WO2019030808A1 - Microorganism capable of producing aromatic compound - Google Patents

Microorganism capable of producing aromatic compound Download PDF

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WO2019030808A1
WO2019030808A1 PCT/JP2017/028650 JP2017028650W WO2019030808A1 WO 2019030808 A1 WO2019030808 A1 WO 2019030808A1 JP 2017028650 W JP2017028650 W JP 2017028650W WO 2019030808 A1 WO2019030808 A1 WO 2019030808A1
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strain
gene
frt
fbr
dna
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PCT/JP2017/028650
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French (fr)
Japanese (ja)
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駒大輔
大本貴士
山中勇人
森芳邦彦
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地方独立行政法人大阪産業技術研究所
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Priority to PCT/JP2017/028650 priority Critical patent/WO2019030808A1/en
Priority to JP2019535461A priority patent/JP7034496B2/en
Publication of WO2019030808A1 publication Critical patent/WO2019030808A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present disclosure relates to microorganisms that produce aromatic compounds, and more specifically to phenylalanine producing bacteria, tyrosine producing bacteria, bacteria producing other aromatic compounds, and their uses.
  • Phenylalanine and tyrosine are used as additives to livestock manure, supplements, pharmaceutical raw materials and the like. Phenylalanine and tyrosine are generally produced by fermentation using microorganisms.
  • Non-Patent Document 1 it is common to try to improve the production amount by eliminating the bottleneck of the synthetic pathway by expressing the key gene on the synthetic pathway with a plasmid (for example, Non-Patent Document 1). .
  • a production bacterium directly introduced into a chromosome without using a plasmid has been reported (for example, Non-Patent Document 2).
  • Non-Patent Document 3 It has also been reported that other aromatic compounds can be produced by introducing additional enzymes into phenylalanine and tyrosine-producing bacteria.
  • Antibiotics need to be added in order to retain the plasmid, but in large-scale fermentation production, significant cost increases, risk of drug spillage into the environment, emergence of drug resistant bacteria, etc. become problems. .
  • microorganisms that do not use a plasmid tend to have low production.
  • the present disclosure provides, in one aspect, a plasmid-free and productivity-enhanced microorganism.
  • the present disclosure relates, in one aspect, to a microorganism belonging to the transformed Escherichia genus, wherein at least the following five genes are introduced together with an expression inducible promoter on a chromosome.
  • aroA aroB
  • aroC aroG fbr or aroF fbr
  • pheA fbr or tyrA fbr aroG fbr or aroF fbr
  • the present disclosure relates to a method of producing an aromatic compound, which comprises culturing the microorganism according to the present disclosure in a culture medium.
  • FIG. 1 is a schematic view of a scheme for producing a microorganism according to the present disclosure.
  • FIG. 2 is a diagram showing an outline of a production scheme of a microorganism according to the present disclosure.
  • the microorganism according to the present disclosure is a transformant of a microorganism belonging to the genus Escherichia, but in one or more embodiments, it is a transformant of Escherichia coli.
  • gene means a gene of E. coli unless otherwise stated.
  • the gene may include an ortholog of the gene of E. coli (homologs derived from other species).
  • the sequences of E. coli genes refer to those of strain K-12 (wild-type) or those registered at the time of filing of the application with a database (such as NCBI GENBANK) unless otherwise specified, but have functions equivalent to those of wild-type
  • the sequence may be different within the scope of the present invention or the range encoding a protein equivalent to that of the wild type, and may be an orthologous sequence as described above.
  • the gene may be codon optimized for expression in the host microorganism.
  • a gene introduced into a chromosome together with an expression-inducible promoter may be a single gene or an operon capable of expressing multiple genes.
  • the expression-inducible promoter is not particularly limited.
  • a promoter capable of inducing expression at a higher expression level than a wild-type (native) promoter such as, for example, a T7 promoter.
  • the host microorganism is preferably a strain having a T7 RNA polymerase gene.
  • a host microorganism for example, a DE3 strain obtained by lysogenizing ⁇ DE3 phage can be mentioned.
  • a method for introducing a gene into a chromosome together with an expression-inducible promoter is not particularly limited, but in one or more embodiments, the method described in the Examples (the method described in Non-patent Document 2) is mentioned Be
  • plasmid-free means that a gene that contributes to the improvement of the productivity of aromatic compounds such as phenylalanine and tyrosine is overexpressed on the chromosome, and the microorganism according to the present disclosure retains the plasmid Well, it does not have to hold the plasmid.
  • phenylalanine and tyrosine may mean L-phenylalanine and L-tyrosine unless otherwise stated.
  • the “aroA” gene is a gene encoding 5-enolpyruvyl shikimate-3-phosphate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 945528.
  • the "aroB” gene is a gene encoding 3-dehydroquinate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 947927.
  • the "aroC” gene is a gene encoding chorismate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 946814.
  • the “aroG” gene is a gene encoding 3-deoxy-D-arabino-7-phosphohepturate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 645605.
  • the “aroG fbr ” gene refers to a desensitized variant of aroG feedback inhibition.
  • the aroG fbr in one or more embodiments, those disclosed in Japanese Patent Application Laid-Open No. 05-236947 can be used.
  • the “aroF” gene is a gene encoding 3-deoxy-D-arabino-7-phosphohepturate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 947084.
  • the “aroF fbr ” gene refers to a desensitized variant of aroF feedback inhibition.
  • aroF fbr can use what is disclosed by Unexamined-Japanese-Patent No. 05-236947 in one or several embodiment.
  • the "pheA” gene is a gene encoding chorismate mutase and prephenate dehydratase, and in one or more embodiments is NCBI GENBANK Gene ID: 947081.
  • the "pheA fbr " gene refers to a desensitized variant of feedback inhibition of pheA.
  • the pheA fbr those disclosed in JP-A-2006-311833 and JP-A-05-344881 can be used in one or more embodiments.
  • the "tyrA” gene is a gene encoding chorismate mutase and prephenate dehydrogenase, which in one or more embodiments is NCBI GENBANK Gene ID: 947115.
  • the "tyrA fbr " gene refers to a desensitized variant of the feedback inhibition of tyrA.
  • the tyrA fbr in one or more embodiments, those disclosed in JP-A-05-076352 can be used.
  • the “ppsA” gene is a gene encoding phosphoenolpyruvate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 946209.
  • the “tktA” gene is a gene encoding transketolase 1, which in one or more embodiments is NCBI GENBANK Gene ID: 947420.
  • the “aroD” gene is a gene encoding 3-dehydroquinate dehydratase, which in one or more embodiments is NCBI GENBANK Gene ID: 946210.
  • the "aroE” gene is a gene encoding dehydroshikimic acid reductase, which in one or more embodiments is NCBI GENBANK Gene ID: 947776.
  • the “ydiB” gene is a gene encoding quinic acid / shikimic acid 5-dehydrogenase, which in one or more embodiments is NCBI GENBANK Gene ID: 946200.
  • the "aroL” gene is a gene encoding shikimate kinase II, which in one or more embodiments is NCBI GENBANK Gene ID: 945031.
  • the “aroK” gene is a gene encoding shikimate kinase I, which in one or more embodiments is NCBI GENBANK Gene ID: 2847759.
  • the "tyrB" gene is a gene encoding a tyrosine aminotransferase, which in one or more embodiments is NCBI GENBANK Gene ID: 945031.
  • aromatic compounds such as phenylalanine and tyrosine.
  • tyrB was often overexpressed.
  • the present inventors have found that overexpression of tyrB has a large negative effect due to metabolic load, and that overexpression of tyrB can improve phenylalanine and tyrosine production.
  • the “ldhA” gene is a gene derived from Cupriavidus necator JCM 20644 encoding lactate dehydrogenase, and in one or more embodiments, is a gene encoding NCBI GENBANK Protein ID: CAJ91827.
  • the "ipdC” gene is a gene derived from Azospirillum brasilense NBRC 102289 which encodes indole-3-pyruvate / phenylpyruvate decarboxylase, and in one or more embodiments, the NCBI GENBANK Protein ID: CAA 67899 Is a gene encoding
  • the first embodiment of the microorganism according to the present disclosure is a microorganism belonging to the genus Escherichia in which the following five genes are introduced together with an expression-inducible promoter on a chromosome.
  • aroA aroB
  • aroC aroG fbr or aroF fbr
  • pheA fbr pheA fbr or tyrA fbr
  • the microorganism according to the present disclosure has the ability to produce phenylalanine, and the fermentation method using the microorganism according to the present disclosure enables the production of phenylalanine .
  • the microorganism according to the present disclosure when the gene of (5) is tyrA fbr , the microorganism according to the present disclosure has an ability to produce tyrosine, and the production of tyrosine becomes possible by a fermentation method using the microorganism according to the present disclosure.
  • the aroG fbr or aroF fbr in the above (4) encodes the same enzyme and can be mutually substituted.
  • a desensitized variant of feedback inhibition of aroH (NCBI GENBANK Gene ID: 946229) can also be used.
  • the present inventors in addition to the above (4) and (5), by overexpressing all of the above (1) to (3), excess of the introduced gene (for example, It has been found that the metabolic load due to expression can be reduced.
  • a second embodiment of the microorganism according to the present disclosure is a microorganism belonging to the genus Escherichia in which the following two genes are introduced together with an expression-inducible promoter on a chromosome in addition to the above-mentioned five genes of the first embodiment. is there. (6) ppsA (7) tktA
  • the above (7) tktA promoter is a mutant T7 promoter having a reduced expression inducibility, from the viewpoint of improving the productivity of the target aromatic compound. Is preferred.
  • At least one of the following three genes is further chromosomally It is a microorganism belonging to the genus Escherichia which has been introduced together with an expression-inducible promoter.
  • aroD (9) aroE or ydiB (10) aroL or aroK Of (8) to (10), preferably two or all of them are introduced.
  • aroE or ydiB in (9) above can be substituted for each other, aroE is preferable as the gene in (9) above.
