Rod-shaped bacterial transformants and methods for producing 2-phenylethanol using the same.

Abstract

The present invention provides a microorganism capable of producing 2-phenylethanol at a high concentration. In addition, the present invention provides a method for efficiently producing 2-phenylethanol using sugars as raw materials. The present invention relates to a coryneform bacterial transformant, which is activated by the shikimic acid pathway and a gene encoding an enzyme is introduced in an expressible manner, wherein the enzyme has phenylpyruvate decarboxylase activity. The present invention also relates to a method for producing 2-phenylethanol, which includes a step of reacting the coryneform bacterial transformant according to the present invention in water containing sugars.

Description

Technical Field

[0001] The present invention relates to a technology for producing 2-phenylethanol. More specifically, the present invention relates to a transformant of Corynebacterium glutamicum obtained by subjecting it to a specific gene manipulation to impart a 2-phenylethanol-producing function, and a technology for efficiently producing 2-phenylethanol using the transformant. Background Art

[0002] 2-Phenylethanol has a rose-like scent and is widely used in cosmetics, spices and fragrances.

[0003] Currently, 2-phenylethanol used in industry is mainly produced by organic synthesis using crude oil as raw material. On the other hand, the extraction and production of 2-phenylethanol from plant raw materials such as roses has problems such as raw material procurement and extraction efficiency, so it is very rare.

[0004] From the perspective of rising crude oil prices, unstable supply, and reducing greenhouse gas emissions from the global environment, fermentation production based on environmentally friendly bioprocesses using microorganisms to convert crude oil into biomass raw materials has attracted much attention. For example, the Ehrlich pathway exists in Kluyveromyces marxianus, which can produce 2-phenylethanol from phenylalanine, one of the amino acids.

[0005] However, phenylalanine, which is a raw material, is used as a food additive and feed additive as an essential amino acid, and is more expensive than sugar raw materials such as glucose. Therefore, it is not suitable for industrial use as a raw material for the production of 2-phenylethanol.

[0006] Non-patent document 1 reports the use of the following yeast gene recombinant to produce 1.3 g / L (about 11 mM) of 2-phenylethanol from glucose, wherein the above-mentioned yeast gene recombinant uses Kluyveromyces marxianus, which is conferred with resistance to phenylalanine analogs, as a host, highly expresses the phenylpyruvate decarboxylase gene (aro10) and the alcohol dehydrogenase gene (adh2), and introduces aroG (fbr) encoding a feedback inhibition mutant of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase from the pathogen Klebsiella pneumoniae.

[0007] Non-patent document 2 discloses a method for producing 2-phenylethanol from glucose using Escherichia coli into which a 2-ketoacid decarboxylase (kdc) gene of Saccharomyces cerevisiae has been introduced. Non-patent document 2 reports the production of about 8 mM of 2-phenylethanol from glucose.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent literature 1: Kim TY, Lee SW, Oh MK. Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus. Enzyme Microb. Technol. 61-62: 44-47 (2014).

[0011] Non-patent literature 2: Guo D, Zhang L, Kong S, Liu Z, Li X, Pan H. Metabolic engineering of Escherichia coli for production of 2-phenylethanol and 2-phenylethyl acetate from glucose. J Agric Food Chem. 66: 5886-5891 (2018). Summary of the invention

[0012] In view of the above background, it is expected that 2-phenylethanol will be produced more efficiently from cheap sugar raw materials such as glucose. However, the biosynthetic pathway of 2-phenylethanol starting from sugar raw materials such as glucose consists of more than dozens of multi-stage steps, and there are problems of being subject to various feedback controls and transcriptional controls. In addition, the strong cytotoxicity exhibited by 2-phenylethanol makes high production using biological methods more difficult.

[0013] The present invention provides a microorganism capable of producing 2-phenylethanol at a high concentration. In addition, the present invention provides a method for efficiently producing 2-phenylethanol using sugar as a raw material.

[0014] In one embodiment, the present invention relates to a coryneform bacterium transformant into which a gene encoding an enzyme having phenylpyruvate decarboxylase activity has been introduced in an expressible manner after activation of the shikimate pathway.

[0015] In another embodiment, the present invention relates to a method for producing 2-phenylethanol, comprising the step of reacting the coryneform bacterium transformant according to the present invention in water containing a sugar.

[0016] According to the present invention, in one or more embodiments, 2-phenylethanol can be efficiently produced from sugars using coryneform bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a graph showing the production of 2-phenylethanol under aerobic culture conditions.

[0018] Figure 2 It is a graph showing the production of 2-phenylethanol based on resin adsorption under aerobic reaction conditions.

[0019] Figure 3 It is a figure which shows the adaptability test as a host for 2-phenylethanol production. DETAILED DESCRIPTION

[0020] The present inventors have found that coryneform bacteria have superior resistance to 2-phenylethanol as compared with Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which is known as a solvent-resistant bacterium. Furthermore, the inventors have found that coryneform bacteria are superior to Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which are known as solvent-resistant bacteria. Furthermore, the inventors have found that coryneform bacteria are superior to Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which are known as solvent-resistant bacteria. Furthermore, the inventors have found that coryneform bacteria are superior to Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which are known as solvent-resistant bacteria. The inventors have found that coryneform bacteria are superior to Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which are known as solvent-resistant bacteria. The inventors have found that coryneform bacteria are superior to Kluyveromyces marxianus, Escherichia coli, and Pseudomonas putida S12, which are known as solvent-resistant bacteria, have superior resistance to 2-phenylethanol ...

[0021] Hereinafter, the present invention will be described in detail.

[0022] (I) Transformant with 2-phenylethanol production ability

[0023] In one embodiment, the transformant of the present invention is a coryneform bacterium transformant that has undergone activation of the shikimate pathway, and a gene encoding an enzyme having phenylpyruvate decarboxylase activity is introduced into the coryneform bacterium transformant in an expressible manner. According to the transformant of the present invention, 2-phenylethanol can be produced.

[0024] In the present invention, the method for introducing a gene into a coryneform bacterium may be introduction into a plasmid or integration into the genome.

[0025] Host

[0026] In one or more embodiments, the coryneform bacteria used as the host is Corynebacterium glutamicum. Corynebacterium glutamicum is a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974).

[0027] Specific examples include strains such as Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, ATCC13869 (DSM1412), ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13655, ATCC13745, ATCC13746, ATCC13761, ATCC14020, ATCC31831, MJ-233 (FERM BP-1497), and MJ-233AB-41 (FERM BP-1498). Among them, the R (FERM BP-18976) strain, the ATCC 13032 strain, and the ATCC 13869 (DSM 1412) strain are preferred, and the R (FERM BP-18976) strain is more preferred.

