Microorganism and method for producing phenylacetaldehyde and phenylethyl alcohol by means of fermentation using said microorganism

Abstract

The present disclosure provides a new microorganism with which productivity of phenylacetaldehyde and / or phenylethyl alcohol can be improved. In one embodiment, the present disclosure relates to a microorganism that has the capacity to produce phenylacetaldehyde and / or phenylethyl alcohol, the microorganism having introduced therein, in an expressible manner, a gene (A) that is derived from Morganella morganii or Shimwellia blattae or is an ortholog thereof, and that encodes an enzyme having activity which produces phenylacetaldehyde from phenylpyruvic acid.

Description

Microorganism and method for producing phenylacetaldehyde and phenylethyl alcohol by fermentation using the same

[0001] The present disclosure relates to a technique for producing phenylacetaldehyde and phenylethyl alcohol. In one aspect, the present disclosure relates to a microorganism subjected to specific genetic manipulation, and a technique for producing phenylacetaldehyde and phenylethyl alcohol by fermentation using the microorganism.

[0002] Phenylacetaldehyde (CAS No.: 122-78-1; α-tolualdehyde, hyacinthin, benzeneacetaldehyde, etc. are also synonyms) has a hyacinth-like aroma, and can be used in applications such as fragrances (for cosmetics, perfumes, etc.) and flavors (for food additives, etc.).

[0003] Phenylethyl alcohol (CAS No.: 60-12-8; phenethyl alcohol, 2-phenylethyl alcohol, 2-phenylethanol, β-phenylethyl alcohol, etc. are also synonyms) is the main component of rose scent, and can be used in applications such as fragrances (for cosmetics, perfumes, etc.) and flavors (for food additives, etc.).

[0004] Phenylethyl alcohol can also be produced by a microbial fermentation method (see, for example, Patent Document 1), and there is a demand for creating a microorganism (transformant) that can produce phenylacetaldehyde and phenylethyl alcohol more efficiently.

[0005] International Publication No. WO 2020 / 130095

[0006] The present disclosure provides a novel microorganism capable of improving the productivity of at least one of phenylacetaldehyde and phenylethyl alcohol, and a method for producing at least one of phenylacetaldehyde and phenylethyl alcohol using the microorganism.

[0007] In one aspect, this disclosure relates to a microorganism having the ability to produce at least one of phenylacetaldehyde and phenylethyl alcohol, through which at least one gene (A) selected from the group consisting of (A1) to (A18) below, which encodes an enzyme having the activity of producing phenylacetaldehyde from phenylpyruvic acid, is introduced in an expressible manner. (A1) A gene derived from Morganella morganii; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 4; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 4; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 1; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1; (A8) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted and / or added per unit, with 100 bases forming one unit, in the nucleotide sequence shown in Sequence ID No. 1; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1; (A10) A gene derived from Shimwellia blattae; (A11) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 12; (A12) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 12; (A13) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in SEQ ID NO: 12;(A14) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 12; (A15) A gene having the nucleotide sequence shown in Sequence ID No. 9; (A16) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 9; (A17) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 9, where 100 nucleotides constitute one unit, with 1 to 10 nucleotides deleted, substituted and / or added per unit; (A18) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 9.

[0008] In other embodiments, the present disclosure relates to a method for producing phenylacetaldehyde and / or phenylethyl alcohol, which includes the step of culturing the microorganisms of the present disclosure to produce phenylacetaldehyde and / or phenylethyl alcohol.

[0009] According to this disclosure, in one embodiment, a novel microorganism capable of improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol can be provided, preferably a microorganism capable of efficiently producing phenylacetaldehyde and / or phenylethyl alcohol using sugars and the like as raw materials. According to this disclosure, in one embodiment, a method for producing phenylacetaldehyde and / or phenylethyl alcohol with improved productivity can be provided.

[0010] Figure 1 schematically illustrates phenylacetaldehyde production in the microorganisms of this disclosure. Figure 2 schematically illustrates another example of phenylacetaldehyde production in the microorganisms of this disclosure. Figure 3 is an overall metabolic pathway diagram illustrating the biosynthetic pathway of phenylacetaldehyde in one form of the microorganisms of this disclosure. Figure 4 schematically illustrates phenylethyl alcohol production in the microorganisms of this disclosure. Figure 5 schematically illustrates another example of phenylethyl alcohol production in the microorganisms of this disclosure. Figure 6 is an overall metabolic pathway diagram illustrating the biosynthetic pathway of phenylethyl alcohol in one form of the microorganisms of this disclosure. Figure 7 is a schematic diagram showing an example of the configuration of an apparatus that can be used in the manufacturing method of this disclosure. Figure 8 is a schematic diagram showing another example of the configuration of an apparatus that can be used in the manufacturing method of this disclosure.

[0011] This disclosure is based on the discovery that high productivity of phenylacetaldehyde and phenylethyl alcohol can be obtained by introducing a gene having the nucleotide sequence shown in SEQ ID NO: 1 into a microorganism (e.g., Corynebacterium glutamicum) that can express the gene. The gene having the nucleotide sequence shown in SEQ ID NO: 1 encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 and encodes an enzyme that has the activity to produce phenylacetaldehyde from phenylpyruvic acid. This disclosure is also based on the discovery that high productivity of phenylacetaldehyde and phenylethyl alcohol can be obtained by introducing a gene having the nucleotide sequence shown in SEQ ID NO: 9 into a microorganism (e.g., Corynebacterium glutamicum) that can express the gene. The gene having the nucleotide sequence shown in SEQ ID NO: 9 encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 12 and encodes an enzyme that has the activity to produce phenylacetaldehyde from phenylpyruvic acid.

[0012] This disclosure relates, in one embodiment, to a microorganism capable of producing at least one of phenylacetaldehyde and phenylethyl alcohol, wherein a gene (A) encoding an enzyme having the activity to produce phenylacetaldehyde from phenylpyruvic acid is introduced in an expressible manner. Gene (A) is selected from the group consisting of (A1) to (A18) below. In one or more embodiments, the microorganism of this disclosure may also be a transformant. In one or more embodiments, the microorganism of this disclosure may also be a transformant capable of producing at least one of phenylacetaldehyde and phenylethyl alcohol, or a transformant in which the ability to produce at least one of phenylacetaldehyde and phenylethyl alcohol is improved, by introducing the above-described gene (A). (A1) A gene derived from Morganella morganii; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 4; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 4; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 1; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1; (A8) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted and / or added per unit, with 100 bases forming one unit, in the nucleotide sequence shown in Sequence ID No. 1; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1; (A10) A gene derived from Shimwellia blattae; (A11) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 12;(A12) A gene encoding a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 12; (A13) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, as shown in the amino acid sequence shown in SEQ ID NO: 12; (A14) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in SEQ ID NO: 12; (A15) A gene having the nucleotide sequence shown in SEQ ID NO: 9; (A16) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 9; (A17) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted, and / or added per unit, with 100 bases forming one unit, as shown in the nucleotide sequence shown in SEQ ID NO: 9; (A18) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9.

[0013] In one or more embodiments, the microorganisms of this disclosure can produce phenylpyruvic acid from sugars within the organism, as shown in Figures 3 and 6, and then, by gene (A), produce phenylacetaldehyde and further phenylethyl alcohol from phenylpyruvic acid.

[0014] In this disclosure, "introducing a gene in an expressible form" means, in one or more embodiments, introducing a gene in a manner that allows the introduced gene to be expressed in a microorganism or transformant. Methods for introducing a gene include, in one or more embodiments, methods using general genetic engineering techniques (e.g., the method described in Michael R. Green & Joseph Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press). Gene introduction methods include, in one or more embodiments, gene introduction using a plasmid vector and integration into the chromosome of a microbial host. Methods for introducing a gene in an expressible form include, in one or more embodiments, introducing the gene together with an expression regulatory sequence such as a promoter capable of inducing increased gene expression. In this disclosure, the gene introduced together with a promoter capable of inducing increased expression may, in one or more embodiments, be a single gene or an operon capable of expressing multiple genes. Known promoters can be used as promoters capable of inducing increased expression.

[0015] In this disclosure, "90% or more identity" with respect to an amino acid sequence or base sequence means at least 90% identity, and in one or more embodiments, means 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity.

[0016] In this disclosure, "identity of amino acid sequence or nucleotide sequence" can be performed using readily available sequence comparison computer programs. Examples of such computer programs in one or more embodiments include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387-395), BLAST (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), and FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448).

[0017] In this disclosure, “stringent conditions” means conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. In one or more embodiments, stringent conditions include conditions under which highly identical base sequences hybridize, but less identical base sequences do not hybridize. In one or more embodiments, high identity between base sequences includes cases where the identity is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more. In one or more embodiments, stringent conditions may also be those described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol2, p11.45. Specifically, hybridization may occur at a temperature 5°C to 10°C lower than the melting temperature (Tm) of the complete hybrid.

[0018] [Gene (A)] Gene (A) is a gene derived from Morganella morganii or an ortholog of a gene derived from Morganella morganii that encodes an enzyme that has the activity to produce phenylacetaldehyde from phenylpyruvic acid. In other embodiments, gene (A) is a gene derived from Shimwellia blattae or an ortholog of a gene derived from Shimwellia blattae that encodes an enzyme that has the activity to produce phenylacetaldehyde from phenylpyruvic acid.

[0019] Genes derived from Morganella morganii include genes encoding polypeptides having the amino acid sequence shown in SEQ ID NO: 4, and genes having the nucleotide sequence shown in SEQ ID NO: 1. Orthologs of genes derived from Morganella morganii include the following genes (A3), (A4), (A5), (A7), (A8), and (A9). (A3) A gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to the amino acid sequence shown in Sequence ID No. 4; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids; (A5) A gene that hybridizes under stringent conditions with a gene having a complementary nucleotide sequence to the gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A7) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in Sequence ID No. 1, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more; (A8) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 1, with 100 nucleotides as one unit, and 1 to 10 nucleotides deleted, substituted and / or added per unit; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1.

[0020] Examples of genes derived from Shimwellia blattae include genes encoding polypeptides having the amino acid sequence shown in SEQ ID NO: 12, and genes having the nucleotide sequence shown in SEQ ID NO: 9. Orthologs of genes derived from Shimwellia blattae include the following genes (A12), (A13), (A14), (A16), (A17), and (A18). (A12) A gene encoding a polypeptide having an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identical to the amino acid sequence shown in Sequence ID No. 12; (A13) A gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids; (A14) A gene that hybridizes under stringent conditions with a gene having a complementary nucleotide sequence to the gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 12; (A16) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in Sequence ID No. 9, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more; (A17) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 9, where 100 nucleotides constitute one unit, and each unit has 1 to 10 nucleotide deletions, substitutions and / or additions; (A18) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 9.

[0021] In one or more embodiments, the microorganisms of this disclosure have reduced or absent aminotransferase (tyrB) function for further improvement in the productivity of phenylacetaldehyde and / or phenylethyl alcohol. In one or more embodiments, the microorganisms of this disclosure have reduced or absent tyrB function due to mutation introduction into the tyrB gene in the host or due to the disruption or deletion of a portion of the tyrB gene.

[0022] In one or more embodiments, the dysfunction or deletion of aminotransferase (tyrB) can be achieved by markerless gene disruption. In one or more embodiments, the dysfunction or deletion of tyrB by markerless gene disruption can be achieved using a known markerless chromosome gene modification (disruption) plasmid. In one or more embodiments, an example of a markerless chromosome gene modification (disruption) plasmid is pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. In one or more embodiments, the dysfunction or deletion of tyrB can be achieved by mutating the first base of the tyrB gene sequence, "adenine (a)" or a corresponding base, to "guanine (g)".