  • aroL or aroK of the above (10) can be replaced with each other, but as the gene of the above (10), aroL is preferable.
  • a fourth embodiment of the microorganism according to the present disclosure in addition to the first, second or third embodiment, the same or different enzymes encoding 4-hydroxyphenylpyruvate, phenylalanine or tyrosine as an enzyme substrate are used. It is a microorganism in which a gene of origin has been introduced together with an expression inducible promoter on a chromosome.
  • the microorganism according to the present disclosure is 2-phenylethanol or 2- (4-hydroxyphenyl) Since it has an ability to produce ethanol, it can be used for the production of these aromatic compounds.
  • heterologous gene ldhA encoding lactate dehydrogenase when introduced on a chromosome together with an expression-inducible promoter, it becomes a microorganism capable of producing phenyl lactic acid or 4-hydroxyphenyl lactic acid, and these aromatic compounds Can be used in the manufacture of That is, when a gene encoding an enzyme having phenylpyruvate, 4-hydroxyphenylpyruvate, phenylalanine or tyrosine as a substrate is further introduced, an aromatic compound can be produced according to the gene.
  • Lactobacillus brevis tyrosine decarboxylase gene (NCBI GENBANK Gene ID: 4413406) or Pseudomonas putida NBRC 100650 aromatic amino acid decarboxylase gene (NCBI GENBANK Gene ID: 1045854) or their orthologous genes can be used to produce phenylethylamine and tyramine.
  • Non-patent Document 3 for example, by introducing a phenylacetaldehyde dehydrogenase gene of Escherichia coli (Gene ID of NCBI GENBANK: 945933) or an ortholog gene thereof together with a phenylpyruvate decarboxylase gene. It becomes possible to produce phenylacetic acid and 4-hydroxyphenylacetic acid.
  • the method for producing an aromatic compound (including phenylalanine and tyrosine) using a microorganism according to the present disclosure is not particularly limited, and an aromatic compound is produced by culturing under the conditions of a common culture method and / or a fermentation method. it can.
  • the culture / fermentation medium is not particularly limited as long as it contains other components such as a carbon source, nitrogen source, phosphate source, sulfur source, minerals, vitamins and the like, and the microorganism can be grown.
  • the methods of the Examples can be referred to.
  • preculture is carried out at 27 ° C., inoculated in minimal medium (eg, M9M2 medium) at a concentration of 1%, and cultured for 4-5 hours at 37 ° C. (OD 660 of 0.3 to 0.4)
  • minimal medium eg, M9M2 medium
  • culture is carried out at 27 ° C. for 43 to 44 hours to obtain the target aromatic compound in the culture solution and / or in the fungus.
  • culture and fermentation conditions are not limited to these.
  • the metabolic load due to overexpression of the introduced gene is reduced in one or more embodiments. Glucose can be added to produce aromatic compounds without causing catabolite repression.
  • the present disclosure may relate to: ⁇ 1> A microorganism belonging to the transformed Escherichia genus, A microorganism in which at least the following five genes are introduced together with an expression inducible promoter on a chromosome. (1) aroA (2) aroB (3) aroC (4) aroG fbr or aroF fbr (5) pheA fbr or tyrA fbr ⁇ 2> The microorganism according to ⁇ 1>, wherein the following two genes are introduced together with an expression inducible promoter on a chromosome.
  • ⁇ 5> The microorganism according to any one of ⁇ 1> to ⁇ 4>, wherein the gene of (5) is pheA fbr and has phenylalanine producing ability.
  • ⁇ 6> The microorganism according to any one of ⁇ 1> to ⁇ 4>, wherein the gene of (5) is tyrA fbr and has a tyrosine producing ability.
  • a gene of the same or different gene encoding an enzyme that uses phenylpyruvate, 4-hydroxyphenylpyruvate, phenylalanine, or tyrosine as a substrate is introduced together with an expression inducible promoter on a chromosome, ⁇ 1
  • the heterologous gene ipdC encoding phenylpyruvate decarboxylase is introduced on a chromosome together with an expression-inducible promoter, and has the ability to produce 2-phenylethanol or 2- (4-hydroxyphenyl) ethanol
  • the heterologous gene ldhA encoding lactate dehydrogenase is introduced on a chromosome together with an expression-inducible promoter, and has the ability to produce phenyllactic acid or 4-hydroxyphenyllactic acid, ⁇ 1> to ⁇ 4>
  • a method for producing an aromatic compound comprising culturing the microorganism according to any one of ⁇ 1> to ⁇ 9> in a culture medium.
  • FIG. 1 shows a scheme for introducing three genes (A, B, C) into a chromosome.
  • strain A strain B, strain C
  • the strain in which the Km cassette has been excised by FLP / FRT recombination in strain A is obtained as acceptor strain A.
  • the lysate B obtained by infecting the donor strain B with P1 vir phage is infected with the acceptor strain A to perform P1 transduction. From the resulting strain, one obtained by excising Km cassette by FLP / FRT recombination is obtained as acceptor strain AB.
  • the lysate C obtained by infecting the donor strain C with P1 vir phage is infected with the acceptor strain AB to perform P1 transduction.
  • the Km cassette is excised by FLP / FRT recombination, it is possible to obtain a strain in which three genes (A, B, C) have been introduced into the chromosome.
  • Fig. 2 Fig. 2 of the same document.
  • pET-21a-FRT has one MCS
  • pETDuet-FRT has two MCS.
  • the linear DNA fragment is amplified by PCR.
  • the primers have a 50 base sequence (H1 or H2) outside the target site and a 20 base sequence (P1 or P2) derived from a plasmid.
  • the amplified fragment is then electroporated into E. coli cells carrying pKD46.
  • the desired gene is introduced into the target site of the chromosome.
  • the Km cassette can be removed by FLP / FRT recombination by introducing the plasmid pCP20 into this recombinant.
  • EcAroG-F and EcAroG-D146N-RM GGGGTGATCATATTATTGAGAAACTC, SEQ ID NO: 05
  • EcAroG-D146N-FM GAGTTTCTCAATATATGATCACCCC, SEQ ID NO: 06
  • EcAroG-R SEQ ID NO: 6
  • overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the two amplified fragments as a template and the EcAroG-Nde (CCAACCATATGAATTATCAGAACGACGATTTACG, SEQ ID NO: 08) and EcAroG-R primer pair, and the 146th asparagine was subjected to overlap extension PCR.
  • AroG-encoding DNA (aroG fbr ) in which the acid was substituted with asparagine was obtained.
  • the amplified DNA and pET21a-FRT vector Non-patent document 2, Koma et al. Appl. Microbiol. Biotechnol.
  • Colony direct PCR was performed using T7 promoter primer (TAATACGACTCACTATAGG, SEQ ID NO: 09, the same applies in the following) and T7 terminator primer (GCTAGTTATTGCTCAGCGG, SEQ ID NO: 10, the same in the following) to select a strain having a desired plasmid.
  • T7 promoter primer TAATACGACTCACTATAGG, SEQ ID NO: 09, the same applies in the following
  • GCTAGTTATTGCTCAGCGG SEQ ID NO: 10
  • the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid is extracted using NucleoSpin Plasmid QuickPure (MACHERY-NAGEL, the same applies hereinafter). Refined.
  • the LB agar medium was adjusted to pH 7.0 with 10 g polypeptone (Nippon Pharmaceutical Co., Ltd.), 5 g dry yeast extract (Nippon Pharmaceutical Co., Ltd.), 10 g sodium chloride, 20 g agar, and sodium hydroxide per liter of pure water. .
  • the LB liquid medium was adjusted to pH 7.0 with 10 g of polypeptone (Nippon Pharmaceutical Co., Ltd.), 5 g of dried yeast extract (Nippon Pharmaceutical Co., Ltd.), 10 g of sodium chloride, and sodium hydroxide per liter of pure water. same as below.
  • EcPheA-F (CCAACCATATGACATCGGAAAACCCGTTAC, SEQ ID NO: 11) and EcPheA-S330P-RM (GAATCGGGCGTGTGGTTCCAGAC, SEQ ID NO: 12) primer pair and EcPheA-S330P-FM (GTCTGGAACCACGCCGCGTC, SEQ ID NO: 13) using the genomic DNA of E. coli MG1655 as a template
  • the two DNA fragments were amplified by Phusion Hot Start II DNA polymerase using the R (CACTCGAGTCAGGTTGATCAACAGGCAC, SEQ ID NO: 14) primer pair.
  • the purified DNA was ligated in a conventional manner, transformed into E. coli DH5 ⁇ , and cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • the conditions for the PCR reaction were based on the basic conditions described in the instruction manual.
  • overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the two amplified fragments as a template and the pps-F and pps-R primer pairs to remove the NcoI site in the sequence of the ppsA gene. DNA was obtained.
  • the amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E.
  • the amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit.
  • the purified DNA was ligated in a conventional manner, transformed into E. coli DH5 ⁇ , and cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • a DNA fragment was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-tktA as a template and a pair of -8TC-FM1 (CGAAATTAATACGACCCACTATAGGGG, SEQ ID NO: 23) and a T7 terminator primer.
  • overlap extension PCR is performed with Phusion Hot Start II DNA polymerase using the pair of F-Bgl and T7 terminator primer with the two amplified fragments as a template, and the sequence of 8 bases upstream from the transcription start origin of T7 promoter is A DNA fragment containing a mutant T7 promoter sequence substituted for T to C was obtained.
  • the DNA fragment and the pET21a-FRT vector were digested with BglII and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from the gel using QIAquick Gel Extraction kit.
  • the purified DNA was ligated in a conventional manner, transformed into E. coli DH5 ⁇ , and cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • EcTyrA-F (CCAACCATATGGTTGCTGAATTGACGC, SEQ ID NO: 24) and EcTyrA-RM1 (CGCGAGGCGAGAGATAGATGCCTCGGC, SEQ ID NO: 25) and a pair of EcTyrA-F1 (GCGAGGCATCTATCTTGTGCTCGGC) and a GlcNAG as a template, using E.