[0028] It should be noted that according to the classification of molecular biology, the names of coryneform bacteria such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum and Corynebacterium lilium are also unified as Corynebacterium glutamicum, and are therefore included in the present invention.

[0029] In addition, from the viewpoint of improving the production of 2-phenylethanol, the coryneform bacteria used as the host preferably block the byproduct production pathway of the 2-phenylethanol production pathway, and more preferably destroy all or part of the ldh (lactate dehydrogenase) gene, qsuB (3-dehydroshikimate dehydrogenase) gene, hdpA (dihydroxyacetone phosphatase) gene and cgR#1237 gene (phenylalanine uptake transporter).

[0030] In addition, from the same viewpoint, the coryneform bacteria as the host preferably reduce the consumption of phosphoenolpyruvate (PEP), and specifically, more preferably, the ptsH (phosphoenolpyruvate-dependent sugar phosphotransferase system) gene, which is one of the subunits of the glucose uptake transporter, is destroyed, and the iolT1 (Myo-inositol facilitator) gene is expressed instead. It is further preferred that iolT1 highly expresses the enzyme gene of glucose kinase (either or both of ATP-dependent and polyphosphate-dependent), which phosphorylates glucose taken into the cell.

[0031] Phenylpyruvate decarboxylase (pdc) gene

[0032] Phenylpyruvate decarboxylase (EC4.1.1.43) is an enzyme that catalyzes the reaction of producing phenylacetaldehyde from phenylpyruvate. Indole-3-pyruvate decarboxylase (EC4.1.1.74) or branched-chain 2-oxoacid decarboxylase (EC4.1.1.72) can be used to produce 2-phenylethanol as long as they can catalyze this reaction.

[0033] As one or more embodiments of genes encoding an enzyme having phenylpyruvate decarboxylase activity, genes such as ipd (indole-3-pyruvate decarboxylase) C, kivd (α-ketoisovalerate decarboxylase), kdc (α-ketoacid decarboxylase) A, aro10 (phenylpyruvate decarboxylase) and pdc (pyruvate decarboxylase) can be cited.

[0034] The source of the gene is a microorganism, a plant containing roses, or an animal, etc., and is not particularly limited. From the viewpoint of the productivity of 2-phenylethanol, the ipdC gene is preferably cited, and the ipdC gene or its homologous gene of Enterobacter bacteria is more preferably cited, and the ipdC gene or its homologous gene of Enterobacter cloacae is further preferably cited. The aro10 gene or its homologous gene of Saccharomyces cerevisiae can also be used. Phenylpyruvate decarboxylase activity can be measured by a known method, for example [Weiss, P., M., Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense. J. Bacteriol. 189: 7626-7633 (2007)].

[0035] One or more embodiments of the ipdC gene of Enterobacter cloacae are genes encoding an enzyme having phenylpyruvate decarboxylase activity, and examples include: (a) a gene having a base sequence as set forth in SEQ ID NO: 3, (b) a gene having a base sequence in which one or more bases are deleted, substituted or added to the base sequence as set forth in SEQ ID NO: 3, and having a base sequence having an identity of 45% or more to the base sequence as set forth in SEQ ID NO: 3, or (c) a gene that hybridizes under stringent conditions with a gene having a base sequence that is complementary to a gene having a base sequence as set forth in SEQ ID NO: 3.

[0036] In one or more embodiments, the consistency may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 98% or more.

[0037] The stringent conditions are general conditions, for example, those described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol 2, p11.45. Specifically, they refer to conditions where hybridization occurs at a temperature 5 to 10°C lower than the melting temperature (Tm) of a perfect hybrid.

[0038] Activation of the shikimate pathway

[0039] From the viewpoint of improving the production of 2-phenylethanol, it is preferred that the shikimate pathway of the coryneform bacteria used as the host is activated. In one or more embodiments, the activation of the shikimate pathway of the present invention is the enhancement of the shikimate pathway, and in another one or more embodiments, it includes heterologous expression and / or hyperexpression caused by the introduction of genes involved in the shikimate pathway.

[0040] In the present invention, in one or more embodiments, activation of the shikimate pathway comprises: introducing at least one gene selected from the following genes into a coryneform bacterium as a host, a gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity, a gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity, and a gene encoding an enzyme having shikimate kinase activity.

[0041] It should be noted that in the present invention, the genes related to the shikimate pathway are not limited to those described above, and other genes related to the shikimate pathway may also be introduced.

[0042] 3-Deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase

[0043] DAHP synthase is an enzyme that catalyzes the reaction of producing DAHP from PEP (phosphoenolpyruvate) and E4P (erythrose-4-phosphate). The genes encoding the enzyme having DAHP synthase activity are not particularly limited to microorganisms, plants including roses, animals, etc., and examples thereof include DAHP synthases aroF, aroG and aroH, and their orthologs. From the viewpoint of the productivity of 2-phenylethanol, preferably, a feedback inhibition-resistant mutant aroG gene or a homologous gene thereof is used, and more preferably, a feedback inhibition-resistant mutant aroG gene or a homologous gene thereof is used, which is derived from Escherichia bacteria such as Escherichia coli.

[0044] DAHP synthase activity can be measured by a known method, for example, [Liu YJ, Li PP, Zhao KX, Wang BJ, Jiang CY, Drake HL, Liu SJ. Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features. Appl Environ Microbiol. 74: 5497-503 (2008).].

[0045] Chorismate mutase / prephenate dehydratase

[0046] Chorismate mutase (EC 5.4.99.5) / prephenate dehydratase (EC 4.2.1.51) is a bifunctional enzyme that catalyzes the reaction of producing phenylpyruvate from chorismate. The gene encoding the enzyme having chorismate mutase / prephenate dehydratase activity is not particularly limited to a microorganism, a plant including rose, an animal, etc., but preferably, from the viewpoint of the productivity of 2-phenylethanol, a feedback inhibition-resistant mutant pheA gene is cited, and more preferably, a gene from Escherichia bacteria such as Escherichia coli is cited.

[0047] Chorismate mutase and prephenate dehydratase genes may be used as separate genes, but more preferably, they are mutants resistant to feedback inhibition.

[0048] Chorismate mutase activity and prephenate dehydratase activity can be measured by known methods, for example [Zhou H, Liao X, Wang T, Du G, Chen J. Enhanced l-phenylalanine biosynthesis by co-expression of pheA(fbr)and aroF(wt). Bioresour Technol. 101: 4151-4156 (2010).].

