JP7083124B2 - Muconic acid-producing transformed microorganisms and their utilization - Google Patents

Description

The present invention relates to a transformed microorganism capable of producing muconic acid and a method for producing muconic acid using the transformed microorganism. In particular, the present invention relates to a transformed microorganism capable of growing and producing muconic acid using an aromatic compound derived from lignin as a single carbon source.

Lignin is an atypical polymer substance that exists as a component of the cell wall of the vascular bundle of plants, and is a complex condensation of phenylpropane-based constituent units. It is a major chemical structural feature that it contains a methoxy group. ing. Lignin acts to adhere woody plant cells to each other and strengthen tissues, and is present in about 18 to 36% in wood and about 15 to 25% in herbs. Therefore, in order to make effective use of wood, various attempts have been made to decompose lignin to obtain useful compounds.

On the other hand, cis, cis-muconic acid (hereinafter, may be simply referred to as muconic acid) is a highly reactive compound due to the presence of two double bonds and two carboxy groups in the molecule. Various muconic acid derivatives using muconic acid as a starting material are known, and examples thereof include lactones, sulfones, polyamides, polyesters, thioesters, and additional polymers. Such muconic acid derivatives are known to have various uses, and can be used, for example, as surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents, and the like.

As described above, muconic acid is used for various purposes in the form of a muconic acid derivative, and if muconic acid can be produced from lignin, resource regeneration is achieved and it 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, in Non-Patent Document 1, Pseudomonas putida is used as a host microorganism, and the pcaH gene and the pcaG gene (hereinafter, may be collectively referred to as the pcaHG gene), the catR gene, and the catB gene on the chromosome. A transformed microorganism that disrupts the catC gene and the catA gene and expresses the inserted catA gene and the aroY gene, or the catA gene, the aroY gene, and the ecdb gene is prepared, and the transformed microorganism is propagated by glucose. Then, it is described that muconic acid was produced by p-kumalic acid.

C.W. Johnson et al., Metabolic Engineering Communications, 3, 111-119, 2016.

Certainly, muconic acid can be produced from p-coumaric acid by using the transformed microorganism described in Non-Patent Document 1. However, 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 it is bad. In addition, since two or more carbon sources such as glucose for cell growth and p-coumaric acid as a substrate for muconic acid production are required, considering the total amount of these carbon sources as a standard, 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.

Therefore, the present invention attempts to solve the problem of providing a microorganism capable of producing muconic acid, which is economical and has a high yield, and a method for producing muconic acid using the microorganism. It is an issue to be done.

In an attempt to solve the above problems, the present inventors first diligently studied muconic acid-producing microorganisms having a high yield.

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 via the protocatechuic acid 3,4-ring cleavage pathway. From this, when muconic acid is efficiently produced by the transformed microorganism described in Non-Patent Document 1, the pcaHG gene and the catB gene on the chromosome of the host microorganism are deleted, and the inserted aroY gene is further expressed. There must be. Only then can lignin-derived aromatic compounds such as p-coumaric acid be once converged to protocatechuic acid and converted to muconic acid.

However, since the transformed microorganism described in Non-Patent Document 1 lacks the pcaHG gene on the chromosome, a substrate other than an aromatic compound derived from lignin such as glucose is required for the growth of the microorganism. do. Therefore, the transformed microorganism described in Non-Patent Document 1 seems to be a high-yield method for producing muconic acid from an aromatic compound derived from lignin, but the actual yield is calculated. Then, the amount of the substrate for growth must be taken into consideration, and as a result, the method using the transformed microorganism described in Non-Patent Document 1 is a method having a low yield as a method for producing muconic acid.

Therefore, as a result of diligent studies, the present inventors have found that a transformed microorganism that overexpresses the pcaHG gene and the aroY gene, which are separately inserted as foreign genes after disrupting the pcaHG gene and the catB gene on the chromosome of the host microorganism. Succeeded in creating. Surprisingly, they have found that the transformed microorganisms produced by the present inventors can produce muconic acid while proliferating using an aromatic compound derived from lignin without using glucose. In this way, the present inventors have succeeded in creating a method for producing muconic acid from an aromatic compound derived from lignin using the transformed microorganisms produced by the present inventors.

Even more surprisingly, the method using the transformed microorganisms prepared by the present inventors requires an aromatic compound derived from lignin as a substrate while using an aromatic compound derived from lignin as the sole carbon source. It has been found that the yield of muconic acid is about the same as or higher than that of the method using the transformed microorganism described in Non-Patent Document 1.

Aromatic compounds derived from lignin can be obtained from biomass such as waste materials and are much cheaper than glucose. Therefore, the method for producing muconic acid using the transformed microorganism produced by the present inventors is an economically advantageous method as compared with the method using the transformed microorganism described in Non-Patent Document 1. The present invention is an invention completed based on these findings and successful examples.

Therefore, according to one aspect of the present invention, the following transformed microorganisms (1) to (4) are provided.
(1) The host microorganism is a Pseudomonas genus microorganism having a pcaH gene, a pcaG gene, a catA gene, and a catB gene on a chromosome.
The pcaH gene, the pcaG gene, and the catB gene on the chromosome are deleted.
It expresses the inserted pcaH gene and pcaG gene, and
Expressing the inserted aroY gene,
Transforming microorganisms.
(2) The transformed microorganism according to (1), wherein the inserted aroY gene and the inserted pcaH gene and the pcaG gene are under the control of the same promoter.
(3) The transformed microorganism according to any one of (1) to (2), wherein the host microorganism is a Pseudomonas microorganism having a pobA gene, a vanA gene and a vanB gene on the chromosome.
(4) The transformed microorganism according to any one of (1) to (3), wherein the host microorganism is Pseudomonas putida .

According to another aspect of the present invention, the following methods (5) and (6) for producing muconic acid are provided.
(5) By allowing an aromatic compound derived from p-hydroxyphenyllignin and / or an aromatic compound derived from guaiacyl lignin to act on the transformed microorganism according to any one of (1) to (4). A method for producing muconic acid, which comprises a step of obtaining muconic acid.
(6) The aromatic compound derived from p-hydroxyphenyllignin and / or the aromatic compound derived from guaiacyl lignin under the condition that the dissolved oxygen concentration is 1 to 13% in the above steps, according to claims 1 to 4. The production method according to (5), which is a step of obtaining muconic acid by acting on the transformed microorganism according to any one item.

According to another aspect of the present invention, the recombinant vector of (7) below is provided.
(7) A recombinant vector containing the aroY gene and the pcaH gene and the pcaG gene.

According to the method for producing the transformed microorganism according to one aspect of the present invention and the muconic acid according to one aspect of the present invention, muconic acid is produced at a lower cost and at a higher cost while maintaining or improving the yield as compared with the conventional method using a microorganism. can do. Therefore, according to the transformed microorganism according to one aspect of the present invention and the method for producing muconic acid according to one aspect of the present invention, production of muconic acid on an industrial scale can be expected as part of the effective utilization of biomass containing lignin. .. According to the recombinant vector of one aspect of the present invention, the transformed microorganism of one aspect of the present invention can be produced.

FIG. 1 shows Fig. 1 of Non-Patent Document 1. It is a schematic diagram of the metabolic pathway of Pseudomonas putida according to 1.

Hereinafter, the details of the transformed microorganism, the method for producing muconic acid, and the recombinant vector, which are one aspect of the present invention, will be described, but the technical scope of the present invention is not limited to the matters of this item. The present invention may take various aspects as long as the object is achieved.

(Overview of transformed microorganisms)
The transformed microorganism according to one aspect of the present invention transforms the host microorganism so that a specific gene on the chromosome of the host microorganism is deleted and the specific gene inserted as a foreign gene is expressed. It is a microorganism that has been transformed.

"Gene deletion" as used herein means that a gene does not function normally, such as that the gene is not transcribed normally, that the protein that should be produced by the expression of the gene is not translated normally, and so on. It means that the expression is impeded. Deletion of a gene can occur, for example, due to changes in the structure of the gene due to destruction, deletion, substitution, insertion, etc. of all or part of the gene. However, gene deletion can also occur if the structure of the gene does not change and the expression of the gene is suppressed by means such as blocking the regulatory region of the gene.

As used herein, "gene expression" means that a protein encoded by a gene is produced in a manner having an original structure and activity through transcription, translation, or the like. Further, the term "gene overexpression" as used herein means that the gene is inserted to produce a protein encoded by the gene in excess of the amount originally expressed by the host microorganism.

(Gene to be deleted or inserted)
For transformed microorganisms, the host microorganism has a pcaH gene, a pcaG gene, a catA gene and a catB gene on the chromosome. Among these genes, the transformed microorganism lacks the pcaH gene, the pcaG gene, and the catB gene on the chromosome of the host microorganism. Regarding the transformed microorganism, it is preferable that the host microorganism has both the pcaH gene and the pcaG gene originally present on the chromosome deleted, but it is probable that either the pcaH gene or the pcaG gene may be deleted. There is.

The pcaH gene and the pcaG gene are not particularly limited as long as they are genes expressing β subunit and α subunit of protocatechuate 3,4-dioxygenase, respectively, but have, for example, the nucleotide sequences of SEQ ID NOs: 29 and 30, respectively. Genes and the like can be mentioned.

The catB gene is not particularly limited as long as it is a gene expressing cis, cis-muconic acid / cycloisomerase, and examples thereof include a gene having the base sequence of SEQ ID NO: 32.

The catA gene is not particularly limited as long as it is a gene expressing catechol 1,2-dioxygenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 31. 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, for example, is possessed by the Pseudomonas putida KT2440 strain, has the activity of catalyzing the reaction to produce cis, cis-muconic acid from catechol, and is a cofactor for Fe ³⁺ . Request as.

It has an Intradiol dioxygenase domain in the amino acid sequence of catechol 1,2-dioxygenase (accession no. Q88I35). The domain is [LIVMF] -x-G-x- [LIVM] -x (4)-[GS] -x (2)-[LIVMA] -x (4)-[LIVM]-[DE]-[LIVMFYC. ] -X (6) -G-x- [FY] (Prosite entry no. P00083), where Y in the sequence is involved in the binding of the cofactor Fe ³⁺ . L137 to Y165 in the amino acid sequence of catechol 1,2-dioxygenase correspond to the above domain.

The transformed microorganism expresses the inserted pcaH gene and pcaG gene. However, if one of the pcaH gene and the pcaG gene that the host microorganism originally has on the chromosome is deleted, there is a possibility that the deleted gene may be inserted and expressed. The transformed microorganism further expresses the inserted aroY gene.