  • overlap extension PCR is performed by Phusion Hot Start II DNA polymerase using the three amplified fragments as a template and a primer pair of EcTyrA-F and EcTyrA-R, and methionine at position 53 is substituted with isoleucine, and 354 A DNA (TyrA fbr ) encoding TyrA in which the second alanine was substituted by valine was obtained.
  • the amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit.
  • the purified DNA was ligated in a conventional manner, transformed into E. coli DH5 ⁇ , and cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • the amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit.
  • the purified DNA was ligated in a conventional manner, transformed into E. coli DH5 ⁇ , and cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • coli DH5 ⁇ is transformed by reacting the DpnI-treated aroB gene DNA with NdeI and XhoI-digested pET21a-FRT using the Gibson assembly system (New England Biolabs, the same shall apply hereinafter), and 20 ⁇ g / ml kanamycin It culture
  • EcAroA-F (CAACAC CATGGA ATCC CTGACGTTACAAC, SEQ ID NO: 34) and EcAroA-R (CAAAGCTTTCAGGCTGCCTGGCTAATCC, SEQ ID NO: 35) primer pair using the genomic DNA of E. coli MG1 655 as a template and EcAroA-F (CAAAAGCTTTAGGCTGCCTGGCTAATCC, SEQ ID NO: 35).
  • DNA of aroA gene and aroC gene is digested with NcoI and HindIII, while DNA of aroL gene is digested with NdeI and XhoI, and DNA is separated by 1% agarose gel electrophoresis, respectively using QIAquick Gel Extraction kit DNA was extracted and purified from the gel.
  • pET21a-FRT vector is digested with NdeI and XhoI, and pET21d-FRT vector (Non-patent document 2, Koma et al. Appl. Microbiol. Biotechnol.
  • Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, extracted and purified using NucleoSpin Plasmid QuickPure, pET21d-FRT-aroA, pET21d -FRT-aroC and pET21a-FRT-aroL were obtained.
  • DNA fragment containing aroA gene by Phusion Hot Start II DNA polymerase using pET21d-FRT-aroA as a template and ALC-F (GAAATTAATACGACTCACTATAGGGGAATTG, SEQ ID NO: 40) and ALC-aroA-R (TCAGGCTGCCTGCGTACTAATC, SEQ ID NO: 41) primer pair was amplified.
  • PET21a-FRT-aroL as a template, ALC-aroL-F (GATTAGCCAGGCACTCACTACTTTGAAAGGAGATACATATATGACAC, SEQ ID NO: 42) and ALC-aroL-R (GGATGGCCTCCTTTAGATCCTCAACAATTGATCGTCTGTGC, SEQ ID NO: 43) using a primer pair by using the Phuaroh Hot Start DNA polymerase The DNA fragment containing the gene was amplified.
  • pET21d-FRT-aroC is used as a template and contains ALC-aroC-F (GGATCTAAAGAGGGCATCCATGGCTGGAACACAATTGG, SEQ ID NO: 44) and ALCorEDB-R (GGATGGCGTCCTTTAGATCCTC AACAATTGATCGTCTGTGC, SEQ ID NO: 45) primer pair containing Phusion Hot Start II DNA aroC by a DNA polymerase The DNA fragment was amplified.
  • overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the primer pair of aroALC-F1 (ATGGAATCCCTGACGTTACAAAC, SEQ ID NO: 46) and aroALC-R1 (TTACCAGCGTGGAATATCAGTCTTC, SEQ ID NO: 47) using the three amplified fragments as a template. Conducted to obtain a DNA fragment containing aroALC. The resulting DNA fragment pCR TM II-Blunt-TOPO TM (Thermo Fisher Scientific, hereinafter the same) is reacted with a reagent of TOPO cloning including, E.
  • coli DH5 ⁇ was transformed, the LB agar medium containing 20 [mu] g / ml of kanamycin It was cultured at 37 ° C. overnight. Colony direct PCR using aroALC-F1 and aroALC-R1 was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and pCR TM II-Blunt-TOPO TM -aroALC is extracted and purified using NucleoSpin Plasmid QuickPure did.
  • the pCR TM II-Blunt-TOPO TM -aroALC as a template, using aroALC-F1 and aroALC-R1 primer pair to amplify a DNA fragment containing the AroALC gene by Phusion Hot Start II DNA polymerase.
  • the pCR TM II-Blunt-TOPO TM -BB-aroALC as a template, using the pET21a-ALC-R and pET21a-ALC-F primer pair, a DNA fragment containing the plasmid backbone by Phusion Hot Start II DNA polymerase
  • the Both DNA fragments obtained were reacted using the Gibson assembly system, transformed into E.
  • DNA of aroE gene and aroD gene is digested with NdeI and XhoI, while DNA of aroB gene is digested with NcoI and HindIII, and DNA is separated by 1% agarose gel electrophoresis, respectively using QIAquick Gel Extraction kit DNA was extracted and purified from the gel.
  • the pET21a-FRT vector is digested with NdeI and XhoI
  • the pET21d-FRT vector is digested with NcoI and HindIII
  • DNA is separated by 1% agarose gel electrophoresis
  • the corresponding DNA is gelled using the QIAquick Gel Extraction kit. It extracted and refined.
  • the DNA of purified aroE gene and pET21a-FRT, the purified DNA of aroD gene and pET21a-FRT, and the purified DNA of aroB gene and pET21d-FRT are respectively transformed into E. coli DH5 ⁇ by ligation.
  • the cells were cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C.
  • DNA fragment containing aroE gene by Phusion Hot Start II DNA polymerase using pET21d-FRT-aroE as template and EDB-F (GAAATTAATACGACTCACTATAGGGGAATTG, SEQ ID NO: 56) and EDB-aroE-R (CATATGTATATCTCCTCTTCTTAAGTCACGCGGACAATTCCTCC, SEQ ID NO: 57) primer pair was amplified.
  • EDB-aroD-F CGTGACTTTAAGAAGGAGATATACATATGAAACCGTAACTGTAAAAGACC, SEQ ID NO: 58
  • EDB-aroD-R GGATGGCCTCCTTTAGATCCTTTATGCCTGTGTAAAATAGTTAATAC, SEQ ID NO: 59
  • a DNA fragment containing the aroB gene was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroB as a template and EDB-aroB-F2 (GGATCTAAAGGAGGCCATCATGGAGCGTATTGTCGTTACTC, SEQ ID NO: 60) and ALCorEDB-R primer pair.
  • overlap extension PCR was performed with Phusion Hot Start II DNA polymerase using the primer pair of aroEDB-F1 (ATGGAAACCTATGCTGTTTTTGG, SEQ ID NO: 61) and aroEDB-R1 (TTACGCTGATTGACAATCGGC, SEQ ID NO: 62) using the three amplified fragments as templates.
  • the resulting DNA fragment is reacted with a reagent of TOPO cloning including pCR TM II-Blunt-TOPO TM , E. coli DH5 ⁇ was transformed, overnight at 37 ° C. using a LB agar medium containing 20 [mu] g / ml kanamycin culture did. Colony direct PCR using pET21a-EDB-R and pET21a-EDB-F was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C.
  • pCR TM II-Blunt-TOPO TM -BB-aroEDB is extracted using NucleoSpin Plasmid QuickPure And refined.
  • the pCR TM II-Blunt-TOPO TM -aroEDB as a template, using aroEDB-F1 and aroEDB-R1 primer pair to amplify a DNA fragment containing the AroEDB gene by Phusion Hot Start II DNA polymerase.
  • the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 50 ⁇ g / ml of ampicillin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
  • the resulting DNA fragment was purified with QIAquick PCR Purification Kit (Qiagen, hereinafter the same), digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara).
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, then self-ligated using DNA ligation kit (Takara, the same in the following), transformed into E. coli DH5 ⁇ , and LB agar medium containing 20 ⁇ g / ml kanamycin. And incubated overnight at 37 ° C.
  • Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid.
  • the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroLC. Obtained.
  • a DNA fragment containing aroAC gene is amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroALC as a template and aroAC-F (CTTAAAGTCAGGCTGCCTGCGC, SEQ ID NO: 67) and aroAC-R (AAGGAGGCCATCATCATGCTGGAAAC, SEQ ID NO: 68) did.
  • the resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara).
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5 ⁇ , and transformed at 37 ° C using LB agar medium containing 20 ⁇ g / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroAC. Obtained.
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5 ⁇ , and transformed at 37 ° C using LB agar medium containing 20 ⁇ g / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroAL. Obtained.
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5 ⁇ , and transformed at 37 ° C using LB agar medium containing 20 ⁇ g / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using an LB liquid medium containing 20 ⁇ g / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroDB. Obtained.
  • pET21a-FRT-aroEB A DNA fragment containing aroEB gene is amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroEDB as a template using aroEB-F (TCACGCGGACAAATTCCTCC, SEQ ID NO: 72) and aroEB-R (GGATCTAAAGAGGGCATCCATG, SEQ ID NO: 73) primer pair did.
  • the resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara).
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5 ⁇ , and transformed at 37 ° C using LB agar medium containing 20 ⁇ g / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroEB. Obtained.
  • the phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5 ⁇ , and transformed at 37 ° C using LB agar medium containing 20 ⁇ g / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 ⁇ g / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroED. Obtained.
  • E. coli MG1655 (DE3) / pKD46 was cultured in LB medium containing 10 mM arabinose and 100 ⁇ g / ml ampicillin until the OD 660 value reached about 0.5. After cooling the culture solution on ice, 3 ml of the culture solution was centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. The cells were suspended in 1 ml of ice-cold sterile water, centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded.
  • the cells were suspended in 50 ⁇ l of sterile water, and a solution containing about 100 ng of DNA cassette was added, followed by electroporation.
