WO2018199111A1 - Micro-organisme transgénique permettant de produire de l'acide muconique et son utilisation - Google Patents

Micro-organisme transgénique permettant de produire de l'acide muconique et son utilisation Download PDF

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WO2018199111A1
WO2018199111A1 PCT/JP2018/016674 JP2018016674W WO2018199111A1 WO 2018199111 A1 WO2018199111 A1 WO 2018199111A1 JP 2018016674 W JP2018016674 W JP 2018016674W WO 2018199111 A1 WO2018199111 A1 WO 2018199111A1
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gene
microorganism
acid
muconic acid
dna
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和典 園木
英司 政井
直史 上村
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国立大学法人弘前大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids

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  • the present invention relates to a transformed microorganism capable of producing muconic acid and a method for producing muconic acid using the transformed microorganism.
  • the present invention relates to a transformed microorganism capable of growing and producing muconic acid using a lignin-derived aromatic compound as a single carbon source.
  • Lignin is an amorphous polymer that exists as a component of plant vascular cell walls. It is a complex condensation of phenylpropane-based structural units, and the main feature of its chemical structure is the inclusion of methoxy groups. ing. Lignin has a function of sticking woody plant cells to each other and strengthening the tissue, and is present in about 18 to 36% in wood and about 15 to 25% in herbs. Therefore, in order to effectively use wood, various attempts have been made to decompose lignin and obtain useful compounds.
  • muconic acid cis, cis-muconic acid
  • muconic acid is a highly reactive compound due to two double bonds and carboxy groups in the molecule.
  • muconic acid derivatives starting from muconic acid include lactone, sulfone, polyamide, polyester, thioester, and addition polymer.
  • Such muconic acid derivatives are known to have various uses, and can be used as, for example, surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents and the like.
  • muconic acid is used in various forms in the form of a muconic acid derivative, and if muconic acid can be produced from lignin, resource regeneration is achieved, which is very useful. Therefore, a method for producing muconic acid from lignin or a substance derived from lignin has been attempted. In particular, bioconversion using microorganisms has been studied as such a method.
  • Non-Patent Document 1 the Pseudomonas putida and (Pseudomonas putida) as a host microorganism, pCAH gene and pcaG gene on chromosome (hereinafter combined And the catR gene, catB gene, catC gene and catA gene are disrupted, and the inserted catA gene and aroY gene, or the catA gene, aroY gene and ecdB gene are expressed. It is described that a transformed microorganism was produced, the transformed microorganism was grown with glucose, and then muconic acid was produced with p-coumaric acid.
  • muconic acid can be produced from p-coumaric acid by using the transformed microorganism described in Non-Patent Document 1.
  • the transformed microorganism described in Non-Patent Document 1 requires expensive glucose as a carbon source for growth
  • the method for producing muconic acid using the transformed microorganism described in Non-Patent Document 1 is economical. There is a problem that is bad.
  • two or more types of carbon sources are required, such as glucose for cell growth and p-coumaric acid as a substrate for muconic acid production, considering the total amount of these carbon sources,
  • the method for producing muconic acid using the transformed microorganism described in Non-Patent Document 1 has a problem that the yield of muconic acid is poor.
  • the present invention is intended to solve the problem of providing a microorganism capable of producing muconic acid having good economic efficiency and high yield, and a method for producing muconic acid using the microorganism. Let's take a challenge.
  • the present inventors have intensively studied a muconic acid-producing microorganism having a high yield.
  • FIG. 1 shows the metabolic pathway of the transformed microorganism described in Non-Patent Document 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 trying to efficiently produce muconic acid by the transformed microorganism described in Non-Patent Document 1, the pcaHG gene and catB gene on the chromosome of the host microorganism are deleted, and the inserted aroY gene is expressed. There must be. Only in this way can an aromatic compound derived from lignin such as p-coumaric acid once converge on protocatechuic acid and be converted to muconic acid.
  • 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. To do. Therefore, it appears that the transformed microorganism described in Non-Patent Document 1 is a high yield method for producing muconic acid from an aromatic compound derived from lignin. Therefore, 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 low yield method for producing muconic acid.
  • the present inventors have made extensive studies, and after destroying the pcaHG gene and the catB gene on the chromosome of the host microorganism, a transformed microorganism that overexpresses the pcaHG gene and the aroY gene separately inserted as foreign genes. Succeeded in creating. Surprisingly, the present inventors have found that the transformed microorganism produced by the present inventors can produce muconic acid while growing using an aromatic compound derived from lignin without using glucose. Thus, the present inventors succeeded in creating a method for producing muconic acid from an aromatic compound derived from lignin, using the transformed microorganism produced by the present inventors.
  • the method using the transformed microorganisms produced by the present inventors requires an aromatic compound derived from glucose and lignin as a substrate while using an aromatic compound derived from lignin as the sole carbon source.
  • the present inventors have found that the yield of muconic acid is the same or higher.
  • Aromatic compounds derived from lignin can be obtained from biomass such as waste materials and are very inexpensive compared to glucose. Therefore, the method for producing muconic acid using the transformed microorganism prepared by the present inventors is an economically advantageous method compared to the method using the transformed microorganism described in Non-Patent Document 1. The present invention has been completed based on these findings and successful examples.
  • the host microorganism is a microorganism belonging to the genus Pseudomonas 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, Expressing the inserted pcaH and pcaG genes; and Expressing the inserted aroY gene; Transformed microorganisms.
  • the following methods (5) and (6) for producing muconic acid are provided.
  • (5) By causing an aromatic compound derived from p-hydroxyphenyl lignin 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, comprising a step of obtaining muconic acid.
  • the following recombinant vector (7) is provided.
  • a recombinant vector comprising an aroY gene, a pcaH gene and a pcaG gene.
  • muconic acid is produced by maintaining or improving the yield at a lower cost than the conventional method using microorganisms. can do. Therefore, according to the transformed microorganism of one embodiment of the present invention and the method for producing muconic acid of one embodiment of the present invention, production of muconic acid on an industrial scale can be expected as part of effective utilization of biomass containing lignin. . According to the recombinant vector of one embodiment of the present invention, the transformed microorganism of one embodiment of the present invention can be produced.
  • FIG. 1 shows FIG. 1 is a schematic diagram of a metabolic pathway of Pseudomonas putida described in 1.
  • the transformed microorganism, the method for producing muconic acid and the recombinant vector which are one embodiment of the present invention, will be described in detail, but the technical scope of the present invention is not limited only to the matters of this item, The present invention can take various forms as long as the object is achieved.
  • the host microorganism is transformed so that the specific gene on the chromosome of the host microorganism is deleted and the specific gene inserted as a foreign gene is expressed.
  • Microorganism is transformed so that the specific gene on the chromosome of the host microorganism is deleted and the specific gene inserted as a foreign gene is expressed.
  • gene deletion means that a gene does not function normally, such as a gene that is not normally transcribed or a protein that is to be produced by gene expression is not translated normally. It means that expression is prevented. Deletion of a gene can occur, for example, when the structure of the gene is changed due to destruction, deletion, substitution, insertion, or the like of all or part of the gene. However, gene deletion can also occur when gene expression is suppressed by means such as blocking the control region of a gene without causing a change in the structure of the gene.
  • gene expression means that a protein encoded by a gene is produced in such a manner as to have an original structure or activity through transcription, translation, or the like.
  • overexpression of a gene in the present specification means that a protein encoded by the gene is produced in excess of the amount originally expressed by the host microorganism by inserting the gene.