[0023] In this disclosure, "reduced or missing enzyme function" may include, in one or more embodiments, a decrease in enzyme activity or expression level of the enzyme, and / or a decrease in transcription level of the gene encoding the target enzyme, compared to a strain in which the function of the enzyme (target enzyme) has not been reduced or missing (or a strain before the function was reduced or missing).

[0024] In one or more embodiments, the microorganisms of this disclosure may have reduced or lost function of one or more of the following enzymes (1) to (3): (1) 3-dehydroschimate dehydratase (qsuB), (2) lactate dehydrogenase (ldhA), and (3) halo acid dehalogenase superfamily phosphatase (hdpA).

[0025] In one or more embodiments, the microorganisms of this disclosure may have a portion of one or more of the qsuB gene, ldhA gene, and hdpA gene, or all three genes, disrupted or missing in the host, for the purpose of further improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol.

[0026] In one or more embodiments, the microorganisms of this disclosure may have enhanced shikimic acid productivity in order to further improve the productivity of phenylacetaldehyde and / or phenylethyl alcohol.

[0027] In one or more embodiments, the productivity of shikimic acid can be increased by increasing the metabolic flux of the shikimic acid pathway. Figures 3 and 6 schematically illustrate an example of the overall carbohydrate metabolic pathway showing the metabolic pathways of phenylacetaldehyde and phenylethyl alcohol using glucose as a raw material. The carbohydrate metabolic pathway in this disclosure refers to various in vivo reaction systems for breaking down and converting sugars such as glucose into raw materials for the biosynthesis of various compounds, and may include, in one or more embodiments, the shikimic acid pathway, the carbohydrate metabolic pathway (iolT1-ppgk), the non-oxidative pentose phosphate pathway, and the TCA cycle. However, the phenylacetaldehyde / phenylethyl alcohol biosynthesis pathway shown in Figures 3 and 6 illustrates an example of the carbohydrate metabolic pathway in this disclosure, and this disclosure is not limited thereto.

[0028] The metabolic modification methods for increasing the metabolic flux of the shikimic acid pathway are not particularly limited, and in one embodiment, any one of the following high-activation methods (1) to (3) may be used, or a combination of two or three of the high-activation methods (1) to (3) may be used. (1) High-activation by enhancing the expression of each enzyme gene constituting the shikimic acid pathway or by using heterologous or mutant genes. Examples of enzymes constituting the shikimic acid pathway in one or more embodiments include 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (e.g., aroG), 3-dehydroquinate synthase (e.g., aroB), 3-dehydroquinate dehydratase (e.g., aroD), shikimic acid dehydrogenase (e.g., aroE), shikimic acid kinase (e.g., aroK), 5-enolpyruvir-shikimic acid-3-phosphate synthase (e.g., aroA), and colismyic acid synthase (e.g., aroC). (2) Increased activation by enhancing the expression of genes constituting the non-PTS sugar transport pathway, which is an alternative sugar transport and metabolic pathway different from the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that is normally used as a sugar transporter in wild-type strains. Examples of genes constituting the non-PTS sugar transport pathway include, in one or more embodiments, the iolT1 and iolT2 genes encoding myo-inositol / glucose transporters, the glk gene encoding glucokinase, and the ppgk gene encoding polyphosphate glucokinase. (3) Increased activation by enhancing the expression of enzyme genes constituting the non-oxidative pentose phosphate pathway. Examples of enzymes constituting the non-oxidative pentose phosphate pathway include, in one or more embodiments, transketolase (e.g., tkt) and transaldolase (e.g., tal).

[0029] Metabolic modification may, in one or more embodiments, be a modification of at least one gene constituting each pathway, a modification of two, three, or four or more genes constituting each pathway, or a modification of all of the genes mentioned above in each pathway.

[0030] In one or more embodiments, the microorganisms of this disclosure may be introduced into a host so as to be expressible at least one or more of the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes, or may be modified to enhance the expression of at least one or all of these genes, in order to further improve the productivity of phenylacetaldehyde / phenylethyl alcohol. This may enhance the activity of at least one or all of the enzymes encoded by these genes.

[0031] In this disclosure, "enhanced gene expression" may include, in one or more embodiments, an increase in the expression level of the polypeptide encoded by the target gene, an increase in the activity of the polypeptide encoded by the target gene, and / or an increase in the transcription level of the target gene, compared to a strain in which the expression of the target gene is not enhanced (or before the enhancement of expression is performed). In one or more embodiments, the gene may be exogenous or may be inherent to the microorganism (host) itself.

[0032] If the microorganism of this disclosure is a microorganism capable of producing phenylacetaldehyde or a microorganism for producing phenylacetaldehyde, then in one or more embodiments, it is preferable that the function of alcohol dehydrogenase is reduced or absent, in order to further improve the productivity of phenylacetaldehyde.

[0033] If the microorganism of this disclosure is a microorganism capable of producing phenylethyl alcohol or a microorganism for producing phenylethyl alcohol, then in one or more embodiments, a gene encoding alcohol dehydrogenase may be expressed and / or its expression may be enhanced, from the viewpoint of further improving the productivity of phenylethyl alcohol.

[0034] In this disclosure, "alcohol dehydrogenase" refers to an enzyme that catalyzes the reaction for the production of phenylethyl alcohol from phenylacetaldehyde. In this disclosure, "alcohol dehydrogenase" can be any enzyme capable of producing phenylethyl alcohol from phenylacetaldehyde, and in one or more embodiments, examples include phenylacetaldehyde reductase (EC 1.1.1.-), aryl alcohol dehydrogenase (or also known as benzyl alcohol dehydrogenase) (EC 1.1.1.90), aldehyde dehydrogenase, and phenylethanol dehydrogenase. In one or more embodiments, examples of genes encoding alcohol dehydrogenase include the adh (alcohol dehydrogenase) gene and the yjgB (alcohol dehydrogenase) gene. Examples of genes encoding alcohol dehydrogenase include, in one or more embodiments, the yjgB gene or its ortholog from the Escherichia genus of bacteria, the yjgB gene from Escherichia coli, the yqhD (alcohol dehydrogenase) gene, the yahK (aldehyde reductase) gene from Escherichia coli, the adhC (alcohol dehydrogenase) gene which is similar to aryl alcohol dehydrogenase from Lactobacillus brevis, the adh1 gene or adh2 gene which are alcohol dehydrogenases from Saccharomyces cerevisiae, or their orthologs.

[0035] If the microorganism of this disclosure is a microorganism capable of producing phenylacetaldehyde or a microorganism for producing phenylacetaldehyde, then in one or more embodiments, from the viewpoint of further improving the productivity of phenylacetaldehyde, the gene encoding alcohol dehydrogenase may be disrupted and / or a mutation may be introduced into said gene that reduces or eliminates the function of alcohol dehydrogenase, and it is preferable that the adh gene is disrupted.

[0036] [Host] In one or more embodiments, the microbial hosts of this disclosure include coryneform bacteria, Escherichia coli (Escherichia species, particularly Escherichia cory), aromatic compound-utilizing bacteria, adipic acid-utilizing bacteria, or yeasts, or transformants thereof. Coryneform bacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology (Vol. 8, 599 (1974)), and are not particularly limited as long as they grow under normal aerobic conditions. Examples of coryneform bacteria in one or more embodiments include Corynebacterium species, Brevibacterium species, Arthrobacter species, Mycobacterium species, and Micrococcus species. Examples of aromatic compound-utilizing bacteria in one or more embodiments include Pseudomonas species and Rosebacter species. Examples of adipic acid-utilizing bacteria include those of the genus Thermobifida in one or more embodiments.

[0037] The microbial host of this disclosure may, in one or more embodiments, be a bacterium of the genus Corynebacterium. Examples of Corynebacterium bacteria in one or more embodiments include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum. The microbial host of this disclosure is not particularly limited, and may, for example, be Corynebacterium glutamicum, from the viewpoint of further improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol.

[0038] The microbial host of this disclosure may, in one or more embodiments, be Corynebacterium glutamicum such as Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), or ATCC13869 (DSM1412), or may be a transformant thereof.

[0039] In one or more embodiments, the microbial host of this disclosure is preferably a microorganism with a risk group of Group 1 (biosafety level 1) based on the Laboratory biosafety manual established by the World Health Organization (WHO).

[0040] One preferred embodiment in this disclosure is a microorganism capable of producing at least one of phenylacetaldehyde and phenylethyl alcohol, wherein, in terms of further improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol, at least one gene selected from the group consisting of (A1) to (A18) above, which encodes an enzyme having the activity to produce phenylacetaldehyde from phenylpyruvic acid, is introduced in an expressible manner, and the function of the aminotransferase (tyrB) is reduced or absent. More preferably, a microorganism is introduced in which at least one gene selected from the group consisting of (A2) to (A9) and (A11) to (A18) is expressible and the function of aminotransferase (tyrB) is reduced or deleted. Even more preferably, a microorganism is introduced in which at least one gene selected from the group consisting of (A2) to (A8) and (A11) to (A17) is expressible and the function of tyrB is reduced or deleted by mutating the first base sequence of the tyrB gene from "adenine (a)" to "guanine (g)". In this embodiment, from the viewpoint of further improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol, in addition to the introduction and mutation of the above genes, it is preferable that at least one or more or all of the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes are introduced in a way that allows expression, and that a part of one or more or all of the qsuB, ldhA, and hdpA genes is destroyed or deleted. More preferably, the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes are introduced in a way that allows expression, and the qsuB, ldhA, and hdpA genes are destroyed.In this embodiment, the microorganism is preferably Corynebacterium glutamicum or a transformant thereof as the host, more preferably Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof, from the viewpoint of further improving the productivity of phenylacetaldehyde and / or phenylethyl alcohol.

[0041] Other preferred embodiments in this disclosure are microorganisms capable of producing phenylethyl alcohol, wherein, in terms of further improving the productivity of phenylethyl alcohol, at least one gene selected from the group consisting of (A1) to (A18) above, which encodes an enzyme having the activity of producing phenylacetaldehyde from phenylpyruvic acid, is introduced in an expressible manner, and the function of the aminotransferase (tyrB) is reduced or absent; more preferably, at least one gene selected from the group consisting of (A2) to (A9) and (A11) to (A18) above, is introduced in an expressible manner, and the function of the aminotransferase (tyrB) is reduced or absent; and even more preferably, at least one gene selected from the group consisting of (A2) to (A8) and (A11) to (A17) above, is introduced in an expressible manner, and the function of tyrB is reduced or absent by mutating the first base sequence of the tyrB gene from "adenine (a)" to "guanine (g)". In this embodiment, from the viewpoint of further improving the productivity of phenylethyl alcohol, in addition to the introduction and mutation of the above genes, it is preferable that at least one or two or more genes, or all of the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes, are introduced in a way that allows expression, and that a part of one or two or more genes, or all three of the qsuB, ldhA, and hdpA genes, is destroyed or deleted. More preferably, the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes are introduced in a way that allows expression, and the qsuB, ldhA, and hdpA genes are destroyed. In this embodiment, the host is preferably Corynebacterium glutamicum or a transformant thereof, and more preferably Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof, from the viewpoint of further improving the productivity of phenylethyl alcohol.