  • the cells were recovered from the electroporation cuvette using 1 ml of LB medium, shaken at 37 ° C. for 2.5 hours, and then cultured overnight at 37 ° C. using LB agar medium containing 20 ⁇ g / ml kanamycin.
  • Colony direct PCR was performed using a pair of primers designed approximately 500 bp downstream from the insertion locus on the chromosome shown in Table 1 with K2 (CGGTGCCCTGAATGA ACTGC, SEQ ID NO: 87, and so forth), and the target DNA on the target locus The strain in which the cassette was inserted was selected.
  • the sequence of the primer designed downstream is: ldhA locus: D-ldhA (CAGCGTTAACTGGTTCGCGGTC, SEQ ID NO: 88) adhE locus: D-adhE (TGCAGGCCGTGCCAGTCATCC, SEQ ID NO: 89) pflDC locus: D-pflDC
  • the pykF locus was D-pykF (GAGCTGCGTCATCTTTAG, SEQ ID NO: 91)
  • the ascF locus was D-ascF (CGTAGCGGCTGAAAAACTCCACC, SEQ ID NO: 92)
  • the ackA-pta locus was D-ackA-pta (CCTTCAAACGGGAAGTTCATCAG, SEQ ID NO: 93).
  • the related genotypes of the prepared strains are shown in Table 3.
  • the GIV -TGs can be used as a pair of G.
  • a GIV tag is used as a pair, we can use the GPTs to: G. TIGGTG TCC TCC TCC TCC.
  • the DNA of -Km-FRT-aroG fbr was amplified. The obtained DNA was purified with QIAquick PCR Purification Kit, then digested with DpnI, and purified again with QIAquick PCR Purification Kit.
  • E. coli MG1655 (DE3) / pKD46 was cultured in LB medium containing 10 mM arabinose and 100 ⁇ g / ml ampicillin until the OD 660 value reached about 0.5.
  • TSS solution (10% polyethylene glycol 3350, 5% dimethyl sulfoxide, 70 mM magnesium chloride, 0.1 M potassium chloride, 30 mM calcium chloride), add 1 ⁇ l of 50 ng / ⁇ l pCP20, and in ice Let stand for 30 minutes.
  • the plate was left in ice for 2 minutes, 900 ⁇ l of LB medium was added, and 100 ⁇ l was applied to LB agar medium containing 50 ⁇ g / ml of ampicillin. After overnight culture at 30 ° C., emerging colonies were streaked on LB agar and cultured overnight at 42 ° C. Next, the emerged colonies were streaked on fresh LB agar and cultured overnight at 37 ° C.
  • this strain is cultured in LB liquid medium containing 20 ⁇ g / ml kanamycin, cultured until the OD 660 value becomes about 0.5, and the kanamycin resistance gene is removed by the method using pCP20 described above. to obtain a MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr stock.
  • This strain was designated as Phe1 strain.
  • the Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S2 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • Colony direct PCR was performed using a primer pair of K2 and D-pflDC, and it was confirmed that FRT-Km-FRT-PT7 (-8TC) -tktA was inserted into the pflDC locus of the obtained strain. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: PT7 (-8TC) -tktA strain was obtained. This strain was named Phe4 strain.
  • the Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S8 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • the Phe4 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S8 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • the Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • the Phe4 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • the Phe5 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • Phe13 has been deposited at the Patent Organism Depositary Center for Patent Products and Technologies, Japan Patent Organism Depositary (# 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) (Accession date July 20, 2017, Accession number: NITE BP-02514).
  • the Phe19 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S6 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • the Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 ⁇ l of 1 M calcium chloride and stirring well, 200 ⁇ l was transferred to a 1.5 ml tube. To this, 20 ⁇ l of P1 phage lysate of S13 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 ⁇ l of 1 M trisodium citrate solution and 700 ⁇ l of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • Tyr13 was deposited at the Patent Organism Depositary Center for Patent Products and Technologies, Japan Patent Organism Depositary (# 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) (consignment date July 20, 2017, Accession number: NITE BP-02513).
  • the bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C.
  • K2 a D-mtlA (GATCAACGACATCATCACCAATGC, SEQ ID NO: 98) using primer pairs, FRT-Km-FRT-P T7 -ipdC to MtIA locus of strains obtained was confirmed that it is inserted.
  • the bacterial solution was applied to LB agar medium containing 20 ⁇ g / ml kanamycin and cultured overnight at 37 ° C. K2 and D-acs (CTCATGCAGGACTTCATTATTAAGACGGTC, SEQ ID NO: 99) using primer pairs, FRT-Km-FRT-P T7 -ldhA the acs locus of strains obtained was confirmed that it is inserted.
  • strain culture and product determination The strain was precultured overnight at 27 ° C. using 5 ml of LB liquid medium or LB liquid medium containing 0.8% glucose. 50 ⁇ l of the preculture liquid was added to M9M2 liquid medium to inoculate, and shake culture was performed at 37 ° C. until the value of OD 660 became about 0.3.
  • M9M2 liquid medium is prepared by adding 10 g of glucose, 6 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride and 4 g of ammonium chloride to 900 ml of distilled water, and further adding 1 M magnesium chloride 1 ml, 1 ml of 10 mg / ml thiamine hydrochloride, 0.5 ml of 100 mM iron sulfate (divalent), 100 ⁇ l of 1 M calcium chloride, 10 ⁇ l of a trace metal salt solution, and the volume up to 1 L is 0.2 ⁇ m filter The filter-sterilized one was used.
  • IPTG Isopropyl- ⁇ -thiogalactopyranoside
  • strains Phe11 to Phe17, Phe19, Phe22 and Phe23 showed good production of phenylalanine. In addition, these strains showed good phenylalanine production even without addition of glucose in the preculture. From the comparison between Phe13 strain and Phe24 strain, it was recognized that overexpression of tyrB had a negative influence on phenylalanine production. Also, according to the results of strains Phe11 to Phe17, Phe19, Phe22 and Phe23, it is considered that at least aroA, aroB, aroC, aroG fbr , and pheA fbr overexpression is necessary for good phenylalanine production. Is recognized.

Abstract

Provided is a microorganism which is plasmid-free and has improved productivity. In one aspect, the present disclosure relates to a transgenic microorganism belonging to the genus Escherichia, in which at least five genes shown below are introduced onto the chromosome thereof along with promoters respectively capable of inducing the expression of the genes: (1) aroA, (2) aroB, (3) aroC, (4) aroGfbr or aroFfbr, and (5) pheAfbr or tyrAfbr. In another aspect, the present disclosure relates to a method for producing an aromatic compound, comprising culturing the microorganism in a culture medium.

Description

芳香族化合物を産生する微生物Microorganisms producing aromatic compounds
　本開示は、芳香族化合物を産生する微生物に関し、より具体的には、フェニルアラニンの生産菌、チロシンの生産菌、その他の芳香族化合物の生産菌、並びにそれらの使用に関する。 TECHNICAL FIELD The present disclosure relates to microorganisms that produce aromatic compounds, and more specifically to phenylalanine producing bacteria, tyrosine producing bacteria, bacteria producing other aromatic compounds, and their uses.
　フェニルアラニンやチロシンは、家畜肥料への添加物、サプリメント、医薬品原料などに用いられる。フェニルアラニンやチロシンは、微生物を用いた発酵法により生産されることが一般的になっている。 Phenylalanine and tyrosine are used as additives to livestock manure, supplements, pharmaceutical raw materials and the like. Phenylalanine and tyrosine are generally produced by fermentation using microorganisms.
　従来は、合成経路上のキーとなる遺伝子をプラスミドで発現させることで、合成経路のボトルネックを解消して生産量の向上を図ろうとするのが一般的である（例えば、非特許文献１）。また、プラスミドを用いずに染色体に直接導入した生産菌が報告されている（例えば、非特許文献２）。フェニルアラニンやチロシンの生産菌にさらなる酵素を導入することでその他の芳香族化合物を産生できることも報告されている（非特許文献３）。 Conventionally, it is common to try to improve the production amount by eliminating the bottleneck of the synthetic pathway by expressing the key gene on the synthetic pathway with a plasmid (for example, Non-Patent Document 1). . In addition, a production bacterium directly introduced into a chromosome without using a plasmid has been reported (for example, Non-Patent Document 2). It has also been reported that other aromatic compounds can be produced by introducing additional enzymes into phenylalanine and tyrosine-producing bacteria (Non-patent Document 3).
　プラスミドを保持するためには抗生物質を添加する必要があるが、大規模な発酵生産においては、大幅なコスト増や、環境中への薬剤流出のリスク、薬剤耐性菌の出現などが問題になる。この問題を解決するため、キーとなる遺伝子を微生物の染色体へ直接導入して「プラスミドフリーな生産微生物」とすることが提案された。しかし、プラスミドを用いない微生物は、生産量が低い傾向がある。 Antibiotics need to be added in order to retain the plasmid, but in large-scale fermentation production, significant cost increases, risk of drug spillage into the environment, emergence of drug resistant bacteria, etc. become problems. . In order to solve this problem, it has been proposed to introduce a key gene directly into the chromosome of the microorganism to make it a "plasmid free producing microorganism". However, microorganisms that do not use a plasmid tend to have low production.
　そこで、本開示は、一態様において、プラスミドフリーでかつ生産性が向上した微生物を提供する。 Thus, the present disclosure provides, in one aspect, a plasmid-free and productivity-enhanced microorganism.
　本開示は、一態様において、形質転換されたエシェリキア属に属する微生物であって、少なくとも下記５つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されている微生物に関する。
（１）ａｒｏＡ
（２）ａｒｏＢ
（３）ａｒｏＣ
（４）ａｒｏＧfbr又はａｒｏＦfbr
（５）ｐｈｅＡfbr又はｔｙｒＡfbr The present disclosure relates, in one aspect, to a microorganism belonging to the transformed Escherichia genus, wherein at least the following five genes are introduced together with an expression inducible promoter on a chromosome.