The aroY gene is not particularly limited as long as it is a gene expressing protocatechuate decarboxylase, and examples thereof include a gene having the base sequence of SEQ ID NO: 33. Protocatechuate decarboxylase (EC 4.1.1.63) is also called 3,4-dihydroxybenzoic acid-carboxylyase (3,4-dihydroxybenzoate carboxy-lyase). The protocatechuate 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-methylgalic acid and a reaction for producing pyrogallol from gallic acid may also be used as protocatechuate decarboxylase. In addition, vanillic acid decarboxylase and 4-hydroxybenzoic acid decarboxylase may also be used as protocatechuic acid / decarboxylase because they may decarboxylate protocatechuic acid. Protocatechuate decarboxylase is structurally classified into a protein group (UbiD superfamily) containing a UbiD domain (Domain architechture ID 104879953).

Specific examples of the protocatechuate decarboxylase include a protein derived from Klebsiella pneumoniae subsp. Pneumoniae A170-40 strain (ATCC 25597 strain) and the like. Can be mentioned. Examples of the protein having an amino acid sequence having high sequence identity with the amino acid sequence of AB479384 protein include protocatechuate decalboxylase (action no. AMJ70686; sequence identity 87.2%) derived from the Enterobacter cloacae MBRL1077 strain. Examples thereof include, but are not limited to, proteins registered as protocatechuate decalboxylase (accession no. CZU76022; sequence identity 85.7%) derived from the clonee e1026 strain. These enzymes are characterized as a family of enzymes that require Mn ²⁺ as a cofactor, a prenylated flavin mononucleotide (prenyl-FMN).

The transformed microorganism may be one in which the kpdB gene is inserted. The kpdB gene is a protein that is presumed to synthesize prenyl-FMN, which is a cofactor for protocatechuic acid decarboxylase, and is supplied with prenyl-FMN by expressing it in one cell together with protocatechuic acid decarboxylase. There is a possibility that the activity of protocatechuic acid decarboxylase will be improved. Therefore, if the kpdB gene can be expressed together with the aroY gene to improve the decarboxylation activity, it is expected that the production yield of muconic acid will increase.

The kpdB gene is a gene expressing 4-hydroxybenzoate decarboxylase subunit B having enzymatic activity as a flavin / prenyl transferase, and examples thereof include a gene having the base sequence of SEQ ID NO: 34.

The transformed microorganism means that the host microorganism has one, two or all three genes of the pobA gene, the vanA gene and the vanB gene on the chromosome in addition to the pcaH gene, the pcaG gene, the catA gene and the catB gene. Is preferable. In the present specification, the vanA gene and the vanB gene may be collectively referred to as the vanAB gene. If the host microorganism does not have the pobA gene, vanA gene and / or vanB gene on the chromosome, it is preferable to insert the gene into the transformed microorganism so as to express these genes. In addition, the transformed microorganisms include vanillin dehydrogenase (vdh) gene, p-hydroxybenzaldehyde dehydrogenase (PP_1948) gene and / or Sphingobium sp. It is preferable to have an aldehyde dehydrogenase (ligV) gene derived from the SYK-6 strain. If the host microorganism does not have the vdh gene, PP_1948 gene and / or ligV gene on the chromosome, it is preferable to insert the gene into the transformed microorganism so as to express these genes.

The pobA gene is not particularly limited as long as it is a gene expressing p-hydroxybenzoic acid / monooxygenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 35. Examples of the p-hydroxybenzoic acid / monooxygenase (EC 1.14.13.2 or EC 1.14.13.33) include PobA (accession no. Q88H28) derived from Pseudomonas putida KT2440 strain. ..

The vanA gene is not particularly limited as long as it is a gene expressing a vanillate demethylase oxygenase component, and examples thereof include a gene having the base sequence of SEQ ID NO: 36.

Examples of the vanillic acid / demethylase / oxygenase component (EC 1.14.13.82) include VanA (accession no. Q88GI6) derived from Pseudomonas putida KT2440 strain. The vanillic acid / demethylase / oxygenase component cleaves the methyl ether bond of vanillic acid by utilizing electrons derived from NADH or NADPH supplied via the Oxidocatechuic compound and oxygen atoms supplied from molecular oxygen. Produces protocatechuic acid, formaldehyde and water.

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 involved in the binding of Fe-S.

The vanB gene is not particularly limited as long as it is a gene expressing a vanillate demethylase oxidoreductase component, and examples thereof include a gene having the base sequence of SEQ ID NO: 37.

Examples of the vanillic acid / demethylase / oxidoreductase component (EC 1.14.13.82) include VanB (accession no. Q88GI5) derived from Pseudomonas putida KT2440 strain. 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 transfers electrons derived from NADH or NADPH to VanA, which is a vanillic acid / demethylase / oxygenase component.

The vanillic acid / demethylase / oxidoreductase component has 2Fe-2S Ferredoxin type iron-sulfur binding domine (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and has C in the amino acid sequence. , C270, C273, C303) are involved in the binding of Fe—S. In addition, the vanillic acid, dimethylase, and oxidoreductase components have NAD-binding domine (L109-D201, Pfam entry no. PF00175) and Ferredoxin reductase typeFAD-bind .PS51384).

The vdh gene is not particularly limited as long as it is a gene expressing vanillin dehydrogenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 43. Examples of the vanillin dehydrogenase (EC 1.2.1.67) include Vdh (accession no. Q88HJ9) derived from the Pseudomonas putida KT2440 strain.

The PP-1948 gene is not particularly limited as long as it is a gene expressing p-hydroxybenzaldehyde dehydrogenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 44. Examples of the p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.64) are p-hydroxybenzaldehyde dehydrogenase (action no. Q88LI4) derived from Pseudomonas putida KT2440 strain and Pseudomonas putida mt-2 strain. Examples thereof include p-hydroxybenzaldehyde dehydrogenase (XylC, accession no. P43503).

The ligV gene is not particularly limited as long as it is a gene that expresses an aldehyde dehydrogenase (aldehyde dehydrogenase) using aromatic aldehydes such as vanillin, p-hydroxybenzaldehyde, syringaaldehyde, protocatechaldehyde, and benzaldehyde as substrates. , Gene having the base sequence of SEQ ID NO: 45 and the like. Examples of the aldehyde dehydrogenase (EC 1.2.1.-) include Sphingobium sp. Aldehyde dehydrogenase (accession no. AB287332) derived from the SYK-6 strain can be mentioned.

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

As used herein, the term "base sequence that hybridizes under stringent conditions" refers to a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization method using a DNA having a wild-type gene base sequence as a probe. It means the base sequence of DNA obtained by using such as.

As used herein, a "stringent condition" is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and the hybridization system to be used, the type and sequence of the probe. And depends on the length. Such conditions can be determined by varying the temperature of hybridization, the temperature of washing and the concentration of salt. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the temperature of hybridization and washing and, if necessary, lowering the salt concentration of washing. If no specific hybrid signal is also detected, the hybrid can be stabilized by lowering the hybridization and wash temperatures and, if necessary, increasing the wash salt concentration.

As a specific example of stringent conditions, for example, a DNA probe is used as a probe, and hybridization is performed by 5 × SSC, 1.0% (w / v) 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 was performed twice with 0.1-0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS for 15 minutes. conduct. The temperature at which hybridization and washing are performed is 65 ° C. or higher, preferably 68 ° C. or higher.

As the DNA having a base sequence that hybridizes under stringent conditions, for example, a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter in which a fragment of the DNA is immobilized is used. After hybridization was performed at 40 to 75 ° C. in the presence of DNA obtained by hybridization under stringent conditions and 0.5 to 2.0 M NaCl, preferably 0.7 to 1. After hybridization was performed at 65 ° C. in the presence of 0 M NaCl, a 0.1 to 1 × SSC solution (1 × SSC solution was 150 mM sodium chloride, 15 mM sodium citrate) was used under 65 ° C. conditions. DNA that can be identified by washing the filter can be mentioned. Methods for probe preparation and hybridization 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) and the like. Can be done. If you are a person skilled in the art, in addition to the conditions such as the salt concentration and temperature of the buffer, other conditions such as the probe concentration, the probe length, and the reaction time are taken into consideration, and the base sequence of the wild-type gene is taken into consideration. Conditions for obtaining a DNA having a base sequence complementary to the above and a base sequence that hybridizes under stringent conditions can be appropriately set.

Examples of the DNA containing a base sequence that hybridizes under stringent conditions include DNA having a certain degree of sequence identity with the base sequence of a DNA having a base sequence of a wild-type gene used as a probe, for example, wild. The base sequence of the type gene and 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 99% or more, more preferably 99.5% or more sequence identity.

As the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild type gene under stringent conditions, for example, 1 to several, preferably 1 to 300, more preferably the base sequence of the wild type gene. Is 1 to 200, more preferably 1 to 100, still more preferably 1 to 50, and particularly preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases. Includes a base sequence with deletions, substitutions, additions, etc. Here, "base deletion" means that a base in the sequence is missing or missing, and "base substitution" means that the base in the sequence is replaced with another base. "Addition of base" means that a new base has been added to be inserted.

The protein encoded by the base sequence complementary to the base sequence of the wild type gene and the base sequence that hybridizes 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. It is probable that it is a protein having an amino acid sequence having deletions, substitutions, additions, etc. of individual amino acids, but it has the same enzymatic activity as a protein encoded by the base sequence of a wild-type gene.

A protein having the same or similar enzymatic properties as a wild-type protein has an amino acid sequence in which the amino acid sequence has one to several amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type protein. It may consist of. Here, the range of "1 to several" in "deletion, substitution, addition of 1 to several amino acids" of the amino acid sequence is not particularly limited, but for example, 1 to 100, preferably 1 to 80. More preferably 1 to 50, still more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. It means about the number. In addition, "amino acid deletion" means the omission or disappearance of an amino acid residue in the sequence, and "amino acid substitution" means that the amino acid residue in the sequence is replaced with another amino acid residue. However, "addition of amino acid" means that a new amino acid residue is added to the sequence so as to be inserted.

As a specific embodiment of "deletion, substitution, addition of one to several amino acids", there is an embodiment in which one to several amino acids are replaced with another chemically similar amino acid. For example, a case where one hydrophobic amino acid is replaced with another hydrophobic amino acid, a case where one polar amino acid is replaced 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 the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged basic amino acids include arginine, histidine, and lysine. Examples of the acidic amino acid having a negative charge include aspartic acid and glutamic acid.