[0042] Further preferred embodiments of this disclosure are microorganisms capable of producing phenylacetaldehyde, wherein, in terms of further improving the productivity of phenylacetaldehyde, at least one gene selected from the group consisting of (A1) to (A18) above, encoding an enzyme having the activity to produce phenylacetaldehyde from phenylpyruvic acid, is expressed, and the function of aminotransferase (tyrB) is reduced or deleted, and the gene encoding alcohol dehydrogenase is disrupted and / or a mutation is introduced into said gene that reduces or deletes the function of alcohol dehydrogenase. Microorganisms are preferred, more preferably microorganisms in which at least one gene selected from the group consisting of (A2) to (A9) and (A11) to (A18) is introduced in an expressible manner, and in which the function of aminotransferase (tyrB) is reduced or deleted, and the adh gene is destroyed, and even more preferably microorganisms in which at least one gene selected from the group consisting of (A2) to (A8) and (A11) to (A17) is introduced in an expressible manner, and in which the function of tyrB is reduced or deleted by mutating the first base sequence of the tyrB gene from "adenine (a)" to "guanine (g)", and the adh gene is destroyed. In this embodiment, from the viewpoint of further improving the productivity of phenylacetaldehyde, in addition to the introduction and mutation of the above genes, it is preferable that at least one or two or more genes, or all of the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes, are introduced in a way that allows expression, and that a part of one or two or more genes, or all three of the qsuB, ldhA, and hdpA genes, is destroyed or deleted. More preferably, the aroG, aroD, aroE, aroC, aroK, aroB, and aroA genes are introduced in a way that allows expression, and the qsuB, ldhA, and hdpA genes are destroyed.In terms of further improving the productivity of phenylacetaldehyde, the host of the microorganism according to this aspect is preferably Corynebacterium glutamicum or a transformant thereof, more preferably Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

[0043] In another aspect, the present disclosure relates to a vector for introducing the above gene (A) into a microorganism. In one or more embodiments, the vector according to the present disclosure may be a plasmid or the like.

[0044] Construction of vector for transformants The introduction of the above gene into a microbial host can be carried out by amplifying the aforementioned gene via PCR, cloning it into an appropriate vector that can replicate in a microbial host such as Coryneform bacteria, and transforming the microbial host with the vector. As a promoter, in one or more embodiments, examples include the promoter of the gapA gene (PgapA) encoding glyceraldehyde 3-phosphate dehydrogenase (also referred to as glyceraldehyde 3-phosphate dehydrogenase) derived from Corynebacterium glutamicum R, the promoter of the mdh gene (Pmdh) encoding malate dehydrogenase, the promoter of the ldhA gene (PldhA) encoding lactate dehydrogenase, and the promoter of the tuf gene (Ptuf) encoding Elongation factor Tu. Among these, PgapA is preferred. As a terminator, in one or more embodiments, examples include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the trpA terminator of Escherichia coli, and the trp terminator of Brevibacterium lactofermentum. Among these, the rrnB T1T2 terminator is preferred.

[0045] [Preparation of Transformants] The transformation method can be any known method without limitation. Such known methods include, in one or more embodiments, the calcium chloride / rubidium chloride method, the calcium phosphate method, DEAE-dextran-mediated transfection, and electroporation (electric pulse method). When the microbial host is a Corynebacterium, the electric pulse method is preferred. The electric pulse method can be carried out by known methods [for example, Kurusu, Y. et al., Electroporation-transformation system for Coryneform bacteria by auxotrophic complementation. Agric. Biol. Chem. 54:443-447 (1990)] and [Vertes AA et al., Presence of mrr- and mcr- like restriction systems in Coryneform bacteria. Res. Microbiol. 144:181-185 (1993)].

[0046] Disruption or Mutation of Host Chromosome Genes: When the microbial host is a Corynebacterium, genes encoding competitive biosynthetic pathways, repressors of biosynthetic pathways, or efflux transporters may be disrupted or deleted as needed. The function of enzyme proteins encoded by specific genes may be improved by introducing mutations into the chromosome. By ligating DNA fragments before and after the target gene to create a DNA fragment in which the entire target gene is deleted, and transforming the bacteria with this DNA to induce homologous recombination on the chromosome, the target gene on the chromosome can be completely deleted. Alternatively, a deletion-type gene can be created by deleting a partial sequence of the target gene and modifying it so that it does not produce a normally functioning enzyme protein. By transforming the bacteria with DNA containing this gene and inducing homologous recombination between the deletion-type gene and the gene on the chromosome, the target gene on the chromosome can be replaced with the deletion-type or disruption-type gene. Enzyme proteins encoded by deletion-type or disruption-type genes, even if produced, have a different three-dimensional structure from wild-type enzyme proteins and have reduced or absent function. Furthermore, mutations can be introduced at specific locations on a chromosome by inducing homologous recombination between a gene fragment into which a specific mutation has been introduced and the chromosomal region in question. Gene deletion or disruption by gene substitution using such homologous recombination is already established, and includes methods such as using plasmids containing temperature-sensitive origins of replication, plasmids that can be transmitted by conjugation, and suicide vectors that do not have origins of replication in the host (U.S. Patent No. 6,303,383, and Japanese Patent Publication No. 05-007491, etc.). Markerless chromosomal gene transfer vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of single crossover strains with homologous regions on chromosomes introduced into plasmid pCRA725, kanamycin resistance is exhibited due to the expression of the kanamycin resistance gene on pCRA725, and lethality in sucrose-containing medium is exhibited due to the expression of the sacR-sacB gene of Bacillus subtilis. In contrast, in the case of double crossover strains, kanamycin sensitivity is exhibited due to the loss of the kanamycin resistance gene on pCRA725, and growth in sucrose-containing medium is exhibited due to the loss of the sacR-sacB gene.Therefore, the marker-less chromosomal gene-introduced strain exhibits kanamycin sensitivity and growth ability in a sucrose-containing medium.

[0047] Growth of Microorganisms In one or more embodiments of the microorganism disclosed herein, it is preferable to culture and proliferate the microorganism under aerobic conditions. In one or more embodiments, culture conditions for proliferation include a temperature of about 25°C to about 38°C and a culture time of about 12 hours to about 48 hours. In one or more embodiments, examples of the growth medium include natural media or synthetic media containing a carbon source, a nitrogen source, inorganic salts, other nutrient substances, and the like. In one or more embodiments, the pH of the medium is about 5 to about 8.

[0048] When the host is a coryneform bacterium, examples of the medium include Medium A [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)], BT medium [Omumasaba, C.A. et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103(2004)], and CGXII medium [Hoffmann, J. et al., Hyaluronic acid production with Corynebacterium glutamicum: effect of media composition on yield and molecular weight. J. Appl. Microbiol. 117:663-678(2014)], and the like. When the host is Escherichia coli, in one or more embodiments, examples of the medium include LB medium and the like.

[0049] Examples of carbon sources in one or more embodiments include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, or gluconic acid; and alcohols such as ethanol or propanol. Hydrocarbons such as normal paraffin may also be used if desired. One carbon source may be used alone, or two or more may be used in combination. The concentration of these carbon sources in the growth medium is about 0.1 (w / v%) to about 10 (w / v%) in one or more embodiments.

[0050] Examples of nitrogen sources in one or more embodiments include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, as well as urea, aqueous ammonia, sodium nitrate, and potassium nitrate. In one or more embodiments, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolysates, and amino acids can also be used as nitrogen sources. One type of nitrogen source may be used alone, or two or more types may be used in combination. The nitrogen source concentration in the growth medium varies depending on the nitrogen compound used, but in one or more embodiments, it is approximately 0.1 (w / v%) to approximately 10 (w / v%).

[0051] Examples of inorganic salts in one or more embodiments include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, ammonium carbonate, and calcium carbonate. These inorganic salts may be used individually or in mixtures of two or more. The concentration of inorganic salts in the growth medium varies depending on the inorganic salt used, but in one or more embodiments, it is approximately 0.01 (w / v%) to approximately 1 (w / v%).

[0052] Other nutrients may include, in one or more embodiments, meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloride hydrolysate, and extracts or decomposition products thereof of animals, plants, or microorganisms. The concentration of nutrients in the culture medium varies depending on the nutrients used, but in one or more embodiments, it is about 0.1 (w / v%) to about 10 (w / v%). Vitamins may be added as needed. Examples of vitamins may include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0053] [Method for Producing Phenylacetaldehyde / Phenylethyl Alcohol] In other embodiments, the present disclosure relates to a method for producing at least one of phenylacetaldehyde and phenylethyl alcohol. The production method of the present disclosure comprises at least the steps of culturing the microorganism of the present disclosure in a culture vessel and recovering a liquid containing at least one of phenylacetaldehyde and phenylethyl alcohol from the culture medium in the culture vessel.

[0054] In one or more embodiments, the culture step includes mixing the microorganism of the present disclosure with a culture medium in a culture vessel, and culturing the microorganism of the present disclosure in the culture medium to produce at least one of phenylacetaldehyde and phenylethyl alcohol, thereby obtaining a culture solution containing at least one of phenylacetaldehyde and phenylethyl alcohol.

[0055] In one or more embodiments, the culture medium preferably contains sugars as a carbon source. In one or more embodiments, the culture medium may also contain other nutrients other than sugars, such as vitamins, yeast extract, and dried yeast.

[0056] In one or more embodiments, the culture medium can be an inorganic salt medium. In one or more embodiments, the inorganic salt medium can be a medium containing one or more inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, calcium carbonate, urea, ammonium sulfate, and ferrous sulfate. Among these, a medium containing ammonium sulfate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate is preferred. Specifically, the inorganic salt medium can be a BT medium. In one or more embodiments, the BT medium is preferred as the culture medium, and a BT medium that substantially does not contain urea is more preferred. The concentration of inorganic salts in the culture medium varies depending on the inorganic salt used, but in one or more embodiments, it can be about 0.01 (w / v%) to about 1 (w / v%).

[0057] Examples of sugars in one or more embodiments include glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol. The concentration of sugars in the culture medium is approximately 0.1 (w / v%) to 20 (w / v%) in one or more embodiments, and may be 1 (w / v%) to 20 (w / v%), 1 (w / v%) to 10 (w / v%), or 5 (w / v%) to 20 (w / v%). In one or more embodiments, there may be one type of sugar or a combination of two or more types.

[0058] Reaction conditions include, in one or more embodiments, reducing conditions and aerobic conditions (for example, conditions with controlled dissolved oxygen concentration), and preferably aerated conditions.

[0059] The reaction temperature (the survival temperature of the transformants during the reaction) is approximately 15°C to approximately 50°C in one or more embodiments. Phenylates and / or phenylethyl alcohol can be produced efficiently within this temperature range. For further improvement of the productivity of phenylacetaldehyde and / or phenylethyl alcohol, the reaction temperature is 16°C or higher, 17°C or higher, 18°C ​​or higher, 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C or higher, or 25°C or higher. Similarly, the reaction temperature is 49°C or lower, 45°C or lower, 40°C or lower, or 35°C or lower.

[0060] In one or more embodiments, the pH of the culture medium is preferably about 5 to about 8, more preferably about 6 to about 8 or about 7. It is preferable to control the pH of the culture medium to about 5 to about 8, about 6 to about 8, or about 7 using a pH controller (for example, Able Co., Ltd., model: DT-1023) with an aqueous ammonia solution and an aqueous sodium hydroxide solution, etc., while carrying out the reaction.

[0061] In one or more embodiments, the culture may be carried out continuously for any length of time. In one or more embodiments, the reaction time may be one day or less, one to 21 days, or 21 days or more.

[0062] In one or more embodiments, the culture may be batch, fed-batch, or continuous. In one or more embodiments, the culture may also be biphase culture.

