Vanillic acid-producing transformed microorganisms and their utilization

Abstract

The purpose of the present invention is to provide a microorganism which can selectively produce and / or accumulate vanillic acid while growing with use of not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds as carbon sources, and a method for using the microorganism to produce vanillic acid. The purpose is achieved by, for example: a transformed microorganism, the host microorganism of which is Pseudomonas species (Pseudomonas sp.) NGC7 strain (accession number: NITE BP-03043), and in which a vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and a vanB4 gene (SEQ ID NO: 2) on the chromosome are deleted; and a method for using the transformed microorganism to produce vanillic acid.

Description

[Technical Field]

[0001] The present invention relates to a transformed microorganism capable of producing vanillic acid and a method for producing vanillic acid using the transformed microorganism. In particular, the present invention relates to p - This invention relates to a transformed microorganism capable of selectively producing vanillic acid from a mixture of aromatic compounds derived from hydroxyphenyl lignin, guaiacyl lignin, and syringyl lignin. [Background technology]

[0002] In order to achieve the United Nations-led Sustainable Development Goals (SDGs) through international cooperation, and to realize Japan's 2050 target of a carbon-neutral society, there is a need to produce functional materials from non-edible biomass instead of petroleum raw materials. In this context, the development of cellulose derivatives and aliphatic polymer raw materials using non-edible biomass is progressing. However, to date, very few polymers that meet the required rigidity, heat resistance, and other properties are known.

[0003] Lignin is a type of non-edible biomass. Lignin is an amorphous polymer that exists as a component of the vascular cell walls of plants. It is a complex condensation of phenylpropane-based units, and its key chemical characteristic is the presence of methoxy groups. Lignin functions to bind lignified plant cells together, strengthening the tissue. It is present in wood at approximately 18%-36% and in herbaceous plants at approximately 15%-25%. Therefore, attempts are being made to decompose lignin and obtain useful compounds in order to effectively utilize wood. Lignin derived from biomass such as wood contains: p It is known that there are three types of lignin: hydroxyphenyl lignin (H-type lignin), guaiacyl lignin (G-type lignin), and syringyl lignin (S-type lignin).

[0004] Some compounds obtained by decomposing lignin can be used as monomers containing aromatic rings within their molecules. Polymers made from such aromatic monomers can exhibit excellent functionality, such as rigidity and heat resistance.

[0005] Vanillic acid is one of the candidate aromatic monomers derived from lignin. Polymers obtained by polymerizing vanillic acid have the characteristic of having a very high melting point. For example, there are reports that high-performance polymers obtained using monomers obtained by modifying vanillic acid as raw materials have high melting points and high functionality (see, for example, Patent Documents 1 and 2 below).

[0006] To date, Sphingobium species have been identified as a microorganism that decomposes lignin or lignin-derived aromatic compounds. Sphingobium The sp.)SYK-6 strain (hereinafter also referred to as the SYK-6 strain) is known. The SYK-6 strain can degrade vanillic acid derived from G-type lignin and syringic acid derived from S-type lignin, and in the presence of methionine, it can degrade vanillic acid derived from H-type lignin. p -Hydroxybenzoic acid can also be broken down. From strain SYK-6. ligM Transformed microorganisms that have been transformed to delete a gene (SYK-6Δ ligM (Co., Ltd.) or SYK-6 Co., Ltd. ligM Genes and desA Transformed microorganisms that have been transformed to delete a gene (SYK-6Δ ligM Δ desA It has been reported that the company (Co., Ltd.) lacks some or all of the degradability of vanillic acid (see, for example, Non-Patent Document 1 below).

[0007] Furthermore, the present inventors have identified lignin-derived aromatic compounds. p - Pseudomonas species that can break down hydroxybenzoic acid, vanillic acid and syringic acid ( Pseudomonas We have successfully isolated the NGC7 strain (deposit number: NITE BP-03043; hereinafter also referred to as the NGC7 strain) and transformed the NGC7 strain to obtain muconic acid from the above-mentioned lignin-derived aromatic compound (see, for example, Patent Document 3 below).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non - Patent Documents

[0009]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] The SYK - 6Δ strain described in Non - Patent Document 1 ligM Δ desA Although the strain does not decompose vanillic acid, it has also lost the ability to decompose syringic acid, which is an analog of vanillic acid, and cannot grow using aromatic compounds derived from H - type lignin as a carbon source in the absence of methionine. Therefore, when attempting to produce vanillic acid using the SYK - 6Δ ligM Δ desA strain, there is a problem that broad - leaf trees containing a large amount of S - type lignin cannot be utilized, and the types of biomass that can be used are limited.

[0011] The SYK - 6Δ strain described in Non - Patent Document 1 ligM strain decomposes syringic acid, but it also decomposes vanillic acid, so vanillic acid cannot be accumulated. Therefore, there is a problem that vanillic acid cannot be selectively produced even when using the SYK - 6Δ ligM strain.

[0012] Furthermore, as stated above, none of the microorganisms described in Non-Patent Document 1 can specifically degrade only vanillic acid or its analog, syringic acid.

[0013] On the other hand, the NGC7 strain and the NGC7 transformed strain described in Patent Document 3 degrade vanillic acid and therefore cannot accumulate vanillic acid. Furthermore, according to the inventors' investigation, in the NGC7 strain, the SYK-6 strain ligM Genes and desA The genes corresponding to these genes have not been found through previous genome analyses.

[0014] If NGC7 strain is SYK-6 strain ligM Genes and desA It possesses genes corresponding to genes, and even if those are destroyed, SYK-6Δ as described in Non-Patent Document 1 ligM Δ desA It may not be able to decompose syringic acid like the strain and therefore cannot be propagated as a carbon source, or it may be SYK-6Δ as described in Non-Patent Document 1. ligM Like a strain, it may propagate using syringic acid as a carbon source, but it also has the potential to decompose vanillic acid.

[0015] As described above, there are virtually no known microbial strains that decompose either vanillic acid or syringic acid and utilize them as a carbon source, while accumulating the other without decomposing it. In other words, there are virtually no known microorganisms that decompose not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds and utilize them as a carbon source, while accumulating vanillic acid without decomposing it. As a result, when using biomass derived from broadleaf trees containing S-type lignin-derived aromatic compounds as raw material, conventional microorganisms lead to the accumulation of aromatic compounds other than vanillic acid in the culture medium, complicating the vanillic acid purification process. Therefore, to date, it has not been possible to selectively produce vanillic acid from a wide range of biomass lignin-derived aromatic compounds using microorganisms.

[0016] Therefore, the problem that the present invention aims to solve is to provide a microorganism capable of selectively producing and / or selectively accumulating vanillic acid while decomposing not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds and utilizing them as a carbon source, and a method for producing vanillic acid using this microorganism. [Means for solving the problem]

[0017] To solve the above problems, the inventors diligently investigated microorganisms that grow using S-type lignin-derived aromatic compounds as a carbon source while exhibiting reduced or absent vanillic acid degradability. Among the many gene groups involved in the degradation of vanillic acid, the inventors identified the oxygenation-type (oxygenase-type) vanillate O -This led to focusing on demethylase. Vanillate O - Demethylase is vanillate O - Demethylase oxygenase component (VanA) and vanillate O - Consists of the demethylase oxidoreductase component (VanB).

[0018] Therefore, the inventors of this invention have determined that strain NGC7 is Pseudomonas ptyida ( P.putida ) is a closely related species, P.putida Codes VanA and VanB derived from strain KT2440 vanA Genes and ​ We attempted to search for genes corresponding to specific genes from the genome sequence of NGC7 strain.

[0019] As a result, unexpectedly, NGC7 strain, ​ It has four types of genes that correspond to genes, and further ​ We discovered that it possesses six types of genes, which correspond to genes. ​ Genes and ​ Microorganisms that possess multiple types of each gene are virtually unknown to date.

[0020] The inventors of the present invention have developed multiple types ​ Genes and multiple types​ Regarding genes, we experimented with different sets of paired genes, resulting in four pairs. ​ Genes and ​ We then selected a set of genes. When we manufactured NGC7 transformants lacking each of these four gene sets, we found that some transformants could degrade syringic acid and grow, while others had poor assimilation of syringic acid; some transformants could degrade vanillic acid, while others hardly degraded it at all.

[0021] Surprisingly, a certain ​ Genes and ​ NGC7 transformants lacking a specific gene set proliferated by degrading syringic acid at a level equal to or greater than that of wild-type NGC7, while hardly degrading vanillic acid.

[0022] Therefore, we found that by using the NGC7 transformed strain, it is possible to produce vanillic acid from aromatic compounds derived from G-type lignin while simultaneously propagating using aromatic compounds derived from S-type lignin. Furthermore, for example, by incorporating a set of genes that enable the conversion of acetovanylon to vanillic acid into the NGC7 transformed strain, we were also able to produce vanillic acid from acetovanylon, a major aromatic compound contained in Kraft pulp and soda pulp, which are currently used as industrial-level lignin degradation methods.

[0023] Based on the above findings, the inventors have finally succeeded in creating a microorganism capable of selectively producing and / or accumulating vanillic acid while decomposing compounds other than G-type lignin-derived aromatic compounds, such as H-type lignin-derived aromatic compounds and S-type lignin-derived aromatic compounds, and growing as a carbon source, as well as a method for producing vanillic acid using this microorganism, which solves the problems of the present invention. The present invention is completed based on the first findings and success stories obtained by these inventors.

[0024] Accordingly, the present invention provides each of the following embodiments. [1] Host microorganisms Pseudomonas species ( ​ sp.) NGC7 strain (Accession number: NITE BP-03043), Located on the chromosome, ​ gene (SEQ ID NO: 1) or the ​ Genes and ​ The gene (Sequence ID 2) is deleted. Transformed microorganisms. [2] Host microorganisms Pseudomonas species ( ​ sp.) NGC7 strain (Accession number: NITE BP-03043), Located on the chromosome ​ gene (SEQ ID NO: 1) or the ​ Genes and ​ The gene (Sequence ID 2) is deleted, and, inserted ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ Genes and ​ Expressing genes Transformed microorganisms. [3] A step of obtaining vanillic acid by reacting an aromatic compound derived from guaiacyl lignin with a transformed microorganism described in [1] or [2]. A method for producing vanillic acid, including the method described above. [4] Aromatic compounds derived from guaiacylignin, p - A step of obtaining vanillic acid by reacting a mixture of an aromatic compound derived from hydroxyphenyl lignin and / or an aromatic compound derived from syringyl lignin with a transformed microorganism described in [1] or [2]. A method for producing vanillic acid, including the method described above. [5] A step to obtain vanillic acid by reacting a mixture of aromatic compounds derived from guaiasyllignin, including acetvanylone, with the transformed microorganism described in [2]. A method for producing vanillic acid, including the method described above. [Effects of the Invention]

[0025] According to the present invention, it is possible to selectively produce and / or accumulate vanillic acid from G-type lignin-derived aromatic compounds microbiologically, while also utilizing H-type lignin-derived aromatic compounds and / or S-type lignin-derived aromatic compounds as a carbon source. According to the present invention, since lignin degradation products derived from either H-type lignin or S-type lignin can be used as a carbon source, it is possible to use lignin degradation products as a carbon source without using carbon sources other than lignin, such as glucose, and vanillic acid can be produced from G-type lignin-derived aromatic compounds inexpensively and without depending on the type of lignin. Therefore, according to the present invention, the production of vanillic acid on an industrial scale is expected as part of the effective utilization of lignin-containing biomass. [Brief explanation of the drawing]

