Method for producing terephthalic acid from biomass resources and method for producing polyester from biomass resources

Abstract

The present invention addresses the problem of providing: a method that enables production of terephthalic acid at low cost from a biomass resource by reducing energy consumption, using less number of steps, and improving the yield of terephthalic acid; and a method for producing polyester.　The method for producing terephthalic acid comprises a step for converting a biomass resource or a compound derived from a biomass resource, into p-tolualdehyde by means of microbes. The method for producing polyester comprises a step for producing polyester by causing a reaction between a diol compound and the terephthalic acid produced from the biomass resource.

Description

Technical Field

[0004] , , ,

[0001] The present invention relates to a method for producing terephthalic acid from biomass resources and a method for producing polyester from biomass resources.

Background Art

[0002] Terephthalic acid, which is a raw material for polyester, has conventionally been produced by subjecting naphtha derived from crude oil, which is a fossil resource, to a thermal decomposition process and then a distillation process to obtain p-xylene, and then chemically oxidizing the p-xylene. Fossil resources not only have a concern of depletion in the future, but also have a problem of increasing the amount of carbon dioxide emissions that cause global warming during combustion.

[0003] On the other hand, biomass resources are renewable resources, and in addition, they can fix carbon dioxide in the atmosphere by photosynthesis. Therefore, terephthalic acid and polyester produced from biomass resources are expected to reduce carbon dioxide emissions compared to terephthalic acid and polyester made from fossil resources. For this reason, methods for producing terephthalic acid and polyester from biomass resources have been proposed (see, for example, Patent Documents 1 to 3 and Non-Patent Documents 1 to 3).

[0004] Polyester is one of the synthetic resins most widely used in the world as various fibers, films, sheets, containers, etc. because it has excellent mechanical strength, chemical stability, transparency, and is inexpensive. Polyester is a high molecular compound obtained by polycondensation of a polyvalent carboxylic acid and a polyvalent alcohol. Polyester using terephthalic acid as the polyvalent carboxylic acid includes polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polybutylene terephthalate succinate (PETS), etc. PET is used for fibers, PET bottles, films, etc. Also, PTT is used for fibers, etc., and PBT is used for injection molded parts, etc. PBAT and PETS are used as biodegradable polymers. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2014-1257 [Patent Document 2] Japanese Patent Publication No. 2015-193647 [Patent Document 3] International Publication No. 2013 / 111782 [Non-patent literature]

[0006] [Non-Patent Document 1] "Suntory Holdings Limited and Suntory Food International Limited decide to construct a demonstration plant in the United States for the development of PET bottles using 100% plant-derived materials," [online], Internet<URL:https: / / www.suntory.co.jp / news / article / 12563.html> [Non-Patent Document 2] "Toray develops 100% plant-based synthetic fiber," [online], Nikkei Inc., [Accessed December 15, 2020], Internet <URL:https: / / www.nikkei.com / nkd / company / article / ?DisplayType=1&ng=DGKKZO55934740R20C20A2TJC000&scode=3401&ba=1> [Non-Patent Document 3] "Development of a method to produce PET resin raw materials from biomass resources unsuitable for food," [online], Japan Science and Technology Agency (JST), Gunma University, [Accessed December 15, 2020], Internet<URL:https: / / www.jst.go.jp / pr / announce / 20150204 / index.html> [Overview of the project] [Problems that the invention aims to solve]

[0007] As described above, methods for producing terephthalic acid using biomass resources as raw materials, and methods for producing polyester using terephthalic acid produced from biomass resources have been disclosed. However, both of these methods have the problem of high energy consumption and a large number of steps during terephthalic acid production.

[0008] Regarding the energy consumption during terephthalic acid production, the methods described in the above-mentioned Patent Documents 1-3 and Non-Patent Documents 1-3 have the problem of high energy consumption because the process for obtaining p-xylene from biomass resources involves thermal decomposition reactions at several hundred degrees Celsius, dehydration reactions, cyclization reactions, aromatization reactions, etc. Furthermore, while Patent Document 3 discloses a method for producing p-tolualdehyde from biomass resources, it also has the problem of high energy consumption because the aromatization reaction is carried out at a high temperature of 400°C, similar to the above.

[0009] Furthermore, the methods described in Patent Documents 1-3 and Non-Patent Documents 1-3 have the problem of having a large number of steps in the process of producing terephthalic acid from biomass resources. In the p-xylene production method disclosed in Patent Document 2, the chemical conversion process is long, with three steps, so large-scale equipment and a significant economic burden are required for the chemical conversion. Also, in the p-xylene production process disclosed in Patent Document 2, the chemical conversion process has two steps, so the equipment and economic burden for the chemical conversion are also large. In Non-Patent Document 3, the chemical conversion process from furfural to terephthalic acid has five steps, so as above, large-scale equipment and a significant economic burden are required. In Patent Documents 2 and 3, which disclose methods for producing p-xylene from biomass resources, at least one further chemical conversion step is required for the production of terephthalic acid. [Means for solving the problem]

[0010] In view of the above problems, the present invention has been diligently studied and has found a method for producing terephthalic acid and polyester that includes a step of converting biomass resources or compounds derived from biomass resources to p-tolualdehyde using microorganisms, with low energy consumption and in a short number of steps. Furthermore, it has been confirmed that terephthalic acid can be biotransformed from p-tolualdehyde produced in microorganisms by introducing a group of genes encoding four enzymes—benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase—into a transformant. The present invention has been completed based on these findings.

[0011] In other words, the present invention is as follows. [1] A method for producing terephthalic acid, comprising the step of converting a biomass resource or a compound derived from a biomass resource into p-tolualdehyde using microorganisms. [2] The method for producing terephthalic acid according to [1] above, wherein the compound derived from the biomass resource is at least one of shikimic acid, 1-p-tolylethanol, p-methylacetophenone, and p-methylbenzyl alcohol. [3] The method for producing terephthalic acid according to [1] or [2] above, wherein the biomass resource is a biomass resource that has been treated by a pretreatment step, a saccharification step, or both. [4] A method for producing terephthalic acid according to any one of [1] to [3] above, wherein the biomass resource is one or more of the following: edible biomass, monosaccharides or polysaccharides derived from edible biomass, non-edible biomass, or monosaccharides or polysaccharides derived from non-edible biomass. [5] The method for producing terephthalic acid according to [4] above, wherein the edible biomass includes at least one type of biomass selected from corn, sweet potato, rice, potato, wheat, barley, tapioca, sugarcane, beet, molasses, and high-test molasses. [6] The method for producing terephthalic acid according to [4] above, wherein the non-edible biomass includes at least one type of biomass selected from herbaceous biomass, woody biomass, paper, pulp, recycled paper, bagasse, and corn stover. [7] A method for producing terephthalic acid according to any one of [1] to [6] above, comprising the step of adding at least one enzyme selected from amylase, cellulase, hemicellulase, and lignin-degrading enzyme to a culture medium. [8] A method for producing terephthalic acid according to any one of [1] to [7] above, wherein p-tolualdehyde is biologically oxidized using microorganisms to produce terephthalic acid. [9] The method for producing terephthalic acid as described in [8] above, wherein the microorganism is the same as the microorganism used to produce p-tolualdehyde, and is a microorganism into which a group of genes encoding four types of enzymes, benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, have been introduced.

[10] A method for producing terephthalic acid according to any one of [1] to [9] above, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

[11] A method for producing polyester, comprising the step of reacting terephthalic acid produced by any of the methods for producing terephthalic acid described in [1] to

[10] above with a diol compound to produce polyester.

[12] A microorganism into which a group of genes encoding four enzymes, benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, has been introduced, and which is capable of producing p-tolualdehyde from biomass resources or compounds derived from biomass resources (hereinafter sometimes referred to as "the microorganism").

[13] The benzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 19 and retains benzaldehyde dehydrogenase activity. The toluate-methyl monooxygenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 and retains toluate-methyl monooxygenase activity. The 4-carboxybenzyl alcohol dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 and retains 4-carboxybenzyl alcohol dehydrogenase activity. The 4-carboxybenzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and retains 4-carboxybenzaldehyde dehydrogenase activity. The microorganisms described in

[12] above.

[14] The microorganism described in

[12] or

[13] above, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

[15] A method for producing a microorganism into which a gene group encoding four enzymes, benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, is introduced, wherein the microorganism is capable of producing p-tolualdehyde from biomass resources or compounds derived from biomass resources (hereinafter sometimes referred to as "the method for producing the microorganism").

[16] The benzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 19 and retains benzaldehyde dehydrogenase activity. The toluate-methyl monooxygenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20 and retains toluate-methyl monooxygenase activity. The 4-carboxybenzyl alcohol dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 and retains 4-carboxybenzyl alcohol dehydrogenase activity. The 4-carboxybenzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and retains 4-carboxybenzaldehyde dehydrogenase activity. The method according to

[15] above.