[0063] The recovery of the liquid containing at least one of phenylacetaldehyde and phenylethyl alcohol can be carried out in one or more embodiments by a flow continuous isolation method applying flow continuous isolation technology, or by a resin adsorption method and a two-phase culture method, etc., since it is possible to produce phenylacetaldehyde and phenylethyl alcohol while maintaining the concentrations of phenylacetaldehyde and phenylethyl alcohol in the culture vessel below a predetermined concentration.

[0064] In one or more embodiments of the present disclosure, the manufacturing method may be carried out using the apparatus shown in Figures 7 and 8, which will be described later.

[0065] In one or more embodiments, the manufacturing method of the present disclosure includes a step of separating at least a portion of the culture medium in a culture vessel into a liquid 1 containing the microorganisms of the present disclosure and a liquid 2 having a lower concentration of the microorganisms of the present disclosure than liquid 1. In one or more embodiments, the quantity of the microorganisms of the present disclosure contained in liquid 2 is less than the quantity of the microorganisms of the present disclosure contained in liquid 1. In one or more embodiments, liquid 2 may not substantially contain the microorganisms of the present disclosure, or may not contain any microorganisms of the present disclosure at all. In one or more embodiments, liquid 2 may contain phenylacetaldehyde and / or phenylethyl alcohol. The separation of the culture medium can be carried out using various devices, not limited to any device capable of separating the culture medium containing the microorganisms of the present disclosure into a liquid 1 with a higher concentration of the microorganisms of the present disclosure than liquid 2 and a liquid 2 with a lower concentration of the microorganisms of the present disclosure than liquid 1, in one or more embodiments. The separation of the culture medium can be carried out using a separation membrane or a module on which a separation membrane is arranged, in one or more embodiments. Examples of separation membranes include MF membranes (micro Filtration membranes) in one or more embodiments.

[0066] In one or more embodiments, the manufacturing method of the present disclosure includes the step of transporting a liquid 1 containing the microorganisms of the present disclosure to a culture vessel. The microorganisms of the present disclosure contained in the liquid 1 are then further cultured in the culture vessel and can be used for the production of phenylacetaldehyde and / or phenylethyl alcohol.

[0067] In one or more embodiments, the manufacturing method of the present disclosure includes a step of mixing liquid 2 and a solvent to produce a mixture. In one or more embodiments, the mixture contains phenylacetaldehyde and / or phenylethyl alcohol, a culture medium in a culture solution, and the solvent. In one or more embodiments, the mixture may contain the microorganisms of the present disclosure. Examples of solvents in one or more embodiments include fatty acid esters, oils and fats, alcohols, alkyl acetates, hexane, and N-isobutyl acetate. Fatty acid esters and oils and fats are preferred because they have a large difference in boiling points with phenylacetaldehyde and phenylethyl alcohol, making it easy to separate phenylacetaldehyde and phenylethyl alcohol. In one or more embodiments, edible oil (MCT oil) mainly composed of medium-chain fatty acids (Medium Chain Triglyceride) is preferred as the solvent. In one or more embodiments, the mixing of liquid 2 and the solvent can be carried out by stirring or by confluence of fluids. In one or more embodiments, mixing by stirring can be carried out in a tank (container). Fluid mixing by confluence is a method of mixing two fluids (liquid 2 and solvent) by confluencering fluids flowing through different pipes into a single pipe, in one or more embodiments, and can be performed using a flow reactor or the like.

[0068] In one or more embodiments, the mixture obtained by mixing liquid 2 and the solvent is separated into a liquid mainly composed of the solvent (upper layer liquid) and a liquid mainly composed of the culture medium (lower layer liquid). The liquid mainly composed of the solvent contains at least one of phenylacetaldehyde and phenylethyl alcohol. In one or more embodiments, the manufacturing method of the present disclosure includes the steps of transporting the lower layer liquid of the obtained mixture to a culture tank and transporting the upper layer liquid of the mixture to a solvent tank. In one or more embodiments, the manufacturing method of the present disclosure may include the steps of: separating a mixture containing liquid 2 and a solvent (a mixture containing phenylacetaldehyde and / or phenylethyl alcohol, culture medium in the culture solution, and the solvent) into a liquid mainly composed of phenylacetaldehyde and phenylethyl alcohol and the solvent, and a liquid mainly composed of the culture medium in the culture solution; transporting the liquid mainly composed of the culture medium in the culture solution to a culture tank; and transporting the liquid mainly composed of phenylacetaldehyde and phenylethyl alcohol and the solvent to a solvent tank. In one or more embodiments, the separation of the mixture can be carried out using a separation settler or a mixer settler that integrates a mixer and a settler.

[0069] In one or more embodiments, the manufacturing method of the present disclosure may include a step of removing the upper liquid of the container and / or the liquid in the solvent tank for the purpose of recovering phenylacetaldehyde and / or phenylethyl alcohol. In one or more embodiments, the step of removing the liquid may be performed when the concentration of phenylacetaldehyde or phenylethyl alcohol contained in the upper liquid separated from the mixture (or a liquid mainly composed of at least one of phenylacetaldehyde and phenylethyl alcohol and a solvent) and / or the concentration of phenylacetaldehyde or phenylethyl alcohol in the solvent tank exceeds a predetermined concentration, in order to maintain the concentrations of phenylacetaldehyde and phenylethyl alcohol in the culture tank below a predetermined concentration and to further improve the productivity of at least one of phenylacetaldehyde and phenylethyl alcohol by the microorganisms of the present disclosure. The predetermined concentrations for extraction are, in one or more embodiments, approximately 1 mM, 1 mM to 5 mM, approximately 5 mM, 5 mM to 10 mM, 5 mM to 15 mM, approximately 10 mM, 10 mM to 20 mM, 15 mM to 25 mM, approximately 20 mM, 20 mM to 30 mM, 25 mM to 35 mM, approximately 30 mM, 30 mM to 40 mM, 35 mM to 45 mM, approximately 40 mM, 40 mM to 50 mM, approximately 50 mM, or 50 mM or more. The concentrations of phenylacetaldehyde and phenylethyl alcohol in the liquid can be measured by the method described in the examples.

[0070] Recovery by resin adsorption can be carried out in one or more embodiments using a separation membrane and a resin column. Recovery by resin adsorption can be carried out in one or more embodiments by supplying at least a portion of the culture medium in a culture tank to a module on which a separation membrane is located to separate microorganisms from the culture medium, supplying the separated microorganisms to the culture tank, supplying the culture medium from which the microorganisms have been separated to a resin column on which an adsorption resin is located to recover phenylacetaldehyde and phenylethyl alcohol from the culture medium using the adsorption resin, and supplying the culture medium from which phenylacetaldehyde and phenylethyl alcohol have been recovered to the culture tank. This makes it possible to produce phenylacetaldehyde and / or phenylethyl alcohol while maintaining the concentrations of phenylacetaldehyde and phenylethyl alcohol in the culture tank below a predetermined concentration. Phenylenacetaldehyde and / or phenylethyl alcohol adsorbed on the adsorption resin can be recovered in one or more embodiments by elution using a solvent. Examples of solvents in one or more embodiments include ethanol, methanol, acetonitrile, 2-propanol, and acetone.

[0071] Recovery by two-phase culture can be performed in one or more embodiments by adding a solvent to a culture tank in which microorganisms are cultured (for the production of phenylacetaldehyde and / or phenylethyl alcohol). Recovery by two-phase culture can be performed in one or more embodiments by culturing the microorganisms of the present disclosure in a culture tank for a predetermined time, adding a solvent to the culture tank and mixing the solvent with the culture solution containing phenylacetaldehyde and / or phenylethyl alcohol produced by the culture, and culturing for a further predetermined time. As a result, phenylacetaldehyde and phenylethyl alcohol produced by the microorganisms can be extracted from the culture solution into the solvent. As the solvent, in one or more embodiments, a solvent capable of extracting (dissolving) phenylacetaldehyde and / or phenylethyl alcohol can be used. Examples of solvents in one or more embodiments include triptyline, oleyl alcohol, dodecane, and vegetable oils.

[0072] In one or more embodiments, the manufacturing method of the present disclosure may include a step of recovering a liquid containing at least one of phenylacetaldehyde and phenylethyl alcohol from the liquid obtained in the extraction step.

[0073] The liquid obtained in the extraction step may, in one or more embodiments, contain phenylacetaldehyde and phenylethyl alcohol. In one or more embodiments, the manufacturing method of the present disclosure may include a step of separating and recovering phenylacetaldehyde and phenylethyl alcohol from the liquid obtained in the extraction step. Separation can be carried out in one or more embodiments by distillation, high-performance liquid chromatography (HPLC), etc.

[0074] An unspecified embodiment of the manufacturing method of the present disclosure will be described below with reference to the drawings.

[0075] [First Embodiment] Figure 7 is a schematic diagram showing an example of the configuration of the apparatus used in the first embodiment. The apparatus 101 is capable of producing phenylacetaldehyde and / or phenylethyl alcohol and comprises a culture tank 11, a cell separation device 21, a solvent tank 12, and containers 13 and 14. The culture tank 11 is connected to the cell separation device 21 by liquid transport paths 31 and 32. The cell separation device 21 is connected to the container 13 by a liquid transport path 33. The container 13 is connected to the solvent tank 12 by a liquid transport path 34 and to the container 14 by a liquid transport path 35. The container 14 is connected to the solvent tank 12 by a liquid transport path 36 and to the culture tank 11 by a liquid transport path 37.

[0076] The culture tank 11 is a container capable of containing microorganisms and culture medium, and is used for culturing microorganisms. Microorganisms and culture medium are supplied to the culture tank 11, and by culturing the microorganisms under predetermined temperature and pH conditions, phenylacetaldehyde and / or phenylethyl alcohol are produced, and a culture solution L11 containing phenylacetaldehyde and / or phenylethyl alcohol is manufactured. The culture tank 11 is connected to a cell separation device 21 by a liquid transport path 31, and at least a portion of the culture solution L11 in the culture tank 11 can be transported to the cell separation device 21 through the liquid transport path 31. The culture solution L11 transported to the cell separation device 21 may contain phenylacetaldehyde and / or phenylethyl alcohol, culture medium, and microorganisms. A liquid transport device (not shown), such as a pump, may be provided in the liquid transport path 31 to transport the culture solution L11 more easily.

[0077] The bacterial cell separation device 21 is a device for separating culture medium L11 into culture medium L21a and culture medium L21b. More specifically, the bacterial cell separation device 21 is a device for separating culture medium L21a, which contains microorganisms and culture medium, and which has a higher microorganism content than culture medium L21b, from culture medium L21b, which has a lower microorganism content than culture medium L21a. Culture medium L21b contains culture medium and contains little to no microorganisms. Culture medium L21a and culture medium L21b may contain phenylacetaldehyde and / or phenylethyl alcohol produced by microorganisms. The bacterial cell separation device 21 is connected to the culture tank 11 by a liquid transport path 32, and the culture medium L21a separated by the bacterial cell separation device 21 can be transported to the culture tank 11 through the liquid transport path 32. The liquid transport path 32 may be provided with a liquid transport device (not shown), such as a pump, to transport the culture medium L21a more easily.

[0078] The bacterial cell isolation tank 21 is connected to the container 13 by a liquid transport path 33, and the culture medium L21b separated by the bacterial cell isolation device 21 can be transported to the container 13 through the liquid transport path 33. The liquid transport path 33 may be equipped with a liquid transport device (not shown), such as a pump, to transport the culture medium L21b more easily.