  • the host microorganism has a pcaH gene, a pcaG gene, a catA gene and a catB gene on the chromosome.
  • the transformed microorganism lacks the pcaH gene, pcaG gene and catB gene on the chromosome of the host microorganism.
  • the host microorganism preferably lacks both the pcaH gene and the pcaG gene originally present on the chromosome, but the probability that either the pcaH gene or the pcaG gene only needs to be deleted. There is.
  • the pcaH gene and the pcaG gene are not particularly limited as long as they are genes that express the ⁇ subunit and ⁇ subunit of protocatechuic acid and 3,4-dioxygenase, respectively.
  • they have the nucleotide sequences of SEQ ID NOs: 29 and 30, respectively. Examples include genes.
  • the catB gene is not particularly limited as long as it is a gene that expresses 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 that expresses catechol 1,2-dioxygenase (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 is possessed by, for example, Pseudomonas putida KT2440 strain, has an activity of catalyzing the reaction of generating cis, cis-muconic acid from catechol, and co-factors Fe 3+ As request.
  • Intradiol dioxygenase domain is contained in the amino acid sequence of catechol 1,2-dioxygenase (accession no. Q88I35).
  • the domain is [LIVMF] -xGx- [LIVM] -x (4)-[GS] -x (2)-[LIVMA] -x (4)-[LIVM]-[DE]-[LIVFMFYC ] -X (6) -Gx- [FY] (Prosite entry no. P00083), and Y in the sequence is related to the binding of Fe 3+ as a cofactor.
  • L137 to Y165 in the amino acid sequence of catechol 1,2-dioxygenase correspond to the above domain.
  • the transformed microorganism expresses the inserted pcaH gene and pcaG gene. However, when one of the pcaH gene and the pcaG gene that the host microorganism originally has on the chromosome is deleted, there is a probability that the deleted gene may be inserted and expressed. The transformed microorganism expresses the further inserted aroY gene.
  • the aroY gene is not particularly limited as long as it is a gene that expresses protocatechuic acid / decarboxylase, and examples thereof include a gene having the base sequence of SEQ ID NO: 33.
  • Protocatechuic acid decarboxylase (EC 4.1.1.63) is also called 3,4-dihydroxybenzoate carboxy-lyase.
  • Protocatechuic acid / decarboxylase is not particularly limited as long as it is an enzyme that catalyzes a reaction for producing catechol from protocatechuic acid.
  • An enzyme having an activity of catalyzing a reaction for producing 3-methoxycatechol from 3-O-methylgallic acid or a reaction for producing pyrogallol from gallic acid may be used as protocatechuic acid / decarboxylase.
  • vanillic acid decarboxylase and 4-hydroxybenzoic acid decarboxylase may also be used as protocatechuic acid / decarboxylase because protocatechuic acid may be decarboxylated.
  • Protocatechuic acid decarboxylase is structurally classified into a protein group (UbiD superfamily) containing a UbiD domain (Domain architecture ID 10487953).
  • protocatechuic acid decarboxylase Klebsiella pneumoniae subsp. Pneumoniae (Klebsiella pneumoniae subsp pneumoniae.) Protein from A170-40 strain (ATCC twenty-five thousand five hundred and ninety-seven strain) (accession no.AB479384; AB479384 protein) etc. Can be mentioned.
  • protocaterate decarboxylase derived from Enterobacter cloacae MBRL1077 accession no. AMJ70686; sequence identity 87.2%
  • E. coli E. coli.
  • Examples include, but are not limited to, proteins registered as protocatechuate decarboxylase such as protocatechuate decarboxylase (accession no. CZU76022; sequence identity 85.7%) derived from cloacae e1026 strain. These enzymes are characterized as a series of enzymes that require Mn 2+ as a cofactor, prenylated flavin mononucleotide (prenyl-FMN).
  • prenyl-FMN prenylated flavin mononucleotide
  • the transformed microorganism may have a kpdB gene inserted therein.
  • the kpdB gene is a protein assumed to synthesize prenyl-FMN, which is a cofactor of protocatechuate decarboxylase, and is supplied together with protocatechuate decarboxylase in one cell to supply prenyl-FMN. There is a probability that protocatechuate decarboxylase activity is improved.
  • the kpdB gene is expressed together with the aroY gene and the decarboxylation activity can be improved, the production yield of muconic acid is expected to increase.
  • the kpdB gene is a gene that expresses 4-hydroxybenzoic acid / decarboxylase / subunit B having enzymatic activity as a flavin / prenyltransferase, and includes, for example, a gene having the base sequence of SEQ ID NO: 34.
  • the transformed microorganism has a host microorganism having one, two, or all three types of genes of the pobA gene, the vanA gene and the vanB gene in addition to the pcaH gene, pcaG gene, catA gene and catB gene on the chromosome. Is preferred.
  • the vanA gene and the vanB gene may be collectively referred to as the vanAB gene.
  • the host microorganism does not have the pobA gene, vanA gene and / or vanB gene on the chromosome, it is preferably inserted into the transformed microorganism so as to express these genes.
  • the transformed microorganism the host microorganism on the chromosome, vanillin dehydrogenase (vdh) gene, p- hydroxybenzaldehyde dehydrogenase (PP_1948) gene and / or sphingomyelin bi um (Sphingobium) sp. It preferably has an aldehyde dehydrogenase (ligV) gene derived from the SYK-6 strain.
  • ligV aldehyde dehydrogenase
  • the pobA gene is not particularly limited as long as it is a gene that expresses p-hydroxybenzoate monooxygenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 35.
  • Examples of p-hydroxybenzoic acid monooxygenase (EC 1.14.13.2 or EC 1.14.13.33) include PobA derived from Pseudomonas putida KT2440 (accession no. Q88H28). .
  • the vanA gene is not particularly limited as long as it is a gene that expresses a vanillic acid, demethylase, oxygenase component (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 include VanA (accession no. Q88GI6) derived from Pseudomonas putida KT2440.
  • the vanillic acid / demethylase / oxygenase component cleaves the methyl ether bond of vanillic acid using an NADH or NADPH-derived electron supplied via Oxidoductase component and an oxygen atom supplied from molecular oxygen, Protocatechuic acid, formaldehyde and water are produced.
  • the vanillic acid / demethylase / oxygenase component has Rieske [2Fe-2S] iron-sulfur domain (W7-V107, PROSITE entry no. PS51296) in its amino acid sequence, and C and H (C47, H49) in the domain , C66, H69) are related to the Fe—S bond.
  • the vanB gene is not particularly limited as long as it is a gene that expresses a vanillate, demethylase, oxidoreductase component (vanillate demethylase oxidoreductase component), and examples thereof include a gene having the base sequence of SEQ ID NO: 37.
  • Examples of the vanillic acid / demethylase / oxidoreductase component include VanB (accession no. Q88GI5) derived from Pseudomonas putida KT2440.
  • the vanillic acid / demethylase / oxidoreductase component is known as one of the oxidoreductases that extract electrons from NADH or NADPH and transfer them to an oxygenase (oxygenase).
  • the vanillic acid / demethylase / oxidoreductase component transmits NADH or NADPH-derived electrons to VanA which is a vanillic acid / demethylase / oxygenase component.
  • the vanillic acid / demethylase / oxidoreductase component has 2Fe-2S ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and C in the amino acid sequence 5 (C26) , C270, C273, and C303) are involved in the Fe—S bond.