[0026] ​ Figure 1A is a copy of the certificate of accession for accession number NITE BP-03043 (Pseudomonas species NGC7 strain). ​ Figure 1B is a copy of the certificate of survival for accession number NITE BP-03043 (Pseudomonas species NGC7 strain). ​ Figure 2A shows the results of measuring OD600 over time when NGC7 strain, NGC7ΔvanA1B1 strain, NGC7ΔvanA2B2 strain, NGC7ΔvanA3B3 strain, and NGC7ΔvanA4B4 strain were cultured using vanillic acid (VA) as the carbon source, as described in the examples below. ​ Figure 2B shows the results of measuring OD600 over time when NGC7 strain, NGC7ΔvanA1B1 strain, NGC7ΔvanA2B2 strain, NGC7ΔvanA3B3 strain, and NGC7ΔvanA4B4 strain were cultured using syringic acid (SA) as a carbon source, as described in the examples below. ​Figure 3A shows the results of evaluating the VA degradation capabilities of NGC7 strain, NGC7ΔvanA1B1 strain, NGC7ΔvanA2B2 strain, NGC7ΔvanA3B3 strain, and NGC7ΔvanA4B4 strain, as described in the examples below. ​ Figure 3B shows the results of evaluating the degradation of SA by NGC7 strain, NGC7ΔvanA1B1 strain, NGC7ΔvanA2B2 strain, NGC7ΔvanA3B3 strain, and NGC7ΔvanA4B4 strain, as described in the examples below. ​ Figure 4A shows the results of measuring the SA concentration and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using SA as a carbon source, as described in the examples below. ​ Figure 4B shows the results of measuring the VA concentration and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using VA as a carbon source, as described in the examples below. ​ Figure 4C shows the results of measuring the HBA concentration and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using p-hydroxybenzoic acid (HBA) as a carbon source, as described in the examples below. ​ Figure 4D shows the results of measuring the SA concentration, VA concentration, and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using a mixture of SA and VA as a carbon source, as described in the examples below. ​ Figure 4E shows the results of measuring the SA concentration, HBA concentration, and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using a mixture of SA and HBA as a carbon source, as described in the examples below. ​ Figure 4F shows the results of measuring the VA concentration, HBA concentration, and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using a mixture of VA and HBA as a carbon source, as described in the examples below. ​Figure 4G shows the results of measuring SA concentration, VA concentration, HBA concentration, and OD600 over time when the NGC7ΔvanA4B4 strain was cultured using a mixture of SA, VA, and HBA as a carbon source, as described in the examples below. ​ Figure 5A shows the results of evaluating the degradability of acetovanylone (AV) by the NGC7[pSEVA241_P lac -acv,pTS093_vceA-B] strain, as described in the examples below, and measuring the AV concentration, vanilloyl acetate (VAA) concentration, and VA concentration over time. ​ Figure 5B shows the results of evaluating the degradation of AV by the NGC7ΔvanA4B4[pSEVA241_P lac -acv,pTS093_vceA-B] strain, as described in the examples below, and measuring the AV concentration, VAA concentration, and VA concentration over time. ​ Figure 6 shows the results of evaluating the degradability of a lignin degradation product model using the NGC7ΔvanA4B4[pSEVA241_P lac -acv,pTS093_vceA-B] strain, as described in the examples below, and measuring AV concentration, VN concentration, VA concentration, glucose concentration, and OD600 over time. ​ Figure 7 shows the results of evaluating the degradability of lignin degradation products by the NGC7ΔvanA4B4[pSEVA241_P lac -acv,pTS093_vceA-B] strain, as described in the examples below, and measuring AV concentration, VN concentration, VA concentration, glucose concentration, and OD600 over time. ​ Figure 8 shows the results of evaluating the degradability of lignin degradation products by the NGC7ΔvanA1B1ΔvanA4B4ΔaphΔvanA2B2ΔvanA3B3[pSEVA241_P lac -acv,pTS093_vceA-B] strain, as described in the examples below, and measuring AV concentration, VN concentration, VA concentration, glucose concentration, and OD600 over time. [Modes for carrying out the invention]

[0027] The details of each aspect of the present invention will be described below, but the present invention is not limited to the matters described in this section and can take various forms insofar as it achieves the objective of the present invention.

[0028] In this specification, unless otherwise specified, each term is used in the sense commonly used by those skilled in the art, such as in the field of microbiology, and should not be interpreted as having an unduly restrictive meaning. Furthermore, since the assumptions and theories made herein are based on the inventors' prior knowledge and experience, the present invention is not limited solely to such assumptions and theories.

[0029] "To include" means that elements other than those explicitly included can be added (synonymous with "at least include"), but it also encompasses "consisting of" and "essentially consisting of." In other words, "to include" can mean that it includes the explicitly included elements and any one or more of those elements, consists of the explicitly included elements, or essentially consists of the explicitly included elements. Examples of elements include components, processes, conditions, parameters, and other limitations. The terms "and / or" mean any one of the listed related items, any combination of two or more, or any combination of all of them.

[0030] "Genetic deletion" means that a gene is not functioning properly and its expression is being hindered, such as when a gene is not transcribed correctly or when the protein that should be produced by gene expression is not translated correctly. Genetic deletion can occur, for example, when the structure of a gene is altered by destruction, deletion, substitution, or insertion of all or part of the gene. However, gene deletion can also occur without any change in the structure of the gene, for example, when gene expression is suppressed by means such as blocking the regulatory region of the gene. "Gene expression" refers to the process by which proteins encoded by genes are produced, through transcription, translation, and other means, so that they have their intended structure and activity.

[0031] (Overview of transformed microorganisms) A transformed microorganism according to one aspect of the present invention is a microorganism obtained by transforming a host microorganism so that a specific gene on the chromosome of the host microorganism is deleted. Furthermore, it is preferable that the transformed microorganism according to one aspect of the present invention is obtained by further transforming the host microorganism so that it expresses a group of acetavanylone-degrading enzyme genes that have been inserted as foreign genes.

[0032] (Deleted or inserted gene) The host microorganism is Pseudomonas species ( ​ The NGC7 strain (accession number: NITE BP-03043) has a chromosome on ​ Gene (SEQ ID NO: 1) and ​ It possesses the gene (Sequence ID 2). Of these, the oxygenase component directly acts on vanillic acid. ​ These are genes. Therefore, in one embodiment of the present invention, the transformed microorganism has, among these genes on the chromosome, ​ A gene is deleted. The transformed microorganism according to one aspect of the present invention has a gene that is originally present on the chromosome of the NGC7 strain. ​ While not particularly limited to those that delete genes, in order to significantly reduce the effect on vanillic acid, ​ Genes and ​ It is preferable that both genes are deleted.

[0033] According to the inventors' research, NGC7 strain is ​ It has four types of genes that are presumed to be genes, and ​ It has six genes that are presumed to be genes. Like the NGC7 strain, it has multiple types ​ Genes and ​ Microorganisms that possess genes that are presumed to be genes are extremely rare.

[0034] ​ Genes vanishO - Demethylase oxygenase component (vanillate O It is a gene that expresses the -demethylase oxygenase component. O - The demethylase oxygenase component (EC 1.14.13.82) utilizes electrons derived from NADH or NADPH supplied via the oxygen reductase component, along with oxygen atoms supplied from molecular oxygen, to cleave the methyl ether bond of vanillic acid, producing protocatechuic acid, formaldehyde, and water.

[0035] Vanish O - The demethylase oxygenase component has a Rieske[2Fe-2S]iron-sulfur domain (W7-V107, PROSITE entry no.PS51296) in its amino acid sequence, and the C and H (C47,H49,C66,H69) in this domain are involved in Fe-S binding.

[0036] ​ Genes vanish O - Demethylase oxidoreductase component (vanillate O It is a gene that expresses the demethylase oxidoreductase component. O - Demethylase oxidoreductase component (EC 1.14.13.82) is known as one of the oxidoreductases that remove electrons from NADH or NADPH and transfer them to oxygenases. O - The demethylase oxidoreductase component is vanillate O - Demethylase transfers electrons derived from NADH or NADPH to VanA, an oxygenase component.

[0037] Vanish O- The demethylase oxidoreductase component has a 2Fe-2S Ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and the C(C265, C270, C273, C303) in this amino acid sequence is involved in Fe-S binding. Also, vanillate O - The demethylase oxidoreductase component has an NAD-binding domain (L109-D201, Pfam entry no. PF00175) and a Ferredoxin reductase type FAD-binding domain (M1-A101, PROSITE entry no. PS51384) in its amino acid sequence.

[0038] ​ Gene expression product (VanA) and ​ The gene expression product (VanB) works together to vanish. O - Constitutes demethylase (VanAB). VanAB is responsible for the demethylation of vanillic acid, but 3- O -Methylgallic acid, 3- O -Methylgallic acid can be converted to gallic acid, so syringic acid O -Demethylase, 3- O -It can also function as methylgallic acid demethylase. ​ sp.SYK-6 strain is syringic acid O -As a gene encoding demethylase ​ It possesses the gene, vanillic acid / 3-O-methylgallic acid O -As a gene encoding demethylase ​ Possesses genes.

[0039] The SYK-6 strain possesses ​ Genes and ​The enzyme encoded by the gene catalyzes the methyl transfer reaction to tetrahydrofolate, and this reaction requires tetrahydrofolate to proceed. Since the regeneration of tetrahydrofolate requires C1 metabolism to supply C1 compounds required for methionine, thymidine, and purine biosynthesis, these enzymes must function coupled with C1 metabolism. In contrast, the vanillate expressed by the NGC7 strain... O - Demethylase is an oxygenated type and consists of an oxygenase component and an oxidoreductase component, thus reducing complexity by eliminating the need for coupling with C1 metabolism.

[0040] As described above, ​ Genes and ​ A gene is thought to function as a set of genes. Therefore, the inventors of the present invention have found that the NGC7 strain possesses ​ Genes and ​ When we searched for genes using the genomic DNA of NGC7 strain, we found unexpected results. ​ Gene (Sequence ID 11), ​ Gene (SEQ ID NO: 12), ​ Gene (SEQ ID NO: 13) and ​ There are four types of genes (Sequence ID 1). ​ Having genes, and ​ Gene (Sequence ID 14), ​ Gene (Sequence ID 15), ​ Gene (SEQ ID NO: 16), ​ Gene (SEQ ID NO: 2), ​ Gene (SEQ ID NO: 17) and ​ Six types of genes (SEQ ID NO: 18) ​ They discovered that it possesses genes. The inventors further investigated and, based on this genetic information, selected a pair of genes for each pair, resulting in a total of four pairs. ​ Genes and ​ We assumed a set of genes. And these four pairs of gene sets, ​ Genes and ​ Gene set of genes, ​ Genes and ​ Gene set of genes, ​ Genes and​ Gene set of genes, and ​ Genes and ​ This was a gene set.

[0041] The inventors created knockout strains of the four gene sets described above and tested their ability to degrade vanillic acid, ​ Genes and ​ We discovered that a specific set of genes is deeply involved in the degradation of vanillic acid, which led to the completion of this invention.

[0042] In light of the above circumstances, the transformed microorganism of one aspect of the present invention is assumed to be of multiple types of vanillate O - Among demethylases, vanillate is deeply involved in the breakdown of vanillic acid. O -In order to inhibit the expression of demethylase, ​ Genes and ​ NGC7-transformed strain in which both genes are deleted (hereinafter referred to as NGC7Δ) ​ It is preferable that it is also called a stock.

[0043] A transformed microorganism according to one aspect of the present invention is ​ Genes and ​ Among the genes, other ​ genes and / or ​ Gene deletions are acceptable. However, ​ Genes and ​ NGC7 transformed strains lacking the gene, and ​ Genes and ​ NGC7-transformed strains lacking the gene are NGC7 strain and NGC7Δ ​ The results show that the assimilation ability of syringic acid is inferior compared to the strain. Therefore, when using S-type lignin-derived aromatic compounds such as syringic acid as a carbon source, the transformed microorganism according to one embodiment of the present invention is ​ gene, ​ gene, ​ Genes and ​It is preferable that one, two, three, or four genes selected from the group of genes are not deleted. On the other hand, when using aromatic compounds derived from H-type lignin and / or G-type lignin or carbon sources such as glucose, it is expected that the yield of vanillic acid will be increased and the degradation of vanillic acid will be further reduced. Therefore, the transformed microorganism in one embodiment of the present invention is ​ Genes and ​ In addition to genes, other ​ Genes and / or other ​ A gene deletion is preferable.

