are not limited thereto.

Protocatechuic acid is a precursor of muconic acid and also a precursor of protocatechuic acid-2,3-, 3,4-, 4,5-ring-cleaving metabolites. Among these metabolites, for example, 2 Some have uses as synthetic resin raw materials such as -pyrone-4,6-dicarboxylic acid (for example, Japanese Patent No. 4658244; the entire description of this document is incorporated herein by reference). Protocatechuic acid is also used as a synthetic raw material for pharmaceuticals, agricultural chemicals, fragrances and the like.

JP 2010-207094 (the entire description of which is incorporated herein by reference) uses Pseudomonas putida as a host microorganism, pcaHG gene on chromosome and protocatechuate 5-position oxidase gene. A transformed microorganism that is disrupted or mutated and expresses the inserted tpaK gene, tpaAa gene, tpaAb gene, tpaB gene, and tpaC gene is produced, and the transformed microorganism is grown with glucose, and then protocatechuic acid with terephthalic acid Is described. However, Japanese Patent Application Laid-Open No. 2010-207094 does not produce protocatechuic acid from lignin or a lignin-derived aromatic compound. In fact, the transformed microorganism described in JP2010-207094 cannot metabolize aromatic compounds derived from syringyl lignin, such as syringic acid.

(Production method)
The production method of one embodiment of the present invention (hereinafter referred to as production method (1)) includes an aromatic compound derived from guaiacyl lignin such as vanillic acid and an aromatic compound derived from syringyl lignin such as syringic acid and syringaldehyde. At least a step of obtaining muconic acid by acting on the transformed microorganism (1). At this time, an aromatic compound derived from p-hydroxyphenyl lignin such as 4-hydroxybenzoic acid is used in the transformed microorganism (1) together with the aromatic compound derived from guaiacyl lignin or instead of the aromatic compound derived from guaiacyl lignin. You may make it act.

In another embodiment of the present invention, a method for producing muconic acid (hereinafter referred to as production method (2)) includes an aromatic compound derived from guaiacyl lignin such as vanillic acid and an aromatic compound derived from syringyl lignin such as syringic acid. At least a step of obtaining protocatechuic acid by acting on the transformed microorganism (2). At this time, an aromatic compound derived from p-hydroxyphenyl lignin such as 4-hydroxybenzoic acid is used in the transformed microorganism (2) together with the aromatic compound derived from guaiacyl lignin or instead of the aromatic compound derived from guaiacyl lignin. You may make it act.

In this specification, when the manufacturing methods (1) and (2) are collectively referred to, they are simply referred to as “manufacturing methods”.

The method of allowing the lignin-derived aromatic compound to act on the transformed microorganism is a method in which the lignin-derived aromatic compound and the transformed microorganism are brought into contact with each other and muconic acid or protocatechuic acid can be produced by the enzyme of the transformed microorganism. Although not particularly limited, for example, the transformed microorganism is cultured under various culture conditions suitable for the transformed microorganism using a medium containing an aromatic compound derived from lignin and suitable for the growth of the transformed microorganism. Depending on the case, a method for producing muconic acid or protocatechuic acid may be mentioned. The culture method is not particularly limited, and examples thereof include a solid culture method and a liquid culture method performed under aerated conditions. The order of contact between the aromatic compound derived from guaiacyl lignin, the aromatic compound derived from p-hydroxyphenyl lignin and the aromatic compound derived from syringyl lignin and the transformed microorganism is not particularly limited, but derived from syringyl lignin Contacting the transformed microorganism with an aromatic compound derived from guaiacyl lignin and / or an aromatic compound derived from p-hydroxyphenyl lignin next to the aromatic compound, an aromatic compound derived from syringyl lignin and an aroma derived from guaiacyl lignin It is preferable to simultaneously act on the transformed microorganism with the aromatic compound and / or the aromatic compound derived from p-hydroxyphenyl lignin.

As the medium, any of a synthetic medium and a natural medium can be used as long as it contains a normal medium for culturing host microorganisms, that is, a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio. Since the host microorganism is a sphingomonadaceae microorganism, a Wx minimal medium or the like as described in Examples described later can be used, but it is not particularly limited. As the carbon source, an aromatic compound derived from lignin, another carbon source such as sugar or organic acid, or a combination thereof can be used. However, the medium component preferably contains a component necessary for activation of an enzyme involved in the production of muconic acid or protocatechuic acid, for example, Fe ²⁺ . Iron ions, magnesium ions and the like can be added to the medium as compounds, but they may be added as mineral-containing materials.

The aromatic compound derived from lignin is not particularly limited as long as it is an aromatic compound that can be derived from lignin of any one of guaiacyl lignin, syringyl lignin and p-hydroxyphenyl lignin, and for example, guaiacyl lignin, for example. And compounds corresponding to degradation products of syringyl lignin and p-hydroxyphenyl lignin, such as p-coumaric acid, ferulic acid, syringic acid, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, etc. Is mentioned. Aromatic compounds derived from lignin include compounds that are modeled on lignin, such as guaiacylglycerol-β-guaiacyl ether. The lignin-derived aromatic compound is preferably a biomass containing lignin or one obtained by subjecting the biomass to pretreatment and extracted, but may be chemically synthesized and purified regardless of the biomass. . The lignin-derived aromatic compound can be used alone or in combination of two or more.

The biomass containing lignin (hereinafter sometimes referred to as lignocellulose) is not particularly limited, and examples thereof include natural products such as grass and trees, those obtained by processing these natural products, and agricultural waste. Specific examples include woody biomass such as conifers and hardwoods. For example, hardwood is known to contain a large amount of syringyl lignin.