  • vanillic acid / demethylase / oxidoreductase components include NAD-binding domain (L109-D201, Pfammentry nono. PF00175) and Ferredoxinreductasetype FAD-BindingPrindoMindPrindoMinder PS 51384).
  • the vdh gene is not particularly limited as long as it is a gene that expresses vanillin dehydrogenase, and examples thereof include a gene having the base sequence of SEQ ID NO: 43.
  • Examples of vanillin dehydrogenase (EC 1.2. 1.67) include Vdh (accession no. Q88HJ9) derived from Pseudomonas putida KT2440.
  • the PP — 1948 gene is not particularly limited as long as it is a gene that expresses p-hydroxybenzaldehyde dehydrogenase (p-hydroxybenzaldehyde dehydrogenase), and examples thereof include a gene having the base sequence of SEQ ID NO: 44.
  • Examples of p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.14) include p-hydroxybenzaldehyde dehydrogenase derived from Pseudomonas putida KT2440 (accession no. Q88LI4) and Pseudomonas putida mt-2.
  • p-hydroxybenzaldehyde dehydrogenase XylC, accession no. P43503 and the like.
  • the ligV gene is not particularly limited as long as it is a gene that expresses aldehyde dehydrogenase using aromatic aldehydes such as vanillin, p-hydroxybenzaldehyde, syringaldehyde, protocatechualdehyde, and benzaldehyde as a substrate. And a gene having the base sequence of SEQ ID NO: 45.
  • aldehyde dehydrogenase EC 1.2.1.-
  • examples thereof include aldehyde dehydrogenase derived from SYK-6 strain (accession no. AB287332).
  • the gene to be inserted may not be completely the same as the gene originally possessed by the organism having the gene (that is, the wild type gene), and at least the protein that is expressed by the wild type gene (that is, the wild type protein), As long as it is a gene that expresses a protein having similar enzymatic properties, it may be a DNA having a base sequence that hybridizes with a base sequence complementary to the base sequence of the wild-type gene under stringent conditions. Good.
  • base sequence that hybridizes under stringent conditions refers to a colony hybridization method, plaque hybridization method, Southern blot hybridization method using a DNA having a base sequence of a wild-type gene as a probe. It means the base sequence of DNA obtained by using.
  • stringent conditions in the present specification is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal.
  • the hybridization system used, the type of probe, and the sequence It depends on the length.
  • Such conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration. If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration.
  • a DNA probe is used as a probe, and hybridization is 5 ⁇ SSC, 1.0% (w / v), a blocking reagent for nucleic acid hybridization (Roche Diagnostics) , 0.1% (w / v) N-lauroyl sarcosine, 0.02% (w / v) SDS, overnight (about 8 to 16 hours). Washing is performed using 0.1 to 0.5 ⁇ SSC, 0.1% (w / v) SDS, preferably 0.1 ⁇ SSC, 0.1% (w / v) SDS twice for 15 minutes. Do.
  • the temperature for performing hybridization and washing is 65 ° C or higher, preferably 68 ° C or higher.
  • the DNA having a base sequence that hybridizes under stringent conditions for example, using a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter on which the DNA fragment is immobilized, After hybridization at 40 ° C. to 75 ° C. in the presence of DNA obtained by hybridization under the above stringent conditions or 0.5 M to 2.0 M NaCl, preferably 0.7 to 1 After hybridization at 65 ° C in the presence of 0.0 M NaCl, 0.1 to 1 ⁇ SSC solution (1 ⁇ SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 65 ° C.
  • DNA containing a base sequence that hybridizes under stringent conditions include DNA having a certain sequence identity with a base sequence of a DNA having a base sequence of a wild-type gene used as a probe. 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more , 98% or more or 99% or more, more preferably 99.5% or more of DNA having sequence identity.
  • the upper limit of the sequence identity is not particularly limited, and is typically 100%.
  • the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild type gene under stringent conditions is, for example, 1 to several, preferably 1 to 300, more preferably, in the base sequence of the wild type gene. Of 1 to 200, more preferably 1 to 100, still more preferably 1 to 50, particularly preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases.
  • a nucleotide sequence having a deletion, substitution, addition or the like is included.
  • base deletion means that there is a deletion or disappearance in the base in the sequence
  • base replacement means that the base in the sequence is replaced with another base
  • Additional of a base means that a new base is added to be inserted.
  • the protein encoded by the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild-type gene under stringent conditions is 1 to several in the amino acid sequence of the protein encoded by the base sequence of the wild-type gene. Although it is likely to be a protein having an amino acid sequence having deletion, substitution, addition, etc. of individual amino acids, it has the same enzyme activity as the protein encoded by the base sequence of the wild-type gene.
  • a protein having the same or similar enzymatic properties as the wild-type protein has an amino acid sequence having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild-type protein It may consist of.
  • the range of “1 to several” in “deletion, substitution and 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 Means about one.
  • amino acid deletion means deletion or disappearance of an amino acid residue in the sequence
  • amino acid substitution means that an amino acid residue in the sequence is replaced with another amino acid residue.
  • “Addition of amino acid” means that a new amino acid residue is added to the sequence.
  • a specific embodiment of “deletion, substitution, addition of 1 to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid.
  • a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned.
  • Such chemically similar amino acids are known in the art for each amino acid.
  • Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • Examples of the basic amino acid having a positive charge include arginine, histidine, and lysine.
  • Examples of acidic amino acids having a negative charge include aspartic acid and glutamic acid.
  • amino acid sequences having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild type protein include amino acid sequences having a certain sequence identity with the amino acid sequence of the wild type protein. For example, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% with the amino acid sequence of the wild-type protein As mentioned above, amino acid sequences having sequence identity of 97% or more, 98% or more, or 99% or more, and more preferably 99.5% or more can be mentioned.
  • the upper limit of the sequence identity is not particularly limited, and is typically 100%.
  • the method for determining the sequence identity of the base sequence or amino acid sequence is not particularly limited.
  • the amino acid sequence of the wild type gene or the wild type protein expressed by the wild type gene and the target base It is obtained by aligning sequences and amino acid sequences and using a program for calculating the coincidence ratio between the sequences.
  • Each of the above methods can be generally used for searching a sequence showing sequence identity from a database.
  • Genetyx network version version 12.0. 1 Genetics
  • This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985; the entire description of this document is incorporated herein by reference).
  • CDS or ORF a region encoding a protein
  • the gene to be inserted is derived from a microorganism or the like carrying the gene to be inserted.
  • Examples of the organism derived from the gene to be inserted include microorganisms that can produce muconic acid from protocatechuic acid, microorganisms that can grow by assimilating protocatechuic acid, and the like.
  • organisms from which the inserted gene is derived include Pseudomonas ptida, Pseudomonas fluorescens, Pseudomonas alkaligenes, Pseudomonas pseudoalkagenes, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas cepacia for the pcaH and pcaG genes.
  • Acinetobacter microorganisms such as genus microorganisms, Acinetobacter bailey and Acinetobacter calcoaceticus; Pseudomonas petitda, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas rennekei, and Acinetobacter radicine cinnamon Microorganisms belonging to the genus Acinetobacter, such as Rhodococcus opacus, Rhodococcus pyridinibolus, Rhodococcus rhodochrous, and the like; Enterobacter genus microorganisms such as Enterobacter aerogenes, Cediment bacter hydroxybenzoicus, etc .; For the pobA gene, Pseudomonas pt. ⁇ Baumanni Klebsiella microorganisms such as Acinetobacter microorganisms, Klebsiella pneumoniae, Klebsi
  • the organism from which the inserted gene is derived is not particularly limited, but the gene expressed in the transformed microorganism is not inactivated by the growth conditions of the host microorganism, or is transformed by inserting the gene so as to exhibit activity.