[0044] Specifically, when the objective is to selectively produce and / or selectively accumulate vanillic acid while decomposing not only H-type lignin-derived aromatic compounds but also S-type lignin-derived aromatic compounds and utilizing them as a carbon source, the transformed microorganism according to one embodiment of the present invention enhances the assimilation ability of S-type lignin-derived aromatic compounds. ​ Genes and ​ The gene is deleted, and ​ Genes and ​ It is preferable to preserve the genes; ​ Genes and ​ The gene is deleted, and ​ Genes and ​ It is preferable to retain the gene; or, ​ Genes and ​ The gene is deleted, and ​ Genes and ​ Genes and ​ Genes and ​ It is preferable to retain the genes.

[0045] Furthermore, in cases where the objective is to selectively produce and / or selectively accumulate vanillic acid while decomposing G-type lignin-derived aromatic compounds and / or H-type lignin-derived aromatic compounds and utilizing them as a carbon source, p -When using aromatic compounds derived from hydroxyphenyl lignin or carbon sources such as glucose, the transformed microorganism according to one embodiment of the present invention increases the recovery rate of vanillic acid. ​ Genes and ​The gene is deleted, and ​ Genes and ​ Deleting the gene is preferable; ​ Genes and ​ The gene is deleted, and ​ Genes and ​ It is preferable that the gene is deleted; ​ Genes and ​ The gene is deleted, and ​ Genes and ​ It is preferable that the gene is deleted; or, ​ Genes and ​ The gene is deleted, and ​ Genes and ​ gene, ​ Genes and ​ Genes and ​ Genes and ​ It is preferable that the gene is deleted. ​ Genes and ​ The gene is deleted, and ​ Genes and ​ gene, ​ Genes and ​ Genes and ​ Genes and ​ The characteristic of a transformed microorganism according to one embodiment of the present invention, which has a gene deletion, that it can produce vanillic acid in high yield, ​ Genes and ​ Transgenic microorganisms with gene deletions, ​ Genes and ​ Transgenic microorganisms lacking genes, and ​ Genes and ​ This is a surprising characteristic that cannot be predicted from transformed microorganisms lacking genes.

[0046] The SYK-6 strain can degrade acetovanylone, one of the aromatic compounds derived from lignin. The acetovanylone degradation ability of the SYK-6 strain is as follows: ​ Gene (Sequence ID 3), ​ Gene (SEQ ID NO: 4), ​ Gene (SEQ ID NO: 5), ​ Gene (Sequence ID 6),​ Gene (Sequence ID 7), ​ Gene (Sequence ID 8), ​ Gene (SEQ ID NO: 9) and ​ The enzyme acetavanylonede, such as the gene (SEQ ID NO: 10), is involved.

[0047] Therefore, in one aspect of the present invention, the transformed microorganism, from the viewpoint of acetvanilone degradation, has an exogenous gene, ​ Gene (Sequence ID 3), ​ Gene (SEQ ID NO: 4), ​ Gene (SEQ ID NO: 5), ​ Gene (Sequence ID 6), ​ Gene (Sequence ID 7), ​ Gene (Sequence ID 8), ​ Gene (SEQ ID NO: 9) and ​ It is preferable that the gene (SEQ ID NO: 10) is inserted and expressed. In one embodiment of the present invention, a transformed microorganism can degrade acetavanylone and produce and accumulate vanillic acid by inserting and expressing a group of acetavanylone-degrading enzyme genes, which are foreign genes.

[0048] Furthermore, the inserted gene does not have to be exactly the same as the gene originally possessed by the SYK-6 strain (i.e., the wild-type gene). As long as it is a gene that expresses a protein having the same or similar enzymatic properties as the protein expressed by the wild-type gene (i.e., the wild-type protein), it may be a nucleotide sequence having a nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene under stringent conditions.

[0049] "Nucleotide sequences that hybridize under stringent conditions" refers to the nucleotide sequences of DNA obtained by using DNA containing the nucleotide sequence of a wild-type gene as a probe, and employing methods such as colony hybridization, plaque hybridization, or Southern blot hybridization.

[0050] "Stringent conditions" are those under which the signal of a specific hybrid is clearly distinguishable from the signal of a nonspecific hybrid, and these conditions vary depending on the hybridization system used, as well as the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature, washing temperature, and salt concentration. For example, if the signal of a nonspecific hybrid is strongly detected, specificity can be increased by raising the hybridization and washing temperatures and, if necessary, lowering the washing salt concentration. Conversely, if the signal of a specific hybrid is not detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration.

[0051] As a specific example of stringent conditions, for instance, a DNA probe is used as the probe, and hybridization is performed overnight (approximately 8 to 16 hours) using 5×SSC, 1.0% (w / v) nucleic acid hybridization blocking reagent (Roche Diagnostics), 0.1% (w / v) N-lauroyl sarcosine, and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5×SSC, 0.1% (w / v) SDS, preferably 0.1×SSC, and 0.1% (w / v) SDS. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.

[0052] Furthermore, examples of DNA having nucleotide sequences that hybridize under stringent conditions include, for example, DNA obtained by hybridizing under the stringent conditions described above using DNA having the nucleotide sequence of a wild-type gene derived from a colony or plaque, or a filter on which a fragment of said DNA is immobilized, and DNA that can be identified by performing hybridization at 40°C to 75°C in the presence of 0.5M to 2.0M NaCl, preferably at 65°C in the presence of 0.7M to 1.0M NaCl, and then washing the filter with a 0.1 to 1×SSC solution (1×SSC solution is 150mM sodium chloride, 15mM sodium citrate) at 65°C. The preparation of probes and hybridization methods can be carried out in accordance with the methods described in Molecular Cloning: A Laboratory Manual, 2nd Edition, 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). Furthermore, those skilled in the art can appropriately set conditions to obtain DNA having a nucleotide sequence complementary to the wild-type gene's nucleotide sequence and a nucleotide sequence that hybridizes under stringent conditions, by taking into account not only the salt concentration and temperature of such buffers, but also other conditions such as probe concentration, probe length, and reaction time.

[0053] Examples of DNA containing nucleotide sequences that hybridize under stringent conditions include DNA having a certain level of sequence identity with the nucleotide sequence of the DNA containing the nucleotide sequence of the wild-type gene used as a probe. For example, DNA having 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, and even more preferably 99.5% or more of sequence identity with the nucleotide sequence of the wild-type gene. The upper limit of this sequence identity is not particularly limited and is typically 100%.

[0054] Nucleotide sequences that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene include, for example, nucleotide sequences in which, if 100 nucleotides in a nucleotide sequence are considered as one unit, the wild-type gene's nucleotide sequence has 1 to several, preferably 1 to 20, more preferably 1 to 15, and even more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide deletions, substitutions, additions, etc., per unit. Here, "nucleotide deletion" means that there is a nucleotide missing or absent in the sequence, "nucleotide substitution" means that a nucleotide in the sequence is replaced with another nucleotide, and "nucleotide addition" means that a new nucleotide is added by insertion.

[0055] Proteins encoded by nucleotide sequences complementary to the wild-type gene's nucleotide sequence and nucleotide sequences that hybridize under stringent conditions are likely to have amino acid sequences with one to several amino acid deletions, substitutions, or additions compared to the amino acid sequence of the protein encoded by the wild-type gene's nucleotide sequence, but they possess the same enzymatic activity as the protein encoded by the wild-type gene's nucleotide sequence.

[0056] A protein having the same or similar enzymatic properties as the wild-type protein may have an amino acid sequence that has one to several amino acid deletions, substitutions, or additions compared to the amino acid sequence of the wild-type protein. Here, the range of "one to several" in "one to several amino acid deletions, substitutions, or additions" in the amino acid sequence is not particularly limited, but for example, if 100 amino acids in the amino acid sequence are considered as one unit, then each unit means approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, preferably approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, more preferably approximately 1, 2, 3, 4, or 5 amino acids. Furthermore, "amino acid deletion" means the absence or disappearance of an amino acid residue in a sequence, "amino acid substitution" means that an amino acid residue in a sequence is replaced by another amino acid residue, and "amino acid addition" means that a new amino acid residue is added to a sequence by insertion.

[0057] Specific examples of "deletion, substitution, or addition of one to several amino acids" include cases where one to several amino acids are replaced with other chemically similar amino acids. For example, this could include substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the relevant field for each amino acid. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0058] Examples of amino acid sequences having one to several amino acid deletions, substitutions, or additions in the amino acid sequence of the wild-type protein include amino acid sequences that have a certain level of sequence identity with the amino acid sequence of the wild-type protein. For example, amino acid sequences having 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, and even more preferably 99.5% or more of sequence identity with the amino acid sequence of the wild-type protein. The upper limit of this sequence identity is not particularly limited and is typically 100%.

[0059] (Means for calculating sequence identity) The method for determining sequence identity between nucleotide and amino acid sequences is not particularly limited, but for example, it can be determined by using a commonly known method to align the nucleotide sequence of the wild-type gene and the amino acid sequence of the wild-type protein expressed by the wild-type gene with the target nucleotide and amino acid sequences, and then using a program to calculate the sequence agreement rate between the two.

[0060] As a program for calculating the degree of agreement between two nucleotide and amino acid sequences, for example, the Karlin and Altschul algorithm (Proc.Natl.Acad.Sci.USA 87:2264-2268, 1990; Proc.Natl.Acad.Sci.USA 90:5873-5877, 1993) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J.Mol.Biol.215:403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res.25:3389-3402, 1997). Therefore, those skilled in the art can use, for example, the above programs to search for sequences that show high sequence identity with a given sequence in a database. These are available, for example, on the website of the U.S. National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0061] While the methods described above are commonly used to search for sequences exhibiting sequence identity within a database, homology analysis using Genetyx Network version 12.0.1 (Genetics Inc.) can also be used to determine the sequence identity of individual sequences. This method is based on the Lipman-Pearson method (Science 227:1435-1441, 1985). When analyzing the sequence identity of nucleotide sequences, protein-coding regions (CDS or ORF) should be used if possible.

[0062] (Origin of the gene to be inserted) The acetvanylase gene group to be inserted is: ​Microorganisms capable of degrading acetovanylon, such as those belonging to the class alpha proteobacteria, order Sphingomonadales, and family Sphingomonadaceae. ​ It is preferable that the microorganisms belong to the genus and are derived from microbial strains that have the ability to decompose lignin-derived aromatic compounds. A "closely related species" of the SYK-6 strain refers to a microorganism of the genus Sphingobium whose nucleotide sequence of the 16S rRNA gene is 99.0% to 99.9% identical to that of the SYK-6 strain. Furthermore, in terms of biological taxonomy Sphingobium While not belonging to the genus, any microbial strain possessing the ability to degrade acetavanylone is preferable as a group of acetavanylone-degrading enzyme genes to be inserted. For example, microbial strains belonging to the class alpha proteobacteria, order Sphingomonadales, family Sphingomonadaceae, that have the ability to degrade acetavanylone and its analog compounds, or from the class gamma proteobacteria, order Pseudomodales, family Pseudomonadaceae, Pseudomonas It is not excluded that the compounds may originate from microbial strains belonging to the genus that have the ability to degrade acetovanylone and its analog compounds.

[0063] (host microorganism) The host microorganism is Pseudomonas species ( Pseudomonas This is strain NGC7 (accession number: NITE BP-03043).

[0064] According to our findings, the genome of strain NGC7 contains what appears to be a genetic factor that gives strain NGC7 resistance to kanamycin. aph The gene (SEQ ID NO: 37) is present. Therefore, when using drug selection with kanamycin for the selection of transformed strains, it is preferable to delete the aph gene on the genome of the NGC7 strain. Thus, in order to improve the efficiency of selecting transformed strains, genes on the genome of the NGC7 strain may be deleted depending on the selection conditions.

[0065] (Genetic engineering techniques for gene cloning) The deleted and inserted genes can be inserted into various known vectors. Furthermore, by introducing this vector into the host microorganism NGC7 strain, transformants (transformed microorganisms) with deleted or inserted genes can be produced. Preferably, the deleted gene is one in which the structure of the wild-type gene has been altered by disruption, deletion, substitution, insertion, etc., in whole or in part. Preferably, the inserted gene is a gene that expresses a protein identical or similar to the wild-type gene.