Examples of the amino acid sequence having deletions, substitutions, additions, etc. of one to several amino acids in the amino acid sequence of the wild-type protein include amino acid sequences having a certain degree of 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 protein. Examples thereof include amino acid sequences having 97% or more, 98% or 99% or more, and more preferably 99.5% or more of sequence identity.

(Means for calculating sequence identity)
The method for determining the sequence identity of a base sequence or an amino acid sequence is not particularly limited, but for example, a commonly known method is used to obtain an amino acid sequence of a wild-type gene or a wild-type protein expressed by a wild-type gene and a target base. It is obtained by aligning a sequence or an amino acid sequence and using a program for calculating the matching rate of both sequences.

As a program for calculating the matching rate between two base sequences and amino acid sequences, for example, Karlin and Altschul's algorithm (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity more sensitively than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can search a database for a sequence showing high sequence identity for a given sequence, for example, by using the above program. These are available, for example, on the National Center for Biotechnology Information website on the Internet (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Each of the above methods can be commonly used to search for sequences showing sequence identity in a database, but as a means of 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). When analyzing the sequence identity of the base sequence, the region encoding the protein (CDS or ORF) is used if possible.

(Origin of the gene to be inserted)
The gene to be inserted is derived from a microorganism or the like that carries the gene to be inserted. Examples of the organism from which the gene to be inserted is derived include a microorganism capable of producing muconic acid from protocatechuic acid and a microorganism capable of growing by assimilating protocatechuic acid.

Specific examples of organisms from which the gene to be inserted are derived include Pseudomonas petitda, Pseudomonas fluorescens, Pseudomonas alkaligenes, Pseudomonas pseudoalkogenes, Pseudomonas mendocina, Pseudomonas erginosa, and Pseudomonas sepasia for the pcaH gene and pcaG gene. Pseudomonas genus microorganisms and Acinetobacter genus microorganisms such as Acinetobacta Bailey and Acinetobacta calcoaceticus; Pseudomonas petitda, Pseudomonas erginosa, Pseudomonas fluorescens, Pseudomonas reinekei and other Pseudomonas genus microorganisms such as Pseudomonas reinekei Pseudomonas genus microorganisms such as Pseudomonas opacus, Pseudomonas pyridinivolas, Pseudomonas genus microorganisms such as Pseudomonas rodocrose; Pseudomonas genus microorganisms such as Bacter aerogenes, Sedimentobacter hydroxybenzoicus, etc .; Pseudomonas petitda, Pseudomonas fluorescens, Pseudomonas erginosa and other Pseudomonas microorganisms such as Pseudomonas bailey, Acinetobacta carcoa secicas, Ashinetobacter Pseudomonas genus microorganisms such as Baumanni, Pseudomonas genus microorganisms such as Klebsiella pneumoniae, Klebsiella baricola; Pseudomonas genus microorganisms such as testosteroni and comamonas thiooxydance, Pseudomonas genus microorganisms such as acetobacta-patuturianus, acetobacta-aceti, acetobacta-tropicalis; Pseudomonas microorganisms such as Shiringe, Rodcoccus josti, Russia Pseudomonas genus microorganisms such as Dococcus erythropolis, Burkholderia genus microorganisms such as Burkholderia sepacia, Burkholderia senosepacia, Sphingobium genus microorganisms such as Sphingobium spices; , Pseudomonas fluorescens, Pseudomonas microorganisms such as Pseudomonas syringe, Pseudomonas microorganisms such as Sphingobium yanoikuyae, Sphingobium spices, Rhodococcus josti, Burkholderia such as Rhodococcus erythropolis, Burkholderia -Includes, but is not limited to, Burkholderia microorganisms such as Sepacia, Burkholderia and Senocepacia.

As described above, the organism from which the gene to be inserted 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 characterized by inserting the gene in order to exhibit activity. It is preferable that the host microorganism to be transformed is a microorganism whose growth conditions are similar to those of the host microorganism.

(Cloning of genes by genetic engineering method)
The gene to be deleted and inserted can be inserted into various known vectors suitable for use. Further, this vector can be introduced into a suitable known host microorganism to prepare a transformant (transformed microorganism) in which the gene is deleted or inserted. It is preferable that the gene to be deleted is a wild-type gene in which all or part of the gene structure is changed due to destruction, deletion, substitution, insertion or the like. The gene to be inserted is preferably a gene expressing a protein that is the same as or similar to the wild-type gene.

Those skilled in the art are involved in 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, and methods for producing transformed microorganisms. Can be selected as appropriate for. Further, in the present specification, the transformant and the transformant include the transductant and the transductant, respectively. An example of cloning of a gene to be deleted and inserted will be described later without limitation.

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

For example, the gene to be inserted can be obtained by cloning using the chromosomal DNA or cDNA of the derived organism having the wild-type gene related to the gene as a template. The organisms from which the wild-type genes are derived are as described above, and specific examples thereof include Pseudomonas putida KT2440 strain and Klebsiella pneumonier subspecies pneumoniae A170-40 strain, depending on the type of gene. Yes, but not limited to these. For example, the Pseudomonas putida KT2440 strain was cultivated, water was removed from the obtained cells, and the cells were physically ground in a mortar while cooling in liquid nitrogen to obtain fine powdery cells. The chromosomal DNA fraction is extracted from the cell piece by a usual method. For the chromosomal DNA extraction operation, a commercially available chromosomal DNA extraction kit such as DNeasy Blood & TissueKit (Qiagen) can be used. As used herein, chromosomal DNA and genomic DNA are synonymous.

The DNA is then amplified by performing a polymerase chain reaction (PCR) using chromosomal DNA as a template and synthetic primers complementary to the 5'end and 3'end sequences. The primer is not particularly limited as long as it can amplify the DNA fragment containing the gene to be inserted. Examples thereof include primers represented by SEQ ID NOs: 9 and 10 designed with reference to the genomic sequence of Pseudomonas putida KT2440 strain as one for amplifying the pcaH gene and the pcaG gene. By using such a primer, the total length of the target gene can be amplified. Alternatively, the DNA containing the gene fragment of interest is amplified by screening the target gene clone from the shotgun library or by appropriate PCR such as Inverse PCR, Next PCR, 5'RACE method, 3'RACE method, etc. , These can be linked to obtain a DNA containing the full-length target gene.

Further, the method for obtaining the gene to be deleted or inserted is not particularly limited as described above, and the gene can be constructed by, for example, a chemical synthesis method without using a genetic engineering method.

The base sequence of the amplified product amplified by PCR or the chemically synthesized gene can be confirmed, for example, as follows. First, the DNA whose sequence is to be confirmed is inserted into an appropriate vector according to a usual method to prepare 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); pUC4K (see Gene, vol. 19, p259-268, 1982). , PEX18Amp (see Gene, vol.212, p77-86, 1998), pPS858 (see Gene, vol.212, p77-86, 1998), pUC118 (Takara Bio), pJB866 (Plasmid, vol.38, See p35-51, 1997), pMCL200 (see Gene, vol. 162, p157-158, 1995), pQE30 (Kiagen), pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio). A known or commercially available plasmid vector such as λEMBL3 (Stratagene) or the like can be used.

When it is desired to obtain a large amount of the constructed recombinant DNA, the recombinant DNA is introduced into, for example, Escherichia coli ( Escherichia coli ), preferably Escherichia coli JM109 strain (Takarabio) or Escherichia coli DH5α strain (Takarabio). Then, the recombinant DNA contained in the obtained transformant can be purified using QIAGEN Plasmamid Mini Kit (Qiagen) or the like.

The base sequence of each gene inserted into the recombinant DNA is determined by the dideoxy method (see Methods in Enzymeology, 101, 20-78, 1983, etc.) and the like. The sequence analysis device 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. Then, based on the determined base sequence, the amino acid sequence of the protein to be translated can be known.

(Construction of recombinant vector containing gene)
A recombinant vector (recombinant DNA) containing a gene to be deleted or inserted is to bind a PCR amplification product containing the gene to be deleted or inserted and various vectors in a form capable of deleting or expressing the gene. Can be constructed by. In the case of a gene to be deleted, the recombinant vector is introduced into the host microorganism, and the gene in the recombinant vector is replaced with the gene in the host microorganism by homologous recombination, so that the recombinant vector is deleted. It preferably contains 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, to cut out a DNA fragment containing any of the genes to be inserted with an appropriate restriction enzyme and cleave the DNA fragment with an appropriate restriction enzyme. It can be constructed by ligating the above-mentioned plasmid vector using a commercially available recombinant vector preparation kit such as In-Fusion HD Cloning Kit (Takara Bio Co., Ltd.). Alternatively, a DNA fragment containing a gene having a sequence homologous to the plasmid vector added to both ends and a DNA fragment derived from the plasmid amplified by inverse PCR can be obtained from a commercially available product such as In-Fusion HD Cloning Kit (Takara Bio). It can be obtained by ligating using a recombinant vector preparation kit.

A recombinant vector containing a gene to be deleted or inserted contains at least a gene to be deleted or inserted and a gene (base sequence) derived from the plasmid vector. An example of a recombinant vector, and another aspect of the present invention is a recombinant vector containing an aroY gene, a pcaH gene, a pcaG gene, and the like. The recombinant vector of one aspect of the present invention is used to prepare the transformed microorganism of one aspect of the present invention. The recombinant vector of one aspect of the present invention is at least one, two, three or four selected from the group consisting of the pobA gene, vanAB gene, vdh gene and PP_1948 gene in addition to the aroY gene and the pcaHG gene. Genes can be included. Further, the recombinant vector of one aspect of the present invention may contain a gene other than the above-mentioned gene as long as it does not interfere with the solution of the problem of the present invention.

The recombinant vector of one embodiment of the present invention preferably contains a heterologous gene or a heterologous nucleic acid sequence. The heterologous gene is not particularly limited as long as it is a gene that does not originally exist in the host microorganism (not naturally occurring), and 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 gene to be inserted and the derived organism are different. Examples include genes derived from, genes derived from other organisms different from the host microorganism, plants, animals, viruses and the like. 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, for example, a lactose promoter region (Plac) and the like.

Specific embodiments of the recombinant vector of one aspect of the present invention include, but are not limited to, the pTS110 plasmid vector and the pTS119 plasmid vector described in Examples described later.