[0079] Container 13 is a device for preparing a mixed solution L13 by mixing the culture medium L21b, which has been transported from the bacterial cell isolation tank 21 via the liquid transport path 33, with the solvent L12. Container 13 is connected to the solvent tank 12 by the liquid transport path 34, and the solvent L12 is transported to container 13 via the liquid transport path 34. The mixed solution L13 contains phenylacetaldehyde and / or phenylethyl alcohol, culture medium, and solvent. A mixer may be used as container 13 to agitate the mixed solution L13. A mixer is, for example, a device equipped with a container and stirring blades. The solvent tank 12 is capable of containing the solvent L12 and can be called a solvent tank or an oil tank. Fatty acid esters and oils and fats can preferably be used as the solvent L12. The liquid transport path 34 may be provided with a liquid transport device (not shown), such as a pump, to transport the solvent L12 more easily. Container 13 is connected to container 14 by a liquid transport path 35, and the mixed liquid L13 prepared in container 13 is transported to container 14 through the liquid transport path 35. The liquid transport path 35 may be equipped with a liquid transport device (not shown), such as a pump, to transport the mixed liquid L13 more easily.

[0080] Container 14 is a device for separating the mixed solution L13 into liquid 14a and liquid 14b. Phenylatealdehyde and / or phenylethyl alcohol contained in the culture solution L21b mixed with solvent L12 in container 13 are extracted into solvent L12 (oil phase) and separated into the upper layer in container 14 (liquid L14a). The remaining components such as the culture medium are separated into the lower layer in container 14 (liquid L14b). Therefore, liquid L14a contains phenylacetaldehyde and / or phenylethyl alcohol and the solvent, with the solvent being the main component, and liquid L14b is mainly composed of the culture medium. A settler can be used as container 14. Container 14 is connected to the solvent tank 12 via a liquid transport path 36 and to the culture tank 11 via a liquid transport path 37. Liquid L14a, containing phenylacetaldehyde and / or phenylethyl alcohol and a solvent, separated in container 14, is transported to solvent tank 12 via liquid transport path 36. Liquid L14b, mainly composed of the culture medium separated in container 14, is transported to culture tank 11 via liquid transport path 37. Liquid transport paths 36 and 37 may be equipped with liquid transport devices (not shown), such as pumps, to transport liquids L14a and L14b more easily. Liquid L14a (containing phenylacetaldehyde and / or phenylethyl alcohol and a solvent) transported to solvent tank 12 can be mixed with solvent L12 contained in solvent tank 12 and supplied to container 13 via liquid transport path 34 as solvent L12.

[0081] We will explain using the apparatus 101 shown in Figure 7 as an example, where phenylethyl alcohol is produced by a flow continuous isolation method that applies flow continuous isolation technology (phenylethyl alcohol continuous extraction technology).

[0082] The manufacturing method in the first embodiment includes at least culturing microorganisms in a culture tank 11 using the apparatus 101 to produce phenylethyl alcohol, and recovering the produced phenylethyl alcohol. Specifically, it can be carried out as follows.

[0083] First, microorganisms are cultured in a culture medium in the culture tank 11 to produce phenylethyl alcohol. This yields a culture solution L11 containing phenylethyl alcohol.

[0084] The microorganism is not particularly limited as long as it has the ability to produce phenylethyl alcohol. For example, it may be a microorganism of this disclosure that has the ability to produce phenylethyl alcohol, in which a gene (A) encoding an enzyme that has the activity of producing phenylacetaldehyde from phenylpyruvic acid has been introduced in an expressible form. The microorganism may also be a microorganism capable of producing phenylalanine, but it is preferable that it is a microorganism in which the function of aminotransferase (tyrB) is reduced or deleted. By reducing or deleting the function of aminotransferase (tyrB), the ability of the microorganism to produce phenylalanine can be suppressed, and it may be easy to keep the concentration of phenylalanine in the culture medium L11 below a predetermined concentration that does not easily affect the cultivation by the microorganism.

[0085] Next, at least a portion of the culture medium L11 in the jar fermenter (culture tank 11) is transported to the microbial cell separation device 21 via the liquid transport path 31. In the microbial cell separation device 21, the culture medium L11 is separated to obtain a culture medium L21a containing microorganisms and a culture medium L21b containing fewer microorganisms than culture medium L21a or substantially no microorganisms. The culture medium L21b contains culture medium and phenylethyl alcohol, and is preferably substantially free of microorganisms from the viewpoint of improving the efficiency of microorganism reuse. The culture medium L21a containing microorganisms is transported to the jar fermenter (culture tank 11) via the liquid transport path 32, and the microorganisms in the culture medium L21a are reused for further cultivation. On the other hand, the culture medium L21b is transported to the mixer (container 13) via the liquid transport path 33 and mixed with the solvent L12 supplied from the solvent tank 12. By mixing the culture medium L21b with the solvent L12, the phenylethyl alcohol contained in the culture medium L21b is extracted into the solvent phase (solvent L12).

[0086] Next, at least a portion of the mixed liquid L13 in the mixer (container 13) is transported to the settler (container 14) via the liquid transport path 35. In the settler (container 14), the mixed liquid L13 is separated into a solvent phase containing phenylethyl alcohol (upper layer, liquid L14a) and a lower layer containing the culture medium (liquid L14b). Liquid L14a is transported to the solvent tank 12 via the liquid transport path 36, and liquid L14b is transported to the jar fermenter (culture tank 11) via the liquid transport path 37. Liquid L14b transported to the jar fermenter (culture tank 11) is used for further cultivation in the culture tank 11. Liquid L14b may contain by-products such as phenylalanine. Since phenylethyl alcohol contained in the culture medium L21b is extracted using solvent L12, the concentration of phenylethyl alcohol in the liquid L14b transported to the jar fermenter (culture tank 11) can be reduced, and the increase in the concentration of phenylethyl alcohol in the culture medium L11 in the jar fermenter (culture tank 11) can be suppressed, allowing the concentration to be kept below a predetermined level that does not significantly affect microbial culture. The concentration of phenylethyl alcohol in the culture medium L11 that does not significantly affect microbial culture is not particularly limited and may be, for example, 40 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less, and from the viewpoint of phenylethyl alcohol production efficiency, for example, 30 mM or less is also preferable.

[0087] The liquid L14a transported to the solvent tank 12 may be mixed with the solvent L12 in the solvent tank 12 and reused as new solvent L12, or the liquid L14a may be recovered to recover phenylethyl alcohol. After transporting the liquid L14a to the solvent tank 12, it may also be recovered together with the solvent L12 in the solvent tank 12. After removing the solvent tank 12, phenylethyl alcohol can be continuously extracted by installing a new solvent tank 12 containing new solvent L12. The method for recovering the liquid L14a and / or solvent L12 is not particularly limited, and may be carried out by providing another liquid transport path (not shown) in the container 14 and / or solvent tank 12, and transporting and removing the liquid L14a and / or solvent L12. At this time, new solvent L12 may be replenished in the solvent tank 12, and the method (production of phenylethyl alcohol) may be carried out continuously.

[0088] The production of phenylethyl alcohol in this embodiment can be carried out continuously for any length of time. The number of days for producing phenylethyl alcohol is not particularly limited; for example, it may be one day or less, one to 21 days, or 21 days or more. By producing phenylethyl alcohol over, for example, 5 to 15 days, it is possible to significantly reduce production costs by reusing microorganisms while maintaining high phenylethyl alcohol productivity.

[0089] The transport of culture medium L11 through liquid transport path 31, transport of culture medium L21a through liquid transport path 32, transport of culture medium L21b through liquid transport path 33, transport of solvent L12 through liquid transport path 34, transport of mixed liquid L13 through liquid transport path 35, transport of liquid L14a through liquid transport path 36, and transport of liquid L14b through liquid transport path 37 may be carried out continuously so that these liquids, such as culture medium, circulate within the apparatus 101.

[0090] In this embodiment, the production of phenylethyl alcohol was described as an example, but the production of phenylacetaldehyde can also be carried out similarly using the apparatus 101 shown in Figure 7, by applying a flow continuous isolation method (phenylacetaldehyde continuous extraction technology).

[0091] [Second Embodiment] Figure 8 is a schematic diagram showing an example of the configuration of the apparatus used in the second embodiment. Apparatus 102 is an apparatus capable of producing phenylacetaldehyde and / or phenylethyl alcohol, and comprises a culture tank 11, a cell isolation device 21, a solvent tank 12, a container 14, a liquid mixer 22, and a flow reactor type device 23. Apparatus 102 of the second embodiment is the same as apparatus 101 of the first embodiment, except that it is equipped with a liquid mixer 22 and a flow reactor type device 23 instead of a container 13, and the same components can be used. For this reason, in apparatus 102, components that have the same function as apparatus 101 are given the same reference numerals and their descriptions are omitted.

[0092] The bacterial cell isolation tank 21 is connected to the liquid mixer 22 by a liquid transport path 33, and the culture medium L21b separated in the bacterial cell isolation device 21 can be transported to the liquid mixer 22 via the liquid transport path 33.

[0093] The liquid mixer 22 is a device for mixing liquids flowing from separate channels into a single channel. The liquid mixer 22 is connected to the bacterial cell isolation tank 21 by a liquid transport channel 33 and to the solvent tank 12 by a liquid transport channel 34. It is a device for mixing the culture medium L21b transported from the bacterial cell isolation tank 21 via the liquid transport channel 33 with the solvent L12 transported from the solvent tank 12 via the liquid transport channel 34. The liquid mixer 22 may also be a device that mixes and discharges the culture medium L21b and solvent L12 while adjusting them to a predetermined amount. The liquid mixer 22 is connected to the flow reactor type device 23 by a liquid transport channel 38, and the liquid containing the culture medium L21b and solvent L12 mixed in the liquid mixer 22 is transported to the flow reactor type device 23 via the liquid transport channel 38. The liquid transport channel 38 may be provided with a liquid transport device (not shown), such as a pump.

[0094] The flow reactor type apparatus 23 is equipped with a tubular container and is a device that mixes substances while supplying liquid into the tubular container to perform reactions, extractions, etc. The liquid transported from the liquid mixer 22 (a liquid containing culture medium L21b and solvent L12) undergoes further mixing as it passes through the tubular container of the flow reactor type apparatus 23, and phenylacetaldehyde and / or phenylethyl alcohol contained in the liquid are extracted into the solvent. The tubular container of the flow reactor type apparatus 23 may be helical or spiral in shape. The flow reactor type apparatus 23 may also be a liquid transport path (liquid transport device) that mixes the culture medium L21b and solvent L12 contained in the liquid discharged from the liquid mixer 22.

[0095] We will explain using the apparatus 102 shown in Figure 8 as an example, where phenylethyl alcohol is produced by a flow continuous isolation method that applies flow continuous isolation technology (phenylethyl alcohol continuous extraction technology).

[0096] The manufacturing method in the second embodiment includes at least culturing microorganisms in a culture tank 11 using the apparatus 102 to produce phenylethyl alcohol, and recovering the produced phenylethyl alcohol, and can be carried out in the same manner as in the first embodiment, except that the mixing of the culture solution L21b and the solvent L12 is performed in a liquid mixer 22 and a flow reactor type apparatus 23.

[0097] In the second embodiment, the culture medium L21b and solvent L12 are mixed using a liquid mixer 22 and a flow reactor type device 23. By using the liquid mixer 22 and the flow reactor type device 23, mixing can be performed using a pipe flow mixing method. By using the flow reactor type device 23, stirring power, temperature control power, and device space can be reduced.