[17] The method according to

[15] or

[16] above, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

Advantages of the Invention

[0012] According to the present invention, by using a fermentation method using a microorganism, biomass resources can be converted into p-tolualdehyde at room temperature, so that the energy consumption can be significantly reduced. Further, in the present invention, p-tolualdehyde having the same carbon skeleton as terephthalic acid can be produced in one step by fermentation of a microorganism from biomass resources, so that the number of steps in producing terephthalic acid can be reduced. In addition, when a transformant obtained by introducing a gene group encoding four kinds of enzymes, benzaldehyde dehydrogenase, toluate methylmonooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, into a microorganism producing p-tolualdehyde is used, terephthalic acid can be biotransformed from p-tolualdehyde produced in the transformant, so that terephthalic acid can be produced in one step from biomass resources and the energy consumption can be significantly reduced. Therefore, the equipment cost can be suppressed as compared with other known methods, and the economic burden is small. Furthermore, it is also possible to provide a method for producing a polyester produced by this method.

Brief Description of the Drawings

[0013] [Figure 1] A diagram for explaining an overview of a method for producing terephthalic acid and polyester. [Figure 2] A diagram showing a first example of a production flow of terephthalic acid and polyester. [Figure 3] A diagram showing a configuration example of a culture device. [Figure 4] A diagram showing a second example of a second production flow of terephthalic acid and polyester. [Figure 5] A diagram showing a third example of a third production flow of terephthalic acid and polyester.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Referring to FIG. 1, an overview of a method for producing terephthalic acid and polyester will be described. FIG. 1(a) is a diagram for explaining a conventional method, and FIG. 1(b) is a diagram for explaining the method of the present invention. Here, as an example of polyester, the production of polyethylene terephthalate (PET) will be described.

[0015] In the conventional method, fossil resources such as crude oil are used as raw materials for terephthalic acid. Naphtha or the like obtained by refining crude oil is used as a raw material, thermally decomposed at a high temperature, fractionated by a fractionating device, and p-xylene is obtained as an intermediate product. p-Xylene is oxidized by supplying air under a catalyst such as cobalt, manganese, and bromine to produce terephthalic acid.

[0016] The produced terephthalic acid is dissolved in ethylene glycol produced from ethylene and polycondensed to produce polyethylene terephthalate (PET).

[0017] On the other hand, the method of the present invention uses biomass resources or compounds derived from biomass resources as raw materials, rather than fossil resources. Specifically, the method of the present invention includes a step of culturing microorganisms in the presence of biomass resources or compounds derived from biomass resources. Such cultivation can be carried out, for example, by solid-state culturing the microorganisms on biomass; by placing them on a culture medium solidified with agar, gellan gum, agarose, etc.; and / or by placing them in a liquid culture medium or shaking them in a liquid culture medium. Furthermore, the cultivation can be carried out under conditions such as an appropriate culture medium, an appropriate culture temperature, and an appropriate culture period. The culture medium is not particularly limited as long as it is a medium in which microorganisms can survive and grow, and examples include potato dextrose agar (PDA) medium, malt extract agar (MA) medium, LB agar medium, LB liquid medium, YM liquid medium, TB liquid medium, etc.

[0018] The culture temperature is not particularly limited, and is, for example, in the range of 15 to 40°C, preferably 20 to 38°C, more preferably 22 to 30°C, and even more preferably 24 to 28°C.

[0019] The above culture period is not particularly limited, but for example, it is in the range of 4 to 40 days, preferably 5 to 35 days, more preferably 5 to 30 days, and even more preferably 10 to 20 days.

[0020] When culturing microorganisms under shaking conditions, the shaking speed is, for example, in the range of 60 to 300 rpm, preferably 80 to 120 rpm.

[0021] Microorganisms decompose or alter biomass resources or compounds derived from biomass resources using their own enzymes, producing p-tolualdehyde, which has the same carbon skeleton as terephthalic acid, as an intermediate product. Therefore, another embodiment of the present invention is a method for producing p-tolualdehyde, which includes the step of culturing microorganisms in the presence of biomass resources or compounds derived from biomass resources. After carrying out such a production method, the culture solution containing p-tolualdehyde can be recovered by heating and then cooling it with a condenser or the like. Alternatively, p-tolualdehyde can be recovered while culturing by cooling the exhaust gas from the culture tank in which the microorganisms are being cultivated using a condenser or the like. Furthermore, p-tolualdehyde can be recovered from the culture solution containing p-tolualdehyde by extraction with an organic solvent. Any organic solvent can be used as long as it is not completely miscible with water; for example, petroleum ether, hexane, ethyl acetate, chloroform, and 4-methyltetrahydropyran can be used. These organic solvents may also be added during cultivation as long as they do not significantly inhibit the growth of the microorganisms. To increase the concentration of p-tolualdehyde to a desired level, purification methods such as chromatography and distillation can be used. The obtained p-tolualdehyde is then oxidized to produce terephthalic acid. While chemical oxidation by supplying air as described above is possible, a biological oxidation method using microorganisms (preferably the same microorganisms used to produce p-tolualdehyde) is preferred from the viewpoint of producing terephthalic acid with low energy consumption and in a short number of steps. When biologically oxidizing p-tolualdehyde to terephthalic acid, the p-tolualdehyde purified by the above method may be oxidized to terephthalic acid, or p-tolualdehyde may be oxidized to terephthalic acid by co-culturing a microorganism that produces p-tolualdehyde with a microorganism that oxidizes p-tolualdehyde to terephthalic acid.

[0022] One method for biologically oxidizing p-tolualdehyde using microorganisms is to biotransform p-tolualdehyde produced in microorganisms through the chemical reactions shown in formulas (I) to (IV) below into terephthalic acid. When producing terephthalic acid using this method, it is preferable that the microorganisms express four types of enzymes (benzaldehyde dehydrogenase [BZDH] which catalyzes the chemical reaction shown in formula (I) below; toluate methylmonooxygenase [TsaMB] which catalyzes the chemical reaction shown in formula (II) below; 4-CBAL dehydrogenase [TsaC] which catalyzes the chemical reaction shown in formula (III) below; and 4-CBA dehydrogenase [TsaD] which catalyzes the chemical reaction shown in formula (IV) below) (Reference "Nat Commun. 2017 May 31;8:15689.").

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] If the microorganism is not one that expresses the four enzymes described above (BZDH, TsaMB, TsaC, and TsaD), then a microorganism into which the gene group encoding the four enzymes has been introduced and which can produce p-tolualdehyde from biomass resources or compounds derived from biomass resources (i.e., the microorganism in question) is preferred. The microorganism in question can be obtained by carrying out a method that includes the step of introducing the gene group (polynucleotide) encoding the four enzymes described above into a microorganism capable of producing p-tolualdehyde from biomass resources or compounds derived from biomass resources (i.e., a method for producing the microorganism in question). Examples of such gene introduction methods include a method using calcium ions, a general competent cell transformation method, a protoplast transformation method, and an electroporation method.

[0028] The gene group encoding the four enzymes described above, which are used to introduce genes into microorganisms, is more specifically contained in a vector (either the same vector or separate vectors) to which a promoter is linked upstream so that it can be activated. Such a vector may further include enhancer regions or ribosome binding site (RBS) sequences to further increase gene expression efficiency, or it may further include drug resistance genes (selection marker genes) such as carboxyne resistance genes, spectinomycin resistance genes, chloramphenicol resistance genes, tetracycline resistance genes, kanamycin resistance genes, and ampicillin resistance genes for screening transformed strains. The enhancer region is usually located upstream of the promoter, and the RBS is usually located between the promoter and the gene group encoding the four enzymes described above.

[0029] In this specification, "promoter" means a region to which RNA polymerase (preferably RNA polymerase and a basic transcription factor) binds and initiates transcription of mRNA encoded by a gene located downstream thereof.

[0030] The origin of the genes encoding the four types of enzymes mentioned above is not particularly limited. For example, in the case of the gene encoding BZDH, examples include a gene encoding BZDH from Pseudomonas putida (preferably a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19 and retaining benzaldehyde dehydrogenase activity [specifically, activity that catalyzes the chemical reaction shown in formula (I)]), a gene encoding BZDH from Novosphingobium (Sphingomonas) aromaticivorans (preferably a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 23 and retaining benzaldehyde dehydrogenase activity [specifically, activity that catalyzes the chemical reaction shown in formula (I)]), or orthologs of these genes. In the case of the gene encoding TsaMB, for example, Comamonas Examples include genes encoding TsaMB derived from testosteroni (preferably a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 20 and retaining toluate-methyl monooxygenase activity [specifically, activity that catalyzes the chemical reaction shown in formula (II) above]), or orthologs of such genes, and in the case of genes encoding TsaC, for example, genes encoding TsaC derived from Comamonas testosteroni (preferably a polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 21 and retaining 4-carboxybenzyl alcohol dehydrogenase activity [specifically, activity that catalyzes the chemical reaction shown in formula (III) above]), or orthologs of such genes, and in the case of genes encoding TsaD, for example, Comamonas Examples include genes encoding TsaD derived from testosteroni (preferably polypeptides having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and retaining 4-carboxybenzaldehyde dehydrogenase activity [specifically, activity to catalyze the chemical reaction shown in formula (IV) above]), or orthologs of such genes.