(Method for producing transformed microorganisms)
The method for producing the transformed microorganism is not particularly limited, and examples thereof include a method of inserting into a host microorganism in a manner in which gene deletion or insertion is realized according to a conventional method. Specifically, a trait that expresses the gene to be inserted is produced by creating a DNA construct in which one of the genes to be inserted is inserted between the expression-inducing promoter and the terminator, and then transforming the host microorganism with the DNA construct. Converted microorganisms are obtained. Alternatively, a transformed microorganism lacking a gene can be obtained by preparing a DNA construct containing 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 a host microorganism are collectively referred to as DNA constructs.

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

In the 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 linked between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome. , Can be inserted into the genome of the host microorganism.

The vector-host system used for producing the transformed microorganism is not particularly limited as long as it is a system in which the gene to be inserted can be expressed or the gene on the chromosome can be deleted in the host microorganism, and for example, pJB866-. Examples include a system of microorganisms of the genus Pseudomonas, pKT230 (Gene, vol.16, p237-247, 1981) -a system of microorganisms of the genus Pseudomonas.

The DNA construct containing the gene to be inserted expresses the gene to be inserted into the chromosome of the host microorganism even if the gene to be inserted is expressed by autonomously amplifying it without introducing it into the chromosome of the host microorganism. However, it doesn't matter which one.

The DNA construct may contain 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 gentamicin, kanamycin, tetracycline, ampicillin, and carbenicillin. The marker gene may be included in the middle of the gene to be deleted or to be replaced with the gene to be deleted.

Depending on the type of gene, the DNA construct containing the gene to be inserted may include promoters and terminators that allow the gene to be expressed in the host microorganism, as well as other regulatory sequences (eg, cis involved in transcriptional regulation by operators, etc.). Can include sequences, etc.).

One embodiment of the DNA construct includes, but is not limited to, the pVTS007 plasmid vector, the pTS108 plasmid vector, the pTS110 plasmid vector, the pTS119 plasmid vector, and the like described in Examples described later.

As a method for transformation into a Pseudomonas microorganism, a method known to those skilled in the art can be appropriately selected, and can be carried out by, for example, an electroporation (electroporation) method, a conjugation transmission method, or the like.

As the medium for selecting and growing the transformed microorganism, an appropriate medium is used depending on the host microorganism to be used and the marker gene. For example, when Pseudomonas petitda is used as the host microorganism and the resistance genes of canamycin, gentamycin and tetracycline are used as the marker genes, the selection and proliferation of the transformed microorganism can be carried out, for example, by using the transformed microorganism in an LB medium containing these agents. It can be carried out by culturing in.

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

To confirm that the transformed microorganism has been produced, chromosomal DNA is extracted from the transformed microorganism and PCR is performed using this as a template to produce a PCR product that can be amplified when transformation occurs. This may be done by confirming the characteristics and the base sequence.

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

When performing transformation by homologous recombination, PCR is performed with a combination of a forward primer located upstream of the homologous region on the upstream side used and a reverse primer located downstream from the homologous region on the downstream side 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 Pseudomonas genus microorganism having a pcaH gene, a pcaG gene, a catA gene and a catB gene on the chromosome, and preferably the pcaH gene, the pcaG gene, the catA gene, the catB gene and the pobA gene on the chromosome. , Pseudomonas genus microorganisms such as Pseudomonas petitda, Pseudomonas fluorescens, Pseudomonas erginosa, having vanA gene, vanB gene, vdh gene and PP_1948 gene, more preferably having these genes and from protocatechuic acid to mucon. Pseudomonas petita capable of producing acids.

(Specific examples of genes to be deleted or inserted)
Specific examples of the gene to be deleted or inserted include the pcaH gene, the pcaG gene, the catA gene, the catB gene, the pobA gene, the vanA gene and the vanB gene. It is a catB gene, a pobA gene, a vanA gene and a vanB gene, and their respective base sequences are those described in SEQ ID NOs: 29 to 32 and 35 to 37, respectively. Similarly, a specific example of the aroY gene is the aroY gene carried by the Klebsiella pneumoniae subspecies pneumoniae A170-40 strain, and the base sequence is that shown in SEQ ID NO: 33. A specific example of the kpdB gene is the kpdB gene possessed by the Klebsiella pneumoniae subspecies pneumonier NBRC14940 strain, and the base sequence is that shown in SEQ ID NO: 34. The amino acid sequences of the proteins expressed by these genes are those described in SEQ ID NOs: 25 to 28 and 38 to 42, respectively.

The method for obtaining a gene to be deleted or inserted from a microorganism other than Pseudomonas petitda or Klebsiera pneumonier is not particularly limited. , VanA gene and vanB gene base sequences (SEQ ID NOs: 29-32 and 35-37); based on the base sequence of the aroY gene (SEQ ID NO: 33) possessed by the Krebsiera pneumonier subspecies pneumonier A170-40 strain. It can be obtained by performing a BLAST homology search on the genomic DNA of other microorganisms and identifying a gene having a base sequence having a high sequence identity with the above base sequence. Further, based on the total protein of other microorganisms, a protein having an amino acid sequence having high sequence identity with the amino acid sequence (SEQ ID NOs: 25 to 28 and 38 to 42) of the protein expressed by the above gene is identified, and the protein is used. It can be obtained by identifying the gene to be expressed. The fact that the obtained gene corresponds to the 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 as compared with the host microorganism. You can check.

Since Pseudomonas petita, Pseudomonas fluorescens, and Pseudomonas erginosa have similar growth conditions, there is a possibility that they can be transformed with each other by inserting the genes possessed by each. For example, a gene obtained from Pseudomonas petita can be introduced into Pseudomonas fluorescens or Pseudomonas erginosa as a host microorganism and transformed. It is also possible to introduce a gene obtained from Pseudomonas putida into a microorganism belonging to the genus Klebsiella, a microorganism belonging to the genus Enterobacter, Escherichia coli or the like as a host microorganism and express it. The gene to be inserted may be a gene whose codon, secondary structure, GC content, etc. are optimized for expression in the host microorganism.

(One Embodiment of Transformed Microorganism)
A specific embodiment of the transformed microorganism is a Pseudomonas genus microorganism in which the host microorganism is selected from the group consisting of Pseudomonas petitda, Pseudomonas fluorescens and Pseudomonas erginosa, and the pcaH gene, pcaG gene and catB gene on the chromosome. Is a transformed Pseudomonas genus microorganism that expresses the inserted pcaH gene and pcaG gene and also expresses the inserted aroY gene. In addition, another specific embodiment of the transformed microorganism expresses at least one gene selected from the group consisting of the inserted pobA gene, catA gene, vanA gene and vanB gene in the transformed Pseudomonas genus microorganism. , Transformed Pseudomonas genus microorganism.

Such a transformed microorganism has an embodiment in which the pcaH gene, the pcaG gene and the catB gene on the chromosome are deleted, the inserted pcaH gene and the pcaG gene are expressed, and the inserted aroY gene is expressed. Proliferation and production of muconic acid using at least an aromatic compound derived from lignin such as vanillic acid and p-hydroxybenzoic acid, which is impossible with the transformed microorganism described in Non-Patent Document 1, as the sole carbon source. Is possible. As described in Examples described later, considering the assimilation property of the aromatic compound derived from lignin, a preferred embodiment of the transformed microorganism is a transformed microorganism that overexpresses the inserted catA gene, vanA gene and vanB gene. Is.

Specific embodiments of the transformed microorganism according to one aspect of the present invention include, but are not limited to, the IDPC / pTS110 strain and the IDPC / pTS119 strain described in Examples described later.

(Manufacturing method of muconic acid)
The method for producing muconic acid according to one aspect of the present invention is a step of obtaining muconic acid by allowing an aromatic compound derived from lignin such as vanillic acid and p-hydroxybenzoic acid to act on the transformed microorganism according to one aspect of the present invention. At least include.

The method of causing the lignin-derived aromatic compound to act on the transformed microorganism is particularly limited as long as the method is such that the lignin-derived aromatic compound and the transformed microorganism come into contact with each other and muconic acid can be produced by the enzyme possessed by the transformed microorganism. However, for example, by culturing the transformed microorganism 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. Examples include a method for producing mucon acid. The culturing method is not particularly limited, and examples thereof include a solid culturing method and a liquid culturing method performed under aeration conditions.

The medium can be either a synthetic medium or a natural medium as long as it contains a normal medium for culturing a host microorganism, that is, a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio. Since the host microorganism is a Pseudomonas microorganism, an MM medium or the like as described in Examples described later can be used, but the host microorganism is not particularly limited. As the carbon source, aromatic compounds derived from lignin, other carbon sources such as sugars and organic acids, or combinations thereof can be used. However, it is preferable that the medium component contains a component necessary for activating the enzyme involved in the production of muconic acid, for example, Fe ²⁺ . Iron ions, magnesium ions and the like can be added to the medium as compounds, but they may also be added as mineral-containing substances.

The aromatic compound derived from lignin is not particularly limited as long as it is a lignin of any one of guaiacyl lignin and p-hydroxyphenyl lignin and an aromatic compound that can be derived from these lignins, and for example, p-hydroxyphenyl lignin and guay. Examples thereof include compounds corresponding to decomposition products of acyllignin, and specific examples thereof include p-kumalic acid, phenolic acid, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, vanillin and p-hydroxybenzaldehyde. , Phenol, benzoic acid, guaiacol and the like. Aromatic compounds derived from lignin include compounds used as model compounds for lignin, for example, guaiacylglycerol-β-guaiacyl ether. The lignin-derived aromatic compound is preferably a biomass containing lignin or a product extracted by subjecting the biomass to pretreatment, but may be chemically synthesized and purified regardless of the biomass. .. As the aromatic compound derived from lignin, the above-mentioned ones can be used alone or in combination of two or more.

Biomass containing lignin (hereinafter, may be referred to as lignocellulosic) 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. However, specific examples include woody biomass such as broad-leaved trees and coniferous trees. For example, conifers are known to be high in p-hydroxyphenyllignin and guaiacyllignin.

Lignocellulosic can be, for example, solid, suspended, liquid or the like, depending on the presence or absence of pretreatment. For example, crushed lignocellulosic can be added to a liquid to obtain a suspension.

Lignocellulosic may be a lignin extract. As the lignin extract, for example, a powdered lignocellulosic substance is lignin so as to have a concentration of 0.1% W / V to 50% W / V, preferably 1% W / V to 20% W / V. Examples thereof include a suspension suspended in a solvent suitable for the extraction of. The lignin extract is prepared by keeping the suspension at 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 it to an extraction treatment and then distilling off the solvent from the liquid lignin extract obtained by removing the solid content from the extraction treatment liquid or the liquid lignin extract and drying it to dryness. And so on.