  • a host microorganism to be converted or a microorganism having similar growth conditions to the host microorganism is preferable.
  • the gene to be deleted and inserted can be inserted into various known vectors. Furthermore, this vector can be introduced into a suitable known host microorganism to produce a transformant (transformed microorganism) having the gene deleted or inserted.
  • the gene to be deleted is preferably one in which the whole or a part of the wild-type gene is altered in its structure due to destruction, deletion, substitution, insertion or the like.
  • the inserted gene is preferably a gene that expresses the same or similar protein as the wild-type gene.
  • a transformation and a transformant include a transduction and a transductant, respectively.
  • chromosomal DNA or mRNA can be extracted from an organism derived from a wild-type gene associated with a gene to be deleted or inserted or various microorganisms by a conventional method, for example, a method described in a reference technical document.
  • CDNA can be synthesized using the extracted mRNA as a template.
  • a chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA thus obtained.
  • the gene to be inserted can be obtained by cloning using a chromosomal DNA or cDNA of a derived organism having a wild-type gene related to the gene as a template.
  • the wild-type gene-derived organism is as described above, and specific examples include Pseudomonas putida KT2440 strain and Klebsiella pneumoniae subspices pneumoniae A170-40 depending on the type of gene. Although it can, it is not limited to these.
  • Pseudomonas putida KT2440 strain is cultured, water is removed from the obtained bacterial cells, and it is physically ground using a mortar while cooling in liquid nitrogen to obtain fine powdered bacterial cell pieces.
  • a chromosomal DNA fraction is extracted from the cell fragments by a conventional method.
  • a commercially available chromosomal DNA extraction kit such as DNeasy Blood® & Tissue Kit (Qiagen) can be used.
  • chromosomal DNA and genomic DNA are synonymous.
  • the DNA is amplified by performing a polymerase chain reaction (PCR) using a chromosomal DNA as a template and a synthetic primer complementary to the 5 'end sequence and the 3' end sequence.
  • the primer is not particularly limited as long as it can amplify a DNA fragment containing a gene to be inserted. Examples thereof include primers represented by SEQ ID NOs: 9 and 10 designed with reference to the genome sequence of Pseudomonas putida KT2440 strain as amplifying the pcaH gene and the pcaG gene. In addition, when such a primer is used, the target gene full length can be amplified.
  • DNA containing a target gene fragment is amplified by screening a target gene clone from a shotgun library, or by appropriate PCR such as Inverse PCR, Nested PCR, 5′RACE method, or 3′RACE method. These can be ligated to obtain DNA containing the full-length target gene.
  • the method for obtaining a gene to be deleted or inserted is not particularly limited as described above, and it is possible to construct a gene using, for example, a chemical synthesis method without using a genetic engineering technique.
  • Confirmation of the base sequence in the amplification product amplified by PCR or the chemically synthesized gene can be performed, for example, as follows.
  • a DNA whose sequence is to be confirmed is inserted into an appropriate vector according to a normal method to produce a recombinant DNA.
  • known or commercially available kits such as In-Fusion HD Cloning Kit (Takara Bio), TA Cloning Kit (Invitrogen); see pUC4K (Gene, vol. 19, p259-268, 1982); The entire description of this document is incorporated herein by reference.), PEX18Amp (see Gene, vol.
  • the recombinant DNA is introduced into, for example, Escherichia coli , preferably E. coli JM109 (Takara Bio) or E. coli DH5 ⁇ (Takara Bio). Then, the recombinant DNA contained in the obtained transformant can be purified using QIAGEN Plasmid Mini Kit (Qiagen) or the like.
  • the sequence analysis apparatus used for determining the base sequence is not particularly limited, and examples thereof include Li-COR MODEL 4200L Sequencer (Aloka), 370 DNA Sequence System (PerkinElmer), and CEQ2000XL DNA Analysis System (Beckman). Can be mentioned. Based on the determined base sequence, the amino acid sequence of the translated protein can be known.
  • Recombinant vector (recombinant DNA) containing a gene to be deleted or inserted is obtained by linking PCR amplification products containing the gene to be deleted or inserted and various vectors in such a manner that the gene can be deleted or expressed. Can be constructed.
  • the recombinant vector is deleted because the gene in the recombinant vector is replaced with the gene in the host microorganism by homologous recombination by introducing the recombinant vector into the host microorganism. It is preferable to include regions upstream and downstream of the gene.
  • a method for producing a recombinant vector containing a gene to be inserted is, for example, excising a DNA fragment containing any of the genes to be inserted with an appropriate restriction enzyme and cleaving the DNA fragment with an appropriate restriction enzyme.
  • the plasmid vector thus constructed can be constructed by ligation using a commercially available recombinant vector production kit such as In-Fusion HD Cloning Kit (Takara Bio Inc.).
  • a DNA fragment containing a gene having a sequence homologous to a plasmid vector added to both ends and a plasmid-derived DNA fragment amplified by inverse PCR are commercially available, such as In-Fusion HD Cloning Kit (Takara Bio Inc.). It can be obtained by ligation using a recombinant vector preparation kit.
  • a recombinant vector containing a gene to be deleted or inserted includes at least a gene to be deleted or inserted and a gene (base sequence) derived from a plasmid vector.
  • An example of a recombinant vector and another embodiment of the present invention includes a recombinant vector containing an aroY gene, a pcaH gene, and a pcaG gene.
  • the recombinant vector of one embodiment of the present invention is used for producing the transformed microorganism of one embodiment of the present invention.
  • the recombinant vector of one embodiment of the present invention is at least one, two, three, or four selected from the group consisting of the pobA gene, the vanAB gene, the vdh gene, and the PP_1948 gene in addition to the aroY gene and the pcaHG gene. Of genes.
  • the recombinant vector of one embodiment of the present invention may include a gene other than the above-described genes as long as the problem of the present invention is not prevented.
  • 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 not naturally occurring in the host microorganism (for example, a synthetic gene that does not depend on the nucleic acid sequence derived from the host microorganism, or an organism in which the inserted gene and the derived organism are different). Examples include genes derived from organisms such as other microorganisms different from host microorganisms, plants, animals, and viruses.
  • heterologous gene when the host microorganism is a Pseudomonas microorganism include, but are not limited to, a DNA fragment derived from pUC118, such as a lactose promoter region (Plac).
  • a DNA fragment derived from pUC118 such as a lactose promoter region (Plac).
  • recombinant vector of one embodiment of the present invention include a pTS110 plasmid vector and a pTS119 plasmid vector described in Examples described later, but are not limited thereto.
  • a method for producing a transformed microorganism is not particularly limited, and examples thereof include a method of inserting into a host microorganism in such a manner that gene deletion or insertion is realized according to a conventional method. Specifically, a DNA construct in which any of the inserted genes is inserted between an expression-inducing promoter and a terminator is prepared, and then a host microorganism is transformed with the DNA construct. A converted microorganism is obtained. Alternatively, a transformed microorganism that lacks the gene can be obtained by preparing a DNA construct that includes the gene to be deleted and regions upstream and downstream of the gene, and then transforming the host microorganism with the DNA construct. In the present specification, recombinant vectors prepared for transforming host microorganisms are collectively referred to as DNA constructs.
  • the method for introducing the DNA construct into the host microorganism is not particularly limited.