[0049] Shikimate kinase

[0050] Shikimate kinase (EC 2.7.1.71) is an enzyme that catalyzes the reaction that produces shikimate-3-phosphate from shikimate.

[0051] As the gene encoding the enzyme, its source is a microorganism, a plant or an animal including a rose, etc., and is not particularly limited. From the viewpoint of the productivity of 2-phenylethanol, preferably aroL (shikimate kinase) gene is cited, and more preferably aroL gene or a homologous gene of Escherichia bacteria such as Escherichia coli is cited, and further preferably aroL gene or a homologous gene of Escherichia coli is cited. It can be aroK (shikimate kinase) from Escherichia coli, aroK gene or a homologous gene from Corynebacterium glutamicum. Shikimate kinase activity can be measured by a known method, for example [R. C. DeFeyter, Purification and properties of shikimate kinase II from Escherichia coli K-12., J. Bacteriol. 165: 331-333 (1986)].

[0052] Alcohol dehydrogenase gene

[0053] From the viewpoint of improving the production of 2-phenylethanol, the transformant of the coryneform bacterium according to the present invention may further be introduced with an alcohol dehydrogenase gene.

[0054] Alcohol dehydrogenase (EC1.1.1.1) is an enzyme that catalyzes the reaction of producing alcohol from aldehyde. Here, it is an enzyme that catalyzes the reaction of producing 2-phenylethanol from phenylacetaldehyde. In addition to alcohol dehydrogenase, it can be phenylacetaldehyde reductase (EC1.1.1.-), aromatic alcohol dehydrogenase (or aliased as benzyl alcohol dehydrogenase) (EC1.1.1.90), aldehyde dehydrogenase and phenylethanol dehydrogenase, etc., as long as it can produce 2-phenylethanol from phenylacetaldehyde.

[0055] The enzyme gene may be derived from microorganisms, plants including roses, animals, etc., but is not particularly limited. From the viewpoint of the productivity of 2-phenylethanol, the yjgB (alcohol dehydrogenase) gene is preferred, the yjgB gene of Escherichia bacteria or a homologous gene thereof is more preferred, and the yjgB gene of Escherichia coli or a homologous gene thereof is further preferred.

[0056] In addition to yqhD (alcohol dehydrogenase) and yahK (aldehyde reductase) derived from Escherichia coli, adhC (alcohol dehydrogenase) similar to aryl alcohol dehydrogenase derived from Lactobacillus brevis, alcohol dehydrogenases derived from Saccharomyces cerevisiae, namely adh1 and adh2, or their orthologs may be used.

[0057] The alcohol dehydrogenase activity can be measured by a known method, for example [Retno Indrati, Purification and properties of alcohol dehydrogenase from a mutant strain of Candida guilliermondii deficient in one form of the enzyme Can. J. Microbiol., 38: 953-957 (1992)].

[0058] (II) Method for producing 2-phenylethanol

[0059] By reacting the transformant of the present invention described above in a reaction solution containing a carbon source, 2-phenylethanol can be produced. Therefore, in one embodiment, the present invention relates to a method for producing 2-phenylethanol, comprising the step of reacting the transformant of the present invention in water containing a sugar.

[0060] Proliferation of microorganisms

[0061] Prior to the reaction, the transformant is preferably cultured under aerobic conditions at a temperature of about 25 to 38°C for about 12 to 48 hours to allow it to proliferate.

[0062] Culture medium

[0063] The medium used for aerobic culture of the transformant before the reaction may be a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutrients, and the like.

[0064] As the carbon source, sugars (monosaccharides such as glucose, fructose, mannose, xylose, arabinose, and galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, and trehalose; polysaccharides such as starch; molasses, etc.), sugar alcohols such as mannitol, sorbitol, xylitol, and glycerol; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol and propanol; hydrocarbons such as normal alkanes, etc. can be used. One carbon source can be used alone, or two or more carbon sources can be used in combination.

[0065] As the nitrogen source, inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, urea, ammonia water, sodium nitrate, or potassium nitrate can be used. In addition, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ amine, protein hydrolyzate, or amino acids can also be used. One nitrogen source can be used alone, or two or more nitrogen sources can be used in combination. The concentration of the nitrogen source in the culture medium varies depending on the nitrogen compound used, and is usually set to about 0.1 to 10 (w / v%).

[0066] Examples of inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. The inorganic salts may be used alone or in combination of two or more. The concentration of the inorganic salts in the culture medium varies depending on the inorganic salt used, but is generally about 0.01 to 1 (w / v%).

[0067] Examples of nutrients include meat extract peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skimmed milk powder, defatted soybean hydrolyzate, extracts of plants, animals or microorganisms, and their decomposition products. The concentration of the nutrient in the culture medium varies depending on the nutrient used, but is generally about 0.1 to 10 (w / v) %.

[0068] Furthermore, vitamins may be added as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid. The pH of the culture medium is preferably about 6 to 8.

[0069] Specific preferred culture media for Corynebacterium glutamicum include A medium [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7: 182-196 (2004)], and BT medium [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 (2004)]. In these culture media, the sugar concentration may be set to the above range and used.

[0070] As the reaction solution, water, a buffer solution, an inorganic salt medium, or the like containing sugars can be used.

[0071] Examples of the buffer include phosphate buffer, Tris buffer, and carbonate buffer, etc. The concentration of the buffer is preferably about 10 to 150 mM.

[0072] In order to prevent the transformant from substantially growing, it is preferred that the reaction solution does not contain biotin, which is a growth factor.

[0073] As inorganic salt culture medium, there can be mentioned culture medium containing one or more of inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate or calcium carbonate. Among them, a culture medium containing magnesium sulfate is preferred. As inorganic salt culture medium, specifically BT culture medium [Omumasaba, CA et al, Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 (2004)] can be mentioned. The concentration of inorganic salts in the culture medium varies depending on the inorganic salt used, and is usually set to about 0.01 to 1 (w / v%).

[0074] The pH of the reaction solution is preferably about 6 to 8. The reaction is preferably carried out while controlling the pH of the reaction solution to near neutral, particularly about 7, using an aqueous ammonia solution, an aqueous sodium hydroxide solution, or the like, using a pH controller (e.g., manufactured by Able Co., Ltd., model: DT-1023).

[0075] Reaction conditions

[0076] The reaction temperature, that is, the survival temperature of the transformant during the reaction, is preferably about 20 to 50° C., more preferably about 25 to 47° C. When the temperature is within the above range, 2-phenylethanol can be produced efficiently.