[0066] Methods for obtaining the genes to be deleted and inserted, methods for obtaining information on the nucleotide sequences of these genes and the amino acid sequences of the proteins expressed by these genes, methods for producing various vectors, and methods for producing transformed microorganisms can be appropriately selected by those skilled in the art. Furthermore, in this specification, transformation and transformant encompass transduction and transduction, respectively. An example of cloning the genes to be deleted and inserted will be described below without limitation.

[0067] For example, chromosomal DNA and mRNA can be extracted from organisms or various microorganisms that possess the wild-type gene related to the gene to be deleted or inserted, using conventional methods, such as those described in the referenced technical literature. cDNA can be synthesized using the extracted mRNA as a template. A library of chromosomal DNA and cDNA can then be constructed using the chromosomal DNA and cDNA obtained in this manner.

[0068] For example, the gene to be inserted can be obtained by cloning using the chromosomal DNA and cDNA of the organism from which the wild-type gene related to the gene is derived as a template. The organisms from which the wild-type gene is derived are the SYK-6 strain and its closely related species as described above. For example, the SYK-6 strain is cultured, water is removed from the resulting bacterial cells, and the cells are physically ground into a fine powder using a mortar and pestle while being cooled in liquid nitrogen. The chromosomal DNA fraction is then extracted from these bacterial cells by a conventional method. Commercially available chromosomal DNA extraction kits such as the DNeasy Blood & Tissue Kit (Qiagen) can be used for the chromosomal DNA extraction procedure. In this specification, chromosomal DNA and genomic DNA are synonymous.

[0069] Next, using chromosomal DNA as a template, a polymerase chain reaction (PCR) is performed using synthetic primers complementary to the 5' and 3' terminal sequences to amplify the DNA. The primers are not particularly limited as long as they can amplify the DNA fragment containing the gene to be inserted. Examples include: vanA4 Examples of primers used to amplify genes include those represented by SEQ ID NOs. 35 and 36, which were designed based on the genome sequence of the NGC7 strain. Using such primers, the entire target gene can be amplified. Alternatively, DNA containing the target gene fragment can be amplified by screening target gene clones from a shotgun library, or by appropriate PCR methods such as Inverse PCR, Nested PCR, 5'RACE, or 3'RACE, and then these fragments can be ligated to obtain DNA containing the entire target gene.

[0070] Furthermore, the method for obtaining the gene to be deleted or inserted is not particularly limited, as described above, and it is possible to construct the gene using, for example, chemical synthesis methods, without resorting to genetic engineering techniques.

[0071] The nucleotide sequences of amplified products and chemically synthesized genes, as a result of PCR, can be confirmed, for example, as follows: First, recombinant DNA is prepared by inserting the DNA whose sequence you want to examine into a suitable vector using standard methods. For cloning into the vector, use the In-Fusion HD Cloning Kit (Takara Bio Inc.) or TA Cloning. Known or commercially available kits such as Kit (Invitrogen); pUC4K (see Gene, vol.19, pp.259-268, 1982), pEX18Amp (see Gene, vol.212, pp.77-86, 1998), pPS858 (see Gene, vol.212, pp.77-86, 1998), pUC118 (Takara Bio), pJB866 (see Plasmid, vol.38, pp.35-51, 1997), pMCL200 (see Gene, vol.162, pp.157-158, 1995), pQE30 (Qiagen), pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio), pAK405 (Andreas Kaczmarczyk et al., Applied and See Environmental Microbiology, 2012, vol.78(10), pp. 3774-3777, p.K18. mobsacB Known or commercially available plasmid vectors such as (see Andreas Schafer et al., Gene, 1994, vol. 145, pp. 69-73); known or commercially available bacteriophage vectors such as λEMBL3 (Stratagene) can be used.

[0072] If you want to obtain a large amount of the constructed recombinant DNA, you can use the recombinant DNA in, for example, E. coli. Escherichia coli Preferably, the recombinant DNA can be introduced into E. coli strain JM109 (Takara Bio Inc.) or E. coli strain DH5α (Takara Bio Inc.) and transformed, and then the recombinant DNA contained in the resulting transformant can be purified using QIAGEN Plasmid Mini Kit (Qiagen Inc.) or the like.

[0073] The nucleotide sequences of each gene inserted into recombinant DNA are determined by methods such as the dideoxy method (see Methods in Enzymology, 101, pp. 20-78, 1983, etc.). The sequencing instrument used for nucleotide sequence determination is not particularly limited, but examples include the Li-COR MODEL 4200L sequencer (Aloka), the 370 DNA sequencing system (PerkinElmer), and the CEQ2000XL DNA analysis system (Beckman). Based on the determined nucleotide sequences, the amino acid sequence of the translated protein can be determined.

[0074] (Construction of recombinant vectors containing genes) Recombinant vectors (recombinant DNA) containing the gene to be deleted or inserted can be constructed by linking a PCR amplification product containing the gene to be deleted or inserted with various vectors in a manner that allows for gene deletion or expression. In the case of a gene to be deleted, it is preferable that the recombinant vector contains the upstream and downstream regions of the deleted gene, so that when the recombinant vector is introduced into a host microorganism, homologous recombination occurs and the gene in the recombinant vector is replaced by the gene in the host microorganism.

[0075] As a non-limiting example, a recombinant vector containing the gene to be inserted can be constructed by, for example, excising a DNA fragment containing one of the genes to be inserted using a suitable restriction enzyme, and then ligating this DNA fragment with a plasmid vector obtained by cutting the DNA fragment with a suitable restriction enzyme, using a commercially available recombinant vector preparation kit such as the In-Fusion HD Cloning Kit (Takara Bio). Alternatively, it can be obtained by ligating a DNA fragment containing a gene with homologous sequences to both ends of the plasmid vector with a plasmid-derived DNA fragment amplified by inverse PCR, using a commercially available recombinant vector preparation kit such as the In-Fusion HD Cloning Kit (Takara Bio).

[0076] A recombinant vector containing a gene to be deleted or inserted contains at least the gene to be deleted or inserted and a gene (nucleotide sequence) derived from the plasmid vector. An example of a recombinant vector is: vanA4 genes and / or vanB4 Examples include recombinant vectors containing genes; recombinant vectors containing acetavanylone-degrading enzyme genes, etc. Furthermore, recombinant vectors may also contain genes other than those mentioned above, as long as they do not hinder the resolution of the problem of the present invention.

[0077] Recombinant vectors may contain heterologous genes or heterologous nucleotide sequences. Heterologous genes are not particularly limited as long as they are genes that do not naturally occur in the NGC7 strain, and include, for example, synthetic genes that do not rely on the nucleotide sequence derived from the NGC7 strain, genes derived from organisms different from the organism of origin of the gene to be inserted, and genes derived from other microorganisms, plants, animals, viruses, etc. that are different from the NGC7 strain. Specific examples of heterologous genes include DNA fragments derived from pUC118, for example, the lactose promoter region (P lac Examples include, but are not limited to, these.

[0078] (Method for producing transformed microorganisms) The method for producing transformed microorganisms is not particularly limited, and examples include inserting a gene into the NGC7 strain in a manner that achieves gene deletion or insertion according to a conventional method. Specifically, a transformed microorganism expressing the inserted gene can be obtained by producing a DNA construct in which one of the genes to be inserted is inserted between the gene expression induction promoter and the terminator, and then transforming the NGC7 strain with the DNA construct. Alternatively, a transformed microorganism lacking the gene can be obtained by producing a DNA construct containing the gene to be deleted and the upstream and downstream regions of the gene, and then transforming a host microorganism with the DNA construct. In this specification, recombinant vectors produced for transforming the NGC7 strain are collectively referred to as DNA constructs.

[0079] The method for introducing a DNA construct into the NGC7 strain is not particularly limited, but examples include, as is known to those skilled in the art, a method of introducing the DNA construct into the NGC7 strain in such a way that the introduced DNA construct autonomously proliferates and expresses genes; and a method of directly inserting the DNA construct onto the chromosomes of the NGC7 strain by utilizing homologous recombination.

[0080] One method for introducing a DNA construct containing the gene to be inserted into the NGC7 strain is to utilize homologous recombination. This method involves ligating the DNA construct between sequences homologous to the upstream and downstream regions of the recombination site on the chromosome, thereby inserting it into the genome of the NGC7 strain.

[0081] The vector-host system used to create the NGC7 strain is not particularly limited as long as it is a system in which the inserted gene can be expressed or a chromosomal gene can be deleted within the NGC7 strain. Examples include the pJB866 (Plasmid, vol.38(1), p35-51, 1997)-Pseudomonas microorganism system, the pKT230 (Gene, vol.16, p237-247, 1981)-Pseudomonas microorganism system, and the pSEVA (Nucleic Acids Research, vol.48(D1), pD1164-D1170, 2020)-Pseudomonas microorganism system.

[0082] The DNA construct containing the gene to be inserted can be either autonomously amplified and expressed without being introduced into the chromosomes of the NGC7 strain, or it can be expressed after being introduced into the chromosomes of the NGC7 strain.

[0083] The DNA construct may include marker genes to enable the selection of transformed cells. These marker genes are not limited to those mentioned above and may include, for example, drug resistance genes to drugs such as gentamicin, kanamycin, tetracycline, ampicillin, and carbenicillin. The marker genes may be included in the middle of the deleted gene or as a substitute for the deleted gene.

[0084] Depending on the type of gene, the DNA construct containing the gene to be inserted may include, in addition to promoters and terminators that enable gene expression in the NGC7 strain, other regulatory sequences (e.g., cis sequences involved in transcriptional regulation, such as operators).

[0085] One embodiment of the DNA construct is, for example, the pvanA1B1del plasmid DNA, pvanA2B2del plasmid DNA, pvanA3B3del plasmid DNA, pvanA4B4del plasmid DNA, and pSEVA241_P described in the examples below. lac Examples include, but are not limited to, -acv plasmid DNA and pTS093_vceA-B plasmid DNA.

[0086] As for the method of transformation into NGC7 strain, a method known to those skilled in the art can be appropriately selected, for example, by electroporation or junction transfer.

[0087] For selecting and growing transformed microorganisms, any medium suitable for the growth of NGC7 strains should be used. For example, if resistance genes for kanamycin, gentamicin, and tetracycline are used as marker genes, the selection and growth of transformed microorganisms can be carried out, for example, by culturing the transformed microorganisms in LB medium containing these drugs.

[0088] Confirmation that transformed microorganisms have been created can be achieved, for example, by culturing the transformed microorganisms under conditions in which only transformed microorganisms with a deleted gene can survive, or under conditions in which only transformed microorganisms expressing the gene to be inserted can survive. Alternatively, the creation of transformed microorganisms can be confirmed by culturing the transformed microorganisms and then confirming that the amount of vanillic acid in the culture obtained after culturing is greater than the amount of vanillic acid in the culture of NGC7 strain cultured under the same conditions.

[0089] Confirmation that transformed microorganisms have been created can be performed by extracting chromosomal DNA from the transformed microorganisms, using it as a template for PCR, and verifying whether a PCR product capable of amplification is produced when transformation occurs, as well as by examining the characteristics and nucleotide sequence of the PCR product.

[0090] For example, PCR is performed using a combination of a forward primer for the nucleotide sequence of the promoter of the gene to be deleted or inserted, and a reverse primer for the nucleotide sequence of the marker gene, to confirm that a product of the expected length is produced.

[0091] When performing transformation by homologous recombination, it is preferable to perform PCR using a combination of a forward primer located upstream of the homologous region used and a reverse primer located downstream of the homologous region used, and to confirm that a product of the expected length is produced when homologous recombination occurs.

[0092] When using acetavanylone-degrading enzyme genes from closely related species of the SYK-6 strain, these genes may be optimized in terms of codons, secondary structure, GC content, etc., for expression in the NGC7 strain.