The method for preparing the lignin extract is not particularly limited, and examples thereof include the following methods. That is, 50 mL of 2M NaOH, 1.5 g of degreased Sugi wood flour, and 3 mL of nitrobenzene were placed in a stainless steel vessel of a small autoclave device (pressure resistant glass industry Co., Ltd., Portable Reactor TVS-1), and 2.5 at 170 ° C. with stirring at 500 rpm. Process time. Allow to cool to 60 ° C or lower, and collect the supernatant by centrifugation (6,000 g, 10 min). Diethyl ether extraction of the obtained supernatant is repeated 3 times (recovering the aqueous layer). After acidifying the aqueous layer with hydrochloric acid, diethyl ether extraction is repeated 3 times (recovering the ether layer). Sodium sulfate is added to the ether layer and dehydrated overnight in the refrigerator. The ether layer is recovered and the extract is dried under reduced pressure. The ether extract is dissolved in ion-exchanged water (pH ≈ 9) while adding sodium hydroxide to prepare a solution of an aromatic compound derived from sugilignin.

The solvent suitable for the extraction and treatment of lignin is not particularly limited, and examples thereof include water, low molecular weight alcohols such as dioxane, methanol and isopropanol, diethyl ether and dimethylformamide.

As the culturing conditions, the culturing conditions of Pseudomonas microorganisms usually known by those skilled in the art may be adopted. For example, the initial pH of the medium is adjusted to 5 to 10, the culturing temperature is 20 to 40 ° C., and the culturing time is several hours to several hours. It can be appropriately set for several days, preferably 1 to 7 days, more preferably 2 to 5 days, and the like. The culture method is not particularly limited, and aeration-stirred deep culture, shaking culture, static culture, etc. can be adopted, but it is preferable to culture under conditions such as aeration so that the dissolved oxygen concentration becomes sufficient. It is more preferable to culture under the condition that the dissolved oxygen concentration is 0.1 to 15%, further preferably to culture under the condition that the dissolved oxygen concentration is 1 to 13%, and the dissolved oxygen concentration is 2. It is even more preferable to culture under conditions such that the content is -10%. For example, as an example of the medium and culture conditions, an MM medium containing an aqueous solution of an aromatic compound derived from vanillic acid, p-hydroxybenzoic acid and / or sugilignin as a carbon source was used as described in Examples described later. Examples thereof include shaking culture and stirring culture for 1 to 5 days at 30 ° C., 180 rpm, and a dissolved oxygen concentration of 5 to 10%. The carbon source and other components can be added as appropriate after the start of culturing.

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

The extraction solvent is not particularly limited as long as it dissolves muconic acid, and examples thereof include organic solvents such as methanol, ethanol, isopropanol, and acetone; and hydrous organic solvents obtained by mixing these organic solvents with water. The extraction temperature is not particularly limited, but can be set from room temperature to 100 ° C., for example.

Specific examples of the method for extracting muconic acid include, for example, the method of Vardon et al. (Green chemistry, vol.18, p3397-3413, 2016) and a method in which the method is partially modified. Specifically, activated carbon (12.5% (w / v), 100 mesh) is added to the culture supernatant, and the mixture is stirred for 1 hour. Activated carbon is removed by suction filtration and the filtrate is collected. Hydrochloric acid is added to the collected filtrate to adjust the pH to ≈2, and then the mixture is allowed to stand at 4 ° C. overnight. The precipitate is collected by suction filtration, washed with ion-exchanged water, collected by suction filtration, and dried under reduced pressure. The dried solid is suspended in ethanol, unnecessary substances are removed by suction filtration, and the filtrate is collected. The filtrate is dried under reduced pressure with an evaporator to obtain purified muconic acid.

The qualitative or quantitative analysis of muconic acid is not particularly limited and can be performed by, for example, HPLC. The HPLC separation conditions can be appropriately selected by those skilled in the art, and can be carried out, for example, under the conditions described in Examples described later.

Muconic acid can be obtained in high yield by using a transforming microorganism. For example, muconic acid can be obtained in a yield of 2.7 wt% in 48 hours of culture with 50 mM vanillic acid as the carbon source; 56 hours of culture with 50 mM p-hydroxybenzoic acid as the carbon source. Muconic acid can be obtained in 11.9 wt% yield; 25 mM vanillic acid and 25 mM p-hydroxybenzoic acid as carbon sources provide muconic acid in 17.7 wt% yield after 48 hours of culture. It can be obtained; when an aqueous solution of phenols derived from sugilignin is used as a carbon source, 8.9 to 24.2 mg / L of muconic acid can be obtained by culturing for 48 hours.

In the production method of the present invention, various steps and operations can be added to the pre-stage or the post-stage or the process of the above-mentioned steps as long as the object of the present invention can be achieved.

(Use of muconic acid)
Muconic acid obtained by utilizing the transformed microorganism of one aspect of the present invention and the production method can be converted into various industrially useful compounds, for example, surfactants, flame retardants, and UV photostabilization. It can be used as a raw material for muconic acid derivatives, which can be expected to be used as agents, thermosetting plastics, coating agents, and the like. Specifically, adipic acid, which is one of the muconic acid derivatives, is actually used as nylon 66 (one of polyamides).

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples, and the present invention may take various aspects as long as the problems of the present invention can be solved. can.

[1. P. Preparation of putida IDPC strain]
It is a mutant strain in which the protocatechuate 3,4-dioxygenase gene (pcaHG gene) and the cis, cis-muconic acid / cycloisomerase gene (catB gene) are disrupted from Pseudomonas putida by the following procedure. P. A putida IDPC strain was produced.

P. Protocatechuate, 3,4-dioxygenase, β subunit (protocatechuate 3,4-) by PCR using the genomic DNA of the putida KT2440 strain as a template and a primer set consisting of primers 1 and 2 of SEQ ID NOs: 1 and 2. A DNA fragment of about 1.2 kbp upstream of the dioxygenase beta subunit (pcaH) gene was amplified. The amplified DNA fragment was digested with EcoRI and SacI and ligated with the pEX18Amp plasmid DNA previously digested with EcoRI and SacI (see Gene, vol. 212, p77-86, 1998) to give the pVTS003 plasmid DNA.

P. Protocatechuate, 3,4-dioxygenase, α subunit (protocatechuate 3,4-) by PCR method using the genomic DNA of the putida KT2440 strain as a template and a primer set consisting of primers 3 and 4 of SEQ ID NOs: 3 and 4. A DNA fragment about 1.2 kbp downstream of the dioygenase subunit; pcaG) gene was amplified. The amplified DNA fragment was digested with SacI and BamHI and ligated with the pVTS003 plasmid DNA previously digested with SacI and BamHI to obtain the pVTS004 plasmid DNA.

About 1.8 kbp of DNA fragment (including gentamicin (Gm) resistance gene) obtained by digesting pPS858 (see Gene, vol.212, p77-86, 1998) with SacI was previously digested with SacI pVTS004. By ligating with plasmid DNA, pVTS007 plasmid DNA was obtained.

Using the pVTS007 plasmid DNA, by electroporation, P. The putida KT2440 strain was transformed. The transformants were selected as Nal-Gm resistant strains capable of growing on LB agar medium containing 25 mg / L of nalidixic acid (Nal) and 50 mg / L of Gm. The obtained Nal-Gm resistant strain was inoculated into an LB liquid medium containing Nal 25 mg / L, Gm 50 mg / L and 10% (w / v) sucrose, and cultured with shaking at 30 ° C. for 16 hours.

A portion of the culture medium is inoculated into an LB liquid medium containing fresh Nal 25 mg / L, Gm 50 mg / L and 10% (w / v) sucrose, and shake-cultured at 30 ° C. for 16 hours two more times. Repeated. Then, the obtained culture solution was smeared on an LB agar medium containing Nal 25 mg / L and Gm 50 mg / L, and cultured at 30 ° C. overnight. Genomic DNA is extracted from each of the grown colonies, and a DNA fragment of about 1.8 kbp containing the Gm resistance gene is inserted into the pcaG and pcaH loci on the genomic DNA by the PCR method and the Southern hybridization method. The transformants were screened as protocatechuic acid 3,4-dioxygenase gene disrupted strains.

P. Using the genomic DNA of the plasmid PpY1100 strain as a template, a PCR method using a primer set consisting of primers 5 and 6 of SEQ ID NOs: 5 and 6 contains a part of the cis, cis-muconic acid / cycloisomerase (catB) gene. The pTS073 plasmid by amplifying a 1.0 kbp DNA fragment and ligating it to pK19movsacB plasmid DNA (see Gene, Vol. 145, p69-73, 1994) previously digested with PstI and XbaI using the Fusion cloning HD Kit. DNA was obtained.

The pUC4K plasmid DNA (see Gene, vol. 19, p259-268, 1982) was digested with SalI, and a DNA fragment of about 1.2 kbp containing the kanamycin (Km) resistance gene was previously digested with SalI with the pTS073 plasmid DNA. By ligation, pTS076 plasmid DNA was obtained.

The pTS076 plasmid DNA was used to transform a protocatechuate 3,4-dioxygenase gene-disrupted strain. Transformants were selected as Nal-Gm-Km resistant strains capable of growing on LB agar medium containing Nal 25 mg / L, Gm 50 mg / L and Km 25 mg / L. The obtained Nal-Gm-Km resistant strain was inoculated into an LB liquid medium containing Nal 25 mg / L, Gm 50 mg / L, Km 25 mg / L and 10% (w / v) sucrose, and inoculated at 30 ° C. for 16 hours. It was cultured with shaking.

Part of the culture medium is inoculated into an LB liquid medium containing fresh Nal 25 mg / L, Gm 50 mg / L, Km 25 mg / L and 10% (w / v) sucrose, and cultured with shaking at 30 ° C. for 16 hours. The operation was repeated two more times. The obtained culture broth was smeared on an LB agar medium containing Nal 25 mg / L, Gm 50 mg / L and Km 25 mg / L, and cultured at 30 ° C. overnight. Genomic DNA was extracted from each of the grown colonies, and transformants in which the Km resistance gene was inserted into the catB gene on the genomic DNA were screened by PCR method and Southern hybridization method. The transformant obtained by screening was a mutant in which both the protocatechuate / 3,4-dioxygenase gene and the cis, cis-muconic acid / cycloisomerase gene were disrupted . It was named putida IDPC strain.