(1) aroA
(2) aroB
(3) aroC
(4) aroG fbr or aroF fbr
(5) pheA fbr or tyrA fbr
　本開示は、その他の態様において、本開示に係る微生物を培地で培養することを含む、芳香族化合物の製造方法に関する。 In another aspect, the present disclosure relates to a method of producing an aromatic compound, which comprises culturing the microorganism according to the present disclosure in a culture medium.
　本開示によれば、一又は複数の実施形態において、フェニルアラニン、チロシン等の芳香族化合物について、プラスミドフリーでかつ生産性が向上した菌株を提供できる。 According to the present disclosure, in one or more embodiments, it is possible to provide a plasmid-free and improved productivity strain of an aromatic compound such as phenylalanine and tyrosine.
図１は、本開示に係る微生物の作製スキームの概略を示す図である。FIG. 1 is a schematic view of a scheme for producing a microorganism according to the present disclosure. 図２は、本開示に係る微生物の作製スキームの概略を示す図である。FIG. 2 is a diagram showing an outline of a production scheme of a microorganism according to the present disclosure.
　本開示に係る微生物は、エシェリキア（Escherichia）属に属する微生物の形質転換体であるが、一又は複数の実施形態において、大腸菌（Escherichia coli）の形質転換体である。 The microorganism according to the present disclosure is a transformant of a microorganism belonging to the genus Escherichia, but in one or more embodiments, it is a transformant of Escherichia coli.
　本開示において、遺伝子は、特に言及がない場合、大腸菌の遺伝子を意味する。本開示において、遺伝子は、大腸菌の当該遺伝子のオーソログ（他生物種由来のホモログ）を含みうる。大腸菌の遺伝子の配列は、特に言及がない場合、Ｋ―12株（野生型）のものあるいはデータベース（ＮＣＢＩ ＧＥＮＢＡＮＫなど）に本願出願時において登録されているものを指すが、野生型と同等の機能を果たす範囲、又は、野生型と同等の蛋白質をコードする範囲で配列が異なっていてもよく、上述のとおりオーソログの配列であってもよい。また、遺伝子は、宿主微生物での発現のため、コドンが最適化されてもよい。 In the present disclosure, gene means a gene of E. coli unless otherwise stated. In the present disclosure, the gene may include an ortholog of the gene of E. coli (homologs derived from other species). The sequences of E. coli genes refer to those of strain K-12 (wild-type) or those registered at the time of filing of the application with a database (such as NCBI GENBANK) unless otherwise specified, but have functions equivalent to those of wild-type The sequence may be different within the scope of the present invention or the range encoding a protein equivalent to that of the wild type, and may be an orthologous sequence as described above. Also, the gene may be codon optimized for expression in the host microorganism.
　本開示において、発現誘導可能なプロモーターともに染色体に導入される遺伝子は、単独の遺伝子であってもよく、複数遺伝子が発現可能なオペロンであってもよい。 In the present disclosure, a gene introduced into a chromosome together with an expression-inducible promoter may be a single gene or an operon capable of expressing multiple genes.
　本開示において、発現誘導可能なプロモーターは、特に限定されない。一又は複数の実施形態において、野生型の（本来の）プロモーターよりも高い発現量の発現誘導が可能なプロモーターであって、例えば、Ｔ７プロモーターが挙げられる。発現誘導にＴ７プロモーターを用いる場合、宿主微生物は、Ｔ７ＲＮＡポリメラーゼ遺伝子を有する株であることが好ましい。宿主微生物としては、例えば、λＤＥ３ファージを溶原化したＤＥ３株が挙げられる。 In the present disclosure, the expression-inducible promoter is not particularly limited. In one or more embodiments, a promoter capable of inducing expression at a higher expression level than a wild-type (native) promoter, such as, for example, a T7 promoter. When T7 promoter is used for induction of expression, the host microorganism is preferably a strain having a T7 RNA polymerase gene. As a host microorganism, for example, a DE3 strain obtained by lysogenizing λDE3 phage can be mentioned.
　本開示において、遺伝子を発現誘導可能なプロモーターともに染色体に導入する方法は、特に限定されないが、一又は複数の実施形態において、実施例に記載の方法（非特許文献２に記載の方法）が挙げられる。 In the present disclosure, a method for introducing a gene into a chromosome together with an expression-inducible promoter is not particularly limited, but in one or more embodiments, the method described in the Examples (the method described in Non-patent Document 2) is mentioned Be
　本開示において、プラスミドフリーとは、フェニルアラニン、チロシン等の芳香族化合物の生産性向上に寄与する遺伝子が染色体上で過剰発現されることをいい、本開示に係る微生物はプラスミドを保有していてもよく、プラスミドを保有していなくてもよい。
　本開示において、フェニルアラニン及びチロシンは、特に言及のない場合、Ｌ－フェニルアラニン及びＬ－チロシンを意味しうる。 In the present disclosure, plasmid-free means that a gene that contributes to the improvement of the productivity of aromatic compounds such as phenylalanine and tyrosine is overexpressed on the chromosome, and the microorganism according to the present disclosure retains the plasmid Well, it does not have to hold the plasmid.
In the present disclosure, phenylalanine and tyrosine may mean L-phenylalanine and L-tyrosine unless otherwise stated.
　本開示において、「ａｒｏＡ」遺伝子は、５－エノールピルビルシキミ酸－３－リン酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：945528である。 In the present disclosure, the “aroA” gene is a gene encoding 5-enolpyruvyl shikimate-3-phosphate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 945528.
　本開示において、「ａｒｏＢ」遺伝子は、３－デヒドロキナ酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947927である。 In the present disclosure, the "aroB" gene is a gene encoding 3-dehydroquinate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 947927.
　本開示において、「ａｒｏＣ」遺伝子は、コリスミ酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：946814である。 In the present disclosure, the "aroC" gene is a gene encoding chorismate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 946814.
　本開示において、「ａｒｏＧ」遺伝子は、３－デオキシ－Ｄ－アラビノ－７－ホスホヘプツロン酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：645605である。
　本開示において、「ａｒｏＧfbr」遺伝子は、ａｒｏＧのフィードバック阻害の脱感作変異型を意味する。ａｒｏＧfbrは、一又は複数の実施形態において、特開平05-236947に開示のものを使用できる。 In the present disclosure, the “aroG” gene is a gene encoding 3-deoxy-D-arabino-7-phosphohepturate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 645605.
In the present disclosure, the “aroG fbr ” gene refers to a desensitized variant of aroG feedback inhibition. As the aroG fbr , in one or more embodiments, those disclosed in Japanese Patent Application Laid-Open No. 05-236947 can be used.
　本開示において、「ａｒｏＦ」遺伝子は、３－デオキシ－Ｄ－アラビノ－７－ホスホヘプツロン酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947084である。
　本開示において、「ａｒｏＦfbr」遺伝子は、ａｒｏＦのフィードバック阻害の脱感作変異型を意味する。ａｒｏＦfbrは、一又は複数の実施形態において、特開平05-236947に開示のものを使用できる。 In the present disclosure, the “aroF” gene is a gene encoding 3-deoxy-D-arabino-7-phosphohepturate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 947084.
In the present disclosure, the “aroF fbr ” gene refers to a desensitized variant of aroF feedback inhibition. aroF fbr can use what is disclosed by Unexamined-Japanese-Patent No. 05-236947 in one or several embodiment.
　本開示において、「ｐｈｅＡ」遺伝子は、コリスミ酸ムターゼ及びプレフェン酸デヒドラターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947081である。
　本開示において、「ｐｈｅＡfbr」遺伝子は、ｐｈｅＡのフィードバック阻害の脱感作変異型を意味する。ｐｈｅＡfbrは、一又は複数の実施形態において、特開2006-311833及び特開平05-344881に開示のものを使用できる。 In the present disclosure, the "pheA" gene is a gene encoding chorismate mutase and prephenate dehydratase, and in one or more embodiments is NCBI GENBANK Gene ID: 947081.
In the present disclosure, the "pheA fbr " gene refers to a desensitized variant of feedback inhibition of pheA. As the pheA fbr , those disclosed in JP-A-2006-311833 and JP-A-05-344881 can be used in one or more embodiments.
　本開示において、「ｔｙｒＡ」遺伝子は、コリスミ酸ムターゼ及びプレフェン酸デヒドロゲナーゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947115である。
　本開示において、「ｔｙｒＡfbr」遺伝子は、ｔｙｒＡのフィードバック阻害の脱感作変異型を意味する。ｔｙｒＡfbrは、一又は複数の実施形態において、特開平05-076352に開示のものを使用できる。 In the present disclosure, the "tyrA" gene is a gene encoding chorismate mutase and prephenate dehydrogenase, which in one or more embodiments is NCBI GENBANK Gene ID: 947115.
In the present disclosure, the "tyrA fbr " gene refers to a desensitized variant of the feedback inhibition of tyrA. As the tyrA fbr , in one or more embodiments, those disclosed in JP-A-05-076352 can be used.
　本開示において、「ｐｐｓＡ」遺伝子は、ホスホエノールピルビン酸シンターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：946209である。 In the present disclosure, the “ppsA” gene is a gene encoding phosphoenolpyruvate synthase, which in one or more embodiments is NCBI GENBANK Gene ID: 946209.
　本開示において、「ｔｋｔＡ」遺伝子は、トランスケトラーゼ１をコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947420である。 In the present disclosure, the “tktA” gene is a gene encoding transketolase 1, which in one or more embodiments is NCBI GENBANK Gene ID: 947420.
　本開示において、「ａｒｏＤ」遺伝子は、３－デヒドロキナ酸デヒドラターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：946210である。 In the present disclosure, the “aroD” gene is a gene encoding 3-dehydroquinate dehydratase, which in one or more embodiments is NCBI GENBANK Gene ID: 946210.