[0077] The reaction time is preferably about 1 to 7 days, more preferably about 1 to 3 days. The culture may be carried out in a batch, fed-batch or continuous manner. Among them, the batch method is preferred.

[0078] Ventilation conditions

[0079] The reaction can be carried out under reducing conditions or microaerobic conditions. Corynebacterium glutamicum reacts without substantially proliferating under any conditions, and thus 2-phenylethanol can be produced more efficiently.

[0080] The reduction conditions are determined by the oxidation-reduction potential of the reaction solution, which is preferably about -200 mV to -500 mV, more preferably -250 mV to -500 mV.

[0081] The reduction state of the reaction solution can be easily estimated using a resazurin indicator (in the reduced state, the color changes from blue to colorless), and can be accurately measured using a redox potential difference meter (eg, ORPElectrodes manufactured by Broadley James).

[0082] The method for preparing the reaction solution under reducing conditions may be any known method without limitation.

[0083] For example, as a liquid medium for preparing a reaction solution, an aqueous solution for a reaction solution can be used instead of distilled water, etc. The method for preparing an aqueous solution for a reaction solution can refer to, for example, a method for preparing a culture solution for absolutely anaerobic microorganisms such as sulfuric acid-reducing microorganisms (Pfennig, N. et al, (1981): The dissimilatory sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria Ed. By Starr, MP et al., p. 926-940, Berlin, Springer Verlag.), "Agricultural Chemistry Experiment Book Vol. 3, Kyoto University Agricultural Department Agricultural Chemistry Classroom, 1990 26th printing, Industrial Book Co., Ltd. Published", etc., to obtain an aqueous solution under the desired reducing conditions.

[0084] Specifically, the aqueous solution for a reaction solution under a reducing condition can be obtained by removing dissolved gases by heating or reducing pressure treatment of distilled water or the like. In this case, the aqueous solution for a reaction solution under a reducing condition can be obtained by removing dissolved gases, particularly dissolved oxygen, by treating distilled water or the like under a reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, and more preferably about 3 mmHg or less for about 1 to 60 minutes, preferably about 5 to 40 minutes.

[0085] Alternatively, an appropriate reducing agent (eg, thioglycolic acid, ascorbic acid, cysteine ​​hydrochloride, mercaptoacetic acid, thioacetic acid, glutathione, or sodium sulfide) may be added to prepare an aqueous solution for a reaction solution under reducing conditions.

[0086] An appropriate combination of these methods is also an effective method for preparing an aqueous solution for a reaction solution under reducing conditions.

[0087] When maintaining the reducing conditions during the reaction, it is preferred to prevent the incorporation of oxygen from outside the reaction system as much as possible, and specifically, a method of sealing the reaction system with an inert gas such as nitrogen, carbon dioxide, etc. As a method for more efficiently preventing the incorporation of oxygen, in order to efficiently exert the metabolic function in the body of the coryneform bacterium transformant of the present invention during the reaction, it is sometimes necessary to add a pH-maintaining adjustment solution to the reaction system, and to appropriately add various nutrient dissolving solutions, but in the above case, it is effective to remove oxygen from the added solution in advance.

[0088] When the reaction is maintained under microaerobic conditions, the reaction can be carried out under the conditions of aeration of 0.5 vvm or less and stirring speed of 500 rpm or less. Depending on the situation, the reaction can be carried out under the conditions of blocking ventilation for an appropriate time after the reaction starts and stirring speed of 100 rpm or less in combination with an increased anaerobic state.

[0089] Recovery of 2-phenylethanol

[0090] By carrying out the cultivation in this way, 2-phenylethanol is produced in the reaction solution. The 2-phenylethanol can be recovered by recovering the reaction solution, or the 2-phenylethanol can be separated from the reaction solution by a known method. Examples of the known methods include distillation, membrane permeation, resin adsorption, and organic solvent extraction.

[0091] In one or more embodiments, the present invention relates to the following:

[0092] <1> A coryneform bacterium transformant in which the shikimate pathway is activated and into which a gene encoding an enzyme having phenylpyruvate decarboxylase activity is introduced in an expressible manner.

[0093] <2> The coryneform bacterium transformant according to <1>, wherein the gene encoding an enzyme having phenylpyruvate decarboxylase activity is an ipdC gene of Enterobacter cloacae.

[0094] <3> A rod-shaped bacterial transformant according to <1> or <2>, wherein the gene encoding an enzyme having phenylpyruvate decarboxylase activity comprises any one of the following genes: (a) a gene having the base sequence of SEQ ID NO: 3, (b) a gene having a base sequence in which one or more bases are deleted, substituted or added to the base sequence of SEQ ID NO: 3 and having a base sequence with an identity of 45% or more to the base sequence of SEQ ID NO: 3, and (c) a gene that hybridizes under stringent conditions with a gene having the following base sequence: the above-mentioned base sequence is a base sequence complementary to the gene having the base sequence recorded in SEQ ID NO: 3.

[0095] <4> A coryneform bacterium transformant according to any one of <1> to <3>, wherein activation of the shikimate pathway comprises: introducing at least one gene selected from the following genes into the coryneform bacterium as a host: a gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity, a gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity, and a gene encoding an enzyme having shikimate kinase activity.

[0096] <5> The coryneform bacterium transformant according to <4>, wherein the gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity and the gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity are resistant to feedback inhibition.

[0097] <6> A coryneform bacterium transformant according to <4> or <5>, wherein the gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity, the gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity, and the gene encoding an enzyme having shikimate kinase activity are derived from Escherichia coli.

[0098] <7> The coryneform bacterium transformant according to any one of <1> to <6>, wherein at least one gene selected from the group consisting of a lactate dehydrogenase gene, a 3-dehydroshikimate dehydratase gene, a dihydroxyacetone phosphatase gene, and a phenylalanine uptake transporter gene is disrupted.

[0099] <8> The coryneform bacterium transformant according to any one of <1> to <7>, wherein the coryneform bacterium as a host is Corynebacterium glutamicum.

[0100] <9> The coryneform bacterium transformant according to any one of <1> to <8>, wherein the coryneform bacterium as a host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 or ATCC13869 (DSM1412).

[0101] <10> A coryneform bacterium transformant, which is Corynebacterium glutamicum 2PE97 strain (Accession No.: NITE BP-02830).

[0102] <11> A method for producing 2-phenylethanol, comprising the step of reacting the coryneform bacterium transformant according to any one of <1> to <10> in water containing a sugar.

[0103] <12> The method for producing 2-phenylethanol according to <11>, wherein the sugar is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose and mannitol.