  • the DNA construct is introduced into the host microorganism so that the introduced DNA construct autonomously proliferates to express the gene, as known to those skilled in the art.
  • Method Examples include a method of directly inserting a DNA construct into the chromosome of a host microorganism by utilizing homologous recombination.
  • the DNA construct is ligated between sequences upstream and downstream of the recombination site on the chromosome. Can be inserted into the genome of the host microorganism.
  • the vector-host system used for the production of the transformed microorganism is not particularly limited as long as the inserted gene can be expressed in the host microorganism or the gene on the chromosome can be deleted.
  • pJB866- Pseudomonas microorganisms pKT230 (Gene, vol. 16, p237-247, 1981; the entire description of this document is incorporated herein by reference)-Pseudomonas microorganisms.
  • the DNA construct containing the gene to be inserted does not introduce into the chromosome of the host microorganism, and autonomously amplifies and expresses the gene to be inserted, but expresses the gene to be inserted in the form introduced into the chromosome of the host microorganism. Or either.
  • the DNA construct may include a marker gene to allow selection of transformed cells.
  • the marker gene is not particularly limited, and examples thereof include drug resistance genes for drugs such as gentamicin, kanamycin, tetracycline, ampicillin, and carbenicillin.
  • the marker gene may be included in the middle of the deleted gene or so as to replace the deleted gene.
  • the DNA construct containing the gene to be inserted is not limited to a promoter and terminator that allow the gene to be expressed in the host microorganism, and other regulatory sequences (for example, cis involved in transcription control such as an operator). An array, etc.).
  • Examples of one embodiment of the DNA construct include, but are not limited to, a pVTS007 plasmid vector, a pTS108 plasmid vector, a pTS110 plasmid vector, and a pTS119 plasmid vector described in Examples described later.
  • an appropriate medium is used according to the host microorganism and marker gene to be used.
  • marker gene for example, when Pseudomonas putida is used as the host microorganism and resistance genes of kanamycin, gentamicin and tetracycline are used as the marker genes, selection and growth of the transformed microorganism can be performed by, for example, transforming the transformed microorganism into an LB medium containing these agents. It can be carried out by culturing in a.
  • Confirmation that a transformed microorganism has been produced can be achieved by, for example, transforming the transformed microorganism under conditions where only the transformed microorganism lacking the gene can survive or under conditions where only the transformed microorganism expressing the inserted gene can survive. It can be achieved by culturing and the like. In addition, by culturing the transformed microorganism and then confirming that the amount of muconic acid in the culture obtained after culturing is larger than the amount of muconic acid in the culture of the host microorganism cultured under the same conditions, etc. It can be confirmed that a transformed microorganism has been produced.
  • Confirmation that a transformed microorganism has been produced can be obtained by extracting chromosomal DNA from the transformed microorganism, performing PCR using this as a template, and generating a PCR product that can be amplified when transformation occurs. You may carry out by confirming a characteristic, a base sequence, etc.
  • PCR is performed with a combination of a forward primer for the promoter base sequence of the gene to be deleted or inserted and a reverse primer for the base sequence of the marker gene to confirm that a product of the expected length is generated.
  • the host microorganism is not particularly limited as long as it is a Pseudomonas microorganism having a pcaH gene, a pcaG gene, a catA gene and a catB gene on the chromosome, and preferably a pcaH gene, a pcaG gene, a catA gene, a catB gene, a pobA gene on the chromosome Pseudomonas ptida, Pseudomonas fluorescens, Pseudomonas aeruginosa and the like, having these genes, and more preferably having these genes and having protocatechuic acid to mucon Pseudomonas putida that can produce acid.
  • genes to be deleted or inserted Specific examples of the pcaH gene, the pcaG gene, the catA gene, the catB gene, the pobA gene, the vanA gene, and the vanB gene include the pcaH gene, pcaG gene, catA gene possessed by Pseudomonas putida KT2440, a catB gene, a pobA gene, a vanA gene and a vanB gene, and the base sequences thereof are those described in SEQ ID NOs: 29 to 32 and 35 to 37, Similarly, a specific example of the aroY gene is the aroY gene possessed by the Klebsiella pneumoniae subspecies pneumoniae A170-40 strain, and the nucleotide sequence thereof is that shown in SEQ ID NO: 33.
  • kpdB gene A specific example of the kpdB gene is the kpdB gene possessed by Klebsiella pneumoniae, subspices pneumoniae NBRC14940, and the nucleotide sequence is that set forth in SEQ ID NO: 34.
  • 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.
  • a method for obtaining a gene to be deleted or inserted from a microorganism other than Pseudomonas putida or Klebsiella pneumoniae is not particularly limited.
  • the pcaH gene, pcaG gene, catA gene, catB gene, and pobA gene possessed by Pseudomonas putida KT2440 Based on the base sequence of the aroY gene (SEQ ID NO: 33) possessed by Klebsiella pneumoniae sub-species pneumoniae A170-40 strain, It can be obtained by performing a BLAST homology search on the genomic DNA of other microorganisms and specifying a gene having a base sequence having a high sequence identity with the above base sequence.
  • a protein having an amino acid sequence having a high sequence identity with the amino acid sequence of the protein expressed by the gene (SEQ ID NOs: 25 to 28 and 38 to 42) is identified, It can be obtained by specifying the gene to be expressed.
  • the reason why the obtained gene corresponds to a gene to be deleted or inserted is that the derived organism is transformed as a host microorganism by the obtained gene, and the production amount of muconic acid is enhanced compared to the host microorganism. I can confirm.
  • Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas aeruginosa have similar growth conditions, there is a probability that they can be transformed into each other by inserting their respective genes.
  • a gene obtained from Pseudomonas putida can be transformed by introducing it into Pseudomonas fluorescens or Pseudomonas aeruginosa as a host microorganism.
  • a gene obtained from Pseudomonas putida can be introduced into a Klebsiella microorganism, Enterobacter microorganism, Escherichia coli or the like as a host microorganism for expression.
  • the gene to be inserted may be a gene whose codon, secondary structure, GC content and the like are optimized for expression in the host microorganism.
  • a specific embodiment of the transformed microorganism is a Pseudomonas microorganism selected from the group consisting of Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas aeruginosa, wherein the pcaH gene, pcaG gene and catB gene are present on the chromosome.
  • a transformed Pseudomonas microorganism that expresses the inserted pcaH and pcaG genes and expresses the inserted aroY gene.
  • Another specific embodiment of the transformed microorganism expresses at least one gene selected from the group consisting of inserted pobA gene, catA gene, vanA gene and vanB gene in the transformed Pseudomonas microorganism.
  • a transformed Pseudomonas microorganism expresses at least one gene selected from the group consisting of inserted pobA gene, catA gene, vanA gene and vanB gene in the transformed Pseudomonas microorganism.
  • the pcaH gene, pcaG gene and catB gene on the chromosome are deleted, the inserted pcaH gene and pcaG gene are expressed, and the inserted aroY gene is expressed.
  • growth and production of muconic acid using lignin-derived aromatic compounds such as vanillic acid and p-hydroxybenzoic acid as a sole carbon source which is impossible with the transformed microorganism described in Non-Patent Document 1. Is possible.
  • a preferred embodiment of the transformed microorganism is a transformed microorganism that overexpresses the inserted catA gene, vanA gene, and vanB gene. It is.
  • transformed microorganism of one embodiment of the present invention include IDPC / pTS110 strain and IDPC / pTS119 strain described in Examples described later, but are not limited thereto.