[0031] A person skilled in the art can specifically and clearly determine the nucleotide sequences corresponding to the amino acid sequences of the four enzymes by referring to the amino acid sequences of the four enzymes and known codon tables corresponding to various microorganisms.

[0032] Conventional methods can be used to produce polyethylene terephthalate (PET) from terephthalic acid. The ethylene glycol that can be used in this invention may be ethylene glycol derived from fossil resources, but in order to reduce carbon dioxide emissions, which are a cause of global warming, it is desirable to use bioethylene glycol derived from bioethanol produced from biomass resources.

[0033] Thus, the method of the present invention uses biomass resources as raw materials and produces terephthalic acid by culturing microorganisms, eliminating the need for conventional high-temperature thermal decomposition reactions and significantly reducing energy consumption. Furthermore, since it does not require expensive equipment such as thermal decomposition furnaces, terephthalic acid and polyester can be produced at low cost.

[0034] The method of the present invention will be described in detail below with reference to the figures. Figure 2 shows a first example of the production flow of terephthalic acid and polyester. In the example shown in Figure 2, edible biomass containing sugars is used as the biomass resource, and sugars are used as the compound derived from the biomass resource. Edible biomass is a biomass resource that grows by photosynthesis and is a biomass resource that can be eaten (food). Sugars are monosaccharides or disaccharides that are nutrients that serve as an energy source for living organisms, such as glucose, fructose, galactose, mannose, arabinose, xylose, sucrose, and maltose. Sugars may also contain oligosaccharides.

[0035] There are no particular restrictions on edible biomass that are rich in sugars; examples include sugarcane, grapes, beets, molasses, and high-test molasses. Only one type of edible biomass containing sugars may be used, or two or more types may be used.

[0036] Starting from step 100, in step 101, edible biomass containing sugars is added to a culture apparatus in which microorganisms are cultured. The culture apparatus includes a reaction vessel having a culture medium for culturing microorganisms. Subsequently, the culture apparatus or culture medium components may be sterilized by autoclave or steam. In addition to edible biomass containing sugars, shikimic acid, 1-p-tolylethanol, p-methylacetophenone, and p-methylbenzyl alcohol derived from biomass resources may also be added to the culture apparatus. As a method for producing p-methylacetophenone from biomass resources, sugars derived from biomass resources can be produced by fermentation of microorganisms according to the present invention, but it can also be prepared by oxidizing citral contained in citrus fruits such as lemons, which are biomass resources.

[0037] In step 102, microorganisms in the culture device produce p-tolualdehyde from sugars contained in edible biomass. The process by which microorganisms produce intermediate products different from sugars, such as p-tolualdehyde shown in Figure 1(b), is called fermentation.

[0038] Any microorganism capable of producing p-tolualdehyde from sugars can be used for cultivation. Additionally, shikimic acid, 1-p-tolylethanol, p-methylacetophenone, and p-methylbenzyl alcohol may be added to the culture apparatus.

[0039] The microorganisms that can be used in the present invention are not particularly limited as long as they are microorganisms that use biomass resources or compounds derived from biomass resources as raw materials and have the ability to produce p-tolualdehyde. Specifically, for example, microorganisms belonging to the family Meruliaceae can be mentioned, and examples of microorganisms belonging to the family Meruliaceae include the genera Abortiporus, Amaurohydnum, Amauromyces, Aquascypha, Aurantiopileus, Aurantiporus, Bjerkandera, Bulbillomyces, Ceriporiopsis, Cerocorticium, Chrysoderma, Climacodon, Columnodontia, Conohypha, Coralloderma, Crustoderma, Crustodontia, C yanodontia, Cymatoderma, Diacanthodes, Elaphroporia, Gyrophanopsis, Hydnophlebia, Hyphoderma, Hyphodontiastra, Hypochnicium, Irpex, Lilaceophlebia, Luteoporia, Merulius, Mycoacia, Mycoaciell Microorganisms belonging to the genus A, Mycoleptodonoides, Niemelaea, Odoria, Phlebia, Phlebiporia, Pirex, Podocypha, Radulodon, Sarcodontia, Scopuloides, Stegiacantha, Uncobasidium can be mentioned; albida, Phlebia aurea, Phlebia brevispora, Phlebia centrifuga, Phlebia chrysocreas, Phlebia coccineofulva, Phlebia floridensis, Phlebia hydnoidea, Phlebia lindtneri, Phlebia lividina, Phlebia ludoviciana, Phlebia nantahaliensis, PhlebiaMicroorganisms belonging to the genus Hydnophlebia (e.g., Hydnophlebia alachuana, Hydnophlebia canariensis, Hydnophlebia gorgonea, Hydnophlebia meloi, Hydnophlebia ominivora, Hydnophlebia sinensis, Hydnophlebia subchrysorhiza, Hydnophlebia chrysorhiza) are preferred, with Phlebia uda (corresponding to "Mycoacia uda" used in this example) and Hydnophlebia chrysorhiza being preferred examples. These microorganisms can be obtained by isolating them from nature. They can also be obtained from sources such as the ATCC (American Type Culture Collection). Alternatively, these microorganisms can be subjected to physical or chemical mutation treatments to create high-productivity strains of p-tolualdehyde, and these high-productivity strains can then be used.

[0040] Microorganisms can be cultured using known fermentation methods such as batch culture, fed-batch culture, and continuous culture. The p-tolualdehyde obtained from the microbial culture can be purified to increase its purity before being converted to terephthalic acid. Any method that increases the purity of p-tolualdehyde can be used for purification. For example, the purification of p-tolualdehyde can be carried out by one or a combination of methods for purifying chemical products, such as solid-liquid separation, distillation, crystallization, decolorization, and desalting. Furthermore, the purification can also be carried out using separation techniques such as membrane separation and chromatography.

[0041] The batch culture method, as shown in Figure 3(a), involves placing a culture medium composition into the reaction vessel 10 when fermentation begins, sterilizing the culture medium components using an autoclave or steam, inoculating microorganisms into the culture medium 11, and culturing the microorganisms while adjusting pH, oxygen concentration, temperature, etc. The reaction vessel 10 is equipped with a stirrer 12 to maintain a uniform concentration of the culture medium 11 and dissolved oxygen concentration, and to supply oxygen to the culture medium. It is also desirable to supply sterilized air from the bottom of the reaction vessel 10. In the batch culture method, no carbon source or other nutrients for the microorganisms are added, so growth stops due to nutrient depletion. Therefore, in the batch culture method, microorganisms go from the induction phase to the logarithmic growth phase and finally enter a stationary phase where the growth rate decreases or stops.

[0042] The induction phase is a period in which microorganisms have not yet adapted to the environment and little growth occurs. To shorten the induction phase, it is desirable to increase the amount of microorganisms by pre-culturing them under appropriate conditions, and then add a few percent of the culture medium containing the microorganisms. The logarithmic growth phase is a period in which microbial growth occurs exponentially or logarithmically. Carbon sources include sugars contained in edible biomass and shikimic acid added to the reaction vessel. After culturing, the microbial cells can be aseptically collected and used in the next batch culture.

[0043] The fed-batch culture method is a method of gradually adding a carbon source as the fermentation process progresses. Therefore, as shown in Figure 3(b), the fed-batch culture method includes a storage tank 13 for storing the carbon source and a pump 14 for supplying the carbon source from the storage tank 13 to the reaction tank 10. The fed-batch culture method is useful when microbial metabolism tends to be suppressed by catabolite inhibition, and it is preferable to limit the amount of carbon source in the culture medium. Here, catabolite inhibition is a phenomenon in which the synthesis rate of a specific enzyme decreases due to the carbon source added to the culture medium.

[0044] The continuous culture method is a method of continuously supplying a predetermined amount of culture medium to a reaction vessel at a constant rate while simultaneously withdrawing the same amount of culture medium. Therefore, as shown in Figure 3(c), the continuous culture method is equipped with a pump 15 for supplying the culture medium 11 and a pump 16 for withdrawing the culture medium from the reaction vessel 10. Furthermore, the continuous culture method allows for the continuous replacement of part or all of the culture medium to replenish nutrients, preventing the accumulation of metabolic by-products and dead cells that may adversely affect the growth of microorganisms.

[0045] The microorganisms used in this invention can be immobilized and utilized using known methods such as carrier binding, crosslinking, and encapsulation. Furthermore, the culture apparatus used in this invention can include aerated and agitated culture tanks, air-lift culture tanks, packed tank culture tanks, and fluidized bed culture tanks.