[0098] The transport of culture medium L11 through liquid transport path 31, transport of culture medium L21a through liquid transport path 32, transport of culture medium L21b through liquid transport path 33, transport of solvent L12 through liquid transport path 34, transport of liquid containing culture medium L21b and solvent L12 through liquid transport paths 38 and 35, transport of liquid L14a through liquid transport path 36, and transport of liquid L14b through liquid transport path 37 may be carried out continuously so that these liquids, such as culture medium, circulate within the apparatus 102.

[0099] In this embodiment, the production of phenylethyl alcohol was described as an example, but the production of phenylacetaldehyde can also be carried out similarly using the apparatus 102 shown in Figure 8, by applying a flow continuous isolation method (phenylacetaldehyde continuous extraction technology).

[0100] The contents of each document described herein are incorporated by reference as constituting part of this disclosure.

[0101] The disclosure further relates to one or more embodiments, not limited to the following: [1] A microorganism having the ability to produce at least one of phenylacetaldehyde and phenylethyl alcohol, wherein at least one gene (A) selected from the group consisting of (A1) to (A18) below, which encodes an enzyme having the activity to produce phenylacetaldehyde from phenylpyruvic acid, is introduced in an expressible manner. (A1) A gene derived from Morganella morganii; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 4; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 4; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 1; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1; (A8) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted and / or added per unit, with 100 bases forming one unit, in the nucleotide sequence shown in Sequence ID No. 1; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1; (A10) A gene derived from Shimwellia blattae; (A11) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 12; (A12) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 12; (A13) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in SEQ ID NO: 12;(A14) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 12; (A15) A gene having the nucleotide sequence shown in Sequence ID No. 9; (A16) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 9; (A17) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 9, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit; (A18) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 9. [2] The microorganism described in [1], wherein the function of the aminotransferase (tyrB) is reduced or deleted, preferably by mutating the first position of the tyrB gene nucleotide sequence from "adenine (a)" to "guanine (g)". [3] The microorganism described in [1] or [2] is characterized in that the function of at least one enzyme selected from the group consisting of (1) to (3) below is reduced or absent, preferably the function of one of the enzymes (1) to (3) below is reduced or absent: (1) 3-dehydrosikimate dehydratase (qsuB), (2) lactate dehydrogenase (ldhA), (3) halo acid dehalogenase superfamily phosphatase (hdpA). [4] The microorganism described in any one of [1] to [3] is characterized in that the expression of at least one enzyme selected from the group consisting of (1) to (7) below is enhanced, preferably the expression of one of the enzymes (1) to (7) below is enhanced, and more preferably the gene encoding one of the enzymes (1) to (7) below has been introduced to enable expression. (1) 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (e.g., aroG), (2) 3-dehydroquinate synthase (e.g., aroB), (3) 3-dehydroquinate dehydratase (e.g., aroD), (4) shikimate dehydrogenase (e.g., aroE), (5) shikimate kinase (e.g., aroK), (6) 5-enolpyruvirshikimate-3-phosphate synthase (e.g., aroA),(7) Corismic acid synthase (e.g., aroC). [5] The microorganism according to any one of [1] to [4], wherein the microorganism is capable of producing phenylacetaldehyde and has been introduced with a mutation that reduces or eliminates the function of alcohol dehydrogenase, preferably with the adh gene disrupted. [6] The microorganism according to any one of [1] to [5], wherein the host of the microorganism is a Corynebacterium or a transformant of a Corynebacterium. [7] The microorganism according to any one of [1] to [6], wherein the host of the microorganism is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum. [8] The microorganism according to any one of [1] to [7], wherein the host of the microorganism is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof. [9] A method comprising at least the steps of: culturing a microorganism described in any of [1] to [8] in a culture tank; and recovering a liquid containing phenylacetaldehyde and / or phenylethyl alcohol from the culture medium in the culture tank.

[10] The method according to [9], further comprising the steps of: separating at least a portion of the culture medium in the culture tank into a liquid 1 containing the microorganism and a liquid 2 having a lower concentration of the microorganism than the liquid 1; transporting the liquid 1 to the culture tank; mixing the liquid 2 with a solvent transported from a solvent tank to produce a mixed solution; transporting the mixed solution to a container; transporting the liquid from the lower layer of the container to the culture tank; transporting the liquid from the upper layer of the container to the solvent tank; and removing the liquid from the upper layer of the container and / or the liquid in the solvent tank for the purpose of recovering phenylacetaldehyde and / or phenylethyl alcohol.

[11] The method according to

[10] , wherein the step of removing the liquid is performed when the concentration of phenylacetaldehyde and / or phenylethyl alcohol contained in the liquid on the upper side of the container exceeds a predetermined concentration.

[12] The method according to

[10] or

[11] , wherein the mixing of the liquid 2 and the solvent transported from the solvent tank is performed by stirring and / or by confluence of fluids.

[13] The method according to [9], further comprising the step of recovering phenylacetaldehyde and / or phenylethyl alcohol from the culture medium by a resin adsorption method and / or a two-phase culture method.

[14] The method according to [9] or

[13] , further comprising supplying at least a portion of the culture medium in the culture tank to a module on which a separation membrane is arranged, supplying microorganisms separated by the separation membrane to the culture tank, supplying the culture medium from which the microorganisms have been separated to a resin column on which an adsorption resin is arranged, and supplying the culture medium from which phenylacetaldehyde and phenylethyl alcohol have been recovered to the culture tank.

[15] The method according to [9] or

[13] , further comprising adding a solvent to the culture tank and further culturing the microorganisms.

[16] The method according to [9] or

[10] , further comprising separating and recovering phenylethyl alcohol from the extracted liquid.

[17] The method according to [9] or

[10] , further comprising separating and recovering phenylacetaldehyde from the extracted liquid.

[18] The method according to [9] or

[10] , comprising separating and recovering phenylethyl alcohol and phenylacetaldehyde from the liquid removed.

[0102] The present disclosure will be further described below using examples. However, the present disclosure shall not be construed as being limited to the following examples.

[0103] [Example 1] Construction of phenylacetaldehyde and / or phenylethyl alcohol producing strains (1) Preparation and acquisition of chromosomal DNA Chromosomal DNA of Morganella morganii NBRC 3848 and Shimwellia blattae NBRC 105725 was prepared using a DNA genome extraction kit (illustra bacteria genomicPrep Mini Spin Kit, Cytiva) after culturing according to the information of the strain acquisition agency. (2) Construction of gene expression plasmids related to phenylacetaldehyde and / or phenylethyl alcohol production Table 1 shows the primer sequences used to isolate the indole-3-pyruvate decarboxylase C gene (gene A). PCR was performed using a VeritiPro thermal cycler (Thermo Fisher Scientific, Inc.) and PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) as the reaction reagent. The PCR-amplified DNA fragments were introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter. Table 1 shows the names of the cloning vectors used and the resulting plasmids. (3) Construction of a plasmid for introducing chromosomal gene mutations into Corynebacterium glutamicum R strain. The DNA region necessary for markerless mutation introduction into the chromosomal genes of Corynebacterium glutamicum R strain was amplified by PCR. Each PCR fragment can be ligated with phosphorylated primers. The obtained DNA fragments were introduced into the markerless chromosomal gene modification plasmid pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. The primer sequences and the obtained plasmid are shown in Table 2. Specifically, in order to reduce the function of tyrB, the nucleotide position 1 of the tyrB gene sequence was mutated from a to g, and the expression of the mutated tyrB gene (tyrB(a1g) gene) was reduced. (4) Construction of chromosome-modified strains for phenylacetaldehyde and / or phenylethyl alcohol production. The markerless chromosome gene modification vector pCRA725 is a plasmid that cannot replicate within Corynebacterium glutamicum R. In the case of a single crossover strain with a homologous region on a chromosome introduced into plasmid pCRA725, it exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality in sucrose-containing medium due to the expression of the sacR-sacB gene derived from Bacillus subtilis. In contrast, in the case of a double crossover strain, it exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth in sucrose-containing medium due to the loss of the sacR-sacB gene. Therefore, markerless chromosome-modified strains exhibit kanamycin sensitivity and growth in sucrose-containing medium. Genetically modified strains were constructed using the chromosome gene mutation introduction plasmids listed in Table 2 by the method described above. The Corynebacterium glutamicum R ldhA knockout strain CRZ1 [Biotechnol Bioeng. Nov;110(11):2938-2948 (2013)] was used as the host strain. In addition, plasmids pCRB284 [WO2017 / 169399] for aroG gene chromosome introduction, pCRB291 [WO2017 / 169399] for aroD gene chromosome introduction, pCRB293 [WO2017 / 169399] for aroE gene chromosome introduction, pCRB286 [WO2017 / 169399] for aroCKB gene chromosome introduction, pCRB289 [WO2017 / 169399] for aroA gene chromosome introduction, pCRB229 [WO2017 / 169399] for qsuB gene disruption, and pCRG32 [WO2020 / 130067] for hdpA gene disruption were used. An overview of this chromosomal recombination is shown in Table 3. (5) Construction of strains with phenylacetaldehyde and / or phenylethyl alcohol production-related gene expression plasmids The phenylacetaldehyde and / or phenylethyl alcohol production-related gene expression plasmid Ppea1 described above was introduced into Corynebacterium glutamicum Rpea1 and Rpea2 strains. In addition, the phenylacetaldehyde and / or phenylethyl alcohol production-related gene expression plasmid Ppea2 described above was introduced into Corynebacterium glutamicum Rpea2 strain. The outlines of these introduced strains are summarized in Table 4.