[0093] (One specific embodiment of transformed microorganisms) One specific example of a transformed microorganism is one in which the host microorganism is strain NGC7 and has chromosomes vanA4 This is a transformed microorganism in which the gene (Sequence ID 1) is deleted. One specific form of the transformed microorganism is when the host microorganism is NGC7 strain and the gene is located on the chromosome. vanA4 In addition to the gene (SEQ ID NO: 1), vanB4 This is a transformed microorganism in which the gene (Sequence ID 2) is deleted. One specific form of the transformed microorganism is one in which the host microorganism is NGC7 strain and the gene is located on the chromosome. vanA4 Gene (SEQ ID NO: 1) and vanB4 In addition to genes, vanA1 Gene (Sequence ID 11), vanA2 Gene (SEQ ID NO: 12), vanA3Gene (SEQ ID NO: 13), vanB1 Gene (Sequence ID 14), vanB2 Gene (Sequence ID 15), ​ Gene (SEQ ID NO: 16), ​ Gene (SEQ ID NO: 17) and ​ These are transformed microorganisms in which one or more genes selected from the group consisting of genes (sequence number 18) are deleted. These transformed microorganisms are collectively called transformed microorganisms (1).

[0094] One specific form of transformed microorganism (hereinafter also referred to as transformed microorganism (2)) is characterized by the deletion of the above gene, in addition to the insertion of ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ Genes and ​ This is a transformed microorganism that expresses a group of acetavanylone-degrading enzyme genes.

[0095] The transformed microorganism (1) is located on the chromosome. ​ gene (SEQ ID NO: 1) or the ​ Genes and ​ By at least adopting a configuration in which the gene (SEQ ID NO: 2) is deleted, the vanillic acid degradation activity is lost while the syringic acid assimilation activity is maintained, which is impossible with the transformed microorganism described in Patent Document 3. p - Vanillic acid can be produced from aromatic compounds derived from G-type lignin, such as ferulic acid and vanillin, while using aromatic compounds derived from H-type lignin, such as hydroxybenzoic acid, and / or aromatic compounds derived from S-type lignin, such as syringic acid, as a carbon source for propagation.

[0096] The transformed microorganism (2) exhibits, in addition to the deletion of the above-mentioned genes, the expression of the inserted acetovanylonase gene group, p- Vanillic acid can be produced from a mixture of aromatic compounds derived from G-type lignin, such as ferulic acid and vanillin, and acetovanylone or aromatic compounds derived from G-type lignin containing acetovanylone, while growing H-type lignin-derived aromatic compounds such as hydroxybenzoic acid and / or S-type lignin-derived aromatic compounds such as syringic acid, using these as a carbon source.

[0097] The specific embodiments of the transformed microorganisms (1) and (2) are described in the examples below in NGC7Δ. ​ Stock, NGC7Δ ​ [pSEVA241_P lac This includes strains such as -acv,pTS093_vceA-B, but is not limited to these.

[0098] (Manufacturing method) A manufacturing method according to one aspect of the present invention (hereinafter referred to as manufacturing method (1)) involves an aromatic compound derived from G-type lignin such as vanillin, p - Hydroxybenzoic acid and p -The process includes a step of obtaining vanillic acid by reacting a transformed microorganism (1) with a mixture of an aromatic compound derived from H-type lignin, such as hydroxybenzaldehyde, and / or an aromatic compound derived from S-type lignin, such as syringic acid or syringaldehyde.

[0099] Another embodiment of the present invention, a manufacturing method (hereinafter referred to as manufacturing method (2)), includes the step of obtaining vanillic acid by acting acetovanylon on a transformed microorganism (2). Since acetovanylon is contained in the decomposition products obtained by alkaline oxidative decomposition of lignin, such as Kraft pulping and soda pulping, the source of acetovanylon may be a mixture of aromatic compounds such as alkaline oxidative decomposition products of coniferous lignin, which mainly consist of G-type lignin.

[0100] In this specification, when referring to manufacturing methods (1) and (2) collectively, they will simply be called "manufacturing method."

[0101] The method for applying lignin-derived aromatic compounds to transformed microorganisms is not particularly limited as long as the lignin-derived aromatic compounds and the transformed microorganisms come into contact and vanillic acid is produced and / or accumulated by enzymes possessed by the transformed microorganisms. For example, one method is to produce vanillic acid by culturing the transformed microorganisms under various culture conditions suitable for the NGC7 strain using a culture medium containing lignin-derived aromatic compounds and suitable for the growth of the NGC7 strain. The culture method is not particularly limited and can include solid culture or liquid culture methods performed under aerated conditions.

[0102] "Production of vanillic acid" means obtaining vanillic acid from a source of vanillic acid, for example, synthesizing and converting vanillic acid from compounds such as vanillic acid precursors; vanillic acid and syringic acid, p -In the presence of S-type lignin-derived aromatic compounds and / or H-type lignin-derived aromatic compounds such as hydroxybenzoic acid, vanillic acid is decomposed while vanillic acid is not, thereby accumulating (concentrating) vanillic acid.

[0103] The culture medium can be any synthetic or natural medium, as long as it is a standard medium for culturing Pseudomonas microorganisms such as strain NGC7, i.e., one that contains carbon sources, nitrogen sources, inorganic substances, and other nutrients in appropriate proportions. For example, MMx-3 medium, minimum Wx medium, etc., as described in the examples below, can be used, but are not particularly limited. The carbon source can be S-type lignin-derived aromatic compounds, H-type lignin-derived aromatic compounds, sugars, organic acids, or other carbon sources or combinations thereof. However, the culture medium components must include components necessary for cell proliferation and enzyme activation, such as Mg 2+ Fe 2+ It is preferable that the culture medium contains the following. Iron ions, magnesium ions, etc., can be added to the culture medium as compounds, but they may also be added as mineral-containing substances.

[0104] Aromatic compounds derived from H-type lignin and aromatic compounds derived from S-type lignin are not particularly limited as long as they are either H-type lignin or S-type lignin, or aromatic compounds that can be derived from these lignins, but examples include compounds corresponding to the decomposition products of S-type lignin and H-type phenyl lignin, and specifically, acetosyringone, p - Hydroxyacetophenone, syringic acid, syringaldehyde, p - Coumaric acid, p Examples include hydroxybenzoic acid and protocatechuic acid.

[0105] The culture medium contains vanillic acid or an aromatic compound derived from G-type lignin that is a precursor to vanillic acid. The aromatic compound derived from G-type lignin is not particularly limited as long as it is G-type lignin or an aromatic compound that can be derived from G-type lignin, but examples include compounds corresponding to the degradation products of G-type lignin, specifically ferulic acid, vanillin, acetovanylone, etc.

[0106] The lignin-derived aromatic compounds are preferably obtained from lignin-containing biomass or from biomass that has been pretreated and extracted; however, they may also be chemically synthesized and purified independently of such biomass. The lignin-derived aromatic compounds can be used individually or in combination of two or more.

[0107] Biomass containing lignin (hereinafter sometimes referred to as lignocellulose) is not particularly limited, but examples include natural products such as grass and trees, products obtained by processing these natural products, and agricultural waste. Specifically, examples include woody biomass such as broad-leaved trees and coniferous trees. For example, broad-leaved trees are known to contain a large amount of S-type lignin, and coniferous trees are known to contain a large amount of G-type lignin.

[0108] Lignocellulose can exist in various forms, such as solid, suspended, or liquid, depending on whether or not it has been pretreated. For example, a suspension can be obtained by adding pulverized lignocellulose to a liquid.

[0109] Lignocellulose may also be a lignin extract. Examples of lignin extracts include a suspension obtained by suspending powdered lignocellulose in a solvent suitable for lignin extraction at a concentration of 0.1% (w / v) to 50% (w / v), preferably 1% (w / v) to 20% (w / v). Alternatively, the lignin extract may be obtained by subjecting the suspension to an extraction treatment 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, and then removing the solids from the extraction solution to obtain a liquid lignin extract, or by distilling off the solvent from the liquid lignin extract and drying it to obtain a solid lignin extract. By using cedar powder as lignocellulose, cedar lignin extract can be obtained, and by using birch powder, birch lignin extract can be obtained.

[0110] The method for preparing aromatic compounds from lignin extracts is not particularly limited, but examples include the following methods. Specifically, 50 mL of 2M NaOH, 0.5 g of lignin extract, and 3 mL of nitrobenzene are placed in a stainless steel vessel of a small autoclave (Taiatsu Glass Industry Co., Ltd., portable reactor TVS-1), and the mixture is treated at 170°C for 2.5 hours while stirring at 500 rpm. After cooling to below 60°C, the supernatant is collected by centrifugation (6,000 x g, 10 min). The obtained supernatant is subjected to diethyl ether extraction three times (the aqueous layer is collected). After acidifying the aqueous layer with hydrochloric acid, diethyl ether extraction is performed three times (the ether layer is collected). Sodium sulfate is added to the ether layer, and the mixture is dehydrated overnight in a refrigerator. The ether layer is collected, and the extract is dried under reduced pressure. The ether extract is dissolved in deionized water with sodium hydroxide added (pH ≈ 9) to obtain a solution of aromatic compounds derived from the lignin extract.

[0111] The solvents suitable for lignin extraction and aromatic compound preparation are not particularly limited and include, for example, water, dioxane, methanol, low molecular weight alcohols such as isopropanol, diethyl ether, and dimethylformamide.

[0112] Furthermore, lignin decomposition products can also be obtained by using metal foams such as copper as catalysts for lignin decomposition. For example, a catalyst layer formed by loading a lignin decomposition catalyst [Cu(OH)2 / CF] into a stainless steel tube is flowed through which an alkaline-treated lignocellulose and oxygen gas are passed. The solution that has passed through the catalyst layer is then adjusted to an alkaline pH, for example, 9-11, with an acid. The solution is then passed through a filtration membrane such as an MF membrane or UF membrane to obtain a filtrate. The obtained filtrate is then adjusted to an acidic pH, for example, 2-4, with an acid. An extraction treatment using ethyl acetate is then performed. The resulting ethyl acetate layer is then concentrated and evaporated to dryness to obtain an extract. The obtained extract is then dissolved in an alkaline aqueous solution and further neutralized to a weakly alkaline to neutral pH with an acid to obtain lignin decomposition products.

[0113] The culture conditions can be those commonly known to those skilled in the art for culturing Pseudomonas microorganisms. For example, the initial pH of the culture medium can be adjusted to 5-10, the culture temperature to 20°C-40°C, and the culture time to several hours-several days, preferably 1-7 days, more preferably 2-5 days, and can be set as appropriate. The culture method is not particularly limited, and deep culture with aeration and stirring, shaking culture, static culture, etc., can be employed, but it is preferable to culture under conditions where the dissolved oxygen concentration is sufficient, such as by aeration. Furthermore, depending on the decrease in carbon source and acetvanylone, and the increase in vanillic acid, a fed-batch culture may be employed in which a carbon source, lignin extract, G-type lignin-derived aromatic compounds, etc., are added.

[0114] For example, as an example of culture medium and culture conditions, syringic acid and / or as a carbon source pExamples of culture methods include using MMx-3 medium containing hydroxybenzoic acid and ferulic acid or vanillin as substrates for vanillic acid, with shaking or stirring culture at 30°C and 180 rpm for 1 hour to 5 days. Another example of a different medium and culture conditions is using LB medium containing acetvanilone as a substrate for vanillic acid, with shaking or stirring culture at 30°C and 180 rpm, with a dissolved oxygen concentration of 5% to 20%, as described in the examples below. Carbon sources and other components can be added as appropriate after the start of culture.

[0115] The method for obtaining vanillic acid from the culture after the culturing is complete is not particularly limited. Since vanillic acid accumulates in the culture medium, the bacterial cells and culture supernatant are separated from the culture by conventional solid-liquid separation operations such as filtration and centrifugation, and vanillic acid is extracted from the recovered culture supernatant by solid-phase extraction using a column or solvent extraction using a solvent in which vanillic acid is soluble.

[0116] The extraction solvent is not particularly limited as long as it is capable of dissolving vanillic acid, and examples include ethyl acetate and diethyl ether.