[2a. Preparation and evaluation of IDPC / pTS108 strain]
By the following procedure, using the pTS108 plasmid DNA, which is a plasmid expressing the pcaG gene and the pcaH gene, P. The putida IDPC strain was transformed to produce an IDPC / pTS108 strain.

Using the pUC118 plasmid DNA as a template, a DNA fragment of about 200 bp containing the lactose promoter region (Plac) was obtained by PCR using a primer set consisting of primers 7 and 8 of SEQ ID NOs: 7 and 8. The obtained DNA fragment was cloned into the NotI site of the pJB866 plasmid DNA by In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the pTS093 plasmid DNA.

P. Using the genomic DNA of the putida KT2440 strain as a template, a DNA fragment of about 1.3 kbp containing the pcaG gene and the pcaH gene was amplified by a PCR method using a primer set consisting of primers 9 and 10 of SEQ ID NOs: 9 and 10. The amplified DNA fragment was digested with SacI and BamHI and cloned into the pUC118 plasmid DNA previously digested with SacI and BamHI to obtain the pTS107 plasmid DNA.

The pTS108 plasmid DNA was obtained by ligating the approximately 1.3 kbp DNA fragment containing the pcaG gene and the pcaH gene obtained by digesting the pTS107 plasmid DNA with SacI and BamHI to the SacI-BamHI site of the pTS093 plasmid DNA. ..

Using the pTS108 plasmid DNA, P. An IDPC / pTS108 strain was prepared by transforming the putida IDPC strain.

[2b. Preparation of IDPC / pTS110 strain]
By the following procedure, using the pTS110 plasmid DNA, which is a plasmid expressing the pcaH gene, the pcaG gene, and the aroY gene, P. The putida IDPC strain was transformed to produce an IDPC / pTS110 strain.

Klebsiella pneumoniae subspecies pneumoniae A170-40 strain of genomic DNA as a template, protocatechuate by PCR using primers 11 and 12 of SEQ ID NOs: 11 and 12. -A DNA fragment of about 1.5 kbp containing the decarboxylase (protocatechuate decarboxylase; aroY) gene was obtained. The obtained DNA fragment was digested with KpnI and cloned into the pMCL200 plasmid DNA previously digested with KpnI to obtain the pTS036 plasmid DNA.

Using the pTS036 plasmid DNA as a template, a DNA fragment of about 1.5 kbp containing the aroY gene was obtained by a PCR method using 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 the pTS093 plasmid DNA by In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the pTS109 plasmid DNA. A DNA fragment of about 1.3 kbp obtained by digesting the pTS107 plasmid DNA with SacI and HindIII was ligated to the SacI-HindIII site of the pTS109 plasmid DNA to obtain the pTS110 plasmid DNA.

Using the obtained pTS110 plasmid DNA, P. An IDPC / pTS110 strain was prepared by transforming the putida IDPC strain.

[3. Production of cis, cis-muconic acid (ccMA) using vanillic acid (VA) as a carbon source (1)]
Each of the IDPC / pTS108 strain and the IDPC / pTS110 strain was inoculated into 5 mL of LB liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and tetracycline (Tc) 20 mg / L for 16 hours at 30 ° C. It was shake-cultured. 50 μL of the obtained culture solution was added to MM liquid medium (Na ₂ HPO ₄ 13.56 g / L) containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L and 25 mM vaniphosphate (VA). KH ₂ PO ₄₆ g / L, NaCl 1 g / L, NH ₄ Cl 2 g / L, 2 mM regsvr ₄ , 100 μM CaCl ₂ and 18 μM FeSO ₄ ) were inoculated into 5 mL and cultured at 30 ° C. with shaking.

After the start of the culture, the optical density (OD) of the culture solution was measured at regular intervals, and the concentrations of VA and ccMA were measured in the culture supernatant obtained by centrifuging the culture solution. A wavelength of 600 nm was used for OD measurement, and the OD600 value was measured using miniphoto 518R (Tietech Co., Ltd.). The concentrations of VA and ccMA were measured using a high performance liquid chromatograph (Agient1200 series; Agilent Technologies, Inc.). The column used was ZORBAX Eclipse Plus C18 volume (diameter 4.6 mm, length 150 mm, particle size 0.5 μm) and kept warm at 40 ° C. Gradient elution mode (solvent A: 5% (v / v) CH ₃ OH, 1% (v / v) CH ₃ COOH, solvent B: 50% (v / v) CH ₃ OH, 1% (v) / V) Using CH ₃ COOH), after equilibration with solvent A, increase the proportion of solvent B to 20% over 8 minutes from the start of analysis, and then increase the proportion of solvent B to 100% over 5 minutes. Raised. The flow rate of the mobile phase was 1.0 mL / min, and the measurement wavelength was 280 nm.

Table 1a summarizes the measurement results of the OD600 value, the VA concentration and the ccMA concentration after the culture times of 0, 48, 56 and 72 hours for the IDPC / pTS108 strain. Table 1b shows the measurement results of the OD600 value, the VA concentration and the ccMA concentration after the culture times of 0, 24, 32 and 48 hours for the IDPC / pTS110 strain.

As shown in Tables 1a and 1b, the IDPC / pTS108 strain propagated using VA but did not produce ccMA, whereas the IDPC / pTS110 strain propagated using VA and ccMA. Was produced over time (yield was 1.4 wt%). In addition, the metabolic rate and specific proliferation rate of VA of the IDPC / pTS110 strain were very high with respect to the IDPC / pTS108 strain. The yield was determined by the amount of ccMA relative to the amount of substrate (VA) consumed.

The IDPC / pTS110 strain was cultured in the same manner as above, except that the concentration of VA contained in the MM liquid medium was changed from 25 mM to 50 mM. Table 1c summarizes the measurement results of the OD600 value, the VA concentration and the ccMA concentration after the culture times of 0, 24, 32 and 48 hours.

As shown in Table 1c, the IDPC / pTS110 strain propagated using VA and produced ccMA (yield 2.7 wt%).

[4. Production of ccMA using p-hydroxybenzoic acid (HBA) as a carbon source (1)]
Each of the IDPC / pTS108 strain and the IDPC / pTS110 strain was inoculated into 5 mL of LB liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and Tc 20 mg / L, and cultured with shaking at 30 ° C. for 16 hours. .. 50 μL of the obtained culture solution was inoculated into 5 mL of MM liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L and 25 mM HBA, and cultured with shaking at 30 ° C.

After the start of the culture, the optical density (OD600) of the culture solution and the HBA and ccMA concentrations in the culture solution were measured at regular intervals. The measurement of OD600 and the measurement of the concentrations of HBA and ccMA were carried out in the same manner as in the method described in 3 above.

Table 2a summarizes the measurement results of the OD600 value, the HBA concentration and the ccMA concentration after the culture times of 0, 24, 48 and 56 hours for the IDPC / pTS108 strain. Table 2b shows the measurement results of the OD600 value, the HBA concentration and the ccMA concentration of the IDPC / pTS110 strain after the culture times of 0, 24, 32 and 48 hours.

As shown in Tables 2a and 2b, the IDPC / pTS108 strain used HBA to grow but did not produce ccMA, whereas the IDPC / pTS110 strain used HBA to grow and ccMA. Was produced over time (yield was 4.1 wt%). In addition, the metabolic rate and specific proliferation rate of HBA of the IDPC / pTS110 strain were very high with respect to the IDPC / pTS108 strain. The yield was determined by the amount of ccMA relative to the amount of substrate (HBA) consumed.

The IDPC / pTS110 strain was cultured in the same manner as above, except that the concentration of HBA contained in the MM liquid medium was changed from 25 mM to 50 mM. Table 2c summarizes the measurement results of the OD600 value, the HBA concentration and the ccMA concentration after the culture times of 0, 24, 32, 48 and 56 hours.

As shown in Table 2c, the IDPC / pTS110 strain propagated using HBA and produced ccMA (yield 11.9 wt%).

[5. CcMA production using a mixture of VA and HBA as a carbon source]
The IDPC / pTS110 strain was inoculated into 5 mL of LB liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and Tc 20 mg / L, and cultured with shaking at 30 ° C. for 16 hours. 50 μL of the obtained culture solution was inoculated into 5 mL of MM liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L, 25 mM VA and 25 mM HBA, and cultured at 30 ° C. with shaking.

After the start of the culture, the optical density (OD600) of the culture solution and the VA, HBA and ccMA concentrations in the culture solution were measured at regular intervals. The OD600 measurement and the concentration measurement of VA, HBA and ccMA were carried out in the same manner as in the method described in 3 above.

Table 3 shows the measurement results of the OD600 value, the VA concentration, the HBA concentration and the ccMA concentration after the culture times of 0, 24, 32 and 48 hours.

As shown in Table 3, the IDPC / pTS110 strain grew using VA and HBA and produced ccMA over time (yield 17.7 wt%). The yield was determined by the amount of ccMA relative to the amount of substrate (VA and HBA) consumed.

[6. CcMA production using phenols derived from sugirignin as a carbon source (1)]
According to a conventional method, Sugi wood flour was subjected to alcohol-benzene extraction treatment, and then 1.5 g of the treated Sugi wood flour was subjected to alkali nitrobenzene oxidative decomposition treatment and diethyl ether extraction treatment (Wood Science Experiment Manual, edited by Japan Wood Society). , See Bunagaido Publishing). The alkaline solution after oxidative decomposition of nitrobenzene was subjected to a diethyl ether extraction treatment, and the obtained aqueous layer was subjected to an acidification treatment and further subjected to a diethyl ether extraction treatment. The diethyl ether extract obtained as an ether layer was used as a sugilignin-derived aromatic compound, and ccMA production was evaluated in an MM medium supplemented with an aqueous solution of a sugirignin-derived aromatic compound (pH ≈ 9) as the sole carbon source.

The IDPC / pTS110 strain was inoculated into 5 mL of LB liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and Tc 20 mg / L, and cultured with shaking at 30 ° C. for 16 hours. 50 μL of the obtained culture solution was inoculated into 5 mL of MM liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and Tc 20 mg / L, and 0.5 mL of an aqueous solution of phenols derived from sugilignin was added as a carbon source. , 30 ° C. with shaking culture. Twenty-four hours after the start of the culture, 0.5 mL of an aqueous solution of an aromatic compound derived from sugilignin was further added, and the cells were cultured with shaking at 30 ° C.

After the start of the culture, the optical density (OD600) of the culture solution and the ccMA concentration in the culture solution were measured at regular intervals. The OD600 measurement and the ccMA concentration measurement were carried out in the same manner as in the method described in 3 above.