  • the method for producing muconic acid of one embodiment of the present invention includes a step of obtaining muconic acid by causing an aromatic compound derived from lignin such as vanillic acid or p-hydroxybenzoic acid to act on the transformed microorganism of one embodiment of the present invention. At least.
  • the method of allowing the lignin-derived aromatic compound to act on the transformed microorganism is particularly limited as long as the lignin-derived aromatic compound and the transformed microorganism are brought into contact with each other and muconic acid can be produced by the enzyme of the transformed microorganism.
  • the culture method is not particularly limited, and examples thereof include a solid culture method and a liquid culture method performed under aerated conditions.
  • any of a synthetic medium and a natural medium can be used as long as it contains a normal medium for culturing host microorganisms, that is, a carbon source, a nitrogen source, an inorganic substance, and other nutrients in an appropriate ratio. Since the host microorganism is a microorganism belonging to the genus Pseudomonas, an MM medium or the like as described in Examples described later can be used, but is not particularly limited.
  • the carbon source an aromatic compound derived from lignin, another carbon source such as sugar or organic acid, or a combination thereof can be used.
  • the medium component preferably contains a component necessary for activation of an 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 be added as mineral-containing materials.
  • 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.
  • p-hydroxyphenyl lignin and Examples include compounds corresponding to degradation products of acyl lignin, and specific examples include p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, vanillin, p-hydroxybenzaldehyde and the like.
  • catechol from phenol, benzoic acid, guaiacol, and the like.
  • Aromatic compounds derived from lignin include compounds that are modeled on lignin, such as guaiacylglycerol- ⁇ -guaiacyl ether.
  • the lignin-derived aromatic compound is preferably a biomass containing lignin or one obtained by subjecting the biomass to pretreatment and extracted, but may be chemically synthesized and purified regardless of the biomass. .
  • the lignin-derived aromatic compound those described above can be used alone or in combination of two or more.
  • the biomass containing lignin (hereinafter sometimes referred to as lignocellulose) is not particularly limited, and examples thereof include natural products such as grass and trees, those obtained by processing these natural products, and agricultural waste. Specific examples include woody biomass such as hardwoods and conifers. For example, it is known that conifers contain a large amount of p-hydroxyphenyl lignin and guaiacyl lignin.
  • Lignocellulose can be, for example, in the form of a solid, suspension, or liquid depending on the presence or absence of pretreatment.
  • a suspension obtained by adding pulverized lignocellulose to a liquid can be used.
  • the lignocellulose may be a lignin extract.
  • a powdered lignocellulose is lignin so that it becomes 0.1% W / V to 50% W / V, preferably 1% W / V to 20% W / V.
  • the lignin extract is obtained by subjecting the suspension to 10 ° C. to 150 ° C., preferably 20 ° C. to 130 ° C., more preferably 20 ° C. to 80 ° C., for several hours to several days, preferably 1 hour to 6 days.
  • a solid lignin extract obtained by subjecting to an extraction treatment and then removing the solid from the liquid lignin extract obtained by removing solids from the extraction treatment liquid and evaporating the solvent to dryness. It may be.
  • a lignin extract is not specifically limited, For example, the following methods etc. are mentioned. That is, 2M NaOH 50 mL, defatted cedar wood powder 1.5 g, and nitrobenzene 3 mL were placed in a stainless steel vessel of a small autoclave apparatus (Pressure Glass Industrial Co., Ltd., portable reactor TVS-1) and stirred at 500 rpm for 2.5 at 170 ° C. Processing time. Cool to 60 ° C. or lower, and collect the supernatant by centrifugation (6,000 g, 10 min). The obtained supernatant is subjected to diethyl ether extraction three times (recovering the aqueous layer).
  • Solvents suitable for extraction and treatment of lignin are not particularly limited, and examples thereof include water, low molecular alcohols such as dioxane, methanol, and isopropanol, diethyl ether, and dimethylformamide.
  • Culture conditions for Pseudomonas microorganisms commonly known by those skilled in the art may be employed.
  • the initial pH of the medium is adjusted to 5 to 10
  • the culture temperature is 20 ° C. to 40 ° C.
  • the culture time is several hours. It can be appropriately set, for example, several days, preferably 1 to 7 days, more preferably 2 to 5 days.
  • the culture means is not particularly limited, and aeration and agitation deep culture, shaking culture, static culture, etc. can be adopted, but it is preferable to culture under conditions such that the dissolved oxygen concentration is sufficient by aeration.
  • the culture is performed under such a condition that the dissolved oxygen concentration is 0.1% to 15%, more preferably the culture is performed under such a condition that the dissolved oxygen concentration is 1% to 13%. It is even more preferable that the culture is performed under such conditions that 2% to 10%.
  • an MM culture medium containing vanillic acid, p-hydroxybenzoic acid and / or an aqueous solution of an aromatic compound derived from cedarignin as a carbon source was used. Examples include shaking culture and stirring culture for 1 to 5 days at 30 ° C., 180 rpm, and dissolved oxygen concentration of 5% to 10%.
  • the carbon source and other components can be added as appropriate after the start of culture.
  • the method for obtaining muconic acid from the culture after completion of the culture is not particularly limited. Since muconic acid accumulates in the culture solution, the cells and the culture supernatant are separated from the culture by normal solid-liquid separation operations 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 can dissolve 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.
  • extraction temperature is not specifically limited, For example, it can set from room temperature to 100 degreeC.
  • the method for extracting muconic acid include, for example, the method of Vardon et al. (Green chemistry, vol. 18, p3397-3413, 2016; the entire description of the document is incorporated herein by reference) and the like.
  • the method may be partially changed. Specifically, activated carbon (12.5% (w / v), 100 mesh) is added to the culture supernatant and stirred for 1 hour. The activated carbon is removed by suction filtration, and the filtrate is recovered. Hydrochloric acid is added to the collected filtrate to adjust the pH to 2, and the mixture is allowed to stand overnight at 4 ° C.
  • the precipitate is collected by suction filtration, and the precipitate is washed with ion exchange water, then collected by suction filtration, and dried under reduced pressure. Suspend the dried solid in ethanol, remove unnecessary substances by suction filtration, and collect the filtrate. The filtrate is dried under reduced pressure using an evaporator to obtain purified muconic acid.
  • the qualitative or quantitative analysis of muconic acid is not particularly limited, and can be performed by, for example, HPLC.
  • HPLC HPLC separation conditions
  • the HPLC separation conditions can be performed under the conditions described in the examples described later.
  • muconic acid can be obtained in high yield.
  • muconic acid when 50 mM vanillic acid is used as a carbon source, muconic acid can be obtained in a yield of 2.7 wt% in 48 hours of culture; when 50 mM p-hydroxybenzoic acid is used as a carbon source, it is cultured for 56 hours.
  • 8.9 to 24.2 mg / L of muconic acid can be obtained after 48 hours of culture.
  • the muconic acid obtained using the transformed microorganism and the production method of one embodiment of the present invention can be converted into various industrially useful compounds, such as surfactants, flame retardants, and UV light stabilization. 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 muconic acid derivatives, is actually used as nylon 66 (one of polyamides).
  • P. Preparation of putida IDPC strain A mutant strain in which protocatechuic acid • 3,4-dioxygenase gene (pcaHG gene) and cis, cis-muconic acid • cycloisomerase gene (catB gene) are disrupted from Pseudomonas putida by the following procedure. P. A putida IDPC strain was produced.
  • a protocatechuate, 3,4-dioxygenase, ⁇ subunit was obtained by PCR using the genomic DNA of 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 region of dioxygenase beta subunit (pcaH) gene was amplified.