Lignocellulose can be, for example, in the form of a solid, suspension, or liquid depending on the presence or absence of pretreatment. For example, a suspension obtained by adding pulverized lignocellulose to a liquid can be used.

The lignocellulose may be a lignin extract. As the lignin extract, for example, a powdered lignocellulose is lignin so that it becomes 0.1% W / V to 50% W / V, preferably 1% W / V to 20% W / V. Suspension suspended in a solvent suitable for extraction of the above. Further, the lignin extract is obtained by subjecting the suspension to 10 ° C. to 150 ° C., preferably 20 ° C. to 130 ° C., more preferably 20 ° C. to 80 ° C., for several hours to several days, preferably 1 hour to 6 days. A solid lignin extract obtained by subjecting to an extraction treatment and then removing the solid from the liquid lignin extract obtained by removing solids from the extraction treatment liquid and evaporating the solvent to dryness. It may be.

Although the preparation method of a lignin extract is not specifically limited, For example, the following methods etc. are mentioned. That is, 2M NaOH 50 mL, degreased birch wood powder 1.5 g, and nitrobenzene 3 mL were placed in a stainless steel vessel of a small autoclave apparatus (pressure-resistant glass industry, portable reactor TVS-1) and stirred at 500 rpm for 2.5 at 170 ° C. Processing time. Cool to 60 ° C. or lower, and collect the supernatant by centrifugation (6,000 g, 10 min). The obtained supernatant is subjected to diethyl ether extraction three times (recovering the aqueous layer). After acidifying the aqueous layer with hydrochloric acid, extraction with diethyl ether is repeated three times (the ether layer is recovered). Add sodium sulfate to the ether layer and dehydrate in the refrigerator overnight. The ether layer is collected and the extract is evaporated to dryness. The ether extract is dissolved in ion-exchanged water with addition of sodium hydroxide (pH≈9) to obtain an aromatic compound solution derived from white birch lignin.

Solvents suitable for extraction and treatment of lignin are not particularly limited, and examples thereof include water, low molecular alcohols such as dioxane, methanol, and isopropanol, nitrobenzene, diethyl ether, and dimethylformamide.

Culture conditions for sphingomonad microorganisms commonly known by those skilled in the art may be employed. For example, the initial pH of the medium is adjusted to 5 to 10, the culture temperature is 20 ° C. to 40 ° C., and the culture time is several times. Time to several days, preferably 1 to 7 days, more preferably 2 to 5 days, etc. can be set as appropriate. The culture means is not particularly limited, and aeration stirring deep culture, shaking culture, stationary culture and the like can be employed. However, when utilizing the enzyme activity of DesA expressed by the desA gene and the enzyme activity of LigM expressed by the ligM gene, the dissolved oxygen concentration is not particularly limited, but the enzyme activities of VanA and VanB expressed by the vanA gene and vanB gene are reduced. When used, it is preferable to culture under conditions that provide sufficient dissolved oxygen. For example, as an example of the culture medium and culture conditions, as described in the examples described later, using Wx minimal medium containing syringic acid and vanillic acid as a carbon source at 30 ° C. and 180 rpm for 1 to 5 days. Examples include shaking culture and stirring culture. The carbon source and other components can be added as appropriate after the start of culture.

The method for obtaining muconic acid or protocatechuic acid from the culture after completion of the culture is not particularly limited. Since muconic acid or protocatechuic acid accumulates in the culture solution, the cells and culture supernatant are separated from the culture by normal solid-liquid separation operations such as filtration and centrifugation, and the column is removed from the collected culture supernatant. Muconic acid or protocatechuic acid is extracted by solid phase extraction or solvent extraction using a solvent in which muconic acid or protocatechuic acid is soluble.

The extraction solvent is not particularly limited as long as muconic acid or protocatechuic acid can be dissolved, and examples thereof include organic solvents such as methanol, ethanol, isopropanol, and acetone; hydrous organic solvents obtained by mixing these organic solvents and water, and the like. Can be mentioned. Although extraction temperature is not specifically limited, For example, it can set from room temperature to 100 degreeC.

Specific embodiments of the method for extracting muconic acid include, for example, the method of Vardon et al. (Green chemistry, vol. 18, p3397-3413, 2016; the entire description of the document is incorporated herein by reference) and the like. For example, the method may be partially changed. Specifically, activated carbon (12.5% (w / v), 100 mesh) is added to the culture supernatant and stirred for 1 hour. The activated carbon is removed by suction filtration, and the filtrate is recovered. Hydrochloric acid is added to the collected filtrate to adjust the pH to 2, and the mixture is allowed to stand overnight at 4 ° C. The precipitate is collected by suction filtration, and the precipitate is washed with ion exchange water, then collected by suction filtration, and dried under reduced pressure. Suspend the dried solid in ethanol, remove unnecessary substances by suction filtration, and collect the filtrate. The filtrate is dried under reduced pressure using an evaporator to obtain purified muconic acid.

As one aspect of the method for extracting protocatechuic acid, for example, hydrochloric acid is added to the culture supernatant to adjust the pH to approximately 2, and then extraction can be performed with an organic solvent such as ethyl acetate. Examples include a method of obtaining protocatechuic acid by recrystallization of the obtained extract or using an ion exchange resin.

The qualitative or quantitative analysis of muconic acid or protocatechuic acid is not particularly limited, and can be performed by, for example, HPLC. Those skilled in the art can appropriately select the HPLC separation conditions, and for example, the HPLC separation conditions can be performed under the conditions described in the examples described later.