[0104] Example

[0105] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

[0106] [Example 1]

[0107] Construction of 2-phenylethanol producing strain

[0108] (1) Preparation and acquisition of chromosomal DNA

[0109] Corynebacterium glutamicum R(FERM BP-18976)、Azospirillum brasilenseNBRC 102289、Beijerinckia indica subsp.indica DSM1715、Bifidobacterium animalissubsp.lactis JCM 10602、Bradyrhizobium diazoefficiens JCM 10833、Bradyrhizobiumelkanii JCM 10832、Bradyrhizobium japonicum JCM 20679、Bradyrhizobium lablabiNBRC 108826、Burkholderia multivorans NBRC 102086、Caldilinea aerophila DSM14535、Chromohalobacter salexigens ATCC BAA-138、Comamonas testosteroni NBRC14951、Corynebacterium aurimucosum JCM 11766、Corynebacterium kroppenstedtiiJCM 11950、Debaryomyces hansenii JCM 1990、Delftia acidovorans JCM 5833、Desulfovibrio magneticus DSM 13731、Enterobacter cloacae NBRC 13535、Enterobacter hormaechei ATCC 49162、Erwinia herbicola NBRC 102470、Escherichiacoli K-12MG1655、Kluyveromyces lactis JCM 22014、Komagataella phaffii ATCC20864、Lachancea thermotolerans JCM 19085、Lactococcus lactis NBRC 100933、Mycobacterium smegmatis MC(2)155ATCC 700084、Nostoc sp.PCC 73102ATCC29133, Pandoraea vervacti NBRC 106088, Polaromonas naphthalenivorans ATCC BAA-779, Providencia rustigianii JCM 3953, Providencia stuartii ATCC 25827, Pseudomonasputida NBRC 14164, Ralstonia eutropha IAM 12368, Rhodococcus jostii JCM 11615, Rhodopseudomonas palustris ATCC BAA-98, Rhodospirillum rubrum ATCC 11170, Saccharomyces cerevisiae NBRC 2376, Saccharopolyspora erythraea JCM 4748, Staphylococcus epidermidis NBRC Chromosomal DNA of Staphylococcus haemolyticus JCSC 1435, Staphylococcus saprophyticus ATCC 15305, and Streptomyces lividans NBRC 15675 was cultured according to the information of the strain acquisition institution and prepared using a DNA genomic extraction kit (trade name: illustra bacteria genomic Prep Mini Spin Kit, GE Healthcare Japan Co., Ltd.). In addition, the pdc gene of Prunus persica and Rosa hybrid cultivar was obtained by GeneArt artificial gene synthesis (Thermo Fisher Scientific Co., Ltd.).

[0110] (2) Construction of 2-phenylethanol production-related gene expression plasmid

[0111] The gene and primer sequences used for isolating the target enzyme gene are shown in Table 1. PCR was performed using a Veriti Thermal Cycler (Thermo Fisher Scientific Inc.), and PrimeSTAR HSDNA Polymerase (Takara Bio Inc.) was used as a reaction reagent.

[0112] The obtained DNA fragment was introduced into a cloning vector containing the PgapA promoter (pCRB207 [Appl Environ Microbiol. 78 (3): 865-875 (2012)], pCRB209 [WO2012 / 033112], pCRB210 [WO2012 / 033112]). In addition, in the pheA gene, the amplified PCR fragments were linked using phosphorylated primers to introduce mutations into the gene. The introduced cloning vectors and the obtained plasmid names are shown in Table 2.

[0113] [Table 1]

[0114] Primers for Isolating Genes Related to 2-Phenylethanol Production

[0115]

[0116] *: Phosphorylated primer

[0117] [Table 2]

[0118] 2-Phenylethanol Production Related Gene Expression Plasmid

[0119] Gene source Enzyme gene Vector for introduction Plasmid name Azospirillum brasiliense pdc pCRB209 P2pe60 Caldilinea aerophila pdc pCRB209 P2pe83 Corynebacterium aurimucosum pdc pCRB209 PGibu50 Enterobacter cloacae iP pCRB210 PGibu37 Rhodopseudomonas palustris pdc1 pCRB209 PGibu52 Rhodospirillum rubrum pdc pCRB209 P2pe85 Escherichia coli aroL pCRB209 Pphe223 Escherichia coli pheA pCRB209 P2pe3

[0120] (3) Construction of plasmid for chromosome introduction of genes related to 2-phenylethanol production

[0121] Based on the sequence reported to be not essential for the proliferation of Corynebacterium glutamicum R strain [Appl.Environ.Microbiol.71:3369-3372 (2005)] (SSI region), the DNA region required for the marker-free introduction of 2-phenylethanol production-related genes into the chromosome of Corynebacterium glutamicum R strain was determined. The DNA region was amplified by the PCR method. The obtained DNA fragment was introduced into the marker-free gene introduction plasmid pCRA725 [J.Mol.Microbiol.Biotechnol.8:243-254 (2004), Japanese Patent Publication 2007-295809]. It should be noted that pCRG40 was introduced into the restriction enzyme site (unique site) for integrating genes in the SSI region by the inverse PCR method. The primer sequences used in the separation and inverse PCR of the SSI region and the obtained chromosome introduction vector are shown in Table 3.

[0122] [Table 3]

[0123] Primer sequences for isolating the SSI region and the resulting vector for chromosome introduction

[0124]

[0125] *: Primers used in inverse PCR

[0126] The PgapA promoter fusion enzyme gene fragment was obtained from the 2-phenylethanol production related gene expression plasmid constructed in Table 2 above and introduced into the above-mentioned chromosome introduction plasmid. In addition, tkt-tal gene expression plasmid pSKM8 [WO2016 / 027870], SSI8-1 region introduction plasmid pCRB278 [WO2017 / 169399], SSI5-1 region introduction plasmid pCRB276 [WO2017 / 169399] and SSI2-2 region introduction plasmid pCRB259 [WO2017 / 146241] were also used. The obtained 2-phenylethanol production related gene chromosome introduction plasmid is shown in Table 4.

[0127] [Table 4]

[0128] Plasmid for chromosome introduction of genes related to 2-phenylethanol production

[0129]

[0130] (4) Construction of plasmid for chromosomal gene disruption of Corynebacterium glutamicum R strain

[0131] Amplify the DNA region required for the chromosomal gene of the R strain of Corynebacterium glutamicum in a marker-free manner by PCR. Each PCR fragment can be linked by overlapping regions. The DNA fragment obtained is introduced into the plasmid pCRA725 [J.Mol.Microbiol.Biotechnol.8:243-254 (2004), Japanese Patent Publication 2007-295809] for marker-free gene disruption. The chromosomal gene disruption plasmid obtained is shown in Table 5.