　本開示において、「ａｒｏＥ」遺伝子は、デヒドロシキミ酸レダクターゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：947776である。 In the present disclosure, the "aroE" gene is a gene encoding dehydroshikimic acid reductase, which in one or more embodiments is NCBI GENBANK Gene ID: 947776.
　本開示において、「ｙｄｉＢ」遺伝子は、キナ酸／シキミ酸５－デヒドロゲナーゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：946200である。 In the present disclosure, the “ydiB” gene is a gene encoding quinic acid / shikimic acid 5-dehydrogenase, which in one or more embodiments is NCBI GENBANK Gene ID: 946200.
　本開示において、「ａｒｏＬ」遺伝子は、シキミ酸キナーゼＩＩをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：945031である。 In the present disclosure, the "aroL" gene is a gene encoding shikimate kinase II, which in one or more embodiments is NCBI GENBANK Gene ID: 945031.
　本開示において、「ａｒｏＫ」遺伝子は、シキミ酸キナーゼＩをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：2847759である。 In the present disclosure, the “aroK” gene is a gene encoding shikimate kinase I, which in one or more embodiments is NCBI GENBANK Gene ID: 2847759.
　本開示において、「ｔｙｒＢ」遺伝子は、チロシンアミノトランスフェラーゼをコードする遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：945031である。
　本開示に係る微生物では、一又は複数の実施形態において、フェニルアラニン、チロシン等の芳香族化合物の産生量を向上させる観点から、ｔｙｒＢは発現誘導可能なプロモーターともに染色体に導入されないことが好ましい。従来、フェニルアラニンやチロシンを産生させる場合、ｔｙｒＢは過剰発現される場合が多かった。しかし、本発明者らは、ｔｙｒＢの過剰発現は代謝負荷によるネガティブな影響が大きく、ｔｙｒＢを過剰発現させないほうが、フェニルアラニンやチロシンの生産を向上できることを見出した。 In the present disclosure, the "tyrB" gene is a gene encoding a tyrosine aminotransferase, which in one or more embodiments is NCBI GENBANK Gene ID: 945031.
In the microorganism according to the present disclosure, in one or more embodiments, it is preferable that tyrB is not introduced into a chromosome together with an expression-inducible promoter from the viewpoint of improving the production amount of aromatic compounds such as phenylalanine and tyrosine. Conventionally, when producing phenylalanine and tyrosine, tyrB was often overexpressed. However, the present inventors have found that overexpression of tyrB has a large negative effect due to metabolic load, and that overexpression of tyrB can improve phenylalanine and tyrosine production.
　本開示において、「ｌｄｈＡ」遺伝子は、乳酸デヒドロゲナーゼをコードするＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ ＪＣＭ２０６４４由来の遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＰｒｏｔｅｉｎ ＩＤ：CAJ91827をコードする遺伝子である。 In the present disclosure, the “ldhA” gene is a gene derived from Cupriavidus necator JCM 20644 encoding lactate dehydrogenase, and in one or more embodiments, is a gene encoding NCBI GENBANK Protein ID: CAJ91827.
　本開示において、「ｉｐｄＣ」遺伝子は、インドール－３－ピルビン酸／フェニルピルビン酸デカルボキシラーゼをコードするＡｚｏｓｐｉｒｉｌｌｕｍ ｂｒａｓｉｌｅｎｓｅ ＮＢＲＣ１０２２８９由来の遺伝子であって、一又は複数の実施形態において、ＮＣＢＩ ＧＥＮＢＡＮＫのＰｒｏｔｅｉｎ ＩＤ：CAA67899をコードする遺伝子である。 In the present disclosure, the "ipdC" gene is a gene derived from Azospirillum brasilense NBRC 102289 which encodes indole-3-pyruvate / phenylpyruvate decarboxylase, and in one or more embodiments, the NCBI GENBANK Protein ID: CAA 67899 Is a gene encoding
　本開示に係る微生物の第１の実施形態は、下記５つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されているエシェリキア属に属する微生物である。
（１）ａｒｏＡ
（２）ａｒｏＢ
（３）ａｒｏＣ
（４）ａｒｏＧfbr又はａｒｏＦfbr
（５）ｐｈｅＡfbr又はｔｙｒＡfbr
　本実施形態において、上記（５）の遺伝子がｐｈｅＡfbrの場合、本開示に係る微生物は、フェニルアラニンの生産能を有し、本開示に係る微生物を用いた発酵法によりフェニルアラニンの製造が可能になる。
　本実施形態において、上記（５）の遺伝子がｔｙｒＡfbrの場合、本開示に係る微生物は、チロシンの生産能を有し、本開示に係る微生物を用いた発酵法によりチロシンの製造が可能になる。
　本実施形態において、上記（４）のａｒｏＧfbr又はａｒｏＦfbrは同じ酵素をコードしており、相互に代替可能である。なお、ａｒｏＧfbr及びａｒｏＦfbrの代わりにａｒｏＨ（ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：946229）のフィードバック阻害の脱感作変異型を使用することもできる。
　本発明者らは、上記（４）及び（５）に加え、上記（１）～（３）のすべて過剰発現させることで、導入した遺伝子（例えば、上記（４）及び（５））の過剰発現による代謝負荷を低減しうることを見出した。 The first embodiment of the microorganism according to the present disclosure is a microorganism belonging to the genus Escherichia in which the following five genes are introduced together with an expression-inducible promoter on a chromosome.
(1) aroA
(2) aroB
(3) aroC
(4) aroG fbr or aroF fbr
(5) pheA fbr or tyrA fbr
In the present embodiment, when the gene of the above (5) is pheA fbr , the microorganism according to the present disclosure has the ability to produce phenylalanine, and the fermentation method using the microorganism according to the present disclosure enables the production of phenylalanine .
In the present embodiment, when the gene of (5) is tyrA fbr , the microorganism according to the present disclosure has an ability to produce tyrosine, and the production of tyrosine becomes possible by a fermentation method using the microorganism according to the present disclosure. .
In the present embodiment, the aroG fbr or aroF fbr in the above (4) encodes the same enzyme and can be mutually substituted. In place of aroG fbr and aroF fbr , a desensitized variant of feedback inhibition of aroH (NCBI GENBANK Gene ID: 946229) can also be used.
The present inventors, in addition to the above (4) and (5), by overexpressing all of the above (1) to (3), excess of the introduced gene (for example, It has been found that the metabolic load due to expression can be reduced.
　本開示に係る微生物の第２の実施形態は、第１の実施形態の上記５つの遺伝子に加え、下記２つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されているエシェリキア属に属する微生物である。
（６）ｐｐｓＡ
（７）ｔｋｔＡ
　ここで、発現誘導可能なプロモーターとしてＴ７プロモーターを使用する場合、目的の芳香族化合物の生産性向上の点から、上記（７）ｔｋｔＡのプロモーターは発現誘導能が低減した変異型Ｔ７プロモーターであることが好ましい。 A second embodiment of the microorganism according to the present disclosure is a microorganism belonging to the genus Escherichia in which the following two genes are introduced together with an expression-inducible promoter on a chromosome in addition to the above-mentioned five genes of the first embodiment. is there.
(6) ppsA
(7) tktA
Here, when a T7 promoter is used as an expression-inducible promoter, the above (7) tktA promoter is a mutant T7 promoter having a reduced expression inducibility, from the viewpoint of improving the productivity of the target aromatic compound. Is preferred.
　本開示に係る微生物の第３の実施形態は、第１及び第２の実施形態の上記（１）～（７）の７つの遺伝子に加え、さらに、下記３つの遺伝子の少なくとも１つが染色体上に発現誘導可能なプロモーターとともに導入されているエシェリキア属に属する微生物である。
（８）ａｒｏＤ
（９）ａｒｏＥ又はｙｄｉＢ
（１０）ａｒｏＬ又はａｒｏＫ
　（８）～（１０）の中で、２つ又は全部が導入されていることが好ましい。
　本実施形態において、上記（９）のａｒｏＥ又はｙｄｉＢは相互に代替可能であるが、上記（９）の遺伝子としては、ａｒｏＥが好ましい。
　本実施形態において、上記（１０）のａｒｏＬ又はａｒｏＫは相互に代替可能であるが、上記（１０）の遺伝子としては、ａｒｏＬが好ましい。 In the third embodiment of the microorganism according to the present disclosure, in addition to the seven genes of the above (1) to (7) of the first and second embodiments, at least one of the following three genes is further chromosomally It is a microorganism belonging to the genus Escherichia which has been introduced together with an expression-inducible promoter.
(8) aroD
(9) aroE or ydiB
(10) aroL or aroK
Of (8) to (10), preferably two or all of them are introduced.
In the present embodiment, although aroE or ydiB in (9) above can be substituted for each other, aroE is preferable as the gene in (9) above.
In the present embodiment, aroL or aroK of the above (10) can be replaced with each other, but as the gene of the above (10), aroL is preferable.