If a transformed microorganism is used, muconic acid or protocatechuic acid can be obtained in high yield. For example, when 5 mM vanillic acid and 5 mM syringic acid are used as a carbon source, muconic acid can be obtained with a yield of 93% (versus the theoretical yield) by culturing for 35 hours; 5 mM p-hydroxybenzoic acid and 5 mM syringic acid. When carbon is used as a carbon source, muconic acid can be obtained in a yield of 75% (versus the theoretical yield) by culturing for 35 hours; 16.5 mg / L of muconic acid can be obtained; when 5 mM vanillic acid and 5 mM syringic acid are used as a carbon source, protocatechuic acid can be obtained with a yield of 83% (versus the theoretical yield) by culturing for 42 hours. Yes; when 5mM p-hydroxybenzoic acid and 5mM syringic acid are used as carbon sources, protocatechuic acid can be obtained in 75% yield (versus the theoretical yield) after 30 hours of culture. Rukoto can.

In the production method of the present invention, as long as the object of the present invention can be achieved, various processes and operations can be added before or after the above-described process.

(Use of muconic acid and protocatechuic acid)
The muconic acid and protocatechuic acid obtained using the transformed microorganism and the production method of one embodiment of the present invention can be converted into various industrially useful compounds, for example, surfactants, flame retardants, UV It can be used as a raw material for muconic acid derivatives that can be expected to be used as light stabilizers, thermosetting plastics, coating agents and the like. Specifically, adipic acid, which is one of muconic acid derivatives, is actually used as nylon 66 (one of polyamides). Protocatechuic acid can be used as a raw material for producing muconic acid, and can also be used as a synthetic raw material for pharmaceuticals, agricultural chemicals, fragrances and the like. Protocatechuic acid is also a precursor of protocatechuic acid-2,3-, 3,4-, 4,5-ring-cleavage metabolites. Among these metabolites, for example, 2-pyrone-4,6- Some have a use as a synthetic resin raw material such as dicarboxylic acid (for example, Japanese Patent No. 4658244; the entire description of this document is incorporated herein by reference).

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. it can.

[1. Sphingobium sp. Production of SME257 strain]
By the following procedure, Sphingobium sp. SME257 strain, which is a mutant strain in which the protocatechuate 4,5-dioxygenase gene (ligAB gene) was disrupted from Sphingobium species SYK-6 strain, was prepared. .

PCR method using a primer set consisting of primers 1 and 2 of SEQ ID NOS: 1 and 2 and a primer set consisting of primers 3 and 4 of SEQ ID NOS: 3 and 4 using the genomic DNA of Sphingobium species SYK-6 as a template The protocatechuic acid 4,5-dioxygenase small subunit (protocatechuate 4,5-dioxygenase; ligA) gene upstream region and the protocatechuic acid 4,5-dioxygenase large subunit (protocatechuate 4,5-dioxygenase) LigB) A downstream region of the gene was obtained.

The obtained DNA fragment and pAK405 plasmid DNA previously digested with BamHI were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain pAKDligAB plasmid DNA.

Using the pAKDligAB plasmid DNA, the Sphingobiium species SYK-6 strain was transformed by the triple parental conjugation method. The transformant was selected as a Nal-Km resistant strain capable of growing on an LB agar medium containing 12.5 mg / L of nalidixic acid (Nal) and 50 mg / L of kanamycin (Km). The obtained Nal-Km resistant strain was inoculated into an LB liquid medium containing Nal 12.5 mg / L and streptomycin (Sm) 100 mg / L, and cultured with shaking at 30 ° C. for 48 hours.

The obtained culture broth was smeared on an LB agar medium containing 100 mg / L of Sm, and statically cultured at 30 ° C. for 72 hours. Genomic DNA was extracted from each of the grown colonies, and then a transformant lacking the ligAB gene on the genomic DNA was screened as Sphingobiium sp. Strain SME257 by PCR.

[2. Protocatechuic acid (PCA) production using syringic acid (SA) and vanillic acid (VA) as carbon sources]
SME257 strain was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30 ° C. for 36 hours. The obtained culture broth was washed with Wx buffer (KH ₂ PO ₄ 1.7 g / L, Na ₂ HPO ₄ · 12H ₂ O 9.8 g / L and (NH ₄ ) ₂ SO ₄ 1 g / L), Wx minimal medium containing 5 mM VA, 5 mM SA and 1 g / L Tryptone (KH ₂ PO ₄ 1.7 g / L, Na ₂ HPO ₄ · 12H ₂ O 9.8 g / L, (NH ₄ ) ₂ SO ₄ 1 g / L _{, MgSO 4 · 7H 2 O 0.1g} / L, FeSO 4 · 7H 2 O 9.5mg / L, MgO 10.75mg / L, CaCO 3 2mg / L, ZnSO 4 · 7H 2 O 1.44mg / L, _{MnSO 4 · 4H 2 O 1.12mg /} L, CuSO 4 · 5H 2 O 0.25mg / L, CoSO 4 · 7H 2 O 0.28mg / L, H 3 BO 3 0.06mg / L and 12N HCl 51. Inoculated into μL / L) 10mL, and cultured with shaking at 30 ° C..

After the start of culture, the optical density (OD) of the culture solution was measured at regular intervals, and the concentrations of SA, VA and PCA were measured for the culture supernatant obtained by further centrifuging the culture solution.