[0117] Vanillic acid can be separated and purified from the culture medium by known methods such as precipitation, extraction, distillation, recrystallization, and adsorbents, or by methods with some modifications thereof. One specific method of purification involves adding hydrochloric acid to the culture supernatant, adjusting the pH to 2-5, and then adding and mixing diethyl ether. The diethyl ether layer is dried under reduced pressure using an evaporator, and the resulting precipitate is washed with deionized water, recovered by suction filtration, and dried under reduced pressure. The dried solid is dissolved in glacial acetic acid and recrystallized to obtain purified vanillic acid. Purified vanillic acid can also be obtained by methods using synthetic adsorbents, such as the method described in the literature by Gomes et al. (Separation and Purification Technology, vol.216, pp.92-101, 2019), or by the method of reduced-pressure distillation described in Japanese Patent Application Publication No. 2017-171591.

[0118] Qualitative or quantitative analysis of vanillic acid may be performed by HPLC as described in the examples below.

[0119] By using a transformed microorganism according to one aspect of the present invention, vanillic acid can be selectively obtained without decomposing it. For example, using a transformed microorganism (1), if 5 mM vanillic acid and 5 mM syringic acid are used as carbon sources, a culture medium containing 4.4 mM vanillic acid and free of syringic acid can be obtained after 48 hours of incubation; 5 mM p -When hydroxybenzoic acid and 5 mM vanillic acid were used as the carbon source, 4.9 mM vanillic acid was obtained after 48 hours of incubation, p -A culture medium free of hydroxybenzoic acid can be obtained; 5 mM vanillic acid, 5 mM p -When hydroxybenzoic acid and 5 mM syringic acid are used as carbon sources, a 48-hour culture yields 4.0 mM vanillic acid, p -A culture medium free of hydroxybenzoic acid and syringic acid can be obtained. The upper limit of the yield is not particularly limited and is typically calculated from the amount of aromatic compounds derived from G-type lignin consumed (e.g., ferulic acid, vanillin, vanillic acid) and the amount of vanillic acid accumulated in the culture medium.

[0120] For example, if a transformed microorganism (2) is used, when 0.2 mM acetvanylone is used as a substrate, acetvanylone is converted to vanillic acid and vanilloylacetic acid within 24 hours of incubation, and by further incubation, vanilloylacetic acid is converted to vanillic acid, and after 30 hours of incubation, a culture medium containing vanillic acid but free of acetvanylone and vanilloylacetic acid can be obtained.

[0121] In the manufacturing method of the present invention, various steps and operations can be added before, after, or during the above-described steps, as long as the objective of the present invention is achieved.

[0122] (Uses of vanillic acid) Vanillic acid obtained using a transformed microorganism and production method according to one aspect of the present invention can be converted into various industrially useful compounds. Vanillic acid has an aromatic ring structure that imparts heat resistance and rigidity to polymers, and is expected to be used in the synthesis of functional polymers such as liquid crystal polyesters. Vanillic acid and vanillic acid derivatives obtained by modifying vanillic acid are expected to be used, for example, on their own or in combination with other components, as heat-resistant plastics, tracking-resistant plastics, liquid crystal copolymer polyester resins, and the like.

[0123] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and can take various forms as long as it can solve the problems of the present invention. [Examples]

[0124] [1. ​ [Biological classification of sp. NGC7 strain] Pseudomonas species ( ​ The NGC7 strain (hereinafter also simply referred to as NGC7 strain) has been deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) on October 4, 2019, with the microbial identification designation "NGC7" and the accession number "NITE BP-03043". A copy of the accession certificate for NGC7 is shown in Figure 1A, and the certificate of survival is shown in Figure 1B.

[0125] Tables 1 to 3 show the test results examining the characteristics of the NGC7 strain.

[0126] [Table 1]

[0127] [Table 2]

[0128] [Table 3]

[0129] Analysis of the 16S rRNA sequence revealed that strain NGC7 is Pseudomonas ptyida ( ​ ​ )NBRC14164 strain, Pseudomonas plecoglossifida ( ​ )FPC951 strain, Pseudomonas taiwanensis ( ​ )BCRC17751 strain and Pseudomonas monteilii ( ​ The 16S rRNA sequence showed 99.2% to 99.5% sequence identity with that of CIP104883 strain (see Shinoda, E. et al., J. Ind. Microbiol. Biotechnol. 46(8), 1071-1080).

[0130] From the results in Tables 1-3, strain NGC7 is a motile Gram-negative bacillus, and tested positive for both catalase and oxidase reactions, oxidizing glucose. These results indicate that strain NGC7 is a motile Gram-negative bacillus. ​ This was consistent with the characteristics of the genus (see, for example, Palleroni NJ. Pseudomonads. In: Bergey's Manual of Systematics of Archeaea and Bacteria).

[0131] Tests using the API20NE kit (biomérieux Japan) showed that strain NGC7 did not reduce nitrates, exhibited arginine dihydrolase activity, did not hydrolyze gelatin, assimilated glucose, potassium gluconate, and n-capric acid, but did not assimilate L-arabinose or adipic acid. Furthermore, it produced a fluorescent dye on King's agar, showed no lecithinase or lipase (Tween 80) activity, grew at 4°C and in the presence of 6% NaCl, but did not grow at 41°C and in the presence of 7% NaCl. The characteristics of these strains NGC7 are as follows: ​ The characteristics were in good agreement. However, from the results of 16S rRNA gene analysis, NGC7 strain ​This suggests that it may be classified as a different species, ​ It has similar properties to ​ It was estimated to be sp.

[0132] [2.NGC7Δ ​ [Creation of stocks] The following procedure will vanillate the NGC7 strain. O - Demethylase gene ( ​ NGC7Δ is a mutant strain that destroys ) ​ I created a stock.

[0133] The genomic DNA of NGC7 strain was prepared using conventional methods, and its entire nucleotide sequence was analyzed. ​ Vanillate derived from strain KT2440 O - Demethylase oxygenase component (VanA) and vanillate O We searched the genome sequence of the NGC7 strain for genes encoding proteins with amino acid sequences that have high sequence identity with the predicted amino acid sequence of the demethylase oxidoreductase component (VanB). We confirmed that the genomic DNA of the NGC7 strain contained four genes encoding amino acid sequences that showed more than 25% sequence identity with VanA (Table 4), and six genes encoding amino acid sequences that showed more than 30% sequence identity with VanB (Table 5).

[0134] Table 4 shows, ​ Table 5 shows the gene group derived from strain NGC7 that encodes an amino acid sequence showing more than 25% sequence identity with the predicted amino acid sequence of VanA derived from strain KT2440. ​ This shows a group of genes from the NGC7 strain that encode amino acid sequences showing more than 30% sequence identity with the predicted amino acid sequence of VanB from the KT2440 strain.

[0135] [Table 4]

[0136] [Table 5]

[0137] Oxygenated vanillate O - Demethylase is composed of an oxygenase component (VanA) and an oxidoreductase component (VanB). ​ and ​ Neither of the two genes contained a region encoding an amino acid sequence identical to that of VanA, an oxygenase component. Therefore, ​ and ​ , ​ and ​ , ​ and ​ ,and ​ and ​ We considered these as paired gene sets. Then, we deleted each gene set into NGC7Δ ​ Stock, NGC7Δ ​ Stock, NGC7Δ ​ Stocks and NGC7Δ ​ The strains were created using the following procedure: by deleting all or nearly all regions of each gene.

[0138] ​ Using the genomic DNA of strain sp. NGC7 as a template, PCR was performed using primer sets consisting of primers 1 and 2 of sequence numbers 19 and 20, and primer sets consisting of primers 3 and 4 of sequence numbers 21 and 22. ​ Upstream of the 5' end of the gene and ​ Each DNA fragment, approximately 1.0 kbp downstream of the 3' end, was amplified. Each fragment was then pre-digested with BamHI and processed using pK18. ​ (See Gene, Vol. 145, pp. 69-73, 1994) and by linking them using a seamless cloning method with NEBuilder HiFi DNA Assembly (New England Biolabs), ​ gene, ​ Plasmid pvanA1B1del was created for generating gene region deletion strains.

[0139] Similarly, using the genomic DNA of the NGC7 strain as a template, by the PCR method using the primer sets consisting of primers 5 and 6 of SEQ ID NO: 23 and 24 and the primer sets consisting of primers 7 and 8 of SEQ ID NO: 25 and 26, respectively, ​ the upstream of the 5'-end of the gene and ​ DNA fragments of approximately 1.0 kbp downstream of the 3'-end of the gene were amplified respectively. Each fragment was ligated by seamless cloning method using pK18 ​ previously digested with BamHI and NEBuilder HiFi DNA Assembly, ​ gene, ​ a plasmid pvanA2B2del for generating a gene region deletion strain was prepared.

[0140] Similarly, using the genomic DNA of the NGC7 strain as a template, by the PCR method using the primer sets consisting of primers 9 and 10 of SEQ ID NO: 27 and 28 and the primer sets consisting of primers 11 and 12 of SEQ ID NO: 29 and 30, respectively, ​ the upstream of the 5'-end of the gene and ​ DNA fragments of approximately 1.0 kbp downstream of the 3'-end of the gene were amplified respectively. Each fragment was ligated by seamless cloning method using pK18 ​ previously digested with BamHI and NEBuilder HiFi DNA Assembly, ​ gene, ​ a plasmid pvanA3B3del for generating a gene region deletion strain was prepared.

[0141] Similarly, using the genomic DNA of the NGC7 strain as a template, by the PCR method using the primer sets consisting of primers 13 and 14 of SEQ ID NO: 31 and 32 and the primer sets consisting of primers 15 and 16 of SEQ ID NO: 33 and 34, respectively, ​ the upstream of the 5'-end of the gene and ​ DNA fragments of approximately 1.0 kbp downstream of the 3'-end of the gene were amplified respectively. Each fragment was ligated by seamless cloning method using pK18 ​By ligating with seamless cloning method using NEBuilder HiFi DNA Assembly, ​ gene, ​ a plasmid pvanA4B4del for creating a gene region deletion strain was created.

[0142] As follows, the prepared plasmids pvanA1B1del, pvanA2B2del, pvanA3B3del and pvanA4B4del were each introduced into ​ sp.NGC7 strain by electroporation.

[0143] sp.NGC7 strain cells obtained by shaking culture in 10 mL of LB liquid medium were washed with 3 mL of 0.5 M sucrose aqueous solution and then suspended in 1 mL of 0.5 M sucrose aqueous solution. The cell suspension and the plasmid were mixed, and Gene Pulser Xcell ​ (Bio-Rad Laboratories) was used to apply at the conditions of 200 Ω, 25 μF, 2.5 kV. Immediately after the application, 1 mL of SOC (20 g / L tryptone, 0.5 g / L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2·6H2O, 10 mM MgSO4·7H2O) liquid medium was added, and the cells were cultured with shaking at 30 °C for 1 hour, and selected as Km-resistant strains that could grow on LB agar medium containing 50 mg / L of kanamycin (Km). TM (Bio-Rad Laboratories) was used to apply at the conditions of 200 Ω, 25 μF, 2.5 kV. Immediately after the application, 1 mL of SOC (20 g / L tryptone, 0.5 g / L yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2·6H2O, 10 mM MgSO4·7H2O) liquid medium was added, and the cells were cultured with shaking at 30 °C for 1 hour, and selected as Km-resistant strains that could grow on LB agar medium containing 50 mg / L of kanamycin (Km).