  • the amplified DNA fragment is digested with EcoRI and SacI and pEX18Amp plasmid DNA previously digested with EcoRI and SacI (see Gene, vol. 212, p77-86, 1998; the entire description of which is incorporated herein by reference) PVTS003 plasmid DNA was obtained.
  • the protocatechuate, 3,4-dioxygenase, ⁇ subunit was obtained by PCR using the genomic DNA of putida KT2440 strain as a template and a primer set consisting of primers 3 and 4 of SEQ ID NOs: 3 and 4.
  • the DNA fragment of about 1.2 kbp downstream of the diogenase alpha subunit (pcaG) gene was amplified.
  • the amplified DNA fragment was digested with SacI and BamHI and ligated with pVTS003 plasmid DNA previously digested with SacI and BamHI to obtain pVTS004 plasmid DNA.
  • the transformant was selected as a Nal-Gm resistant strain capable of growing on an 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.
  • Part of the culture solution is inoculated into an LB liquid medium containing fresh Nal 25 mg / L, Gm 50 mg / L, and 10% (w / v) sucrose, and further subjected to shaking culture at 30 ° C. for 16 hours. Repeated. Subsequently, the obtained culture solution was smeared on an LB agar medium containing 25 mg / L of Nal and 50 mg / L of Gm, and statically cultured at 30 ° C. overnight. Genomic DNA was extracted from each of the grown colonies, and a DNA fragment of about 1.8 kbp containing the Gm resistance gene was inserted into the pcaG locus and pcaH locus on the genomic DNA by PCR and Southern hybridization. Transformants were screened as protocatechuic acid • 3,4-dioxygenase gene disruption strains.
  • cis-muconic acid / cycloisomerase (catB) gene is obtained by PCR using a putida PpY1100 strain genomic DNA as a template and a primer set consisting of primers 5 and 6 of SEQ ID NOs: 5 and 6.
  • a pK19mobsacB plasmid DNA amplified from a 1.0 kbp DNA fragment and previously digested with PstI and XbaI see Gene, Vol. 145, p69-73, 1994; the entire description of this document is incorporated herein by reference).
  • pUC4K plasmid DNA (see Gene, vol. 19, p259-268, 1982; the entire description of this document is incorporated herein by reference) is digested with SalI and contains about 1. the kanamycin (Km) resistance gene.
  • the 2 kbp DNA fragment was ligated with pTS073 plasmid DNA previously digested with SalI to obtain pTS076 plasmid DNA.
  • the pTS076 plasmid DNA was used to transform a protocatechuic acid / 3,4-dioxygenase gene disruption strain.
  • the transformant was selected as a Nal-Gm-Km resistant strain capable of growing on an LB agar medium containing 25 mg / L of Nal, 50 mg / L of Gm, and 25 mg / L of Km.
  • 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 at 30 ° C. for 16 hours. Cultured with shaking.
  • a part of the culture solution is inoculated into 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, followed by stationary culture 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 the PCR method and the Southern hybridization method.
  • the transformant obtained by screening was a mutant strain in which both the protocatechuic acid • 3,4-dioxygenase gene and the cis, cis-muconic acid • cycloisomerase gene were disrupted .
  • Putida IDPC strain was named.
  • a DNA fragment of about 200 bp containing a lactose promoter region (Plac) was obtained by PCR using pUC118 plasmid DNA as a template and a primer set consisting of primers 7 and 8 of SEQ ID NOs: 7 and 8.
  • the obtained DNA fragment was cloned into the NotI site of pJB866 plasmid DNA by In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain pTS093 plasmid DNA.
  • a DNA fragment of about 1.3 kbp including the pcaG gene and the pcaH gene was amplified by PCR using the putida KT2440 strain genomic DNA as a template and a primer set consisting of primers 9 and 10 of SEQ ID NOs: 9 and 10.
  • the amplified DNA fragment was digested with SacI and BamHI and cloned into pUC118 plasmid DNA previously digested with SacI and BamHI to obtain pTS107 plasmid DNA.
  • a 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 pTS107 plasmid DNA with SacI and BamHI to the SacI-BamHI site of the pTS093 plasmid DNA. .
  • the IDPC / pTS108 strain was produced by transforming the putida IDPC strain.
  • the obtained DNA fragment was digested with KpnI and cloned into pMCL200 plasmid DNA previously digested with KpnI to obtain pTS036 plasmid DNA.
  • a DNA fragment of about 1.5 kbp containing the aroY gene was obtained by PCR using pTS036 plasmid DNA as a template and a primer set consisting of primers 13 and 14 of SEQ ID NOs: 13 and 14. The obtained DNA fragment was cloned into the NotI site of pTS093 plasmid DNA by In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain pTS109 plasmid DNA.
  • a pTS110 plasmid DNA was obtained by ligating a DNA fragment of about 1.3 kbp obtained by digesting pTS107 plasmid DNA with SacI and HindIII to the SacI-HindIII site of pTS109 plasmid DNA.
  • the IDPC / pTS110 strain was produced by transforming the putida IDPC strain.
  • MM liquid medium Na 2 HPO 4 13.56 g / L, Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L and 25 mM vanillic acid (VA), KH 2 PO 4 6 g / L, NaCl 1 g / L, NH 4 Cl 2 g / L, 2 mM MgSO 4 , 100 ⁇ M CaCl 2 and 18 ⁇ M FeSO 4 ) 5 mL were inoculated and cultured at 30 ° C. with shaking.
  • MM liquid medium Na 2 HPO 4 13.56 g / L, Nal 25 mg / L, Km 25 mg / L, Gm 50 mg / L, Tc 20 mg / L and 25 mM vanillic acid (VA), KH 2 PO 4 6 g / L, NaCl 1 g / L, NH 4 Cl 2 g / L, 2 mM MgSO 4 , 100 ⁇ M CaCl
  • the optical density (OD) of the culture solution was measured at regular intervals, and the concentrations of VA and ccMA were measured for the culture supernatant obtained by centrifuging the culture solution.
  • the wavelength of 600 nm was used for the OD measurement, and the OD600 value was measured using a miniphoto 518R (Tytec Corporation).
  • the concentrations of VA and ccMA were measured using a high performance liquid chromatograph (Agilent 1200 series; Agilent Technology Co., Ltd.).
  • the column used was ZORBAX Eclipse Plus C18 column (diameter 4.6 mm, length 150 mm, particle size 0.5 ⁇ m) and kept at 40 ° C.
  • Table 1a summarizes the measurement results of OD600 value, VA concentration and ccMA concentration after 0, 48, 56 and 72 hours of culture time for the IDPC / pTS108 strain.
  • Table 1b shows the summary of measurement results of OD600 value, VA concentration and ccMA concentration after 0, 24, 32 and 48 hours of culture time for the IDPC / pTS110 strain.
  • the IDPC / pTS108 strain grew using VA but did not produce ccMA, whereas the IDPC / pTS110 strain grew using VA and ccMA Was produced over time (yield 1.4 wt%).
  • the metabolic rate and specific growth rate of VA of the IDPC / pTS110 strain were very large compared to the IDPC / pTS108 strain.
  • the yield was determined by the amount of ccMA relative to the amount of substrate (VA) consumed.
  • IDPC / pTS110 strain was cultured in the same manner as described 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 OD600 values, VA concentrations, and ccMA concentrations after 0, 24, 32, and 48 hours of culture time.