The wavelength of 600 nm was used for OD measurement, and the OD600 value was measured using GeneQuant 100 (GE Healthcare Japan Co., Ltd.). The concentrations of SA, VA and PCA were measured using a high performance liquid chromatograph (Nihon Waters Co., Ltd.). The column used was TSKgel ODS-140HTP column (diameter 2.1 mm, length 100 mm, particle size 2.3 μm; Tosoh Corporation) and kept at 30 ° C. Gradient elution mode (solvent A: 99.9% (v / v) H ₂ O, 0.1% (v / v) HCOOH, solvent B: 99.9% (v / v) CH ₃ CN, 0.1% (v / v) HCOOH), equilibrated with 99% solvent A and 1% solvent B, and after 3 to 6 minutes from the start of the analysis, Increasing to 25%, then reducing the proportion of solvent B to 1% over 1 minute. The mobile phase flow rate was 0.5 mL / min, the measurement wavelength was 270 nm for SA, and 260 nm for VA and PCA.

Table 1 shows a summary of the measurement results of OD600 value, SA concentration, VA concentration, and PCA concentration after 0, 6, 18, 30, and 42 hours of culture time.

As Table 1 shows, the SME257 strain grew using SA and produced PCA from VA over time (83% yield relative to the theoretical yield).

[3. PCA production using SA and 4-hydroxybenzoic acid (HBA) as carbon source]
SME257 strain was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30 ° C. for 36 hours. The obtained culture broth was washed with Wx buffer, then inoculated into 10 mL of Wx minimal medium containing 5 mM HBA, 5 mM SA and 1 g / L Tryptone, and cultured at 30 ° C. with shaking.

After initiation of the culture, the OD600 of the culture solution was measured at regular intervals, and the concentrations of SA, HBA and PCA were measured for the culture supernatant obtained by further centrifuging the culture solution. The OD600 measurement and the SA, HBA, and ccMA concentration measurements were performed in the same manner as described in 2 above.

Table 2 shows a summary of the measurement results of OD600 value, SA concentration, HBA concentration, and PCA concentration after 0, 6, 18, and 30 hours of culture time.

As Table 2 shows, SME257 strain grew using SA and produced PCA from HBA over time (75% yield over theoretical yield).

[4. Production of SME257 / pTS084 strain]
According to the following procedure, Sphingobium sp. SME257 strain is transformed with pTS084 plasmid DNA, which is a plasmid expressing aroY gene, kpdB gene, catA gene, vanA gene and vanB gene, and SME257 / pTS084 strain is prepared. did.

Klebsiella pneumoniae subspecies pneumoniae ( Klebsiella pneumoniae subspecies pneumoniae ) A partial fragment of the genomic DNA of A170-40 strain was used as a template, and PCR was performed by PCR using a primer set consisting of primers 5 and 6 of SEQ ID NOs: 5 and 6. An about 1.5 kbp DNA fragment containing the acid decarboxylase (aroY) gene was obtained. The obtained DNA fragment was digested with KpnI and cloned into pMCL200 plasmid DNA previously digested with KpnI to obtain pTS036 plasmid DNA.

Klebsiella pneumoniae sub-species pneumoniae DNA of NBRC14190 strain was used as a template by PCR using the primer set of primers 7 and 8 of SEQ ID NOS: 7 and 8, 4-hydroxybenzoic acid decarboxylase subunit B (4 -A DNA fragment of about 0.6 kbp containing a hydroxylbenzoate decarboxylase subunit B (kpdB) gene was amplified. Both ends of the amplified DNA fragment were subjected to a blunting treatment, then digested with XbaI, and then ligated to a preblunted pTS036 plasmid DNA to obtain pTS052 plasmid DNA. The pTS052 plasmid DNA was obtained by selecting as a clone in which the aroY gene contained in the pTS036 plasmid DNA and the kpdB gene were linked in the forward direction.

A DNA fragment of about 2.2 kbp containing the aroY gene and the kpdB gene was amplified by PCR using pTS052 plasmid DNA as a template and a primer set consisting of primers 9 and 10 of SEQ ID NOs: 9 and 10. The amplified DNA fragment was ligated to pJB866 plasmid DNA previously digested with BamHI and EcoRI using Infusion HD Cloning Kit to obtain pTS074 plasmid DNA.

Pseudomonas putida ( Pseudomonas putida ) A catechol 1,2-dioxygenase (catechol 1,2) was obtained by PCR using a genomic DNA of the KT2440 strain as a template and a primer set consisting of primers 11 and 12 of SEQ ID NOs: 11 and 12. -Dioxygenase; catA) A DNA fragment of about 1.0 kbp containing the gene was obtained. The obtained DNA fragment was ligated to pTS074 plasmid DNA previously digested with SacI using Infusion HD Cloning Kit to obtain pTS079 plasmid DNA.

A DNA fragment of about 200 bp containing a lactose promoter region (Plac) was obtained by PCR using pUC118 plasmid DNA as a template and a primer set consisting of primers 13 and 14 of SEQ ID NOs: 13 and 14. The obtained DNA fragment was cloned into the NotI site of pTS079 plasmid DNA using Infusion HD Cloning Kit to obtain pTS082 plasmid DNA.

Pseudomonas putida KT2440 genomic DNA as a template, and a PCR method using a primer set consisting of primers 15 and 16 of SEQ ID NOS: 15 and 16, vanillate demethylase oxygenase component (vanA) gene and vanillate -About 2.0 kbp DNA fragment containing a demethylase oxidoreductase component (vanillate demethylase oxidoreductase component; vanB) gene was obtained. The obtained DNA fragment was digested with SacI and SmaI and ligated with pQE30 plasmid DNA previously digested with SacI and SmaI to obtain pKY001 plasmid DNA.