[0144] <00009​​​​​​​​​​​​​​[Evaluation of the growth of the NGC7ΔvanAB strain using vanillic acid (VA) and syringic acid (SA) as substrates] The NGC7 strain, NGC7Δ ​ strain, NGC7Δ ​ strain, NGC7Δ ​ strain and NGC7Δ ​ strain were each inoculated into 10 mL of LB liquid medium and subjected to shaking culture at 30 °C for 18 hours. The obtained culture broth was centrifuged to recover the cells, and the obtained cells were washed with carbon source-free Wx-solution [9.8 g / L Na2HPO4·12H2O, 1.7 g / L KH2PO4, 1.0 g / L (NH4)2SO4], and then 5 mM vanillic acid (VA) or 5 mM syringic acid (SA) as the sole carbon source was added to 0.2 mL of Wx liquid medium [9.8 g / L Na2HPO4·12H2O, 1.7 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 100 mg / L MgSO4·7H2O, 9.5 mg / L FeSO4·7H2O, 10.75 mg / L MgO, 2 mg / L CaCO3, 1.44 mg / L ZnSO4·7H2O, 1.​​​​​​​​​​​​​​​​​​​​​The strain multiplied, but NGC7Δ ​ The stock did not multiply.

[0148] However, as shown in Figure 2B, under culture conditions where SA is the sole carbon source, NGC7Δ ​ The strain showed a similar growth profile to the NGC7 strain. That is, ​ Genes and ​ Gene-encoded vanillates O - Demethylase is involved in the oxygenated vanillate degradation of NGC7 strain. O - It is a demethylase, and ​ Genes and ​ It was found that the ability to degrade SA can be maintained even when the gene is deleted.

[0149] [4.NGC7Δ ​ [Evaluation of the degradability of VA and SA contained in the stock] NGC7 strain, NGC7Δ ​ Stock, NGC7Δ ​ Stock, NGC7Δ ​ Stocks and NGC7Δ ​ Each strain was inoculated into 10 mL of LB liquid medium and subjected to overnight shaking incubation at 30°C. 0.1 mL of the resulting culture was inoculated into 10 mL of fresh LB liquid medium and subjected to 18 hours of shaking incubation at 30°C.

[0150] The obtained culture medium was subjected to centrifugation to collect the bacterial cells, and the obtained bacterial cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) medium, and OD 600 A 1 mL cell suspension was prepared such that the ratio was 2. To the obtained cell suspension, VA or SA was added to achieve a final concentration of 0.1 mM, and then the suspension was subjected to shaking culture at 30°C.

[0151] After the start of culture, samples were taken at regular intervals, and the concentrations of SA and VA were measured in the culture supernatant obtained by centrifugation of the culture medium.

[0152] OD 600 The "GeneQuant 100" (GE Healthcare Japan) was used for the measurements.

[0153] The concentrations of SA and VA were measured using a high-performance liquid chromatograph ("Acquity ultraperformance liquid chromatography system", Waters Japan). The column used was a TSKgel ODS-140HTP column (diameter 2.1 mm, length 100 mm, particle size 2.3 μm; Tosoh Corporation), and it was maintained at 30°C. In isocratic mode, the solvent used was 90% (v / v) H2O, 0.1% (v / v) HCOOH, and 10% (v / v) CH3CN. The flow rate of the mobile phase was 0.5 mL / min, the measurement wavelength for SA was 270 nm, and the measurement wavelength for VA was 260 nm.

[0154] Figure 3A and Figure 3B respectively show the reduction rates of the SA concentration and VA concentration at each measurement time based on the SA concentration and VA concentration at the start of cultivation.

[0155] As shown in Figure 3A, under the condition of adding VA, the NGC7 strain decomposed all the added VA, but the NGC7Δ ​ strain hardly decomposed VA. On the other hand, as shown in Figure 3B, under the condition of adding SA, the NGC7Δ ​ strain decomposed SA, similar to the NGC7 strain.

[0156] From the above results, ​ the gene and ​ the vanillate O -demethylase encoded by the gene is an oxygen-added type of vanillate O -demethylase involved in the VA decomposition of the NGC7 strain, and ​ the NGC7 strain lacking the gene and ​ the gene was found to be able to maintain the decomposability of SA.

[0157] [5. Evaluation of VA accumulation from lignin-derived aromatic compounds using the NGC7Δ ​ strain] NGC7Δ ​The strain was inoculated into 10 mL of LB liquid medium and incubated overnight at 30°C with shaking. 0.1 mL of the resulting culture was inoculated into 10 mL of fresh LB liquid medium and incubated at 30°C for 16 hours with shaking.

[0158] The obtained culture medium is subjected to centrifugation to collect the bacterial cells, the obtained bacterial cells are washed with physiological saline, and then resuspended in physiological saline, and OD 600 A cell suspension of 1 mL was prepared so that the ratio was 5. 0.2 mL of the resulting cell suspension was then mixed with SA and VA as carbon sources. p -Hydroxybenzoic acid (HBA), a mixture of SA and VA (SA-VA), a mixture of SA and HBA (SA-HBA), a mixture of VA and HBA (VA-HBA), or a mixture of SA, VA and HBA (SA-VA-HBA) was added to 10 mL of MMx-3 liquid medium [34.2 g / L Na2HPO4·12H2O, 6.0 g / L KH2PO4, 1.0 g / L NaCl, 2.5 g / L (NH4)2SO4, 49.3 mg / L MgSO4·7H2O, 15 mg / L CaCl2·2H2O, 5 mg / L FeSO4·7H2O] (each compound having a final concentration of 5 mM) and subjected to shaking culture at 30°C.

[0159] After the start of cultivation, samples are taken at regular intervals, and the OD of the culture medium is measured. 600 The following measurements were taken, and the concentrations of SA, VA, and HBA were measured in the culture supernatant obtained by centrifugation of the culture medium.

[0160] OD 600 The "OD-MonitorC&T" (Tytec Co., Ltd.) was used for the measurements.

[0161] The concentrations of SA, VA, and HBA were measured using a high-performance liquid chromatograph ("Agilent 1200 series," Agilent Technologies). A ZORBAX Eclipse Plus C18 column (4.6 mm diameter, 150 mm length, 0.5 μm particle size) was used and incubated at 40°C. A gradient elution mode was used (solvent A: 5% (v / v) CH3OH, 1% (v / v) CH3COOH; solvent B: 50% (v / v) CH3OH, 1% (v / v) CH3COOH). After equilibration with solvent A, the proportion of solvent B was increased to 20% over 8 minutes from the start of analysis, and then increased to 100% over 5 minutes. The mobile phase flow rate was 1.0 mL / min, and the measurement wavelengths were 254 nm and 280 nm.

[0162] The results of measurements using SA, VA, HBA, SA-VA, SA-HBA, VA-HBA, and SA-VA-HBA as carbon sources are shown in Figures 4A to 4G.

[0163] As shown in Figures 4A to 4G, NGC7Δ ​ The strains grew when at least one of SA and HBA was used as the carbon source, but did not grow when VA was used as the sole carbon source.

[0164] In particular, as shown in Figure 4F, when VA-HBA is used as the carbon source, NGC7Δ ​ Although the strain decomposed HBA, more than 98% of VA remained undecomposed and accumulated. As shown in Figure 4D, when SA-VA was used as the carbon source, SA was preferentially decomposed, and more than 90% of VA remained undecomposed and accumulated. Furthermore, as shown in Figure 4G, even when SA-VA-HBA was used as the carbon source, more than 80% of VA remained undecomposed and accumulated.

[0165] [6.NGC7Δ ​ [Evaluation of VA productivity from lignin-derived aromatic compounds using a strain] ​The sp.SYK-6 strain (hereinafter also referred to as the SYK-6 strain) possesses the ability to break down acetovanylone (AV) and acetosylingone, which are aromatic compounds derived from lignin. Furthermore, the SYK-6 strain has a group of AV-degrading enzyme genes involved in the degradation of AV, ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ gene, ​ Genes and ​ It possesses the gene. However, there are no reports of producing VA from AV using these enzyme genes. Therefore, as follows, NGC7Δ ​ We attempted to produce VA from AV by introducing a group of AV-degrading enzyme genes into the strain.

[0166] Following standard procedures, from the group of AV-degrading enzyme genes derived from the SYK-6 strain, the genomic DNA of the SYK-6 strain was selected. ​ gene, ​ gene, ​ gene, ​ gene, ​ Genes and ​ A nucleic acid fragment containing a gene is obtained, and this nucleic acid fragment is ligated under the control of an E. coli-derived lactose promoter inserted into pSEVA241 plasmid DNA, and plasmid pSEVA241_P lac -acv was obtained.

[0167] Similarly, from the genomic DNA of the SYK-6 strain ​ Genes and ​ A nucleic acid fragment containing the gene was obtained and ligated under the control of an E. coli-derived lactose promoter inserted into the pJB866 plasmid DNA to obtain plasmid pTS093_vceA-B.

[0168] pSEVA241_P lac - Using plasmid DNA from acv and pTS093_vceA-B, NGC7 strain and NGC7Δ ​ Transform NGC7[pSEVA241_P lac-acv,pTS093_vceA-B] strain and NGC7Δ ​ [pSEVA241_P lac Strains [-acv,pTS093_vceA-B] were obtained. These strains were inoculated into 10 mL of LB liquid medium containing 25 mg / L kanamycin (Km) and 15 mg / L tetracycline (Tc), and subjected to overnight shaking culture (180 rpm) at 30°C.

[0169] The obtained culture medium (0.1 mL) was inoculated into 10 mL of fresh LB liquid medium (containing 25 mg / L Km and 15 mg / L Tc) and subjected to shaking incubation at 30°C for 16 hours.

[0170] The obtained culture medium is subjected to centrifugation to collect the bacterial cells, the obtained bacterial cells are washed with MMx-3 medium, and then resuspended in MMx-3 medium, and OD 600 A cell suspension of 1 mL was prepared so that the ratio was 10. AV was added to the resulting cell suspension to a final concentration of 0.2 mM, and then the suspension was subjected to shaking culture at 30°C (1500 rpm).

[0171] After the start of culture, samples were taken at regular intervals, and the concentrations of AV, vanilloyl acetate (VAA), and VA were measured in the culture supernatant obtained by centrifugation of a portion of the culture medium. The measurements were performed using a high-performance liquid chromatograph with the apparatus and conditions described in Example 5 above. [NGC7[pSEVA241_P] lac Figure 5A shows the results measured using the strain [-acv,pTS093_vceA-B], and NGC7Δ ​ [pSEVA241_P lac The results using the strain [-acv,pTS093_vceA-B] are shown in Figure 5B.

[0172] As shown in Figure 5A, the NGC7 strain did not possess the ability to degrade AV, but it acquired the ability to degrade AV by introducing AV-degrading enzyme genes. However, because it possessed the ability to degrade VA, VA did not accumulate.

[0173] In contrast, as shown in Figure 5B, NGC7Δ lacks the decomposability of VA.​ It was found that by introducing a group of AV-degrading enzyme genes, the strain can degrade AV, accumulate VA, and consequently produce VA from AV.

[0174] [7. Preparation of lignin hydrolysates] Copper foam (1.7 cm × 5 cm, 0.5 g; Xiamen TOB New Energy Technology), washed with acetone and distilled water, was immersed for 1 hour at 30°C in a mixture of 100 mL of 0.12 M ammonium persulfate aqueous solution and 100 mL of 3 M sodium hydroxide aqueous solution. The copper foam after the above treatment was washed with distilled water and ethanol to prepare [Cu(OH)2 / CF] as a catalyst for lignin decomposition.

[0175] The obtained lignin-decomposing catalyst was cut into 10 mm diameter discs, and 10 of these discs were loaded into a stainless steel tube with an inner diameter of 10 mm to form a catalyst layer. A lignin solution, obtained by dissolving 10 g of sulfite lignin (Tokyo Chemical Industries Co., Ltd.) in 900 mL of 2 M sodium hydroxide aqueous solution, was introduced into this catalyst layer, which had been heated to 180 °C using a heater, at a rate of 0.225 mL / mL using a liquid delivery pump. In addition to the lignin solution, oxygen gas was introduced into the catalyst layer at a rate of 2 mL / min (at standard conditions). A back pressure valve was installed at the outlet of the catalyst layer, and the pressure inside the catalyst layer was adjusted to be maintained at 8 atmospheres as measured by a gauge.

[0176] The pH of the solution obtained from the catalyst layer was adjusted to 10 using hydrochloric acid. The adjusted solution was then passed sequentially through an MF membrane, a UF membrane ("MWCO 5,000Da"; MT, Synder), and a UF membrane ("MWCO 1,000Da"; GE, Suez) using a flat membrane testing machine "HP4750" (Steritech), and the filtrate was collected.