　本開示に係る微生物の第４の実施形態は、第１、第２、又は第３の実施形態にさらに、４－ヒドロキシフェニルピルビン酸、フェニルアラニン、又はチロシンを基質とする酵素をコードする同種又は異種起源の遺伝子が染色体上に発現誘導可能なプロモーターとともに導入された微生物である。
　例えば、フェニルピルビン酸デカルボキシラーゼをコードする異種起源の遺伝子ｉｐｄＣが染色体上に発現誘導可能なプロモーターとともに導入されると、本開示に係る微生物は、２－フェニルエタノール又は２－（４－ヒドロキシフェニル）エタノールの生産能を有するから、これらの芳香族化合物の製造に使用できる。
　また、例えば、乳酸デヒドロゲナーゼをコードする異種起源の遺伝子ｌｄｈＡが染色体上に発現誘導可能なプロモーターとともに導入されると、フェニル乳酸又は４－ヒドロキシフェニル乳酸の生産能を有する微生物となり、これらの芳香族化合物の製造に使用できる。
　すなわち、フェニルピルビン酸、４－ヒドロキシフェニルピルビン酸、フェニルアラニン、又はチロシンを基質とする酵素をコードする遺伝子をさらに導入すると、その遺伝子に応じて芳香族化合物が製造できる。
　また、非特許文献２に記載されているように、例えば、Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓのチロシンデカルボキシラーゼ遺伝子（ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：4413406）やＰｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ ＮＢＲＣ１００６５０の芳香族アミノ酸デカルボキシラーゼ遺伝子（ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：1045854）又はそれらのオーソログ遺伝子を導入することで、フェニルエチルアミンやチラミンを製造することが可能となる。一方、非特許文献３に記載されているように、例えば、フェニルピルビン酸デカルボキシラーゼ遺伝子と共に、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉのフェニルアセトアルデヒドデヒドロゲナーゼ遺伝子（ＮＣＢＩ ＧＥＮＢＡＮＫのＧｅｎｅ ＩＤ：945933）又はそのオーソログ遺伝子を導入することで、フェニル酢酸や４－ヒドロキシフェニル酢酸を製造することが可能となる。 In a fourth embodiment of the microorganism according to the present disclosure, in addition to the first, second or third embodiment, the same or different enzymes encoding 4-hydroxyphenylpyruvate, phenylalanine or tyrosine as an enzyme substrate are used. It is a microorganism in which a gene of origin has been introduced together with an expression inducible promoter on a chromosome.
For example, when the heterologous gene ipdC encoding phenylpyruvate decarboxylase is introduced on a chromosome together with an expression-inducible promoter, the microorganism according to the present disclosure is 2-phenylethanol or 2- (4-hydroxyphenyl) Since it has an ability to produce ethanol, it can be used for the production of these aromatic compounds.
Also, for example, when the heterologous gene ldhA encoding lactate dehydrogenase is introduced on a chromosome together with an expression-inducible promoter, it becomes a microorganism capable of producing phenyl lactic acid or 4-hydroxyphenyl lactic acid, and these aromatic compounds Can be used in the manufacture of
That is, when a gene encoding an enzyme having phenylpyruvate, 4-hydroxyphenylpyruvate, phenylalanine or tyrosine as a substrate is further introduced, an aromatic compound can be produced according to the gene.
In addition, as described in Non-Patent Document 2, for example, Lactobacillus brevis tyrosine decarboxylase gene (NCBI GENBANK Gene ID: 4413406) or Pseudomonas putida NBRC 100650 aromatic amino acid decarboxylase gene (NCBI GENBANK Gene ID: 1045854) or their orthologous genes can be used to produce phenylethylamine and tyramine. On the other hand, as described in Non-patent Document 3, for example, by introducing a phenylacetaldehyde dehydrogenase gene of Escherichia coli (Gene ID of NCBI GENBANK: 945933) or an ortholog gene thereof together with a phenylpyruvate decarboxylase gene. It becomes possible to produce phenylacetic acid and 4-hydroxyphenylacetic acid.
本開示に係る微生物を用いた芳香族化合物（フェニルアラニン、チロシンを含む）の製造方法は、特に制限されず、通常の培養方法及び／又は発酵法の条件で培養することで、芳香族化合物を製造できる。培養・発酵の培地は、炭素源、窒素源、リン酸源、硫黄源、ミネラル、ビタミンなどのその他の成分を含むもので、当該微生物が生育できるものなら特に限定されない。本開示に係る微生物を用いた芳香族化合物の産生方法は、例えば、実施例の方法を参照できる。簡単には、２７℃で前培養を行い、１％の濃度で最小培地（例えば、Ｍ９Ｍ２培地）に植菌し、３７℃で４－５時間培養し（ＯＤ６６０が０．３～０．４）、発現誘導して（Ｔ７プロモーターであれば、例えば１ｍＭ　ＩＰＴＧを添加して）、２７℃で４３～４４時間培養し、培養液及び／又は菌内に目的の芳香族化合物を得る。但し、培養・発酵条件は、これらに限定されない。
　なお、本開示に係る微生物を用いたフェニルアラニン、チロシンを含む芳香族化合物の製造方法では、一又は複数の実施形態において、導入した遺伝子の過剰発現による代謝負荷が低減されているから、前培養にグルコースを添加してカタボライトリプレッションを引き起こさなくても芳香族化合物を産生させることができる。 The method for producing an aromatic compound (including phenylalanine and tyrosine) using a microorganism according to the present disclosure is not particularly limited, and an aromatic compound is produced by culturing under the conditions of a common culture method and / or a fermentation method. it can. The culture / fermentation medium is not particularly limited as long as it contains other components such as a carbon source, nitrogen source, phosphate source, sulfur source, minerals, vitamins and the like, and the microorganism can be grown. For the method of producing an aromatic compound using the microorganism according to the present disclosure, for example, the methods of the Examples can be referred to. Briefly, preculture is carried out at 27 ° C., inoculated in minimal medium (eg, M9M2 medium) at a concentration of 1%, and cultured for 4-5 hours at 37 ° C. (OD 660 of 0.3 to 0.4) After induction of expression (if T7 promoter, for example, 1 mM IPTG is added), culture is carried out at 27 ° C. for 43 to 44 hours to obtain the target aromatic compound in the culture solution and / or in the fungus. However, culture and fermentation conditions are not limited to these.
In the method of producing an aromatic compound containing phenylalanine and tyrosine using the microorganism according to the present disclosure, the metabolic load due to overexpression of the introduced gene is reduced in one or more embodiments. Glucose can be added to produce aromatic compounds without causing catabolite repression.
　本開示は、一又は複数の実施形態において、以下に関しうる；
　＜１＞　形質転換されたエシェリキア属に属する微生物であって、
　少なくとも下記５つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されている微生物。
（１）ａｒｏＡ
（２）ａｒｏＢ
（３）ａｒｏＣ
（４）ａｒｏＧfbr又はａｒｏＦfbr
（５）ｐｈｅＡfbr又はｔｙｒＡfbr
　＜２＞　さらに、下記２つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されている、＜１＞に記載の微生物。
（６）ｐｐｓＡ
（７）ｔｋｔＡ
　＜３＞　さらに、下記３つの遺伝子の少なくとも１つが染色体上に発現誘導可能なプロモーターとともに導入されている、＜１＞又は＜２＞に記載の微生物。
（８）ａｒｏＤ
（９）ａｒｏＥ又はｙｄｉＢ
（１０）ａｒｏＬ又はａｒｏＫ
　＜４＞　染色体上に発現誘導可能なプロモーターとともに導入される遺伝子にｔｙｒＢが含まれない、＜１＞から＜３＞のいずれかに記載の微生物。
　＜５＞　前記（５）の遺伝子が、ｐｈｅＡfbrであり、フェニルアラニン生産能を有する＜１＞から＜４＞のいずれかに記載の微生物。
　＜６＞　前記（５）の遺伝子が、ｔｙｒＡfbrであり、チロシン生産能を有する、＜１＞から＜４＞のいずれかに記載の微生物。
　＜７＞　さらに、フェニルピルビン酸、４－ヒドロキシフェニルピルビン酸、フェニルアラニン、又はチロシンを基質とする酵素をコードする同種又は異種起源の遺伝子が染色体上に発現誘導可能なプロモーターとともに導入された、＜１＞から＜４＞のいずれかに記載の微生物。
　＜８＞　さらに、フェニルピルビン酸デカルボキシラーゼをコードする異種起源の遺伝子ｉｐｄＣが染色体上に発現誘導可能なプロモーターとともに導入され、２－フェニルエタノール又は２－（４－ヒドロキシフェニル）エタノールの生産能を有する、＜１＞から＜４＞のいずれかに記載の微生物。
　＜９＞　さらに、乳酸デヒドロゲナーゼをコードする異種起源の遺伝子ｌｄｈＡが染色体上に発現誘導可能なプロモーターとともに導入され、フェニル乳酸又は４－ヒドロキシフェニル乳酸の生産能を有する、＜１＞から＜４＞のいずれかに記載の微生物。
　＜１０＞　＜１＞から＜９＞のいずれかに記載の微生物を培地で培養することを含む、芳香族化合物の製造方法。 The present disclosure, in one or more embodiments, may relate to:
<1> A microorganism belonging to the transformed Escherichia genus,
A microorganism in which at least the following five genes are introduced together with an expression inducible promoter on a chromosome.
(1) aroA
(2) aroB
(3) aroC
(4) aroG fbr or aroF fbr
(5) pheA fbr or tyrA fbr
<2> The microorganism according to <1>, wherein the following two genes are introduced together with an expression inducible promoter on a chromosome.
(6) ppsA
(7) tktA
<3> The microorganism according to <1> or <2>, wherein at least one of the following three genes is introduced together with an expression-inducible promoter on a chromosome.
(8) aroD
(9) aroE or ydiB
(10) aroL or aroK
<4> The microorganism according to any one of <1> to <3>, wherein the gene introduced together with an expression-inducible promoter on a chromosome does not contain tyrB.
<5> The microorganism according to any one of <1> to <4>, wherein the gene of (5) is pheA fbr and has phenylalanine producing ability.
<6> The microorganism according to any one of <1> to <4>, wherein the gene of (5) is tyrA fbr and has a tyrosine producing ability.
<7> Furthermore, a gene of the same or different gene encoding an enzyme that uses phenylpyruvate, 4-hydroxyphenylpyruvate, phenylalanine, or tyrosine as a substrate is introduced together with an expression inducible promoter on a chromosome, <1 The microorganism according to any one of> to <4>.
<8> Furthermore, the heterologous gene ipdC encoding phenylpyruvate decarboxylase is introduced on a chromosome together with an expression-inducible promoter, and has the ability to produce 2-phenylethanol or 2- (4-hydroxyphenyl) ethanol The microorganism according to any one of <1> to <4>.
<9> Furthermore, the heterologous gene ldhA encoding lactate dehydrogenase is introduced on a chromosome together with an expression-inducible promoter, and has the ability to produce phenyllactic acid or 4-hydroxyphenyllactic acid, <1> to <4> The microorganism described in any one.