A DNA fragment of about 2.0 kbp containing the vanA gene and the vanB gene was amplified by PCR using pKY001 plasmid DNA as a template and a primer set consisting of primers 17 and 18 of SEQ ID NOs: 17 and 18. The amplified DNA fragment was ligated using pTS082 plasmid DNA previously digested with NotI and Infusion HD Cloning Kit to obtain pTS084 plasmid DNA.

Using the obtained pTS084 plasmid DNA, the SME257 / pTS084 strain was produced by transforming the Sphingobiium species SME257 strain.

[5. Production of cis, cis-muconic acid (ccMA) using SA and VA as carbon sources]
SME257 / pTS084 strain was inoculated into 10 mL of LB liquid medium containing 12.5 mg / L of tetracycline (Tc), and cultured with shaking at 30 ° C. for 36 hours. The obtained culture broth was washed with Wx buffer (KH ₂ PO ₄ 1.7 g / L, Na ₂ HPO ₄ · 12H ₂ O 9.8 g / L and (NH ₄ ) ₂ SO ₄ 1 g / L), Wx minimal medium containing 12.5 mg / L of Tc, 5 mM VA, 5 mM SA and 1 g / L Tryptone (KH ₂ PO ₄ 1.7 g / L, Na ₂ HPO ₄ · 12H ₂ O 9.8 g / L, (NH _{4 ) 2 SO 4 1g / L,} MgSO 4 · 7H 2 O 0.1g / L, FeSO 4 · 7H 2 O 9.5mg / L, MgO 10.75mg / L, CaCO 3 2mg / L, ZnSO 4 · 7H 2 _{O 1.44mg / L, MnSO 4 ·} 4H 2 O 1.12mg / L, CuSO 4 · 5H 2 O 0.25mg / L, CoSO 4 · 7H 2 O 0.28mg / L, H 3 BO 3 0.06mg / L and And 12N HCl (51.3 μL / L) was inoculated into 10 mL, and cultured at 30 ° C. with shaking.

After initiation of the culture, the OD600 of the culture solution was measured at regular intervals, and the concentrations of SA, VA and ccMA were measured for the culture supernatant obtained by further centrifuging the culture solution. OD600 measurement and SA, VA, and ccMA concentration measurements were performed in the same manner as described in 2 above.

Table 3 summarizes the measurement results of OD600 value, SA concentration, VA concentration and ccMA concentration after 0, 5, 10, 20 and 35 hours of culture time.

As Table 3 shows, the SME257 / pTS084 strain grew using SA, and produced ccMA over time from VA (yield with respect to the theoretical yield was 93%).

[6. CcMA production using SA and HBA as carbon sources]
SME257 / pTS084 strain was inoculated into 10 mL of LB liquid medium containing 12.5 mg / L of Tc, and cultured with shaking at 30 ° C. for 36 hours. The obtained culture solution was washed with Wx buffer, then inoculated into 10 mL of Wx minimal medium containing 12.5 mg / L of Tc, 5 mM HBA, 5 mM SA and 1 g / L Tryptone, and cultured at 30 ° C. with shaking.

After initiation of the culture, the OD600 of the culture solution was measured at regular time intervals, and the concentrations of SA, HBA and ccMA were measured for the culture supernatant obtained by centrifuging the culture solution. The OD600 measurement and the SA, HBA, and ccMA concentration measurements were performed in the same manner as described in 2 above.

Table 4 summarizes the measurement results of OD600 value, SA concentration, HBA concentration and ccMA concentration after 0, 5, 10, 20 and 35 hours of culture time.

As Table 4 shows, SME257 / pTS084 strain was grown using SA, and ccMA was produced from HBA over time (yield based on theoretical yield was 75%).

[7. Production of ccMA using birch lignin-derived aromatic compound as a carbon source]
According to a conventional method, birch wood flour was subjected to an alcohol-benzene extraction treatment, and then 1.5 g of the treated birch wood flour was subjected to alkali nitrobenzene oxidative decomposition treatment and diethyl ether extraction treatment (wood science experiment manual, edited by the Japan Wood Society) , See Buneidou Publishing; the full description of this document is hereby incorporated by reference.) The alkaline solution after nitrobenzene oxidative decomposition was subjected to diethyl ether extraction treatment, and the resulting aqueous layer was subjected to acidification treatment and further to diethyl ether extraction treatment. Diethyl ether extract obtained as an ether layer was evaluated as ccMA production in a Wx medium supplemented with a white birch lignin-derived aromatic compound and a white birch arginine-derived aromatic compound aqueous solution (pH≈9) as a carbon source.

SME257 / pTS084 strain was inoculated into 10 mL of LB liquid medium containing 12.5 mg / L of Tc and cultured with shaking at 30 ° C. for 36 hours. The obtained culture solution was washed with Wx buffer, then inoculated into 10 mL of Wx minimal medium containing 12.5 mg / L of Tc and 0.1 g / L of Tryptone, and 5 μL of an aqueous solution of shiraka barignin-derived aromatic compound was added as a carbon source. And cultured with shaking at 30 ° C. Every 4 hours of cultivation, 5 μL of a white birch arginine-derived aromatic compound aqueous solution was added.