Table 4 shows the measurement results of the OD600 value and the ccMA concentration after the culture times of 0, 24 and 48 hours.

As shown in Table 4, the IDPC / pTS110 strain was grown using an aromatic compound derived from lignin obtained from real biomass, and ccMA was produced over time.

[7. Preparation of IDPC / pTS119 strain]
Using the pTS119 plasmid DNA, which is a plasmid expressing the pcaH gene, pcaG gene, aroY gene, catA gene vanA gene and vanB gene, according to the following procedure . The putida IDPC strain was transformed to produce an IDPC / pTS119 strain.

P. Using the genomic DNA of the putida KT2440 strain as a template, the catechol 1,2-dioxygenase (catA) gene was obtained by a PCR method using a primer set consisting of primers 15 and 16 of SEQ ID NOs: 15 and 16. A DNA fragment containing about 1 kbp was obtained. The obtained DNA fragment was digested with KpnI and SmaI and cloned into the pUC118 plasmid previously digested with KpnI and SmaI to obtain pNI001 plasmid DNA.

Using the pNI001 plasmid DNA as a template, amplification was performed by the PCR method using a primer set consisting of primers 17 and 18 of SEQ ID NOs: 17 and 18, to obtain a DNA fragment of about 1 kbp containing the catA gene. The obtained DNA fragment was ligated to pTS109 plasmid DNA previously digested with NotI using an Infusion cloning HD kit to obtain pTS115 plasmid DNA.

P. Using the genomic DNA of the putida KT2440 strain as a template, the vanillate demethylase oxygenase component (vanA) is amplified by a PCR method using a primer set consisting of primers 19 and 20 of SEQ ID NOs: 19 and 20. A DNA fragment of about 2.0 kbp containing the gene and the vanillate demethylase oxygenodoreductase component (vanB) gene was obtained. The obtained DNA fragment was digested with SacI and SmaI, digested with SacI and SmaI in advance, and ligated with the pQE30 plasmid DNA to obtain pKY001 plasmid DNA.

Using the pKY001 plasmid DNA as a template, amplification was performed by a PCR method using a primer set consisting of primers 21 and 22 of SEQ ID NOs: 21 and 22, to obtain a DNA fragment of about 2.0 kbp containing the vanA gene and the vanB gene. The obtained DNA fragment was ligated with the pTS115 plasmid DNA previously digested with NotI using the Infusion HD Cloning Kit to obtain the pTS116 plasmid DNA.

Using the pTS107 plasmid DNA as a template, amplification was performed by PCR using a primer set consisting of primers 23 and 24 of SEQ ID NOs: 23 and 24 to obtain a DNA fragment of about 1.3 kbp containing the pcaG gene and the pcaH gene. The obtained DNA fragment was ligated with pTS116 plasmid DNA previously digested with HindIII using an Infusion HD cloning kit to obtain pTS119 plasmid DNA.

Using the obtained pTS119 plasmid DNA, P. An IDPC / pTS119 strain was prepared by transforming the putida IDPC strain.

[8. CcMA production using aromatic compounds derived from sugirignin as a carbon source (2)]
CcMA production was evaluated in MM medium supplemented with an aqueous solution of an aromatic compound derived from sugilignin (pH ≈9) as the sole carbon source in the same manner as in 6 above except that the IDPC / pTS119 strain was used.

After the start of the culture, the optical density (OD600) of the culture solution and the ccMA concentration in the culture solution were measured at regular intervals. The OD600 measurement and the ccMA concentration measurement were carried out in the same manner as in the method described in 3 above.

Table 5 summarizes the measurement results of the OD600 value and the ccMA concentration after the culture times of 0, 24 and 48 hours.

As shown in Table 5, the IDPC / pTS119 strain was grown using an aromatic compound derived from lignin obtained from real biomass, and ccMA was produced over time.

[9. CcMA production using a mixture of VA and HBA as a carbon source (2)]
The IDPC / pTS110 strain was inoculated into 10 mL of LB liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L and Tc 20 mg / L, and cultured with shaking at 30 ° C. overnight. 10 mL of the obtained culture medium was inoculated into 1.0 L of MM liquid medium containing Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L, 25 mM VA and 25 mM HBA, and 30 ° C., pH 7 , Aeration stirring culture was carried out for 24 to 100 hours while maintaining the dissolved oxygen concentration (DO) during the culture period within a certain range.

The VA, HBA and ccMA concentrations after culturing were measured in the same manner as in the method described in 3 above. From the obtained measurement results, the yield of ccMA was calculated by the amount of ccMA with respect to the amount of the consumed substrate (VA and HBA). Table 6 shows a summary of the ccMA yield, aeration volume, and DO value.

As shown in Table 6, the yield of ccMA produced was not affected by the aeration amount, but good results were obtained under the condition that the dissolved oxygen concentration in the culture solution was 5 to 10%. When the IDPC / pTS119 strain was used, the yield of ccMA produced was about 30% under the condition that the dissolved oxygen concentration in the culture solution was 2.5% and 5.0%.

The sequences listed in the sequence listing are:
[SEQ ID NO: 1] Primer 1
ATGAATTCCTGATTCGTGCAGGGGTTAT
[SEQ ID NO: 2] Primer 2
GCTTCACCTCAGAAGAGCTCGGGCATGCCGGTTTCCTCTC
[SEQ ID NO: 3] Primer 3
GAGCTCTTCTGAGGTGAAGCTTGGG
[SEQ ID NO: 4] Primer 4
ATGGATCCTCCATCCTGTTCGTCATCAA
[SEQ ID NO: 5] Primer 5
ACAAGCTTGCATGCCGATGCACACCATGCAGCAG
[SEQ ID NO: 6] Primer 6
CCGGGGATCCTCTAGACAGCTCTGTACCCCAGGTG
[SEQ ID NO: 7] Primer 7
ATGTGAATTGCGGCCAGCGCCCAATACGCAAACC
[SEQ ID NO: 8] Primer 8
TCTGGTACCGCGGCCGCGTAATCATGGTCATAGCTGTTTC
[SEQ ID NO: 9] Primer 9
ATGCGAGCTCATTAAAGAGGAGAAATTAACTATGCCCGCCCAGGACAAC
[SEQ ID NO: 10] Primer 10
ATGCGGATCCCCCAAGCTTCACCTCAGAAG
[SEQ ID NO: 11] Primer 11
AGCTGGTACCATTAAAGAGGAGAAATTAACTATGACCGCACCGATTCAG
[SEQ ID NO: 12] Primer 12
AGCTGGTACCTTATTTTGCGCTACCCTGGTT
[SEQ ID NO: 13] Primer 13
CATGATTACGCGGCCGCATTAAAGAGGAGAAATTAACTATGACCGCACCGATT
[SEQ ID NO: 14] Primer 14
TCTGGTACCGCGGCCTTATTGCGCGCTACCCTGGTT
[SEQ ID NO: 15] Primer 15
ATGCGGTACCTTAAAGAGGAGAAATTAACTATGACCGTGAAAATTTCCA
[SEQ ID NO: 16] Primer 16
AGCTCCCGGGTCAGCCCTCCTGCAACGCCC
[SEQ ID NO: 17] Primer 17
CATGATTACGCGGCCGCTTAAAGAGGAGAAATTAACTATGACCGTGA
[SEQ ID NO: 18] Primer 18
CTCTTTAATGCGGCCTCAGCCCTCCTGCAACGC
[SEQ ID NO: 19] Primer 19
ATGCGAGCTCATGTACCCCAAAAACACCT
[SEQ ID NO: 20] Primer 20
ATGCCCGGGTCAGATGTCCAGCACCAGCA
[SEQ ID NO: 21] Primer 21
CATGATTACGCGGCCATTAAAGAGGAGAAATTAAC
[SEQ ID NO: 22] Primer 22
CCTCTTTAAGCGGCCGCTCAGATGTCCAGCACCAG
[SEQ ID NO: 23] Primer 23
TCACCATGGGAAGCTATTAAAGAGGAGAAATTAACTAT
[SEQ ID NO: 24] Primer 24
TGGATCCACGAAGCTCCCAAGCTTCACCTCAGA
[SEQ ID NO: 25] KpdB
MKLIIGMTGATGAPLGVALLQALRDMPEVETHLVMSKWAKTTIELETPWTAREVAALADFSHSPADQAATISSGSFRTDGMIVIPCSMKTLAGIRAGYAEGLVGRAADVVLKEGRKLVLVPREMPLSTIHLENMLALSRMGVAMVPPMPAYYNHPETldapITNHIVTRVLDQFGL
[SEQ ID NO: 26] PobA
MKTQVAIIGAGPSGLLLGQLLHKAGIDNIIVERQTAEYVLGRIRAGVLEQGTVDLLREAGVAERMDREGLVHEGVELLVGGRRQRLDLKALTGGKTVMVYGQTEVTRDLMQAREASGAPIIYSAANVQPHELKGEKPYLTFEKDGRVQRIDCDYIAGCDGFHGISRQSIPEGVLKQYERVYPFGWLGLLSDTPPVNHELIYAHHERGFALCSQRSQTRSRYYLQVPLQDRVEEWSDERFWDELKARLPAEVAADLVTGPALEKSIAPLRSLVVEPMQYGHLFLVGDAAHIVPPTGAKGLNLAASDVNYLYRILVKVYHEGRVDLLAQYSPLALRRVWKGERFSWFMTQLLHDFGSHKDAWDQKMQEADREYFLTSPAGLVNIAENYVGLPFEEVA
[SEQ ID NO: 27] VanA
MYPKNTWYVACTPDEIATKPLGRQICGEKIVFYRARENQVAAVEDFCPHRGAPLSLGYVEDGNLVCGYHGLVMGCDGKTVSMPGQRVRGFPCNKTFAAVERYGFIWVWPGDQAQADPALIPHLEWAVSDEWAYGGGLFHIGCDYRLMIDNLMDLTHETYVHASSIGQKEIDEAPPVTTVTGDEVVTARHMENIMAPPFWRMALRGNGLADDVPVDRWQICRFTPPSHVLIEVGVAHAGKGGYHAEAQHKASSIVVDFITPESDTSIWYFWGMARNFAAHDQTLTDNIREGQGKIFSEDLEMLERQQQNLLAHPERNLLKLNIDAGGVQSRKVLERIIAQERAPQPQLIATSANPA
[SEQ ID NO: 28] VanB
MIDAVVVSRNDEAQGICSFELAAADGSLLPAFSAGAHIDVHLPDGLVRQYSLCNHPEERHRYLIGVLNDPASRGGSRSLHEQVQAGARLRISAPRNLFPLAEGAQRSLLFAGGIGITPILCMAEQLSDSGQAFELHYCARSSERAAFVERIRSAPFADRLFVHFDEQPETALDIAQVLGNPQDDVHLYVCGPGGFMQHVLDSAKGLGWQEANLHREYFAAAPVDASNDGSFAVQVGSTGQVFEVPADRTVVQVLEENGIEIAMSCEQGICGTCLTRVLQGTPDHRDLFLTEEEQALNDQFTPCCSRSKTPLLVLDI
[SEQ ID NO: 29] pcaH gene