[0132] [Table 5]

[0133] Plasmid for chromosome gene disruption of Corynebacterium glutamicum R strain

[0134]

[0135] *: Primers containing overlapping regions

[0136] (5) Construction of 2-phenylethanol production strain based on chromosomal gene recombination

[0137] The marker-free chromosomal gene introduction vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of a single crossover strain with a homologous region on the chromosome introduced into the plasmid pCRA725, kanamycin resistance based on the expression of the kanamycin resistance gene on pCRA725 and lethality in a sucrose-containing medium based on the expression of the sacR-sacB gene of Bacillus subtilis are shown, whereas in the case of a double crossover strain, kanamycin sensitivity based on the shedding of the kanamycin resistance gene on pCRA725 and proliferative properties in a sucrose-containing medium based on the shedding of the sacR-sacB gene are shown. Therefore, the marker-free chromosomal gene introduction strain shows kanamycin sensitivity and proliferative properties in a sucrose-containing medium.

[0138] By the above method, a gene recombinant strain was constructed using the above 2-phenylethanol production related gene chromosome introduction plasmid and chromosomal gene disruption plasmid. As host strains, Corynebacterium glutamicum R strain and ldhA disruption strain CRZ1 [Biotechnol Bioeng. Nov; 110(11): 2938-2948 (2013)] were used. In addition, plasmids pCRB285 for chromosome introduction of aroG (S180F) gene [WO2017 / 169399], plasmids pCRB292 for chromosome introduction of aroD gene [WO2017 / 169399], plasmids pCRB294 for chromosome introduction of aroE gene [WO2017 / 169399], plasmids pCRB289 for chromosome introduction of aroA gene [WO2017 / 169399], plasmids pCRB287 for chromosome introduction of aroCKB gene [WO2017 / 169399], plasmids pCRB294 for chromosome introduction of gapA gene [Appl Environ Microbiol. 78 (12): 4447-4457 (2012)], iolT1 gene chromosome introduction plasmid pSKM14 [WO2016 / 027870], qsuB gene disruption plasmid pSKM26 [WO2016 / 027870], hdpA gene disruption plasmid pSKM28 [WO2016 / 027870], and ptsH gene disruption plasmid pCRC809 [Microbiology. 155 (Pt11): 3652-3660 (2009)]. The outline of the chromosomal gene recombination is summarized in Tables 6 and 7.

[0139] [Table 6]

[0140] Construction of 2-phenylethanol production strain based on chromosomal gene recombination

[0141]

[0142] [Table 7]

[0143] Summary of 2-phenylethanol producing strain based on chromosomal gene recombination

[0144]

[0145] x3: represents the number of genes introduced into the chromosome.

[0146] (6) Construction of 2-phenylethanol production gene expression plasmid-introduced strain

[0147] A 2-phenylethanol producing strain was constructed by introducing the pdc gene and the ipdC gene into the above-mentioned chromosomal gene recombinant strain. The summary of the producing strain is shown in Table 8.

[0148] [Table 8]

[0149] Summary of 2-phenylethanol-producing strains

[0150] Construct strain name Host strain name Import plasmid Gene source 2PE97 LHglc1449 PGibu37 Enterobacter cloacae 2PE143 LHglc1449 P2pe60 Azospirillum brasilense 2PE144 LHglc1449 PGibu50 Corynebacterium au rimucosum 2PE145 LHglc1449 PGibu52 Rhodopseudomonas palustris 2PE146 LHglc1449 P2pe83 Caldilinea aerophila 2PE147 LHglc1449 P2pe85 Rhodospirillum rubrum

[0151] These strains were cultured in test tubes, and the following production test using tank culture was performed on strains 2PE97 and 2PE143 to 2PE147 that produced 38 mM or more of 2-phenylethanol after 48 hours at 33°C.

[0152] [Test on production of 2-phenylethanol from glucose by strains introduced with a gene encoding an enzyme having phenylpyruvate decarboxylase activity]

[0153] The 2-phenylethanol production gene-introduced strain (2PE97, 143-147 strain) was spread on A agar medium containing 50 μg / ml of kanamycin [(NH2)2CO 2g, (NH4)2SO4 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4·7H2O0.5g, 0.06% (w / v) FeSO4·7H2O+0.042% (w / v) MnSO4·2H2O 1ml, 0.02% (w / v) biotin solution 1ml, 0.01% (w / v) thiamine solution 2ml, yeast extract 2g, vitamin analysis casein hydrolyzate 7g, glucose 40g, and agar 15g were suspended in distilled water 1L] and cultured statically at 33°C for 18 hours.

[0154] The 2-phenylethanol-producing gene-introduced strain (2PE97, 143-147 strain) proliferated in one platinum loop on the above plate was inoculated into a test tube containing 10 ml of A liquid medium [(NH2)2CO 2 g, (NH4)2SO4 7 g, KH2PO4 0.5 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, 0.06% (w / v) FeSO4·7H2O+0.042% (w / v) MnSO4·2H2O 1 ml, 0.02% (w / v) biotin solution 1 ml, 0.01% (w / v) thiamine solution 2 ml, yeast extract 2 g, vitamin analysis casein hydrolyzate 7 g, and glucose 40 g dissolved in distilled water 1 L] containing 50 μg / ml of kanamycin, and cultured aerobically at 33°C for 18 hours.

[0155] The culture solution thus obtained was transferred to a 100 mL tank fermentation device so that the initial OD610 was 0.8, glucose (initial concentration 100 g / L) was added as a substrate to the A medium, and the pH of the culture reaction solution was adjusted to not less than 7.0 using 2.5 N ammonia water at 33°C, and cultured under aeration conditions. Glucose was appropriately added when the glucose concentration decreased.

[0156] The sampled culture medium was centrifuged (4°C, 10,000×g, 5 minutes), and the obtained supernatant was used for product analysis. The glucose concentration in the culture medium was measured by a glucose sensor (OSI BF-5D). The concentration of 2-phenylethanol was measured using an HPLC system manufactured by Shimadzu Corporation, and the separation column was Cosmosil C18-AR-II from Nacalai Tesque. The separation conditions based on HPLC were: 40% methanol and 0.069% perchloric acid as the mobile phase, a flow rate of 1.0 ml / min, and a column temperature of 40°C.