After initiation of the culture, the OD600 of the culture solution was measured at regular intervals, and the ccMA concentration was measured for the culture supernatant obtained by centrifuging the culture solution. The concentration of ccMA was measured in the same manner as described in 2 above, and the separation conditions in the high performance liquid chromatograph were equilibrated with 99% solvent A and 1% solvent B, and 3 minutes after starting the analysis. 6 minutes later, the proportion of solvent B is increased to 25%, then the proportion of solvent B is increased to 99% over 1 minute and held for 1 minute, and then the proportion of solvent B is increased to 1% over 1 minute. Lowered to. The mobile phase flow rate was 0.5 mL / min and the measurement wavelength was 260 nm for ccMA.

Table 5 summarizes the measurement results of the ccMA concentration after 0, 12, 24 and 48 hours of culture time.

As Table 5 shows, the SME257 / pTS084 strain grew using a birch lignin-derived aromatic compound and produced ccMA over time.

The sequences listed in the sequence listing are as follows:
[SEQ ID NO: 1] Primer 1
ATCCGCCCTAGTGGACGAATGGTCCTCTCTTTAGTGATTTCG
[SEQ ID NO: 2] Primer 2
CGACTCTAGAGGATCAGATTTCGGACGACGAGATCC
[SEQ ID NO: 3] Primer 3
TCGTCCACTAGGGCGGATCGACATTC
[SEQ ID NO: 4] Primer 4
CGGTACCCGGGGATCAGGTGCCGAGCGGCCCG
[SEQ ID NO: 5] Primer 5
AGCTGGTACCATTAAAGAGGAGAAATTAACTATGACCGCACCGATTCAG
[SEQ ID NO: 6] Primer 6
AGCTGGTACCTTATTTTGCGCTACCCTGGTT
[SEQ ID NO: 7] Primer 7
AGCTGAATTCATTAAAGAGGAGAAATTAACTATGAAACTGATTATTGGGATGACG
[SEQ ID NO: 8] Primer 8
AGCTTCTAGATTATTCGATCTCCTGTGCAAAT
[SEQ ID NO: 9] Primer 9
GAAGCTTCGTGGATCATTAAAGAGGAGAAATTAACTATGACCGCACCGATT
[SEQ ID NO: 10] Primer 10
ACGTCTCGAGGAATTCTTATTCGATCTCCTGTGCAAAT
[SEQ ID NO: 11] Primer 11
CGGCCGCGGTACCAGTTAAAGAGGAGAAATTAACTATGACCGTGA
[SEQ ID NO: 12] Primer 12
CTAGATACCTAGGTGTCAGCCCTCCTGCAACGCC
[SEQ ID NO: 13] Primer 13
ATGTGAATTGCGGCCAGCGCCCAATACGCAAACC
[SEQ ID NO: 14] Primer 14
ACTGGTACCGCGGCCGCGTAATCATGGTCATAGCTGTTTC
[SEQ ID NO: 15] Primer 15
ATGCGAGCTCATGTACCCCAAAAACACCT
[SEQ ID NO: 16] Primer 16
ATGCCCGGGTCAGATGTCCAGCACCAGCA
[SEQ ID NO: 17] Primer 17
CATGATTACGCGGCCATTAAAGAGGAGAAATTAAC
[SEQ ID NO: 18] Primer 18
ACTGGTACCGCGGCCTCAGATGTCCAGCACCAGCA
[SEQ ID NO: 19] ligA gene
ATGACCGAGAAGAAAGAGAGAATCGACGTTCACGCCTATCTCGCCGAGTTTGACGACATTCCCGGCACCCGCGTGTTCACCGCCCAGCGCGCGCGCAAGGGCTATAATCTCAACCAGTTCGCGATGAGCCTGATGAAGGCCGAGAACCGCGAGCGGTTCAAGGCCGACGAGAGCGCCTATCTGGACGAGTGGAACCTCACGCCCGCCGCCAAGGCCGCCGTGCTGGCCCGCGACTACAATGCGATGATCGACGAGGGCGGGAATGTCTATTTCCTGTCCAAGCTGTTCTCGACCGACGGCAAGAGCTTCCAGTTCGCCGCCGGCTCGATGACCGGCATGACTCAGGAAGAATATGCACAGATGATGATCGATGGCGGCCGTTCGCCCGCGGGTGTGCGCTCGATCAAGGGAGGCTACTGA
[SEQ ID NO: 20] ligB gene