[SEQ ID NO: 30] pcaG gene

[SEQ ID NO: 31] catA gene

[SEQ ID NO: 32] catB gene

[SEQ ID NO: 33] aroY gene

[SEQ ID NO: 34] kpdB gene

[SEQ ID NO: 35] pobA gene

[SEQ ID NO: 36] vanA gene

[SEQ ID NO: 37] vanB gene

[SEQ ID NO: 38] PcaH
MPAQDNSRFVIRDRNWHPKALTPDYKTSVARSPRQALVSIPQSISETTGPDFSHLGFGAHDHDLLLNFNNGGLPIGERIIVAGRVVDQYGKPVPNTLVEMWQANAGGRYRHKNDRYLAPLDPNFGGVGRCLTDRDGYYSFRTIKPGPYPWRNGPND
[SEQ ID NO: 39] PcaG
MPIELLPETPSQTAGPYVHIGLALEAAGNPTRDQEIWNCLAKPDAPGEHILLIGHVYDGNGHLVRDSFLEVWQADANGEYQDAYNLENAFNSFGRTATTFDAGEWTLQTVKPGVVNNAAGVPMAPHINISLFARGINIHLHTRLYFDDEAQANAKCPVLNLIEQPQRRET
[SEQ ID NO: 40] CatA
MTVKISHTADIQAFFNRVAGLDHAEGNPRFKQIILRVLQDTARLIEDLEITEDEFWHAVDYLNRLGGRNEAGLLAAGLGIEHFLDLLQDAKDAEAGLGGGTPRTIEGPLYVAGAPLAQGEARMDDGTDPGVVMFLQGQVFDADGKPLAGATVDLWHANTQGTYSYFDSTQSEFNLRRRIITDAEGRYRARSIVPSGYGCDPQGPTQECLDLLGRHGQRPAHVHFFISAPGHRHLTTQINFAGDKYLWDDFAYATRDGLIGELRFVEDAAAARDRGVQGERFAELSFDFRLQGAKSPDAEARSHRPRALQEG
[SEQ ID NO: 41] CatB
MTSVLIEHIDAIIVDLPTIRPHKLAMHTMQQQTLVVLRLRCSDGVEGIGEATTIGGLAYGYESPEGIKANIDAYLAPALIGLPADNINAAMLKLDKLAKGNTFAKSGIESALLDAQGKRLGLPVSELLGGRVRDSLEVAWTLASGDTARDIAEAQHMLDIRRHRVFKLKIGANPVAQDLKHVVAIKRELGDSASVRVDVNQYWDESQAIRACQVLGDNGIDLIEQPISRINRAGQVRLNQRSPAPIMADESIESVEDAFSLAADGAASIFALKIAKNGGPRAVLRTAQIAEAAGIALYGGTMLEGSIGTLASAHAFLTLRQLTWGTELFGPLLLTEEIVNEPPQYRDFQLHIPHTPGLGLTLDEQRLARFARR
[SEQ ID NO: 42] AroY
MTAPIQDLRDAIALLQQHDNQYLETDHPVDPNAELAGVYRHIGAGGTVKRPTRIGPAMMFNNIKGYPHSRILVGMHASRQRAALLLGCEASQLALEVGKAVKKPVAPVVVPASSAPCQEQIFLADDPDFDLRTLLPAPTNTPIDAGPFFCLGLALASDPVDASLTDVTIHRLCVQGRDELSMFLAAGRHIEVFRQKAEAAGKPLPITINMGLDPAIYIGACFEAPTTPFGYNELGVAGALRQRPVELVQGVSVPEKAIARAEIVIEGELLPGVRVREDQHTNSGHAMPEFPGYCGGANPSLPVIKVKAVTMRNNAILQTLVGPGEEHTTLAGLPTEASIWNAVEAAIPGFLQNVYAHTAGGGKFLGILQVKKRQPADEGRQGQAALLALATYSELKNIILVDEDVDIFDSDDILWAMTTRMQGDVSITTIPGIRGHQLDPSQTPEYSPSIRGNGISCKTIFDCTVPWALKSHFERAPFADVDPRPFAPEYFARLEKNQGSAK
[SEQ ID NO: 43] vdh gene

[SEQ ID NO: 44] PP_1948 gene

[SEQ ID NO: 45] ligV gene
ATGGACTCAGCGCGGATCGCGCCAGACCAGGAGAGAGACAGAATGGAATTTACCCGGCTTAACCCCATGACCGGCGAAGTCGCTTCGTCGGCGCCCGCGCTCAAGGCGGGTGACATTCCCGCGATCGCCCGCAAGGCCCGCGAAGGCTTCACCGCCTGGTCGGTGATGGGCCCCAATGCGCGCCGCGCCGTGCTGATGAAAGCCGCGACGGCGCTGGAAGCCCGCGCGGACGCGTTCGTCGATGCGATGATGGGAGAGATCGGCGCCACCAAGGGCTGGGCTCTGTTCAATCTCGGACTGGCGGCCAGCATGGTGCGCGAGGCAGCGGCCCTGACGACGCAGATCAATGGCGAAGTCATCCCCTCCGACAAGCCCGGCTGCCTCGCCATGGCGCTGCGCGAACCGGTGGGCGTCATTCTCGGCATCGCGCCCTGGAATGCGCCGATCATCCTCGGCGTGCGCGCCATCGCCGTGCCGCTGGCTTGCGGCAACAGCGTGATCCTGAAGGCGAGCGAGACCTGCCCGCGCACCCATGCCCTCATCATCGAGGCATTCGCAGATGCGGGGTTCCCCGAGGGAGTCGTCAACGTCGTCACCAATGCGCCAGCCGATGCGGGCGAAGTGGTCGGCGCGCTGATCGACGCGCCGGAAGTCAAGCGGATCAATTTCACCGGCTCCACCGGGGTCGGCAAGATCATCGCCAAGCGTGCCGCCGAGCATCTCAAGCCCGTGCTGCTGGAGCTGGGTGGCAAAGCCCCGCTCATTGTCCTGGAGGACGCCGATCTGGATGAAGCCGTCAAGGCGGCCGCCTTCGGCGCTTTCATGAACCAGGGGCAGATCTGCATGTCCACGGAGCGGATCATCGTGGTGGATGCGGTGGCCGACGAATTCGCCGCCCGGTTCAAGGCAAAGGTTTCGGCCATGCCTGTTGGCGATCCCCGGCAGGGAAGCACGCCGCTGGGAGCGGTCGTCGACACCAAGACTGTCGCGCATTGCCTGT CCTTGATCGAGGATGCGCTTGGAAAGGGCGCGGAGCAGCTGACGGGCGGCGAGACGACGCAGAATGTGCTGATGCCGGCGCATGTGATCGACCGCGTCACGCCCGACATGAAGCTCTTCCGGGACGAGAGCTTCGGTCCTGTCGTGGGGATCATCCGCGCACGCGACGCCGAGCATGCGATCGAACTGGCCAACGACACCGAATATGGTCTCTCGGCCTCGGTCTTCACGCGCGACACGGCCAAGGGCCTCAGCGTCGCCCGGCGGATCGAATCCGGGATCTGCCATGTCAACGGGCCGACGGTCCATGACGAGGCGCAGATGCCCTTCGGCGGCGTGAAGGCTTCGGGCTATGGTCGTTTCGGCGGCAAGGCCGGCATCGACAGCTTCACGGAGCTGCGCTGGATCACCATCGAGACCCAGCCGGGACATTTCCCGATCTGA

According to the transformation microorganism of one aspect of the present invention and the method for producing muconic acid, muconic acid can be obtained from lignin-derived aromatic compounds and biomass containing lignin. Muconic acid can be converted into various industrially useful compounds, for example, raw materials for muconic acid derivatives having uses such as surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents and the like. Can be used as.

Claims (6)

The host microorganism is a Pseudomonas microorganism having a pcaH gene, a pcaG gene, a catA gene and a catB gene on the chromosome.
The pcaH gene, the pcaG gene, and the catB gene on the chromosome are deleted.
It expresses the inserted pcaH gene and pcaG gene, and
Expressing the inserted aroY gene,
It is a transforming microorganism
The inserted aroY gene, the inserted pcaH gene and the pcaG gene are contained in a DNA construct that autonomously amplifies without being introduced into the chromosome of the host microorganism.
The inserted aroY gene is a gene that expresses protocatechuate decarboxylase and has a base sequence encoding an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 42.
The inserted pcaH gene expresses the β subunit of protocatechuic acid 3,4-dioxygenase and has a base sequence encoding an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 38. The pcaG gene, which is a gene having and is inserted, expresses the α subunit of protocatechuic acid 3,4-dioxygenase, and has an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 39. Is a gene having a base sequence encoding
The transformed microorganism. The transformed microorganism according to claim 1, wherein the inserted aroY gene and the inserted pcaH gene and the pcaG gene are under the control of the same promoter. The transformed microorganism according to any one of claims 1 to 2, wherein the host microorganism is a Pseudomonas microorganism having a pobA gene, a vanA gene and a vanB gene on the chromosome. The transformed microorganism according to any one of claims 1 to 3, wherein the host microorganism is Pseudomonas putida. A step of obtaining muconic acid by allowing an aromatic compound derived from p-hydroxyphenyllignin and / or an aromatic compound derived from guaiacyllignin to act on the transformed microorganism according to any one of claims 1 to 4. A method for producing muconic acid, including. In the step, under the condition that the dissolved oxygen concentration is 1 to 13%, the aromatic compound derived from p-hydroxyphenyllignin and / or the aromatic compound derived from guaiacyl lignin is obtained from any one of claims 1 to 4. The production method according to claim 5, which is a step of obtaining muconic acid by acting on the transformed microorganism according to the above item.

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