[0157] As described above, the gene encoding the enzyme having phenylpyruvate decarboxylase activity was cloned and introduced into the coryneform bacterium 2-phenylethanol production host LHglc1449 strain for investigation. Table 9 shows the host, culture, and recombinant strains showing a productivity of 25 mM or more of 2-phenylethanol concentration after 48 hours of culture under the reaction conditions. The 2PE97 strain produced the highest concentration of 2-phenylethanol.

[0158] [Table 9]

[0159] Comparison of 2-phenylethanol production concentrations in strains introduced with various enzyme genes having Pdc activity

[0160] Genetic recombinant strain 2-Phenylethanol concentration (mM) 2PE97 55.6 2PE143 47.7 2PE144 48.9 2PE145 48.2 2PE146 39.6 2PE147 26.1

[0161] Corynebacterium lutamicum 2PE97 strain was deposited in the National Patent Microorganism Depository of Japan (NPMD), KAZUSA, 2-5-8, Room 122, Kisarazu, Chiba Prefecture, Japan (Postal Code: 292-0818) (Deposit date: November 22, 2018, deposit number: NITE BP-02830).

[0162] [Example 2]

[0163] <2-phenylethanol production experiment using a 2-phenylethanol-producing gene-introduced Corynebacterium glutamicum strain>

[0164] The 2-phenylethanol-producing gene-introduced strain of Corynebacterium glutamicum (strain 2PE97) was spread on an A agar medium (same as above) containing 50 μg / ml of kanamycin, and statically cultured at 33°C for 18 hours.

[0165] One platinum loop of the 2PE97 strain proliferated in the plate was inoculated into a test tube containing 10 ml of liquid medium A (same as above) containing 50 μg / ml of kanamycin, and cultured aerobically at 33°C for 18 hours.

[0166] The culture solution thus obtained was transferred to a 100 mL tank fermentation device so that the initial OD610 was 0.8, glucose (initial concentration 100 g / L) was added as a substrate to the A medium, and the pH of the culture solution was adjusted to not less than 7.0 using 2.5 N ammonia water at 33°C, and cultured under aeration. Glucose was appropriately added when the glucose concentration decreased.

[0167] The sampled culture medium was centrifuged (4°C, 10,000×g, 5 minutes), and the obtained supernatant was used to analyze the product. The glucose concentration in the culture medium was measured by a glucose sensor (OSI BF-5D). The concentration of 2-phenylethanol was determined using an HPLC system manufactured by Shimadzu Corporation, and the separation column was Cosmosil C18-AR-II manufactured by Nacalai Tesque. For the HPLC-based separation conditions, 40% methanol and 0.069% perchloric acid were used as the mobile phase, the flow rate was set to 1.0 ml / min, and the column temperature was set to 40°C. After reacting for 48 hours, the yield of 2-phenylethanol was 6.5 g / L ( Figure 1 ). On the other hand, the recombinant strain into which the phenylpyruvate decarboxylase gene was not introduced and used as a control did not produce 2-phenylethanol at all.

[0168] The 2PE97 strain prepared in Example 1 was spread on A agar medium (same as above) containing 50 μg / ml of kanamycin, and allowed to stand at 33°C for 16 hours.

[0169] One platinum loop of the 2PE97 strain proliferated in the plate was inoculated into a test tube containing 10 ml of liquid medium A (same as above) containing 50 μg / ml of kanamycin, and cultured aerobically at 33°C with shaking for 16 hours.

[0170] The 2PE97 strain grown under the above conditions was inoculated into a 2 L conical flask containing 500 ml of liquid medium A (same as above) containing 50 μg / ml of kanamycin, and cultured aerobically at 33°C with shaking for 16 hours.

[0171] The bacterial cells cultured and proliferated in this way were recovered by centrifugation (4°C, 5000×g, 10 minutes). The obtained bacterial cells were suspended in 400 ml of BT (-urea) liquid medium [0.7% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4·7H2O, 0.06% (w / v) FeSO4·7H2O+0.000042% (w / v) MnSO4·2H2O, 0.00002% (w / v) thiamin solution] in such a manner that the final bacterial cell concentration was 5%. The bacterial suspension was transferred to a 1L jar fermentation device, glucose (initial concentration 100 g / L) was added as a substrate, and the pH of the reaction solution was adjusted to not less than 7.0 using 5.0N ammonia water at 33°C, and the reaction was carried out under the conditions of aeration of 1.0 vvm and stirring speed of 900 rpm without causing bacterial proliferation. For the purpose of adsorbing 2-phenylethanol, synthetic adsorption resin XAD2 (Organo Co., Ltd.) was added so that the final concentration became 5% (w / v).

[0172] The sampled reaction solution was centrifuged (4°C, 10,000×g, 5 minutes), and the obtained supernatant was used for product analysis. The glucose concentration in the culture medium was measured by a glucose sensor (OSI BF-5D). The concentration of 2-phenylethanol was measured using an HPLC system manufactured by Shimadzu Corporation, and the separation column was Cosmosil C18-AR-II from Nacalai tesque. The separation conditions based on HPLC were: 40% methanol and 0.069% perchloric acid as the mobile phase, a flow rate of 1.0 ml / min, and a column temperature of 40°C. 2-phenylethanol adsorbed on XAD2 was also analyzed by HPLC after elution with acetone.

[0173] As a result of the production reaction in which the transformant did not substantially proliferate, accumulation of 2-phenylethanol was observed ( Figure 2The left side shows the time-dependent change in the concentration of 2-phenylethanol remaining in the culture medium. At 53.5 hours of reaction, the resin adsorbed component was combined to produce a total of 12.8 g / L (105 mM) of 2-phenylethanol. The details are: the resin adsorbed component was 78 mM, the residual component in the culture medium was 27 mM ( Figure 2 right).

[0174] [Example 3]

[0175] Adaptability test of the plant as a host for 2-phenylethanol production

[0176] Effects of 2-phenylethanol on aerobic proliferation

[0177] The growth inhibition test of 2-phenylethanol in aerobic culture was carried out on Corynebacterium glutamicum, Escherichia coli, Pseudomonas putida and Kluyveromyces marxianus.

[0178] Corynebacterium glutamicum R was spread on A agar medium (same as above) and allowed to stand at 33°C in the dark for 15 hours.

[0179] One platinum loop of Corynebacterium glutamicum R grown in the above plate was inoculated into a test tube containing 10 ml of liquid medium A (same as above), and cultured aerobically at 33°C with shaking for 13 hours.