[SEQ ID NO: 21] catA gene

[SEQ ID NO: 22] aroY gene

[SEQ ID NO: 23] kpdB gene

[SEQ ID NO: 24] ligM gene

[SEQ ID NO: 25] vanA gene

[SEQ ID NO: 26] vanB gene

[SEQ ID NO: 27] LigA
MTEKKERIDVHAYLAEFDDIPGTRVFTAQRARKGYNLNQFAMSLMKAENRERFKADESAYLDEWNLTPAAKAAVLARDYNAMIDEGGNVYFLSKLFSTDGKSFQFAAGSMTGMTQEEYAQMMIDGGRSPAGVRSIKGG
[SEQ ID NO: 28] LigB
MARVTTGITSSHIPALGAAIQTGTSDNDYWGPVFKGYQPIRDWIKQPGNMPDVVILVYNDHASAFDMNIIPTFAIGCAETFKPADEGWGPRPVPDVKGHPDLAWHIAQSLILDEFDMTIMNQMDVDHGCTVPLSMIFGEPEEWPCKVIPFPVNVVTYPPPSGKRCFALGDSIRAAVESFPEDLNVHVWGTGGMSHQLQGPRAGLINKEFDLNFIDKLISDPEELSKMPHIQYLRESGSEGVELVMWLIMRGALPEKVRDLYTFYHIPASNTALGAMILQPEETAGTPLEPRKVMSGHSLAQA
[SEQ ID NO: 29] CatA
MTVKISHTADIQAFFNRVAGLDHAEGNPRFKQIILRVLQDTARLIEDLEITEDEFWHAVDYLNRLGGRNEAGLLAAGLGIEHFLDLLQDAKDAEAGLGGGTPRTIEGPLYVAGAPLAQGEARMDDGTDPGVVMFLQGQVFDADGKPLAGATVDLWHANTQGTYSYFDSTQSEFNLRRRIITDAEGRYRARSIVPSGYGCDPQGPTQECLDLLGRHGQRPAHVHFFISAPGHRHLTTQINFAGDKYLWDDFAYATRDGLIGELRFVEDAAAARDRGVQGERFAELSFDFRLQGAKSPDAEARSHRPRALQEG
[SEQ ID NO: 30] AroY
MTAPIQDLRDAIALLQQHDNQYLETDHPVDPNAELAGVYRHIGAGGTVKRPTRIGPAMMFNNIKGYPHSRILVGMHASRQRAALLLGCEASQLALEVGKAVKKPVAPVVVPASSAPCQEQIFLADDPDFDLRTLLPAPTNTPIDAGPFFCLGLALASDPVDASLTDVTIHRLCVQGRDELSMFLAAGRHIEVFRQKAEAAGKPLPITINMGLDPAIYIGACFEAPTTPFGYNELGVAGALRQRPVELVQGVSVPEKAIARAEIVIEGELLPGVRVREDQHTNSGHAMPEFPGYCGGANPSLPVIKVKAVTMRNNAILQTLVGPGEEHTTLAGLPTEASIWNAVEAAIPGFLQNVYAHTAGGGKFLGILQVKKRQPADEGRQGQAALLALATYSELKNIILVDEDVDIFDSDDILWAMTTRMQGDVSITTIPGIRGHQLDPSQTPEYSPSIRGNGISCKTIFDCTVPWALKSHFERAPFADVDPRPFAPEYFARLEKNQGSAK
[SEQ ID NO: 31] KpdB
MKLIIGMTGATGAPLGVALLQALRDMPEVETHLVMSKWAKTTIELETPWTAREVAALADFSHSPADQAATISSGSFRTDGMIVIPCSMKTLAGIRAGYAEGLVGRAADVVLKEGRKLVLVPREMPLSTIHLENMLALSRMGVAMVPPMPAYYNHPETVDDITNHAETRVLDQGLGLRTQLD
[SEQ ID NO: 32] LigM
MSAPTNLEQVLAAGGNTVEMLRNSQIGAYVYPVVAPEFSNWRTEQWAWRNSAVLFDQTHHMVDLYIRGKDALKLLSDTMINSPKGWEPNKAKQYVPVTPYGHVIGDGIIFYLAEEEFVYVGRAPAANWLMYHAQTGGYNVDIVHDDRSPSRPMGKPVQRISWRFQIQGPKAWDVIEKLHGGTLEKLKFFNMAEMNIAGMKIRTLRHGMAGAPGLEIWGPYETQEKARNAILEAGKEFGLIPVGSRAYPSNTLESGWIPSPLPAIYTGDKLKAYREWLPANSYEASGAIGGSFVSSNIEDYYVNPYEIGYGPFVKFDHDFIGRDALEAIDPATQRKKVTLAWNGDDMAKIYASLFDTEADAHYKFFDLPLANYANTNADAVLDAAGNVVGMSMFTGYSYNEKRALSLATIDHEIPVGTELTVLWGEENGGTRKTTVEPHKQMAVRAVVSPVPYSVTARETYEGGWRKAAVTA
[SEQ ID NO: 33] VanA
MYPKNTWYVACTPDEIATKPLGRQICGEKIVFYRARENQVAAVEDFCPHRGAPLSLGYVEDGNLVCGYHGLVMGCDGKTVSMPGQRVRGFPCNKTFAAVERYGFIWVWPGDQAQADPALIPHLEWAVSDEWAYGGGLFHIGCDYRLMIDNLMDLTHETYVHASSIGQKEIDEAPPVTTVTGDEVVTARHMENIMAPPFWRMALRGNGLADDVPVDRWQICRFTPPSHVLIEVGVAHAGKGGYHAEAQHKASSIVVDFITPESDTSIWYFWGMARNFAAHDQTLTDNIREGQGKIFSEDLEMLERQQQNLLAHPERNLLKLNIDAGGVQSRKVLERIIAQERAPQPQLIATSANPA
[SEQ ID NO: 34] VanB
MIDAVVVSRNDEAQGICSFELAAADGSLLPAFSAGAHIDVHLPDGLVRQYSLCNHPEERHRYLIGVLNDPASRGGSRSLHEQVQAGARLRISAPRNLFPLAEGAQRSLLFAGGIGITPILCMAEQLSDSGQAFELHYCARSSERAAFVERIRSAPFADRLFVHFDEQPETALDIAQVLGNPQDDVHLYVCGPGGFMQHVLDSAKGLGWQEANLHREYFAAAPVDASNDGSFAVQVGSTGQVFEVPADRTVVQVLEENGIEIAMSCEQGICGTCLTRVLQGTPDHRDLFLTEEEQALNDQFTPCCSRSKTPLLVLDI
[SEQ ID NO: 35] pcaH gene