[0046] The nutrients used for culturing microorganisms are not limited to carbon sources; they may also include nitrogen sources, various salts, vitamins, minerals, etc. Examples of nitrogen sources include yeast extract, peptone, various amino acids, soybean meal, corn steep liquor, urea, and various inorganic nitrogen compounds.

[0047] Step 103 involves producing terephthalic acid from p-tolualdehyde. Terephthalic acid is produced from p-tolualdehyde by either a chemical oxidation method or a biological oxidation method. A chemical oxidation method is, for example, oxidation with an oxygen-containing gas using a heavy metal salt or bromine compound as a catalyst (see, for example, Japanese Patent Publication No. 51-86437). A biological oxidation method is, for example, a method using genetically modified Escherichia coli (see, for example, Luo, Z., Lee, S. Biotransformation of p-xylene into terephthalic acid by engineered Escherichia coli. Nat Common 8, 15689(2017)(http: / / doi.org / 10.1038 / ncomms15689)).

[0048] In step 104, terephthalic acid is purified to improve its purity. The purification method can be one or a combination of any of the following chemical purification methods: solid-liquid separation, distillation, crystallization, decolorization, desalting, etc. Alternatively, the purification method can be carried out using separation techniques such as membrane separation or chromatography.

[0049] In step 105, polyester is produced by polymerization of purified terephthalic acid with a diol compound, and the process is completed in step 106. The polyesters produced include polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), and polybutylene terephthalate succinate (PETS). Known methods can be used to produce these polyesters by polymerization of terephthalic acid and a diol compound.

[0050] Figure 4 shows a second example of the production flow for terephthalic acid and polyester. In the example shown in Figure 4, edible biomass containing polysaccharides is used as the biomass resource, and polysaccharides are used as the compounds derived from the biomass resource. Examples of polysaccharides include cellulose, starch, hemicellulose, and pectin. Edible biomass containing polysaccharides is, for example, edible biomass containing starch, which is one type of polysaccharide (starch-based edible biomass).

[0051] The starch-based edible biomass is not particularly limited and can include, for example, corn, sweet potato, rice, potato, wheat, barley, tapioca, etc. Only one of the above starch-based edible biomass may be used, or two or more may be used. It is preferable to gelatinize the starch-based edible biomass by adding water and heating it to increase the hydrolysis reaction rate.

[0052] In the example shown in Figure 4, starting from step 200, step 201 hydrolyzes the polysaccharides contained in the edible biomass into monosaccharides. While microorganisms can be used as raw materials even in their polysaccharide form, it is preferable to hydrolyze the polysaccharides into monosaccharides before adding them to the culture medium, as this increases the rate of p-tolualdehyde production by the microorganisms in this invention. Furthermore, oligosaccharides may be present after the hydrolysis of the starch-based edible biomass.

[0053] The hydrolysis of polysaccharides to monosaccharides may be carried out using acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, or by enzymes produced by microorganisms. Examples of enzymes produced by microorganisms include amylase. When preparing glucose for use in this invention from starch-based edible biomass, it is desirable that the amylase used includes at least one type, such as liquefied amylase (α-amylase), saccharifying amylase (β-amylase), pullulanase, or glucoamylase. Depending on the type of enzyme, a pH and temperature suitable for the enzymatic reaction can be selected. There are no restrictions on the microorganisms that produce amylase; for example, microorganisms of the genera Aspergillus, Bacillus, and Pseudomonas can be used. Hydrolysis may be carried out in a separate tank from the reaction tank, or in the reaction tank itself. When carried out in the reaction tank, enzymes such as amylase can be added to the culture medium for the microorganisms.

[0054] Steps 202 onward are the same as those shown in Figure 2, from step 101 onward, so the explanation is omitted here. Note that in step 202, when adding edible biomass to the culture apparatus, microorganisms that produce enzymes such as amylase may be added. Alternatively, microorganisms that produce enzymes such as amylase may be cultured together with microorganisms that produce p-tolualdehyde from sugars.

[0055] Figure 5 shows a third example of the production flow for terephthalic acid and polyester. In the example shown in Figure 5, non-edible biomass is used as the biomass resource. Non-edible biomass, unlike edible biomass, is biomass that cannot be eaten. There are no particular restrictions on non-edible biomass, and examples include woody biomass and herbaceous biomass. Examples of woody biomass include coniferous trees such as pine, cedar, fir, spruce, Douglas fir, and radiata pine, and hardwoods such as beech, birch, alder, maple, eucalyptus, poplar, acacia, lauan, aspen, and rubber. Examples of herbaceous biomass include kenaf, Manila hemp, corn stover, corn cob, bamboo, bagasse, rice straw, rice husks, wheat straw, cotton linters, reeds, flax, Erianthus, paper mulberry, Mitsumata, soybean residue, and sweet potato stems and leaves. Similar to edible biomass, non-edible biomass is not limited to the use of only one type; multiple types may be used.

[0056] When using non-edible biomass as a raw material, it is preferable to use wood and other materials in a physically crushed state rather than using them as they are. This is because it allows chemicals to penetrate more easily when reducing the lignin content, which is bound to cellulose, one of the polysaccharides that make up wood and other materials, using chemicals.

[0057] In the example shown in Figure 5, starting from step 300, step 301 involves pretreatment of the non-edible biomass.

[0058] In the pretreatment stage, woody biomass is mechanically crushed into pieces approximately 2-3 cm in size and 5 mm thick to form wood chips. Paper, recycled paper, and pulp can also be used as raw materials. Paper, recycled paper, and pulp can be produced by known methods such as chemical pulping, mechanical pulping, and semi-chemical pulping (see, for example, Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press (1989)). It is desirable that the pulp contains a large amount of cellulose and hemicellulose, and may also contain lignin.

[0059] In the pretreatment, the material can be broken down into smaller pieces as described above to increase the specific surface area and facilitate chemical penetration. However, any other method that can reduce the lignin content can be employed. For example, known chemical pulp manufacturing methods can be used (see, for example, the Paper and Pulp Manufacturing Technology Series, Vol. 1 Kraft Pulp, Vol. 2 Mechanical Pulp, edited by the Japan Paper and Pulp Technology Association).

[0060] Furthermore, any chemical that can reduce the lignin content can be used during pretreatment. For example, sodium hydroxide, sodium sulfide, sodium sulfite, calcium sulfite, ozone, oxygen, chlorine, hypochlorous acid, hydrogen peroxide, chlorine dioxide, or a combination of at least two of these can be used.

[0061] In the pretreatment, a method may be employed in which non-edible biomass is treated under high temperature and pressure, either in the presence or absence of chemicals, in order to improve the separation and recovery of lignin. In addition, chemicals having a quinone structure, such as anthraquinones, or surfactants may be added simultaneously during pretreatment to increase the separation and recovery rate of lignin.

[0062] Next, in step 302, the polysaccharides in the non-edible biomass are hydrolyzed to monosaccharides, similar to the polysaccharide-containing polysaccharide biomass shown in Figure 4. The non-edible biomass can also be hydrolyzed using acids such as hydrochloric acid, sulfuric acid, and phosphoric acid. Alternatively, it can be hydrolyzed using enzymes produced by microorganisms. In the case of non-edible biomass, microorganisms that produce enzymes such as cellulase, hemicellulase, and lignin-degrading enzymes can be used.

[0063] Hydrolysis may be carried out in a separate tank from the reaction tank, or it may be carried out in the reaction tank. If carried out in the reaction tank, enzymes such as cellulase, hemicellulase, and lignin-degrading enzymes may be added to the culture medium for the microorganisms. Only one type of these enzyme may be added, or two or more types may be added.

[0064] Steps 303 onward are the same as those shown in Step 101 in Figure 2 and Steps 202 onward in Figure 4, so the explanation is omitted here. Note that when adding non-edible biomass in Step 303, microorganisms that produce enzymes such as cellulase, hemicellulase, and lignin-degrading enzymes may be added. Alternatively, microorganisms that produce enzymes such as cellulase, hemicellulase, and lignin-degrading enzymes may be cultured together with microorganisms that produce p-tolualdehyde from sugars.

[0065] Examples of microorganisms that produce cellulase include bacteria such as Acetobacter xylinum, Cellulomonas fimi, and Clostridium thermocellum. Examples of filamentous fungi include Aspergillus aculeatus, Aspergillus niger, Humicola grisea, Humicola insolens, Trichoderma reesei, and Trichoderma viridie. Furthermore, the cellulase produced by these microorganisms can also be used as an enzyme.

[0066] Examples of enzymes that make up cellulase include endoglucanase, cellobiohydrolase, cellobiose dehydrogenase, and β-glucosidase. The enzyme may consist of only one of these types, or it may consist of two or more types.

[0067] As the cellulase, commercially available cellulases such as Cellic Ctec3 (Novozemes) and Accellerase (DuPont Industrial Biosciences) can be used.