[0180] The Corynebacterium glutamicum R grown under the above conditions was inoculated into 100 ml of liquid medium A (same as above) to make the initial bacterial concentration OD610 = 0.05, and 2-phenylethanol was added to make the final concentration 0, 10, 20, 30, 40, 50 mM, and cultured with aerobic shaking at 33° C. The bacterial growth was carried out by measuring the absorbance of OD610.

[0181] Escherichia coli MG1655 was spread on LB agar medium [1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride and 1.5% agar] and allowed to stand at 37°C in the dark for 15 hours.

[0182] A platinum loop of Escherichia coli MG1655 grown in the above plate was inoculated into a test tube containing 10 ml of LB liquid medium [1% polypeptone, 0.5% yeast extract and 0.5% sodium chloride] and cultured aerobically at 37°C for 13 hours.

[0183] Escherichia coli MG1655 grown under the above conditions was inoculated into 100 ml of LB liquid medium to an initial bacterial concentration of OD610 = 0.05, and 2-phenylethanol was added to a final concentration of 0, 10, 20, 30, 40, and 50 mM, and aerobic shaking culture was performed at 33° C. The growth of the bacteria was measured by measuring the absorbance of OD610.

[0184] Pseudomonas putida S12 was spread on LB agar medium [1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride and 1.5% agar] and allowed to stand at 30°C in a dark place for 15 hours.

[0185] A platinum loop of Pseudomonas putida S12 proliferated in the above plate was inoculated into a test tube containing 10 ml of LB (+ glucose) liquid medium [1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride and 0.4% glucose] and cultured aerobically at 33°C for 13 hours.

[0186] The Pseudomonas putida S12 strain proliferated under the above conditions was inoculated into 100 ml of LB (+ glucose) liquid medium at an initial bacterial concentration of OD610 = 0.05, and 2-phenylethanol was added to a final concentration of 0, 10, 20, 30, 40, and 50 mM, and aerobic shaking culture was performed at 30° C. The bacterial growth was measured by measuring the absorbance of OD610.

[0187] Kluyveromyces marxianus was spread on a YPD agar medium [2% polypeptone, 1% yeast extract, 2% glucose and 1.5% agar] and allowed to stand at 30°C in the dark for 15 hours.

[0188] One platinum loop of Kluyveromyces marxianus grown in the plate was inoculated into a test tube containing 10 ml of YPD liquid medium [2% polypeptone, 1% yeast extract and 2% glucose] and cultured aerobically at 40°C for 13 hours.

[0189] Kluyveromyces marxianus grown under the above conditions was inoculated into 100 ml of YPD liquid medium to an initial bacterial concentration of OD610 = 0.05, and 2-phenylethanol was added to a final concentration of 0, 10, 20, 30, 40, and 50 mM, and cultured aerobically with shaking at 40° C. The bacterial growth was measured by measuring the absorbance of OD610.

[0190] The results of the analysis of the effect of adding 2-phenylethanol to the culture medium on aerobic proliferation are shown in Figure 3 The growth of Escherichia coli was significantly inhibited in the presence of 20 mM 2-phenylethanol, and the growth was almost completely inhibited in the presence of 30 mM 2-phenylethanol.

[0191] Pseudomonas putida S12, which was reported as a solvent-resistant bacterium, showed a similar trend, and its growth was completely inhibited at 30 mM 2-phenylethanol.

[0192] In contrast, at a concentration of 20 mM, which was strongly inhibited in Escherichia coli, Pseudomonas putida, and Kluyveromyces marxianus, Corynebacterium glutamicum did not affect proliferation at all. In addition, in Escherichia coli, Pseudomonas putida, and Kluyveromyces marxianus, even in the presence of 30 mM 2-phenylethanol, which almost completely inhibited proliferation, Corynebacterium glutamicum proliferation was delayed, but good proliferation was shown after 24 hours.

[0193] The above results show that Corynebacterium glutamicum has high resistance to 2-phenylethanol compared with Escherichia coli, Pseudomonas putida and Kluyveromyces marxianus and has high adaptability as a host for producing 2-phenylethanol.

[0194] Industrial Applicability

[0195] According to the present invention, 2-phenylethanol can be produced from a sugar raw material with practical efficiency.

[0196]

Claims

1. A transformant of Corynebacterium glutamicum, characterized in that The transformant of Corynebacterium glutamicum has undergone activation of the shikimate pathway, and a gene encoding an enzyme having phenylpyruvate decarboxylase activity is introduced into the transformant of Corynebacterium glutamicum in an expressible manner, The gene encoding an enzyme having phenylpyruvate decarboxylase activity is selected from the following (1) to (4): (1) an ipdC gene derived from Enterobacter cloacae, which has the base sequence shown in SEQ ID NO: 3; (2) a pdc gene derived from Azospirillum brasilense, which has the base sequence shown in SEQ ID NO: 1; (3) a pdc gene derived from Corynebacterium aurimucosum, which has the base sequence shown in SEQ ID NO: 2; (4) The pdc gene derived from Rhodopseudomonas palustris, which has the base sequence shown in SEQ ID NO:

4.

2. The transformant of Corynebacterium glutamicum according to claim 1, wherein Activation of the shikimate pathway comprises: introducing at least one gene selected from the following genes into a coryneform bacterium as a host, a gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity, a gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity, and A gene encoding an enzyme having shikimate kinase activity.

3. The transformant of Corynebacterium glutamicum according to claim 2, wherein A gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity and a gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity are resistant to feedback inhibition.

4. The transformant of Corynebacterium glutamicum according to claim 2, wherein A gene encoding an enzyme having 3-deoxy-D-arabinoheptulonic acid-7-phosphate (DAHP) synthase activity, a gene encoding an enzyme having at least one of chorismate mutase activity and prephenate dehydratase activity, and a gene encoding an enzyme having shikimate kinase activity are derived from Escherichia coli.

5. The transformant of Corynebacterium glutamicum according to any one of claims 1 to 4, wherein At least one gene selected from the group consisting of a lactate dehydrogenase gene, a 3-dehydroshikimate dehydratase gene, a dihydroxyacetone phosphatase gene, and a phenylalanine uptake transporter gene is disrupted.

6. The transformant of Corynebacterium glutamicum according to any one of claims 1 to 4, wherein The Corynebacterium glutamicum used as the host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869 (DSM1412).

7. Corynebacterium glutamicum 2PE97 strain, whose deposit number is NITE BP-02830.

8. A method for producing 2-phenylethanol, characterized in that include: A step of reacting the transformant of Corynebacterium glutamicum according to any one of claims 1 to 7 in water containing a sugar.

9. The method for producing 2-phenylethanol according to claim 8, wherein The sugar is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose and mannitol.

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