[0177] The obtained filtrate was adjusted to a pH of 3 using hydrochloric acid, and then subjected to three extraction treatments with ethyl acetate. The resulting ethyl acetate layer was evaporated to dryness to obtain an extract, and then the lignin degradation product was obtained by dissolving the obtained extract in a small amount of 2M NaOH and adjusting the pH of the solution with hydrochloric acid to 8.

[0178] The concentrations of the major aromatic monomers in the lignin degradation products were measured using a high-performance liquid chromatograph under the apparatus and conditions described in Example 5 above. The major aromatic monomers in the lignin degradation products were acetovanylone (AV), vanillin (VN), and vanillic acid (VA), with concentrations of 51.1 mM, 296.4 mM, and 113.8 mM, respectively.

[0179] [8.NGC7Δ ​ [Evaluation of VA productivity from a lignin degradation product model using strains] As a lignin degradation product model, a solution (pH 8.0) containing 81.5 mM AV, 292.0 mM VN, and 126.5 mM VA was prepared.

[0180] NGC7Δ as described in Example 6 above ​ [pSEVA241_P lac The strain [-acv,pTS093_vceA-B] was inoculated into 10 mL of LB liquid medium containing 25 mg / L Km and 15 mg / L Tc, and subjected to overnight shaking culture (180 rpm) at 30°C. 0.1 mL of the resulting culture was inoculated into 10 mL of fresh LB liquid medium (containing 25 mg / L Km and 15 mg / L Tc), and subjected to shaking culture at 30°C for 16 hours. The resulting culture was centrifuged to collect the cells, the obtained cells were washed with MMx-3 medium, and then resuspended in MMx-3 medium. 600 A 1 mL cell suspension was prepared so that the ratio was 10.

[0181] 10 mL of MMx-3 medium (containing 25 mg / L Km and 15 mg / L Tc) containing 0.75 mL of 200 g / L glucose solution and 0.1 mL of lignin degradation product model, initial OD 600 Cell suspension was added until the ratio was 0.1. The culture medium containing the obtained cells was subjected to shaking culture at 30°C. After the start of culture, samples were taken at regular intervals and the OD of the culture medium was measured. 600 The following measurements were taken, and the concentrations of AV, VN, VA, and glucose were measured in the culture supernatant obtained by centrifugation of the culture medium. 600The concentrations of AV, VN, and VA were measured using a spectrophotometer ("BioSpec-mini", Shimadzu Corporation). The concentrations of AV, VN, and VA were measured using a high-performance liquid chromatograph under the apparatus and conditions described in Example 5 above. The glucose concentration was measured using a "Biosensor BF-5" (Oji Instruments Co., Ltd.).

[0182] The measurement results are shown in Figure 6. As shown in Figure 6, NGC7Δ ​ [pSEVA241_P lac The strain [-acv,pTS093_vceA-B] produced VA in 97.3% yield by degrading acetovanilone and vanillin in the lignin degradation product model. The VA yield was calculated as [VA amount after 78 hours of culture (mol)] / [AV amount (mol) + VN amount (mol) + VA amount (mol) at the start of culture] × 100 (%).

[0183] [9.NGC7Δ ​ [Evaluation of VA productivity from lignin degradation products using strains] NGC7Δ as described in Example 6 above ​ [pSEVA241_P lac The strain [-acv,pTS093_vceA-B] was inoculated into 10 mL of LB liquid medium containing 25 mg / L Km and 15 mg / L Tc, and subjected to overnight shaking culture (180 rpm) at 30°C. 0.1 mL of the resulting culture was inoculated into 10 mL of fresh LB liquid medium (containing 25 mg / L Km and 15 mg / L Tc), and subjected to shaking culture at 30°C for 16 hours. The resulting culture was centrifuged to collect the cells, the obtained cells were washed with MMx-3 medium, and then resuspended in MMx-3 medium. 600 A 1 mL cell suspension was prepared so that the ratio was 10.

[0184] 10 mL of MMx-3 medium (containing 25 mg / L Km and 15 mg / L Tc) containing 0.75 mL of 200 g / L glucose solution and 0.108 mL of lignin hydrolysate prepared in Example 7 above, 600Cell suspension was added until the ratio was 0.1. The culture medium containing the obtained cells was subjected to shaking culture at 30°C. After the start of culture, samples were taken at regular intervals and the OD of the culture medium was measured. 600 The following measurements were taken, and the concentrations of AV, VN, VA, and glucose were measured in the culture supernatant obtained by centrifugation of the culture medium. 600 This was measured in the same manner as in Example 8 above.

[0185] The results are shown in Figure 7. As shown in Figure 7, NGC7Δ ​ [pSEVA241_P lac The strain [-acv,pTS093_vceA-B] produced VA in 90.7% yield by degrading acetovanylon and vanillin in lignin degradation products. The VA yield was calculated as [VA amount (mol) after 68 hours of culture] / [AV amount (mol) + VN amount (mol) + VA amount (mol) at the start of culture] × 100 (%).

[0186] [10.NGC7Δ ​ Δ ​ Δ ​ Δ ​ Δ ​ [Evaluation of VA productivity from lignin degradation products using strains] Using the genomic DNA of strain NGC7 as a template, PCR was performed using primer sets consisting of primers 17 and 18 for sequence numbers 38 and 39, and primer sets consisting of primers 19 and 20 for sequence numbers 40 and 41. ​ Upstream of the 5' end of the gene and ​ Approximately 1.2 kbp DNA fragments downstream of the 3' end of each gene were amplified. Each fragment was pre-digested with HindIII and BamHI to produce pK18. mobsacB By linking them using a seamless cloning method with NEBuilder HiFi DNA assembly, a new vanA2 gene vanB2 Plasmid pvanA2B2del2 was created for generating gene region deletion strains.

[0187] Similarly, using the genomic DNA of NGC7 strain as a template, PCR was performed using primer sets consisting of primers 21 and 22 for SEQ ID NOs. 42 and 43, and primer sets consisting of primers 23 and 24 for SEQ ID NOs. 44 and 45, respectively. vanA4 Upstream of the 5' end of the gene and vanB4 Approximately 1.2 kbp DNA fragments downstream of the 3' end of each gene were amplified. Each fragment was pre-digested with HindIII and BamHI to produce pK18. mobsacB By linking them using a seamless cloning method with NEBuilder HiFi DNA assembly, a new vanA4 gene vanB4 Plasmid pvanA4B4del2 was created for generating gene region deletion strains.

[0188] The genome of NGC7 strain contains Klebsiella pneumoniae The gene encoding the amino acid sequence PSN_1511( showed 50.9% sequence identity with the derived aminoglycoside-3'-phosphotransferase (P00552) aph The presence of the gene (sequence number 37) was confirmed. aph Since it was hypothesized that the NGC7 strain would acquire Km resistance due to the function of this gene, a mutant strain was created in which this gene was disrupted.

[0189] Similarly to the above, using the genomic DNA of NGC7 strain as a template, PCR was performed using primer sets consisting of primers 25 and 26 for SEQ ID NOs. 46 and 47, and primer sets consisting of primers 27 and 28 for SEQ ID NOs. 48 and 49. aph Approximately 1.2 kbp DNA fragments from the upstream 5' end and downstream 3' end of the gene were amplified. Each fragment was pre-digested with HindIII and BamHI to produce pK18. mobsacB By linking them using a seamless cloning method with NEBuilder HiFi DNA assembly, aph We created the plasmid paphdel for generating gene region deletion strains.

[0190] Plasmid pvanA4B4del2 to NGC7Δ vanA1B1 Introduced into the strain, on the genomic DNA vanA4 Genes and vanB4 NGC7Δ with a deletion in the internal region of the gene vanA1B1 Δ vanA4B4 A strain was prepared. Similarly, the obtained NGC7Δ vanA1B1 Δ vanA4B4 Plasmid paphdel was introduced into the strain, and the genomic DNA aph NGC7Δ with a deletion in the internal region of the gene vanA1B1 Δ vanA4B4 Δ aph A strain was created. Furthermore, the obtained NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Plasmid pvanA2B2del2 was introduced into the strain, and the genomic DNA vanA2 Genes and vanB2 NGC7Δ with a deletion in the internal region of the gene vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 A strain was created. And the resulting NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Plasmid pvanA3B3del was introduced into the strain, and the genomic DNA vanA3 Genes and vanB3 NGC7Δ with a deletion in the internal region of the gene vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 I created a stock.

[0191] In accordance with the standard method, plasmid pSEVA241_P described in 6 above lac -acv, using plasmid pTS093_vceA-B, NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 The strain was transformed into NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 [pSEVA241_P lac-acv,pTS093_vceA-B] strain was obtained.

[0192] NGC7Δ vanA4B4 [pSEVA241_P lac -acv,pTS093_vceA-B] Replace NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 [pSEVA241_P lac Except for using the strain [-acv,pTS093_vceA-B], the VA production capacity from lignin degradation products was evaluated using the method described in Example 9 above.

[0193] The results are shown in Figure 8. As shown in Figure 8, NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 [pSEVA241_P lac The strain [-acv,pTS093_vceA-B] degraded AV and VN in lignin degradation products to produce VA in a yield of 92.8%. The VA yield was calculated as [VA amount (mol) after 68 hours of culture] / [AV amount (mol) + VN amount (mol) + VA amount (mol) at the start of culture] × 100 (%). Note that in Figure 2A, Δ vanA1B1 Stocks, Δ vanA2B2 Shares and Δ vanA3B3 Despite the strain growing using VA as a carbon source, Δ in Figure 8 vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 The fact that the stock produced VA at a high yield is a surprising result.

[0194] [11. Sequence Listing] The sequences listed in the sequence listing are shown in Tables 6A and 6B below.

[0195] [Table 6A]

[0196] [Table 6B] [Industrial applicability]

[0197] According to one embodiment of the present invention, vanillic acid can be obtained from biomass containing lignin-derived aromatic compounds and vanillic acid by a transformed microorganism and a method for producing it. Vanillic acid can be converted into various industrially useful compounds and can be used, for example, as a raw material for vanillic acid derivatives that have applications such as heat-resistant and rigid plastics and coatings. Cross-reference to related applications

[0198] This application claims priority to Japanese Patent Application No. 2021-059621, filed on 31 March 2021, the entirety of which is incorporated herein by reference. Furthermore, the entirety of all documents cited in the detailed description of the invention of this application is incorporated herein by reference.

Claims

1. The host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), The vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and vanB4 gene (SEQ ID NO: 2) located on the chromosome are deleted. Transformed microorganisms.

2. The host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), The vanA4 gene (SEQ ID NO: 1) or the vanA4 gene and vanB4 gene (SEQ ID NO: 2) located on the chromosome are deleted, and The inserted acvA gene (SEQ ID NO: 3), acvB gene (SEQ ID NO: 4), acvC gene (SEQ ID NO: 5), acvD gene (SEQ ID NO: 6), acvE gene (SEQ ID NO: 7), acvF gene (SEQ ID NO: 8), vceA gene (SEQ ID NO: 9), and vceB gene (SEQ ID NO: 10) are expressed. Transformed microorganisms.

3. A step of obtaining vanillic acid by reacting a transformed microorganism according to Claim 2 with at least one aromatic compound derived from guaiacyllignin, selected from the group consisting of ferulic acid, vanillin, and acetovanylone. A method for producing vanillic acid, including the method described above.

4. A step to obtain vanillic acid by reacting a transformed microorganism according to Claim 2 with a mixture of at least one aromatic compound derived from guaiacyllignin selected from the group consisting of ferulic acid, vanillin, and acetovanylone, and at least one aromatic compound derived from p-hydroxyphenyllignin and / or syringyllignin selected from the group consisting of acetosyringone, p-hydroxyacetophenone, syringic acid, syringaldehyde, p-coumaric acid, p-hydroxybenzoic acid, and protocatechuic acid. A method for producing vanillic acid, including the method described above.

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