[0068] Regarding hemicellulase, if the non-edible biomass is broad-leaved trees or herbaceous non-edible biomass, it is desirable to primarily contain xylanase. If the non-edible biomass is coniferous trees, it is desirable to primarily contain mannanase.

[0069] As lignin-degrading enzymes, manganese peroxidase, lignin peroxidase, laccase, and versatile peroxidase derived from basidiomycetes can be used. When using manganese peroxidase, lignin peroxidase, or versatile peroxidase, it is desirable to add hydrogen peroxide to carry out the enzymatic reaction efficiently.

[0070] In this specification, "at least 90% sequence identity" means that the sequence identity with respect to the entire sequence of interest is 90% or more, preferably 91% or more, more preferably 92% or more, even more preferably 93% or more, even more preferably 94% or more, particularly preferably 95% or more, particularly more preferably 98% or more, and most preferably 100% identity.

[0071] In this specification, the term “sequence identity” means the degree of similarity between amino acid sequences (which is determined by matching the query sequence with other preferably identical sequences (protein sequences)). Preferred computer programs for calculating and determining "sequence identity" include, but are not limited to, GCG BLAST (Basic Local Alignment Search Tool) (Altschul et al., J.Mol.Biol.1990,215:403-410; Altschul et al., Nucleic Acids Res.1997,25:3389-3402; Devereux et al., Nucleic Acid Res.1984,12:387), BLASTN 2.0 (Gish W., http: / / blast.wustl.edu,1996-2002), and FASTA (Pearson and Lipman, Proc. Natl.Acad.Sci.USA 1988,85:2444-2448).

[0072] (Example 1) Mycoacia uda (ATCC76971) was cultured in the presence of edible biomass rich in sugars to produce p-tolualdehyde, after which terephthalic acid and polyester were produced by chemical synthesis.

[0073] (preculture) If isolated microorganisms are inoculated into a large volume of culture medium from the beginning, the induction period becomes longer and it takes time for the microorganisms to grow. Therefore, they are initially inoculated into a small volume of culture medium, allowed to grow, and then the volume of culture medium is gradually increased. The process of inoculating a small volume of culture medium and allowing the microorganisms to grow before inoculating into a large volume of culture medium is called pre-culturing.

[0074] Mycoacia uda (ATCC76971) was used as the microorganism, and as a pre-culture, it was cultured on potato dextrose agar (PDA) at 26°C for 1 week (7 days). Then, 10 cells were punched out using a 7 mm diameter cork porer, and inoculated into three 300 mL Erlenmeyer flasks containing 100 mL of YM liquid medium (0.3% yeast extract, 0.3% malt extract, 1% glucose, 0.5% peptone, pH 6.2), which had been sterilized in an autoclave (121°C, 20 minutes). Subsequently, the medium was stirred in a shaking incubator (26°C, 100 rpm) for 7 days to perform pre-culture.

[0075] (main culture) The culture medium after pre-culturing was filtered with Miracross (Merck), and the resulting bacterial cells were crushed with 100 mL of sterile water using a Waring blender. The crushed bacterial cell lysate was cultured for 2 weeks at 26°C in a 3 L jar fermenter (Able) using a medium containing 5% molasses and yeast nitrogen base (amino acid-free) as a nitrogen source.

[0076] (Extraction and purification of p-tolualdehyde) The culture medium after the initial culture was subjected to p-tolualdehyde extraction using an essential oil quantification device (compliant with the 16th edition of the Japanese Pharmacopoeia, manufactured by Shibata Chemical Co., Ltd.). The extraction method for p-tolualdehyde followed the 16th edition of the Japanese Pharmacopoeia, except that 2 mL of toluene was used instead of xylene in the grading tube of the quantification device. The extracted p-tolualdehyde contained toluene. Therefore, in order to separate the toluene, toluene was removed by vacuum distillation (vacuum distillation) using a rotary evaporator (manufactured by Buchi). Furthermore, p-tolualdehyde was purified by silica gel chromatography using a mixture of hexane and ethyl acetate in a 9:1 ratio as the developing solvent.

[0077] (Oxidation of p-tolualdehyde to terephthalic acid) Terephthalic acid is produced by oxidizing p-tolualdehyde. Therefore, purified p-tolualdehyde is oxidized, and the method described in Japanese Patent Publication No. 51-86437 was adopted as the oxidation method. Specifically, continuous oxidation of p-tolualdehyde was carried out in a continuous oxidation reactor equipped with a reflux condenser, a stirring device, and a heating device, and having a titanium pressure reactor (capacity 2.5 L) with a raw material inlet, a raw material gas inlet, a gas outlet, and a reactant outlet, and two reactant receivers connected in series to the reactant outlet.

[0078] A predetermined amount of catalyst metal and bromine compound-containing acetic acid (containing 5 wt% water) was pre-filled into the reactor, and after pressurizing with nitrogen to approximately 1 MPa, the temperature was raised to 210°C. After raising the temperature to 210°C, under conditions of approximately 1.8 MPa, a reaction solution consisting of p-tolualdehyde and acetic acid (water content 0.03 wt%) containing 0.0283 wt% cobalt, 0.0567 wt% manganese, and 0.121 wt% bromine as catalysts was continuously supplied at a supply rate of 225 g / hr of p-tolualdehyde and 1120 g / hr of acetic acid. The acetic acid containing cobalt, manganese, and bromine was prepared using cobalt acetate tetrahydrate, manganese acetate, and tetrabromoethane.

[0079] The reaction product was continuously withdrawn from the reactor in slurry form, filtered, and the cake was washed twice each with acetic acid and water, then dried to obtain terephthalic acid. The yield of the terephthalic acid obtained in this way was calculated to be 5.2% of the sugars contained in the molasses. The yield is the ratio of the amount of terephthalic acid actually obtained (yield) to the maximum amount of terephthalic acid that can be obtained theoretically (theoretical yield), multiplied by 100 and expressed as a percentage.

[0080] (Preparation of dimethyl terephthalate) Polyethylene terephthalate (PET), a type of polyester, is produced from terephthalic acid via dimethyl terephthalate.

[0081] 105 g of terephthalic acid derived from the biomass resources obtained above, 200 g of methanol, 15 mL of 98 wt% sulfuric acid, and 2.25 g of copper(II) sulfate pentahydrate were charged into a 500 mL autoclave, and an esterification reaction was carried out at 110 °C and 1.5 MPa for 1 hour. The reaction product was filtered off, and the esterification reaction was repeated under the same conditions. After that, the reaction product was filtered off and washed repeatedly with methanol. This yielded dimethyl terephthalate in a yield of 87 mol%. 0.018 wt% sodium carbonate was added to this dimethyl terephthalate, and simple distillation was carried out at 185 °C and 56 hPa to obtain dimethyl terephthalate derived from biomass resources.

[0082] (Preparation of polyethylene terephthalate (PET)) Biomass-derived ethylene glycol (manufactured by India Glycol) was refluxed under 1 hour with 1 wt% metallic sodium added and purified by distillation. 15.5 g of the biomass-derived dimethyl terephthalate obtained above, 11.8 g of purified ethylene glycol, 0.025 g of calcium acetate, and 0.01 g of antimony trioxide were weighed into a 100 mL round-bottom flask. A Claisen tube and an air condenser were attached to this flask. Nitrogen gas was introduced into the reaction system, and after removing the air, the mixture was heated to 180°C to melt the contents. A capillary tube was then inserted, and nitrogen gas was introduced into the reaction system. Methanol was then distilled off for 1 hour.

[0083] The mixture was heated at 200°C for 2 hours to remove methanol, then the temperature was raised to 220°C to distill off excess ethylene glycol. After 20 minutes at 220°C, the temperature was raised to 280°C and maintained for 15 minutes. A vacuum adapter was then attached, and the pressure was gradually reduced to 0.4 hPa. After 3 hours, the reactor was cooled while introducing nitrogen gas. After cooling, the mixture was vacuum dried at 150°C for 12 hours.

[0084] After drying, the material was melted at a spinning temperature of 285°C, extruded from a spinneret with a pore diameter of 0.18 mmφ, and taken up by a take-up roller to obtain undrawn yarn. The obtained undrawn yarn was drawn using a hot-roll drawing machine and heat-treated to obtain PET fibers. The obtained biomass resource-derived PET exhibited good polymer properties, spinning stability, and dye exhaustion rate.

[0085] (Example 2) Terephthalic acid and polyester were produced using starch-based edible biomass. When using starch-based edible biomass, it is desirable to hydrolyze polysaccharides into monosaccharides by saccharification treatment, so saccharification treatment was performed.

[0086] (Enzymatic saccharification of starch) Cornstarch (manufactured by Oji Cornstarch Co., Ltd.) was used as the starch-based edible biomass. Cornstarch was added to deionized water at a concentration of 30 wt% and sterilized by autoclaving (121°C, 20 minutes). After that, it was cooled to 90°C, and 0.2 wt% of heat-resistant α-amylase (Termamyl 120, manufactured by Novozymes) was added and the mixture was reacted with stirring for 3 hours. After that, it was cooled to 60°C, and after cooling, 0.35 wt% of glucoamylase (Allcoholase II L400, manufactured by Alltech) was added with stirring and the mixture was reacted with stirring for 2 hours to saccharify the starch.