[0104] [Example 2] <Phenylethyl Alcohol Production Experiment> A strain producing phenylacetaldehyde and / or phenylethyl alcohol (PEA-2 strain) was inoculated onto A agar medium containing 50 μg / ml kanamycin [(NH2)2CO2 2g, (NH4)2SO4 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・7H2O 0.5g, 0.06%(w / v) FeSO4・7H2O + 0.042%(w / v) MnSO4・2H2O 1ml, 0.02%(w / v) biotin solution 1ml, 0.01%(w / v) thiamin solution 2ml, yeast extract 2g, vitamin assay casamino acid 7g, glucose 40g, agar 15g suspended in 1L of distilled water] and incubated at 33°C for 18 hours. Phenylatealdehyde and / or phenylethyl alcohol producing strain (PEA-2 strain) grown on the above plate was inoculated into a test tube containing 10 ml of liquid medium A containing 50 μg / ml kanamycin [(NH2)2CO2 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) thiamin solution 2 ml, yeast extract 2 g, vitamin assay casamino acid 7 g, glucose 40 g dissolved in 1 L of distilled water] using a platinum loop, and cultured aerobically with shaking at 33°C for 20 hours. The culture medium obtained in this way was concentrated by centrifugation (room temperature, 3,000 × g, 10 minutes). The concentrated bacterial cells were then placed in 600 ml of liquid medium A containing 50 μg / ml kanamycin to the initial OD. 610The culture medium was inoculated into a 1 L jar fermenter after adding the necessary amount to reach 0.8–1.0. Glucose (initial concentration 73 g / L) was added as a substrate, and the culture medium was cultured at 33°C with a 10 N sodium hydroxide solution to adjust the pH to 7.0, stirring for 22 hours under aerated conditions. The cultured cells were collected by centrifugation (20°C, 5,000 × g, 10 min). The obtained cells were suspended in 600 ml of BT(-urea) liquid medium [0.7% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4・7H2O, 0.00006% (w / v) FeSO4・7H2O + 0.000042% (w / v) MnSO4・2H2O, 0.00002% (w / v) thiamin solution]. The bacterial suspension was transferred to a 1 L jar fermenter, glucose (initial concentration 73 g / L) was added as a substrate, the pH of the reaction solution (culture medium) in the jar fermenter was adjusted to 7.0 using a 10 N sodium hydroxide aqueous solution, the temperature inside the jar fermenter was adjusted to 33°C, and the culture was stirred under aerated conditions. Specifically, the culture was performed using the apparatus 101 shown in Figure 7. In this example, a jar fermenter was used as the culture tank 11, a liquid-liquid separator (settler) was used as the container 14, a mixing device (mixer) was used as the container 13, and an MF membrane (Micro Filtration membrane) module was used as the bacterial cell isolation device 21. An oil tank was used as the solvent tank 12 and filled with MCT oil (solvent L12). 100% medium-chain triacylglycerides were used as the MCT oil. After the start of cultivation, the culture medium containing the bacterial cells was sent to an MF membrane (Micro Filtration membrane) module (bacterial cell isolation device 21) connected to a jar fermenter (culture tank 11) and continuously filtered. The culture medium containing the bacterial cells (L21a) that passed through the MF membrane module (bacterial cell isolation device 21) was returned to the jar fermenter (culture tank 11), and the culture medium (L21b) from which the bacterial cells had been filtered by the MF membrane module (bacterial cell isolation device 21) was sent to a mixer (container 13) and mixed with MCT oil (solvent L12) supplied from an oil tank (solvent tank 12).A mixture (L13) of culture medium (L21b) without bacterial cells and MCT oil (solvent L12) was sent to a settler (container 14) and separated. The separated MCT oil (liquid L14a on the upper layer of the settler (container 14)) was sent to an oil tank (solvent tank 12), and the culture medium from which the MCT oil had been separated (liquid L14b on the lower layer of the settler (container 14)) was sent to a jar fermenter (culture tank 11). The MCT oil (solvent L12) in the oil tank (solvent tank 12) was replaced as needed, and continuous operation was performed for 48 hours. During continuous operation, the culture medium L11 was continuously delivered through the liquid transport path 31, the culture medium L21a through the liquid transport path 32, the culture medium L21b through the liquid transport path 33, the solvent L12 through the liquid transport path 34, the mixed liquid L13 through the liquid transport path 35, the liquid L14a through the liquid transport path 36, and the liquid L14b through the liquid transport path 37, circulating within the apparatus 101 during operation. The MCT oil was replaced (liquid L14a was removed) when the concentration of phenylethyl alcohol in liquid L14a exceeded 50 mM. The replaced (removed from the oil tank) MCT oil was recovered, and the concentrations of phenylethyl alcohol and phenylalanine were measured in the recovered MCT oil during sampling at 24 hours and 48 hours of culture, as described later. At 24 and 48 hours of incubation, samples were taken from the culture medium L11 in the jar fermenter (culture tank 11), the lower culture medium (liquid L14b) in the settler (container 14), and the MCT oil (solvent L12) in the oil tank (solvent tank 12). The concentrations of phenylethyl alcohol and phenylalanine were measured in the culture medium, and the concentration of phenylethyl alcohol was measured in the MCT oil. The concentrations of phenylethyl alcohol and phenylalanine in the sampled culture medium were analyzed using a Shimadzu HPLC system, with a Nacalai Tesque Cosmoseal C18-AR-II column used for separation. The HPLC separation conditions were as follows: 40% methanol and 0.069% perchloric acid were used as the mobile phase, the flow rate was 1.0 ml / min, and the column temperature was 40°C.The phenylethyl alcohol concentration in the sampled MCT oil was analyzed using a Shimadzu GC system, with a Restek Rxi-5MS micropolar column. The GC analysis conditions involved heating from 60°C to 250°C at 10°C / min, and holding at 250°C for 20 minutes. Concentration measurements were performed on the sampled culture medium L11, liquid L14b, solvent L12, and MCT oil recovered from the oil tank during MCT oil replacement, and the obtained concentrations were summed. The results of this experiment showed that phenylethyl alcohol was produced at a concentration of 18.4 g / L after 24 hours of incubation and 34.3 g / L after 48 hours of incubation. Phenylalanine was produced as a by-product at a concentration of 0.26 g / L after 24 hours of incubation and 0.03 g / L after 48 hours of incubation.

[0105] [Example 3] <Phenylacetaldehyde Production Experiment> A strain producing phenylacetaldehyde and / or phenylethyl alcohol (PEA-2 strain) was inoculated onto A agar medium containing 50 μg / ml kanamycin [(NH2)2CO2 2g, (NH4)2SO4 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・7H2O 0.5g, 0.06%(w / v) FeSO4・7H2O + 0.042%(w / v) MnSO4・2H2O 1ml, 0.02%(w / v) biotin solution 1ml, 0.01%(w / v) thiamin solution 2ml, yeast extract 2g, vitamin assay casamino acid 7g, glucose 40g, agar 15g suspended in 1L of distilled water] and incubated at 33°C for 18 hours. Phenylatealdehyde and / or phenylethyl alcohol producing strain (PEA-2 strain) grown on the above plate was inoculated into a test tube containing 10 ml of liquid medium A containing 50 μg / ml kanamycin [(NH2)2CO2 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) thiamin solution 2 ml, yeast extract 2 g, vitamin assay casamino acid 7 g, glucose 40 g dissolved in 1 L of distilled water] using a platinum loop, and cultured aerobically with shaking at 33°C for 20 hours. The culture medium obtained in this way was concentrated by centrifugation (room temperature, 3,000 × g, 10 minutes). The concentrated bacterial cells were then placed in 600 ml of liquid medium A containing 50 μg / ml kanamycin to the initial OD. 610The culture medium was inoculated into a 1 L jar fermenter after adding the necessary amount to reach 0.8–1.0. Glucose (initial concentration 73 g / L) was added as a substrate, and the culture medium was cultured at 33°C with a 10 N sodium hydroxide solution to adjust the pH to 7.0, stirring for 22 hours under aerated conditions. The cultured cells were collected by centrifugation (20°C, 5,000 × g, 10 min). The obtained cells were suspended in 600 ml of BT(-urea) liquid medium [0.7% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4・7H2O, 0.00006% (w / v) FeSO4・7H2O + 0.000042% (w / v) MnSO4・2H2O, 0.00002% (w / v) thiamin solution]. The bacterial suspension was transferred to a 1 L jar fermenter, glucose (initial concentration 73 g / L) was added as a substrate, the pH of the reaction solution (culture medium) in the jar fermenter was adjusted to 7.0 using a 10 N sodium hydroxide aqueous solution, the temperature inside the jar fermenter was adjusted to 33°C, and the culture was stirred under aerated conditions. Specifically, the culture was performed using the apparatus 101 shown in Figure 7. In this example, a jar fermenter was used as the culture tank 11, a liquid-liquid separator (settler) was used as the container 14, a mixing device (mixer) was used as the container 13, and an MF membrane (Micro Filtration membrane) module was used as the bacterial cell isolation device 21. An oil tank was used as the solvent tank 12 and filled with MCT oil (solvent L12). 100% medium-chain triacylglycerides were used as the MCT oil. After the start of cultivation, the culture medium containing the bacterial cells was sent to an MF membrane (Micro Filtration membrane) module (bacterial cell isolation device 21) connected to a jar fermenter (culture tank 11) and continuously filtered. The culture medium containing the bacterial cells (L21a) that passed through the MF membrane module (bacterial cell isolation device 21) was returned to the jar fermenter (culture tank 11), and the culture medium (L21b) from which the bacterial cells had been filtered by the MF membrane module (bacterial cell isolation device 21) was sent to a mixer (container 13) and mixed with MCT oil (solvent L12) supplied from an oil tank (solvent tank 12).A mixture (L13) of culture medium (L21b) without bacterial cells and MCT oil (solvent L12) was sent to a settler (container 14) and separated. The separated MCT oil (liquid L14a on the upper layer of the settler (container 14)) was sent to an oil tank (solvent tank 12), and the culture medium from which the MCT oil had been separated (liquid L14b on the lower layer of the settler (container 14)) was sent to a jar fermenter (culture tank 11). The MCT oil (solvent L12) in the oil tank (solvent tank 12) was replaced as needed, and continuous operation was performed for 48 hours. During continuous operation, the culture medium L11 was continuously delivered through liquid transport path 31, culture medium L21a through liquid transport path 32, culture medium L21b through liquid transport path 33, solvent L12 through liquid transport path 34, mixed liquid L13 through liquid transport path 35, liquid L14a through liquid transport path 36, and liquid L14b through liquid transport path 37, circulating within the apparatus 101 during operation. The MCT oil was replaced (liquid L14a was removed) every 24 hours of culture. The replaced (removed from the oil tank) MCT oil was recovered, and the concentration of phenylacetaldehyde in the recovered MCT oil was measured. The phenylacetaldehyde concentration in the MCT oil was analyzed using a Shimadzu GC system, with a micropolar column, Restek Rxi-5MS, being used. The GC system analysis conditions involved raising the temperature from 60°C to 250°C at a rate of 10°C / min, and then holding at 250°C for 20 minutes. The results of this experiment showed that phenylacetaldehyde was produced at a concentration of 8.9 mg / L after 48 hours of incubation.

[0106] [Example 4] <Experiment in the production of phenylethyl alcohol by resin adsorption method> A strain that produces phenylacetaldehyde and / or phenylethyl alcohol (PEA-2 strain) was spread onto A agar medium containing 50 μg / ml kanamycin [(NH2)2CO2 2g, (NH4)2SO4 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・7H2O 0.5g, 0.06%(w / v) FeSO4・7H2O + 0.042%(w / v) MnSO4・2H2O 1ml, 0.02%(w / v) biotin solution 1ml, 0.01%(w / v) thiamin solution 2ml, yeast extract 2g, vitamin assay casamino acid 7g, glucose 40g, agar 15g suspended in 1L of distilled water] and incubated at 33°C for 18 hours. Phenylatealdehyde and / or phenylethyl alcohol-producing strains grown on the above plates were inoculated into test tubes containing 10 ml of liquid medium A [(NH2)2CO2 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) thiamin solution 2 ml, yeast extract 2 g, vitamin assay casamino acid 7 g, glucose 40 g dissolved in 1 L of distilled water] using a platinum loop, and cultured aerobically with shaking at 33°C for 20 hours. The culture medium obtained in this way was concentrated by centrifugation (room temperature, 3,000 × g, 10 minutes). The concentrated bacterial cells were then placed in 600 ml of liquid medium A containing 50 μg / ml kanamycin to the initial OD. 610The culture medium was inoculated into a 1 L jar fermenter after adding the necessary amount to reach 0.8–1.0. Glucose (initial concentration 73 g / L) was added as a substrate, and the culture medium was cultured at 33°C with a 10 N sodium hydroxide solution to adjust the pH to 7.0, stirring for 22 hours under aerated conditions. The cultured cells were collected by centrifugation (20°C, 5,000 × g, 10 min). The obtained cells were suspended in 600 ml of BT(-urea) liquid medium [0.7% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4・7H2O, 0.00006% (w / v) FeSO4・7H2O + 0.000042% (w / v) MnSO4・2H2O, 0.00002% (w / v) thiamin solution]. The bacterial suspension was transferred to a 1 L jar fermenter, glucose (initial concentration 73 g / L) was added as a substrate, the pH of the reaction solution (culture medium) in the jar fermenter was adjusted to 7.0 using a 10 N sodium hydroxide aqueous solution, the temperature inside the jar fermenter was adjusted to 33°C, and the culture was stirred under aerated conditions. After the start of cultivation, the culture medium containing the bacterial cells was sent to an MF membrane (Micro Filtration membrane) module connected to the jar fermenter and continuously filtered. The culture medium containing the bacterial cells that passed through the MF membrane was returned to the jar fermenter, and the culture medium filtered by the MF membrane was passed through a resin column to adsorb phenylethyl alcohol onto the resin column. The culture medium that passed through the resin was returned to the jar fermenter. The resin column was replaced as needed, and continuous operation was performed for 48 hours. The phenylethyl alcohol adsorbed on the resin column was eluted with ethanol. The concentration of phenylethyl alcohol eluted from the resin with ethanol was analyzed using a Shimadzu HPLC system, with a Nacalai Tesque Cosmoseal C18-AR-II column. The HPLC separation conditions were as follows: 40% methanol and 0.069% perchloric acid were used as the mobile phase, the flow rate was 1.0 ml / min, and the column temperature was 40°C.