  • the optical density (OD600) of the culture solution and the HBA and ccMA concentrations in the culture solution were measured at regular intervals. OD600 measurement and HBA and ccMA concentration measurements were performed in the same manner as described in 3 above.
  • Table 2a shows a summary of the OD600 values, HBA concentrations, and ccMA concentrations measured after culture times 0, 24, 48, and 56 hours for the IDPC / pTS108 strain.
  • Table 2b shows the summary of measurement results of OD600 value, HBA concentration and ccMA concentration after 0, 24, 32 and 48 hours of culture time for the IDPC / pTS110 strain.
  • IDPC / pTS108 strain grew using HBA but did not produce ccMA
  • IDPC / pTS110 strain grew using HBA and ccMA was produced over time (yield 4.1 wt%).
  • the metabolic rate and specific growth rate of HBA of the IDPC / pTS110 strain were very large compared to the IDPC / pTS108 strain.
  • the yield was determined by the amount of ccMA relative to the amount of substrate (HBA) consumed.
  • IDPC / pTS110 strain was cultured in the same manner as described 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 OD600 values, HBA concentrations and ccMA concentrations after 0, 24, 32, 48 and 56 hours of culture time.
  • the optical density (OD600) of the culture solution and the VA, HBA, and ccMA concentrations in the culture solution were measured at regular intervals. OD600 measurement and VA, HBA, and ccMA concentration measurements were performed in the same manner as described in 3.
  • Table 3 summarizes the measurement results of the OD600 value, VA concentration, HBA concentration and ccMA concentration after 0, 24, 32 and 48 hours of culture time.
  • 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 consumed substrate (VA and HBA).
  • cedar wood powder was subjected to an alcohol-benzene extraction treatment, and then 1.5 g of the treated cedar wood flour was subjected to an alkali nitrobenzene oxidative decomposition treatment and a diethyl ether extraction treatment (wood science experiment manual, edited by the Wood Society of Japan).
  • 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 ⁇ L of the obtained culture broth 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 cedarignin-derived phenol aqueous solution was added as a carbon source And cultured at 30 ° C. with shaking. 24 hours after the start of the culture, 0.5 mL of a cedarignin-derived aromatic compound aqueous solution was further added, followed by shaking culture at 30 ° C.
  • 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 performed in the same manner as described in 3 above.
  • Table 4 summarizes the measurement results of OD600 values and ccMA concentrations after 0, 24, and 48 hours of culture time.
  • the catechol 1,2-dioxygenase (catA) gene was obtained by PCR using the genomic DNA of putida KT2440 strain as a template and a primer set consisting of primers 15 and 16 of SEQ ID NOs: 15 and 16. A DNA fragment of about 1 kbp containing 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.
  • the pNI001 plasmid DNA was used as a template to amplify by PCR using a primer set consisting of primers 17 and 18 of SEQ ID NOs: 17 and 18, thereby obtaining 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 Infusion cloning HD kit to obtain pTS115 plasmid DNA.
  • a vanillate demethylase oxygenase component VA
  • a DNA fragment of about 2.0 kbp containing the gene and the vanillate demethylase oxidoreductase 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 pQE30 plasmid DNA to obtain pKY001 plasmid DNA.
  • telomere sequence was obtained by PCR using a primer set consisting of primers 21 and 22 of SEQ ID NOs: 21 and 22, thereby obtaining a DNA fragment of about 2.0 kbp containing the vanA gene and vanB gene.
  • the obtained DNA fragment was ligated using pTS115 plasmid DNA previously digested with NotI and Infusion HD Cloning Kit to obtain pTS116 plasmid DNA.
  • the pTS107 plasmid DNA was used as a template to amplify 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 using pTS116 plasmid DNA previously digested with HindIII and Infusion HD cloning kit to obtain pTS119 plasmid DNA.
  • the IDPC / pTS119 strain was produced by transforming the putida IDPC strain.
  • 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 performed in the same manner as described in 3 above.
  • Table 5 summarizes the results of measurement of OD600 values and ccMA concentrations after 0, 24 and 48 hours of culture time.
  • the IDPC / pTS119 strain grew using lignin-derived aromatic compounds obtained from real biomass and produced ccMA over time.
  • the VA, HBA and ccMA concentrations after the culture 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 from the amount of ccMA relative to the amount of consumed substrates (VA and HBA). Table 6 summarizes the yield of ccMA, the air flow rate, and the DO value.
  • the yield of ccMA produced was good under the condition that the dissolved oxygen concentration in the culture broth was 5 to 10%, although there was almost no influence by the aeration rate.
  • the yield of ccMA produced was about 30% under conditions where the dissolved oxygen concentration in the culture solution was 2.5% and 5.0%.
  • muconic acid is obtained from a biomass containing an aromatic compound derived from lignin or lignin.
  • Muconic acid can be converted into various industrially useful compounds, for example, raw materials for muconic acid derivatives that have applications such as surfactants, flame retardants, UV light stabilizers, thermosetting plastics, coating agents, etc. Can be used as

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Abstract

L'objet de la présente invention est de fournir : un micro-organisme qui permet la production d'acide muconique avec une bonne efficacité économique et un rendement élevé ; et un procédé de production d'acide muconique faisant appel au micro-organisme. L'objet peut être atteint au moyen : d'un micro-organisme transgénique dont un micro-organisme hôte est un micro-organisme appartenant au genre Pseudomonas et qui possède un gène pcaH, un gène pcaG, un gène catA et un gène catB sur son chromosome et dans lequel le gène pcaH, le gène pcaG et le gène catB sont supprimés sur le chromosome, et le gène pcaH inséré et le gène pcaG inséré peuvent être exprimés et le gène aroY inséré est également exprimé ; d'un procédé de production d'acide muconique faisant appel au micro-organisme transgénique ; et autres.
PCT/JP2018/016674 2017-04-25 2018-04-24 Micro-organisme transgénique permettant de produire de l'acide muconique et son utilisation WO2018199111A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2012106257A1 (fr) * 2011-01-31 2012-08-09 Los Alamos National Security, Llc Production de composés intéressants au plan industriel dans des organismes prokaryotes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012106257A1 (fr) * 2011-01-31 2012-08-09 Los Alamos National Security, Llc Production de composés intéressants au plan industriel dans des organismes prokaryotes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JOHNSON, C. W. ET AL.: "Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin", METABOLIC ENGINEERING, vol. 28, pages 240 - 247, XP055537233 *
JOHNSON, C. W. ET AL.: "Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity", METABOLIC ENGINEERING COMMUNICATIONS, vol. 3, 2016, pages 111 - 119, XP055537227 *
JUNG, HM. ET AL.: "Metabolic engineering of Klebsiella pneumoniae for the production of cis, cis-muconic acid", APPL MICROBIOL BIOTECHNOL, vol. 99, 2015, pages 5217 - 5225, XP002780985 *
KIKUCHI, AKIHIRO ET AL.: "Production of muconic acid using lignin-derived phenols as raw materials", LECTURE ABSTRACTS OF THE CONFERENCE OF JAPAN SOCIETY OF BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, 2016 *
SONOKI, T. ET AL.: "Glucose-Free cis, cis-Muconic Acid Production via New Metabolic Design Corresponding to the Heterogeneity of lignin", ACS SUSTAINABLE CHEM. ENG., vol. 6, no. 1, 4 December 2017 (2017-12-04), pages 1256 - 1264, XP055537236 *
SONOKI, TOMONORI ET AL.: "Muconic acid production from lignin without glucose", BIOSCIENCE & INDUSTRY, vol. 76, 2018, pages 139 - 141 *

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