[0087] After saccharification, in the same culture as in Example 1, enzymatically saccharified starch was added instead of molasses to a glucose concentration of 10 wt%. Otherwise, terephthalic acid and polyester were produced using the same method as in Example 1. The yield of terephthalic acid obtained in this way was 5.1% of the added starch. Furthermore, the obtained biomass resource-derived PET exhibited good polymer properties, spinning stability, and dye exhaustion rate.

[0088] (Example 3) Terephthalic acid and polyester were produced using non-edible biomass. When using non-edible biomass, the lignin content was reduced and polysaccharides were hydrolyzed to monosaccharides through pretreatment and saccharification.

[0089] In the main culture in Example 1, instead of molasses, deligned hardwood pulp (10 wt%), whose lignin content had been reduced using the chemical and physical treatments and lignin-degrading enzymes described above, was added. In addition, commercially available cellulase (Cellic CTec3, Novozymes) was added to the culture medium so that the enzyme activity was 10 U / mL. For reference, 1 U (enzyme unit) is the amount of enzyme that catalyzes a change of 1 μmol of substrate per minute under specified conditions. Terephthalic acid and polyester were produced in the same manner as in Example 1. The yield of terephthalic acid obtained in this way was 3.8% of the deligned hardwood pulp. Furthermore, the obtained biomass resource-derived PET had good polymer properties, spinning stability, and dye exhaustion rate.

[0090] (Example 4) Terephthalic acid and polyester were produced from p-methylacetophenone, a raw material derived from biomass resources.

[0091] Pre-culturing was carried out in the same manner as in Example 1, and in the main culture, p-methylacetophenone derived from biomass resources was added to the culture medium at a concentration of 1 wt%. The p-methylacetophenone derived from biomass resources was obtained by oxidizing citral obtained from lemon, a biomass resource. The oxidation of citral was carried out by the method described in the literature (Ueno et al., Formation Mechanism of p-Methylacetophenone from Citral via a tert-Alkoxy Radical Intermediate. J.Agric. Food Chem., 52, 5677-5684 (2004)). As a result, the yield of terephthalic acid was 1.5% of the added citral. Furthermore, the obtained biomass-derived PET had good polymer properties, spinning stability, and dye exhaustion rate.

[0092] While no literature has been found that has produced terephthalic acid from biomass resources and calculated its yield, there is literature disclosing the yield of a method for producing p-tolualdehyde from biomass-derived materials (Patent Document 3 above) and literature disclosing the yield of a method for producing terephthalic acid from p-tolualdehyde (Japanese Patent Publication No. 51-86437). Calculating the overall yield for obtaining terephthalic acid from biomass resources from these documents, it is approximately 3.8-4.9%.

[0093] In the method of the present invention, as shown in Examples 1 to 4, the yield is 1.5 to 5.2%, and although microorganisms are not used, it was found that terephthalic acid can be produced in a yield almost equivalent to that when terephthalic acid is produced from biomass resource-derived materials via p-tolualdehyde.

[0094] (Example 5) Genes for benzaldehyde dehydrogenase, toluate methylmonooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase were prepared for the basidiomycete [Mycoacia uda].

[0095] (5-1) Preparation of the benzaldehyde dehydrogenase gene for basidiomycetes [Mycoacia uda] The nucleotide and amino acid sequences of the benzaldehyde dehydrogenase gene from Pseudomonas putida are publicly known (accession number: AAN67564). To efficiently express benzaldehyde dehydrogenase (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 19) in basidiomycetes, the nucleotide sequence of the benzaldehyde dehydrogenase gene from Pseudomonas putida was optimized using geneious prime (Biomatters) based on codon usage frequency in basidiomycetes, and then the DNA was chemically synthesized and named MuBZDH. MuBZDH was cloned into pUC18 (Takara Bio Inc.) and named pMuBZDH.

[0096] (5-2) Preparation of toluate-methyl monooxygenase gene, 4-carboxybenzyl alcohol dehydrogenase gene, and 4-carboxybenzaldehyde dehydrogenase gene for basidiomycete [Mycoacia uda] The toluate-methyl monooxygenase gene for basidiomycete [Mycoacia uda], the 4-carboxybenzyl alcohol dehydrogenase gene for basidiomycete [Mycoacia uda], and the 4-carboxybenzaldehyde dehydrogenase gene for basidiomycete [Mycoacia uda] were prepared using the same method as described in item (5-1) above. The nucleotide and amino acid sequences of the toluate-methyl monooxygenase gene (tsaMB), the 4-carboxybenzyl alcohol dehydrogenase gene (tsaC), and the 4-carboxybenzaldehyde dehydrogenase gene (tsaD) from Comamonas testosteroni are publicly known [Reference (Junker F, Kiewitz R, Cook AM. Characterization of the p-toluenesulfonate operon tsaMBCD and tsaR in Comamonas testosteroni T-2. J Bacteriol. 179(3). 919-927. 1997)]. To efficiently express toluate-methyl monooxygenase (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20), 4-carboxybenzyl alcohol dehydrogenase (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 21), and 4-carboxybenzaldehyde dehydrogenase (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22) in basidiomycetes, the nucleotide sequences of the toluate-methyl monooxygenase gene (tsaMB), the 4-carboxybenzyl alcohol dehydrogenase gene (tsaC), and the 4-carboxybenzaldehyde dehydrogenase gene (tsaD) derived from Comamonas testosteroni were optimized using geneious prime (Biomatters) based on codon usage frequency in basidiomycetes, and then the DNA was chemically synthesized.The DNA encoding toluate-methyl monooxygenase optimized for basidiomycetes was named MutsaMB, the DNA encoding 4-carboxybenzyl alcohol dehydrogenase optimized for basidiomycetes was named MutsaC, and the DNA encoding 4-carboxybenzaldehyde dehydrogenase optimized for basidiomycetes was named MutsaD. MutsaMB, MutsaC, and MutsaD were cloned into pUC18 (manufactured by Takara Bio Inc.) and named pMutsaMB, pMutsaC, and pMutsaD, respectively.

[0097] (Example 6) Expression vectors for MuBZDH, MutsaMB, MutsaC, and MutsaD were constructed using the promoter and 3' terminal region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) derived from the basidiomycete Mycoacia uda.

[0098] (6-1) Construction of a benzaldehyde dehydrogenase (MuBZDH) expression vector using the promoter and 3' terminal region of the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene derived from the basidiomycete [Mycoacia uda]. The MuBZDH expression vector was constructed using the well-known double-joint PCR method in Mycoacia uda. Specifically, it was constructed according to the method described in the literature (Yu, JH, Hamari, Z., Han, KH, Seo, JA, Reyes-Dominguez, Y., Scazzocchio, “C.Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi.” Fungal Genetics and Biology. 41(11). 973-981. (2004).).

[0099] To express the benzaldehyde dehydrogenase gene (MuBZDH) prepared in (5-1) above in the basidiomycete Mycoacia uda, the promoter region of the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Mycoacia uda, which is known to be constitutively expressed, was used as the promoter. To obtain the promoter region of the GPD gene of Mycoacia uda, a PCR reaction was performed using Mycoacia uda genomic DNA as a template, with primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 2. The enzyme used for PCR was KOD plus, sold by Toyobo, and the PCR reaction was performed under the conditions of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes for 30 cycles. The 1 kbp DNA fragment amplified by the PCR reaction was purified using the QIAquick PCR Purification Kit (QIAGEN).

[0100] Next, using the pMuBZDH prepared in (5-1) above (i.e., a plasmid containing the nucleotide sequence of the benzaldehyde dehydrogenase gene (MuBZDH) optimized for basidiomycetes) as a template, a PCR reaction was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 3 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 4. The enzyme used for PCR was KOD plus, sold by Toyobo, and the PCR reaction was performed under the conditions of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes for 30 cycles. The DNA fragments amplified by the PCR reaction were purified using the QIAquick PCR Purification Kit (QIAGEN).

[0101] Furthermore, to stabilize MuBZDH expression, the 3' terminal region (0.8 kbp) of the GPD gene of Mycoasia uda (M. uda) was used. To amplify this region, PCR was performed using Mycoasia uda (M. uda) genomic DNA as a template, with primers consisting of the nucleotide sequence shown in SEQ ID NO: 5 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 6. The enzyme used for PCR was KOD plus, sold by Toyobo, and the PCR reaction was performed under the conditions of 30 cycles of PCR reaction at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The obtained 0.8 kbp DNA fragment was purified using the QIAquick PCR Purification Kit (QIAGEN).