[SEQ ID NO: 36] pcaG gene

[SEQ ID NO: 37] prA gene

[SEQ ID NO: 38] desA gene
ATGGCGAAAAGTCTTCAAGATGTGCTGGACAATGCCGGAAATGCAGTCGATTTCCTGCGCAACCAGCAGACCGGCCCGAACGTCTATCCCGGCGTCCCGGCGGAATATTCCAACTGGCGCAACGAGCAGCGCGCATGGGCCAAGACCGCCGTGCTCTTCAACCAGAGCTACCACATGGTCGAGCTGATGGTCGAAGGCCCCGACGCCTTCGCCTTCCTCAACTATCTCGGCATCAACAGCTTCAAGAACTTCGCGCCCGGCAAGGCCAAGCAGTGGGTTCCGGTGACGGCCGAGGGCTATGTCATCGGCGACGTGATCCTGTTCTATCTCGCCGAGAACCAGTTCAACCTCGTCGGCCGCGCGCCGGCCATCGAGTGGGCCGAGTTCCATGCCGCCACCGGCAAGTGGAACGTGACGCTCACCCGTGACGAGCGCACCGCGCTGCGCACCGACGGCGTGCGTCGCCACTATCGCTTCCAGCTGCAGGGCCCCAACGCCATGGCGATCCTAACGGACGCGATGGGCCAGACCCCGCCGGACCTCAAATTCTTCAACATGGCGGACATCCAGATCGCCGGGAAGACCGTCGGCGCGCTGCGTCACGGCATGGCCGGTCAGCCGGGCTATGAGCTCTATGGTCCCTGGGCGGATTATGAGGCGGTTCATTCGGCGCTGGTCGCGGCCGGCAAGAACCATGGGCTGGCGCTCGTCGGCGGCCGTGCCTATTCGTCCAACACGCTGGAATCCGGCTGGGTGCCCTCGCCGTTCCCGGGCTATCTCTTCGGCGAAGGCTCGGCCGACTTCCGCAAGTGGGCCGGCGAGAACAGCTATGGCGCCAAGTGCTCCATCGGCGGTTCCTATGTGCCCGAGAGCCTGGAAGGCTATGGCCTGACGCCCTGGGACATCGGCTATGGCATCATCGTCAAGTTCGACCATGACTTCATCGGCAAGGAAGCGCTGGAGAAGATGGCGAACGAGCCGCACCTCGAGAAGGTGACGC TGGCGCTGGACGACGAGGACATGCTGCGCGTGATGAGCAGCTATTTCTCGGACTCCGGTCGTGCGAAATATTTCGAGTTCCCGAGCGCGGTCTACTCGATGCACCCCTATGACTCGGTGCTGGTCGACGGCAAGCATGTCGGCGTCTCGACCTGGGTCGGCTACTCGTCGAACGAGGGCAAGATGCTCACGCTCGCGATGATCGATCCCAAATATGCCAAGCCCGGCACGGAAGTCTCGCTGCTCTGGGGCGAGCCCAATGGCGGCACCTCCAAGCCGACCGTCGAGCCGCACGAGCAGACGGAGATCAAGGCGGTCGTGGCGCCGGTGCCGTACTCGGCCGTGGCGCGCACGGGCTATGCCGACAGCTGGCGCACCAAGAAGGCCTGA

According to the transformed microorganism and the production method of one embodiment of the present invention, muconic acid and protocatechuic acid can be obtained from a biomass containing an aromatic compound derived from syringyl lignin or a hardwood derived syringyl lignin. Muconic acid and protocatechuic acid can be converted into various industrially useful compounds, such as surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents, pharmaceuticals, agricultural chemicals, fragrances, etc. It can be used as a raw material.

Claims (9)

1. Host microorganism has a protocatechuic acid degrading enzyme gene on a chromosome, and a Sphingomonas monad (Sphingomonad) family microorganism assimilates aromatic compounds from syringyl lignin,
   The protocatechuate degrading enzyme gene on the chromosome is deleted,
   Expressing the inserted catA gene, and
   Expressing the inserted aroY gene or aroY gene and kpdB gene;
   Transformed microorganisms.
2. The transformed microorganism according to claim 1, wherein the inserted aroY gene, the kpdB gene, and the catA gene are under the control of the same promoter.
3. The transformed microorganism according to claim 1, wherein the transformed microorganism further expresses the inserted vanA gene and vanB gene.
4. The transformed microorganism according to claim 3, wherein the inserted aroY gene, the kpdB gene, the catA gene, the vanA gene and the vanB gene are under the control of the same promoter.
5. The transformed microorganism according to claim 1, wherein the protocatechuate degrading enzyme gene is a gene selected from the group consisting of a ligA gene, a ligB gene, a pcaG gene, a pcaH gene and a prA gene.
6. The transformed microorganism according to claim 1, wherein the host microorganism is Sphingobium species SYK-6 strain.
7. The transformed microorganism according to any one of claims 1 to 6, wherein an aromatic compound derived from p-hydroxyphenyl lignin and / or an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from syringyl lignin are used. A method for producing muconic acid, which comprises a step of obtaining muconic acid by acting on the above.
8. The host microorganism is a sphingomonad family microorganism having a protocatechuate degrading enzyme gene on a chromosome and assimilating an aromatic compound derived from syringyl lignin; and
   The protocatechuate degrading enzyme gene on the chromosome is deleted,
   Transformed microorganisms.
9. A protocatechu by treating a transformed microorganism according to claim 8 with an aromatic compound derived from p-hydroxyphenyl lignin and / or an aromatic compound derived from guaiacyl lignin and an aromatic compound derived from syringyl lignin. A method for producing protocatechuic acid, comprising a step of obtaining an acid.
   
   
   
   

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