[0107] As a comparative example, a strain was prepared by introducing Enterobacter cloacae-derived ipdC into Corynebacterium glutamicum Rpea1 strain, referring to WO2020 / 130095, and phenylethyl alcohol production experiments were conducted using the resin adsorption method in the same manner as described above.

[0108] The results of this experiment showed that the comparative strain (Enterobacter cloacae-derived ipdC-introduced strain) produced 17.6 g / L of phenylethyl alcohol after 48 hours of culture, while the example strain PEA-2 (Morganella morganii-derived ipdC-introduced tyrB-weakened strain) produced 31.8 g / L of phenylethyl alcohol after 48 hours of culture.

[0109] [Example 5] <Phenylethyl alcohol production experiment using apparatus 101> Instead of strain PEA-2, a strain that produces phenylacetaldehyde and / or phenylethyl alcohol (strain PEA-3) was used, and a phenylethyl alcohol production experiment was conducted using apparatus 101 in the same manner as in Example 2. As a result of this experiment, 19.3 g / L of phenylethyl alcohol was produced after 24 hours of culture, and 32.2 g / L after 48 hours of culture. Phenylalanine was produced as a by-product at a rate of 0.44 g / L after 24 hours of culture, and 0.47 g / L after 48 hours of culture.

[0110] [Example 6] <Phenylethyl alcohol production experiment by resin adsorption method> Instead of strain PEA-2, a strain that produces phenylacetaldehyde and / or phenylethyl alcohol (strain PEA-3) was used, and the phenylethyl alcohol production experiment was carried out in the same manner as in Example 4. The amount of phenylethyl alcohol produced by strain PEA-3 (a tyrB weakened strain with ipdC introduced from Shimwellia blattae) was 31.9 g / L after 48 hours of culture.

[0111] [Example 7] Construction of a phenylacetaldehyde-producing strain (1) Construction of a plasmid for disrupting the chromosomal genes of Corynebacterium glutamicum R strain. The DNA region necessary for markerless gene disruption of the chromosomal genes of Corynebacterium glutamicum R strain was amplified by PCR. Each PCR fragment can be introduced into a vector by seamless cloning. The obtained DNA fragments were introduced into the markerless chromosomal gene modification plasmid pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. The primer sequences and the obtained plasmid are shown in Table 5. (2) Construction of a chromosomally modified strain for phenylacetaldehyde production A recombinant strain was constructed using a plasmid for chromosomal gene disruption in the same manner as in Example 1. The Corynebacterium glutamicum Rpea2 strain constructed in Example 1 was used as the host strain. An overview of this chromosomal gene recombination is shown in Table 6. (3) Construction of a strain with a phenylacetaldehyde production-related gene expression plasmid The phenylacetaldehyde production-related gene expression plasmid described above was introduced into Corynebacterium glutamicum Rpea3 strain. An overview of this strain is shown in Table 7.

[0112] [Example 8] <Phenylacetaldehyde Production Experiment by Two-Phase Culture Method> The phenylacetaldehyde-producing strain (PAD-1 strain) was inoculated onto A agar medium containing 50 μg / ml kanamycin [(NH2)2CO2 2g, (NH4)2SO4 7g, KH2PO4 0.5g, K2HPO4 0.5g, MgSO4・7H2O 0.5g, 0.06%(w / v) FeSO4・7H2O + 0.042%(w / v) MnSO4・2H2O 1ml, 0.02%(w / v) biotin solution 1ml, 0.01%(w / v) thiamin solution 2ml, yeast extract 2g, vitamin assay casamino acid 7g, glucose 40g, agar 15g suspended in 1L of distilled water] and cultured statically at 33°C for 18 hours. The phenylacetaldehyde-producing strains grown on the above plate were inoculated into test tubes containing 10 ml of liquid medium A [(NH2)2CO2 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) thiamin solution 2 ml, yeast extract 2 g, vitamin assay casamino acid 7 g, glucose 40 g dissolved in 1 L of distilled water] using a platinum loop, and cultured aerobically with shaking at 33°C for 20 hours. The culture medium obtained in this manner was concentrated by centrifugation (room temperature, 3,000 × g, 10 minutes). The concentrated bacterial cells were then placed in 60 ml of modified liquid medium A containing 50 μg / ml kanamycin [(NH4)2SO4 24 g, KH2PO4 1.5 g, MgSO4・7H2O 1.0 g, 0.06% (w / v) FeSO4・7H2O + 0.042% (w / v) MnSO4・2H2O 2 ml, 0.02% (w / v) biotin solution 2 ml, 0.01% (w / v) thiamin solution 4 ml, yeast extract 4 g dissolved in 1 L of distilled water] with an initial OD (Oral Dissociation) level. 610The culture medium was inoculated into a 150 ml jar fermenter after adding a solution to a value of 1.0–1.5. Glucose (initial concentration 73 g / L) was added as a substrate, and the culture medium was cultured at 33°C with a 5 N sodium hydroxide aqueous solution to adjust the pH to 7.0, under aerated conditions with stirring. Sixteen hours after the start of stirring, 80% v / v (48 ml) of solvent (tributyline) was added to the jar fermenter, and stirring was continued for another 32 hours under the same conditions, continuing until 48 hours from the start of culture. The solvent phase (solvent containing phenylacetaldehyde) was recovered from the jar fermenter. The concentration of phenylacetaldehyde in the solvent phase was analyzed using a Shimadzu HPLC system after dilution with ethanol, and a Nacalai Tesque Cosmoseal C18-AR-II column was used for separation. The HPLC separation conditions were as follows: 40% methanol and 0.069% perchloric acid were used as the mobile phase, the flow rate was 1.0 ml / min, and the column temperature was 40°C.

[0113] As a comparative example, a phenylacetaldehyde production experiment was conducted using the Corynebacterium glutamicum Rpea2 strain (a strain without gene A) constructed in Example 1, in the same two-phase culture method as described above.

[0114] The results of this experiment showed that the comparative example, strain Rpea2 (without gene A introduction and without adh gene knockout), produced 0.0 g / L of phenylacetaldehyde after 48 hours of culture, while the example, strain PAD-1 (with gene A introduction and adh gene knockout), produced 5.9 g / L of phenylacetaldehyde after 48 hours of culture. Phenylalanine was produced as a by-product at a concentration of 4.3 g / L after 48 hours of culture. In addition, strain PAD-1 produced 0 g / L of phenylethyl alcohol after 48 hours of culture.

[0115] The following lists the compound names and their CAS numbers as shown in Figure 3 (overall metabolic pathway diagram illustrating the biosynthesis pathway of phenylacetaldehyde) or Figure 6 (overall metabolic pathway diagram illustrating the biosynthesis pathway of phenylethyl alcohol). The "compound names" listed below are examples, and it goes without saying that synonymous names may exist. Furthermore, it goes without saying that the "CAS numbers" listed below are not necessarily exhaustive.

[0116] The enzymes encoded by the genes shown in Figure 3 (overall metabolic pathway diagram illustrating the biosynthesis pathway of phenylacetaldehyde) or Figure 6 (overall metabolic pathway diagram illustrating the biosynthesis pathway of phenylethyl alcohol) and their EC numbers are shown below. The "enzyme names" shown below are examples, and it goes without saying that synonymous names may exist. Furthermore, the "enzymes" encoded by the "genes" shown below are examples, and it goes without saying that there may be "genes" that encode "enzymes" with multiple functions.

[0117] 11 Culture tank 12 Solvent tank 13 Container 14 Container 21 Cell isolation device 22 Liquid mixer 23 Flow reactor type device 31 Liquid transport path 32 Liquid transport path 33 Liquid transport path 34 Liquid transport path 35 Liquid transport path 36 Liquid transport path 37 Liquid transport path 38 Liquid transport path 101 Device 102 Device L11 Culture medium L12 Solvent L13 Mixture L14a Liquid L14b Liquid L21a Culture medium L21b Culture medium

[0118]

Claims

1. A microorganism having the ability to produce at least one of phenylacetaldehyde and phenylethyl alcohol, wherein at least one gene (A) selected from the group consisting of (A1) to (A18) below, which encodes an enzyme having the activity to produce phenylacetaldehyde from phenylpyruvic acid, is introduced in an expressible form. (A1) A gene derived from Morganella morganii; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 4; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 4; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 4; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 1; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1; (A8) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted and / or added per unit, with 100 bases forming one unit, in the nucleotide sequence shown in Sequence ID No. 1; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1; (A10) A gene derived from Shimwellia blattae; (A11) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 12; (A12) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 12; (A13) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in SEQ ID NO: 12;(A14) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 12; (A15) A gene having the nucleotide sequence shown in Sequence ID No. 9; (A16) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 9; (A17) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 9, where 100 nucleotides constitute one unit, with 1 to 10 nucleotides deleted, substituted and / or added per unit; (A18) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No.

9.

2. The microorganism according to claim 1, wherein the microorganism has reduced or absent aminotransferase (tyrB) function.

3. The microorganism according to claim 1 or 2, wherein the function of at least one enzyme selected from the group consisting of (1) to (3) below is reduced or absent: (1) 3-dehydroschimate dehydratase (qsuB), (2) lactate dehydrogenase (ldhA), (3) halo acid dehalogenase superfamily phosphatase (hdpA).

4. The microorganism according to any one of claims 1 to 3, wherein the host of the microorganism is a Corynebacterium or a transformed Corynebacterium.

5. The microorganism according to any one of claims 1 to 4, wherein the host of the microorganism is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum.

6. The microorganism according to any one of claims 1 to 5, wherein the host of the microorganism is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

7. A method comprising at least the steps of: culturing a microorganism according to any one of claims 1 to 6 in a culture tank; and recovering a liquid containing phenylacetaldehyde and / or phenylethyl alcohol from the culture medium in the culture tank.

8. The method according to claim 7, further comprising: separating at least a portion of the culture medium in the culture tank into a liquid 1 containing the microorganisms and a liquid 2 having a lower concentration of the microorganisms than the liquid 1; transporting the liquid 1 to the culture tank; mixing the liquid 2 with a solvent transported from a solvent tank to produce a mixed solution; transporting the mixed solution to a container; transporting the liquid from the lower layer of the container to the culture tank; transporting the liquid from the upper layer of the container to the solvent tank; and removing the liquid from the upper layer of the container and / or the liquid in the solvent tank.

9. The method according to claim 8, wherein the step of removing the liquid is performed when the concentration of phenylacetaldehyde and / or phenylethyl alcohol contained in the upper layer of the container exceeds a predetermined concentration.

10. The method according to claim 8 or 9, wherein the mixing of the liquid 2 and the solvent transported from the solvent tank is carried out by stirring and / or by fluid merging.

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