[0102] Next, PCR reactions were performed using (1) the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of Mycoacia uda, (2) the benzaldehyde dehydrogenase gene optimized for basidiomycetes, and (3) the 3' terminal region of the GPD gene of Mycoacia uda, all amplified by the above PCR. KOD plus, sold by Toyobo, was used as the enzyme for PCR, and the PCR reaction was carried out under the conditions of 94°C for 1 minute, 60°C for 10 minutes, and 72°C for 5 minutes for 30 cycles. The DNA fragments after PCR were purified using the QIAquick PCR Purification Kit (QIAGEN).

[0103] Next, the purified PCR-treated DNA fragment was used as a template, and a PCR reaction was performed using primers consisting of SEQ ID NO: 1 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 6. KOD plus, sold by Toyobo, was used as the PCR enzyme, and the PCR reaction was performed under conditions of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 5 minutes for 30 cycles. The PCR-treated DNA fragment was purified using the QIAquick PCR Purification Kit (QIAGEN). Cloning was then performed using the TOPO TA Cloning Kit (Invitrogen). The resulting vector was named pGPDMuBZDH. To determine the nucleotide sequence, the obtained DNA fragment was subjected to a PCR reaction using nucleotide sequence analysis reagents from Applied Biosystems, and the reaction product was analyzed using an Applied Biosystems ABI PRISM 310 automated nucleotide sequencer.

[0104] (6-2) Construction of toluate-methyl monooxygenase gene expression vector, 4-carboxybenzyl alcohol dehydrogenase gene expression vector, and 4-carboxybenzaldehyde dehydrogenase gene expression vector using the promoter and 3' terminal region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) derived from the basidiomycete [Mycoacia uda]. The promoters of the GPD genes derived from the basidiomycete [Mycoacia uda] are ligated to the 5' ends of the toluate methyl monooxygenase gene (MutsaMB) for the basidiomycete [Mycoacia uda], the 4-carboxybenzyl alcohol dehydrogenase gene (MutsaC) for the basidiomycete [Mycoacia uda], and the promoters of the GPD genes derived from the basidiomycete [Mycoacia uda] are ligated to the 3' ends of each of these genes. The 3' terminal region of the GPD gene derived from uda) was ligated to construct toluate methyl monooxygenase gene expression vector (pGPDMutsaMB), 4-carboxybenzyl alcohol dehydrogenase gene expression vector (pGPDMutsaC), and 4-carboxybenzaldehyde dehydrogenase gene expression vector (pGPDMutsaD), respectively. In constructing these gene expression vectors, the PCR reaction was performed using the toluate methyl monooxygenase gene-containing vector (pMutsaMB), the 4-carboxybenzyl alcohol dehydrogenase gene-containing vector (pMutsaC), and the 4-carboxybenzaldehyde dehydrogenase gene-containing vector (pMutsaD) as templates, respectively, in the method for producing pGPDMuBZDH described in (6-1) above, instead of pMuBZDH.

[0105] In the process of constructing the toruate-methyl monooxygenase gene expression vector (pGPDMutsaMB), the amplification of the gpd promoter of Mycoacia uda was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 7. The amplification of the toruate-methyl monooxygenase gene was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 8 and SEQ ID NO: 9. The amplification of the 3' terminal region (0.8kbp) of the gpd gene of Mycoacia uda was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 10. The amplification of GPDMutsaMB was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 6. The PCR reaction conditions other than the primers were the same as in (6-1) above. After the PCR reaction, the DNA fragments were purified using the QIAquick PCR Purification Kit (QIAGEN) and then cloned using the TOPO TA Cloning Kit (Invitrogen). The resulting vector was named pGPDMutsaMB.

[0106] In the process of constructing the 4-carboxybenzyl alcohol dehydrogenase gene expression vector (pGPDMutsaC), the amplification of the gpd promoter of Mycoacia uda was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 11. The amplification of the 4-carboxybenzyl alcohol dehydrogenase gene was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 12 and SEQ ID NO: 13. The amplification of the 3' terminal region (0.8kbp) of the gpd gene of Mycoacia uda was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 6 and SEQ ID NO: 14. The amplification of GPDMutsaC was performed using primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 6. The PCR reaction conditions other than the primers were the same as in (6-1) above. The DNA fragments obtained after the PCR reaction were purified using the QIAquick PCR Purification Kit (QIAGEN), and then cloned using the TOPO TA Cloning Kit (Invitrogen). The resulting vector was named pGPDMutsaC.

[0107] In the process of constructing the 4-carboxybenzaldehyde dehydrogenase gene expression vector (pGPDMutsaD), the following primers were used to amplify the gpd promoter of Mycoacia uda: primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 15; the following primers were used to amplify the 4-carboxybenzaldehyde dehydrogenase gene: primers consisting of the nucleotide sequence shown in SEQ ID NO: 16 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 17; the following primers were used to amplify the 3' terminal region (0.8kbp) of the GPD gene of Mycoacia uda: primers consisting of the nucleotide sequence shown in SEQ ID NO: 6 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 18; and the following primers were used to amplify GPDMutsaD: primers consisting of the nucleotide sequence shown in SEQ ID NO: 1 and primers consisting of the nucleotide sequence shown in SEQ ID NO: 6. The PCR reaction conditions other than the primers were the same as in (6-1) above. The DNA fragments after the PCR reaction were purified using the QIAquick PCR Purification Kit (QIAGEN). Furthermore, cloning was performed using the TOPO TA Cloning Kit (Invitrogen). The resulting vector was named pGPDMutsaD.

[0108] (Example 7) We created a transformed strain of Mycoacia uda for the biosynthesis of terephthalic acid from biomass resources.

[0109] (7-1) Culture of mycelium In a 500 mL Erlenmeyer flask containing approximately 30 glass beads with a diameter of about 6 mm, 100 mL of SMY medium (1% sucrose, 1% malt extract, 0.4% yeast extract) was dispensed and sterilized. Then, 5 mm diameter agar pieces were punched out from Mycosia uda (ATCC76971) agar plates using a cork borer, inoculated onto the SMY medium, and incubated at 26°C for 7 days (pre-culture). However, the mixture was shaken 1-2 times a day to subdivide the hyphae. Next, 200 mL of SMY medium was dispensed into a 1 L Erlenmeyer flask, a stirrer was added, and after sterilization, the pre-cultured hyphae were filtered through a nylon mesh (pore size 30 μm), and the entire volume was inoculated and incubated at 26°C. The hyphae were subdivided by stirring with a stirrer for about 2 hours a day. This culture was carried out for 7 days.

[0110] (7-2) Preparation of protoplasts The above liquid cultured mycelium was filtered through a nylon mesh (pore size 30 μm) and washed with an osmotic pressure adjustment solution (0.5 M MgSO4, 50 mL maleate buffer (pH 5.6)). Next, 100 mg of wet cells were suspended in 1 mL of cell wall-degrading enzyme solution [5 mg of cellulase ONOZUKA RS (manufactured by Yakult Co., Ltd.) and 10 mg of Yatalase (manufactured by Takara Shuzo Co., Ltd.) dissolved in 1 mg of the above osmotic pressure adjustment solution], and incubated at 26°C for 3 hours with gentle shaking to release protoplasts.

[0111] (7-3) Purification of protoplasts After removing hyphae fragments from the enzyme reaction solution containing the protoplasts using a nylon mesh (pore size 30 μm), the remaining hyphae fragments and protoplasts on the nylon mesh were washed once with the osmotic pressure adjustment solution to increase the protoplast recovery rate. The resulting protoplast suspension was centrifuged (1,000 g, 5 minutes), the supernatant was removed, and the suspension was resuspended in 20 mM MOPS buffer (pH 6.3) containing 4 mL of 1 M sucrose. The centrifugation procedure was repeated, and the suspension was washed twice with the 1 M sucrose solution. The precipitate was suspended in 500 μL of a solution of 20 mM MES buffer (pH 6.4) containing 1 M sorbitol and 40 mM calcium chloride to obtain the protoplast suspension. This suspension was stored at 4°C. The protoplast concentration was determined by direct microscopy using a hemocytometer. All centrifugation operations were performed at room temperature using a swing rotor at 1,000 g for 5 minutes.

[0112] (7-4) Transformation of Mycoacia uda using benzaldehyde dehydrogenase gene expression vector (pGPDMuBZDH), toluate methyl monooxygenase gene expression vector (pGPDMutsaMB), 4-carboxybenzyl alcohol dehydrogenase gene expression vector (pGPDMutsaC), and 4-carboxybenzaldehyde dehydrogenase gene expression vector (pGPDMutsaD) To 100 μL of the protoplast suspension containing approximately 100 cells / 100 μL purified in (7-3) above, 2 μg each of the benzaldehyde dehydrogenase gene expression vector (pMuBZDH), toruate methyl monooxygenase gene expression vector (pGPDMutsaMB), 4-carboxybenzyl alcohol dehydrogenase gene expression vector (pGPDMutsaC), and 4-carboxybenzaldehyde dehydrogenase gene expression vector (pGPDMutsaD) constructed in (6-1) and (6-2) above, and a carboxyne resistance gene from Mycoasia uda prepared by a known method were added, and the mixture was cooled on ice for 30 minutes. Next, an equal volume of PEG solution (20 mM MOPS buffer (pH 6.4) containing 50% PEG3400) was added to the protoplast DNA mixture, and the mixture was cooled on ice for another 30 minutes. Next, the mixture was gently mixed into 10 mL of minimal agar medium (1% agar) containing 0.5 M sucrose and allowed to solidify. The mixture was then cultured at 26°C for several days. After 3 days of culture, 10 mL of minimal agar medium containing 2 μg / mL carboxyne was added as a top layer, and the culture was continued. After top layering, the grown transformants were selected.

[0113] (Example 8) Transformed organisms into which benzaldehyde dehydrogenase gene expression vectors (pGPDMuBZDH), toluate methyl monooxygenase gene expression vector (pGPDMutsaMB), 4-carboxybenzyl alcohol dehydrogenase gene expression vector (pGPDMutsaC), and 4-carboxybenzaldehyde dehydrogenase gene expression vector (pGPDMutsaD) were introduced using GPD promoters derived from the basidiomycete Mycoacia uda were cultured in the presence of edible biomass rich in sugars to produce terephthalic acid.

[0114] The transformants prepared in (7-4) above were cultured on potato dextrose agar at 26°C and then stored at 4°C. Pre-culture and main culture were performed in the same manner as in Example 1. Terephthalic acid content in the culture medium during the main culture was quantified by HPLC analysis according to a known method [Reference (Luo, Z., Lee, S. Biotransformation of p-xylene into terephthalic acid by engineered Escherichia coli. Nat Commun 8, 15689 (2017)).].

[0115] As a result, the yield of terephthalic acid was 5.0% of the sugars contained in molasses. Furthermore, the obtained biomass resource-derived PET exhibited good polymer properties, spinning stability, and dyeability. On the other hand, in the transformant prepared as a comparative example, which was introduced only with the carboxyne resistance gene from Mycoasia uda, terephthalic acid was not produced.

[0116] These results demonstrate that when microorganisms expressing four types of enzymes (benzaldehyde dehydrogenase [BZDH], toluate methyl monooxygenase [TsaMB], 4-carboxybenzyl alcohol dehydrogenase [TsaC], and 4-carboxybenzaldehyde dehydrogenase [TsaD]) are cultured in the presence of biomass resources, terephthalic acid can be produced from biomass-derived materials via p-tolualdehyde.

[0117] (Example 9) Hydnophlebia chrysorhiza was cultured in the presence of edible biomass rich in sugars to produce p-tolualdehyde, after which terephthalic acid and polyester were produced by chemical synthesis.

[0118] Instead of the microorganism used in Example 1 (Mycoacia uda ATCC76971), Hydnophlebia chrysorhiza FD-282 was used, and terephthalic acid and polyester were produced using molasses as a raw material in the same manner as in Example 1.

[0119] As a result, the yield of terephthalic acid was 4.8% of the sugars contained in molasses. Furthermore, the obtained biomass resource-derived PET exhibited good polymer properties, spinning stability, and dyeability.

[0120] From the above, the method of the present invention can produce terephthalic acid in a relatively high yield, can be produced at a milder temperature compared to conventional methods, and can reduce energy consumption. Furthermore, compared to conventional methods, the method of the present invention can produce terephthalic acid in fewer steps, such as the step of converting biomass resources or compounds derived from biomass resources to p-tolualdehyde using microorganisms, or can produce terephthalic acid in a single step by biotransformation of p-tolualdehyde produced in microorganisms into terephthalic acid. Since it does not use expensive furnaces or fractional distillation equipment required for pyrolysis, it can be produced at a low cost. As p-tolualdehyde and terephthalic acid are produced by culturing microorganisms, it is also possible to provide cultured and proliferated microorganisms.

[0121] Furthermore, the present invention also provides a method for producing terephthalic acid and a method for producing polyester, as well as terephthalic acid and polyester produced by these methods.

[0122] The present invention has been described in detail with reference to the embodiments described above regarding the method for producing terephthalic acid, the method for producing polyester, and the terephthalic acid and polyester produced by these methods. However, the present invention is not limited to the embodiments described above, and can be modified in any way that is conceivable by those skilled in the art, including additions, changes, and deletions. Any embodiment that achieves the function and effects of the present invention is included within the scope of the present invention. [Explanation of symbols]

[0123] 10…Reaction vessel 11…Culture medium 12…Agitator 13…Storage tank 14-16... Pump

Claims

1. A method for producing terephthalic acid, comprising the steps of: converting a biomass resource or a compound derived from a biomass resource into p-tolualdehyde using a microorganism capable of producing p-tolualdehyde; and biologically oxidizing the converted p-tolualdehyde using the microorganism to produce terephthalic acid, The microorganisms used in the production of terephthalic acid The microorganisms used to produce p-tolualdehyde are the same, and This microorganism has been genetically modified to contain a set of genes encoding four enzymes: benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase. The biomass resources include at least one edible biomass selected from corn, sweet potato, rice, potato, wheat, barley, tapioca, sugarcane, beet, molasses, and high-test molasses; at least one non-edible biomass selected from herbaceous biomass, woody biomass, paper, pulp, recycled paper, bagasse, and corn stover; the edible biomass; or monosaccharides or polysaccharides derived from the non-edible biomass. One or more of the following: The compound derived from the biomass resource is at least one of shikimic acid, 1-p-tolylethanol, or p-methylacetophenone. A method for producing the aforementioned terephthalic acid.

2. The method for producing terephthalic acid according to claim 1, wherein the biomass resource is a biomass resource that has been treated by a pretreatment step, a saccharification step, or both.

3. A method for producing terephthalic acid according to claim 1 or 2, comprising the step of adding at least one enzyme selected from amylase, cellulase, hemicellulase, and lignin-degrading enzyme to a culture medium.

4. A method for producing terephthalic acid according to any one of claims 1 to 3, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

5. A microorganism for use in a method for producing terephthalic acid according to any one of claims 1 to 3, wherein a gene group encoding four types of enzymes, benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, has been introduced, and which can produce p-tolualdehyde from biomass resources or compounds derived from biomass resources. The biomass resources include at least one edible biomass selected from corn, sweet potato, rice, potato, wheat, barley, tapioca, sugarcane, beet, molasses, and high-test molasses; at least one non-edible biomass selected from herbaceous biomass, woody biomass, paper, pulp, recycled paper, bagasse, and corn stover; the edible biomass; or monosaccharides or polysaccharides derived from the non-edible biomass. One or more of the following: The compound derived from the biomass resource is at least one of shikimic acid, 1-p-tolylethanol, or p-methylacetophenone. The aforementioned microorganism.

6. The benzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 19, and retains benzaldehyde dehydrogenase activity. The toluate-methyl monooxygenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and retains toluate-methyl monooxygenase activity. The 4-carboxybenzyl alcohol dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 and retains 4-carboxybenzyl alcohol dehydrogenase activity. The 4-carboxybenzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and retains 4-carboxybenzaldehyde dehydrogenase activity. The microorganism described in claim 5.

7. The microorganism according to claim 5 or 6, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

8. A method for producing a microorganism into which a gene group encoding four enzymes, benzaldehyde dehydrogenase, toluate methyl monooxygenase, 4-carboxybenzyl alcohol dehydrogenase, and 4-carboxybenzaldehyde dehydrogenase, is introduced, wherein the microorganism is capable of producing p-tolualdehyde from biomass resources or compounds derived from biomass resources, and is a microorganism for use in the method for producing terephthalic acid according to any one of claims 1 to 3. The biomass resources include at least one edible biomass selected from corn, sweet potato, rice, potato, wheat, barley, tapioca, sugarcane, beet, molasses, and high-test molasses; at least one non-edible biomass selected from herbaceous biomass, woody biomass, paper, pulp, recycled paper, bagasse, and corn stover; the edible biomass; or monosaccharides or polysaccharides derived from the non-edible biomass. One or more of the following: The compound derived from the biomass resource is at least one of shikimic acid, 1-p-tolylethanol, or p-methylacetophenone. The aforementioned method.

9. The benzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 19, and retains benzaldehyde dehydrogenase activity. The toluate-methyl monooxygenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20, and retains toluate-methyl monooxygenase activity. The 4-carboxybenzyl alcohol dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 and retains 4-carboxybenzyl alcohol dehydrogenase activity. The 4-carboxybenzaldehyde dehydrogenase has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 and retains 4-carboxybenzaldehyde dehydrogenase activity. The method according to claim 8.

10. The method according to claim 8 or 9, wherein the microorganism is Phlebia uda or Hydnophlebia chrysorhiza.

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