<10> A method for producing an aromatic compound, comprising culturing the microorganism according to any one of <1> to <9> in a culture medium.
　以下、実施例により本開示をさらに詳細に説明するが、これらは例示的なものであって、本開示はこれら実施例に制限されるものではない。 Hereinafter, the present disclosure will be described in more detail by way of examples, but these are illustrative and the present disclosure is not limited to these examples.
　［大腸菌の染色体に部位特異的に複数の遺伝子を導入した菌の作製］
　大腸菌の染色体に部位特異的に複数の遺伝子を導入する方法は、非特許文献２（Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．９３：８１５－８２９，２０１２）に従った。簡単には、同文献Ｆｉｇ．１（図１）に記載のように、Ｒｅｄ相同組み換え、ＦＬＰ／ＦＲＴ部位特異的組み換え、及びＰ１トランスダクションを利用して作製した。
　図１は、３つの遺伝子（Ａ，Ｂ，Ｃ）を染色体に導入する場合のスキームを示す。まず、Ｒｅｄ相同組み換えにより、ＦＲＴ－Ｋｍ－ＦＲＴ－Ａ、ＦＲＴ－Ｋｍ－ＦＲＴ－Ｂ、ＦＲＴ－Ｋｍ－ＦＲＴ－Ｃを染色体の所望の位置に有する３つの株（株Ａ，株Ｂ，株Ｃ）を作製する。株Ａ内でＦＬＰ／ＦＲＴ組み換えによりＫｍカセットが切り出されたものをアクセプター株Ａとして得る。次にドナー株ＢをＰ１ｖｉｒファージに感染させて得た溶菌液Ｂをアクセプター株Ａに感染させてＰ１トランスダクションを行う。得られた株からＦＬＰ／ＦＲＴ組み換えによりＫｍカセットが切り出されたものをアクセプター株ＡＢとして得る。ドナー株ＣをＰ１ｖｉｒファージに感染させて得た溶菌液Ｃをアクセプター株ＡＢに感染させてＰ１トランスダクションを行う。最後に、ＦＬＰ／ＦＲＴ組み換えによりＫｍカセットが切り出されたものを選択すれば、３つの遺伝子（Ａ，Ｂ，Ｃ）が染色体に導入された株を得ることができる。 [Preparation of bacteria in which a plurality of genes are introduced site-specifically into the chromosome of E. coli]
The method for introducing a plurality of genes site-specifically into the chromosome of E. coli was according to Non-Patent Document 2 (Koma et al. Appl. Microbiol. Biotechnol. 93: 815-829, 2012). For simplicity, refer to Fig. As described in FIG. 1 (FIG. 1), it was generated using Red homologous recombination, FLP / FRT site-specific recombination, and P1 transduction.
FIG. 1 shows a scheme for introducing three genes (A, B, C) into a chromosome. First, three strains having FRT-Km-FRT-A, FRT-Km-FRT-B, and FRT-Km-FRT-C at desired positions of chromosomes by Red homologous recombination (strain A, strain B, strain C )). The strain in which the Km cassette has been excised by FLP / FRT recombination in strain A is obtained as acceptor strain A. Next, the lysate B obtained by infecting the donor strain B with P1 vir phage is infected with the acceptor strain A to perform P1 transduction. From the resulting strain, one obtained by excising Km cassette by FLP / FRT recombination is obtained as acceptor strain AB. The lysate C obtained by infecting the donor strain C with P1 vir phage is infected with the acceptor strain AB to perform P1 transduction. Finally, if the Km cassette is excised by FLP / FRT recombination, it is possible to obtain a strain in which three genes (A, B, C) have been introduced into the chromosome.
　所望の遺伝子を大腸菌の染色体に部位特異的に導入する方法も、非特許文献２（Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．９３：８１５－８２９，２０１２）に従った。簡単には、そのスキームを図２に示す（同文献のＦｉｇ．２）。
　Ｒｅｄ相同組み換えで導入する遺伝子断片を作製するために、ＦＲＴ－Ｋｍ－ＦＲＴカセットをマルチクローニングサイト（ＭＣＳ）の上流に備える２つのプラスミドを利用した。ｐＥＴ－２１ａ－ＦＲＴは、ＭＣＳが１つ、ｐＥＴＤｕｅｔ－ＦＲＴは、ＭＣＳが２つである。これらのプラスミドはＴ７プロモーターによって遺伝子発現を誘導できる。次に、直鎖ＤＮＡ断片をＰＣＲで増幅する。プライマーには、ターゲット部位の外側の５０塩基の配列（Ｈ１又はＨ２）と、プラスミド由来の２０塩基の配列（Ｐ１又はＰ２）を有する。そして、増幅断片を、ｐＫＤ４６を有する大腸菌細胞にエレクトロポレートする。得られる組み替え体は、染色体のターゲット部位に所望の遺伝子が導入される。この組み替え体にプラスミドｐＣＰ２０を導入することで、ＦＬＰ／ＦＲＴ組み換えによりＫｍカセットを除去できる。 The method for site-specifically introducing a desired gene into the chromosome of E. coli also follows non-patent document 2 (Koma et al. Appl. Microbiol. Biotechnol. 93: 815-829, 2012). Briefly, the scheme is shown in Fig. 2 (Fig. 2 of the same document).
In order to generate a gene fragment to be introduced by Red homologous recombination, two plasmids were used which provided the FRT-Km-FRT cassette upstream of the multiple cloning site (MCS). pET-21a-FRT has one MCS, and pETDuet-FRT has two MCS. These plasmids can induce gene expression by the T7 promoter. Next, the linear DNA fragment is amplified by PCR. The primers have a 50 base sequence (H1 or H2) outside the target site and a 20 base sequence (P1 or P2) derived from a plasmid. The amplified fragment is then electroporated into E. coli cells carrying pKD46. In the resulting recombinant, the desired gene is introduced into the target site of the chromosome. The Km cassette can be removed by FLP / FRT recombination by introducing the plasmid pCP20 into this recombinant.
　［プラスミドの作製］
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＧfbrの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型として、ＥｃＡｒｏＧ－Ｆ（ＡＴＧＡＡＴＴＡＴＣＡＧＡＡＣＧＡＣＧＡＴＴＴＡＣＧ，配列番号０１）とＡｒｏＧ－ＲＭ－Ｎｄｅ（ＧＣＴＡＴＴＡＴＣＣＡＴＧＴＧＣＧＧＡＴＣＧ，配列番号０２）プライマーペア及びＡｒｏＧ－ＦＭ－Ｎｄｅ（ＣＧＡＴＣＣＧＣＡＣＡＴＧＧＡＴＡＡＴＡＧＣ，配列番号０３）とＥｃＡｒｏＧ－Ｒ（ＣＡＡＡＧＣＴＴＴＴＡＣＣＣＧＣＧＡＣＧＣＧＣＴＴＴＴＡＣ，配列番号０４）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ、以下同じ）により２つのＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。つぎに、２つの増幅断片を鋳型として、ＥｃＡｒｏＧ－ＦとＥｃＡｒｏＧ－Ｒのプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、配列中のＮｄｅＩサイトを消失させたａｒｏＧ遺伝子のＤＮＡを得た。さらに、増幅されたＤＮＡ断片を鋳型として、ＥｃＡｒｏＧ－ＦとＥｃＡｒｏＧ－Ｄ１４６Ｎ－ＲＭ（ＧＧＧＧＴＧＡＴＣＡＴＡＴＴＧＡＧＡＡＡＣＴＣ，配列番号０５）及びＥｃＡｒｏＧ－Ｄ１４６Ｎ－ＦＭ（ＧＡＧＴＴＴＣＴＣＡＡＴＡＴＧＡＴＣＡＣＣＣＣ，配列番号０６）とＥｃＡｒｏＧ－Ｒ（ＣＡＡＡＧＣＴＴＴＴＡＣＣＣＧＣＧＡＣＧＣＧＣＴＴＴＴＡＣ，配列番号０７）のプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより２つのＤＮＡ断片を得た。最後に、２つの増幅断片を鋳型として、ＥｃＡｒｏＧ－Ｎｄｅ（ＣＣＡＡＣＣＡＴＡＴＧＡＡＴＴＡＴＣＡＧＡＡＣＧＡＣＧＡＴＴＴＡＣＧ，配列番号０８）とＥｃＡｒｏＧ－Ｒのプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、１４６番目のアスパラギン酸がアスパラギンに置換されたＡｒｏＧをコードするＤＮＡ（ａｒｏＧfbr）を得た。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクター（非特許文献２、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．９３：８１５－８２９，２０１２、以下同じ）をそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔ（Ｑｉａｇｅｎ、以下同じ）を用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマー（ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧ，配列番号０９、以下同じ）とＴ７ターミネータープライマー（ＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧＣＧＧ，配列番号１０、以下同じ）を用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅ（ＭＡＣＨＥＲＹ－ＮＡＧＥＬ、以下同じ）を用いて目的のプラスミドを抽出し精製した。ＬＢ寒天培地は、純水１Ｌに対して、１０ｇポリペプトン（日本製薬株式会社）、５ｇ乾燥酵母エキス（日本製薬株式会社）、１０ｇ塩化ナトリウム、２０ｇ寒天、水酸化ナトリウムでｐＨ７．０に調整したもの。ＬＢ液体培地は、純水１Ｌに対して、１０ｇポリペプトン（日本製薬株式会社）、５ｇ乾燥酵母エキス（日本製薬株式会社）、１０ｇ塩化ナトリウム、水酸化ナトリウムでｐＨ７．０に調整したもの。以下同じ。 [Preparation of a plasmid]
[Preparation of pET21a-FRT-aroG fbr ]
EcAroG-F (ATGAATTATCAGAACGACGATTTACG, SEQ ID NO: 01) and AroG-RM-Nde (GCATTATTATCATGTGCGGATCG, SEQ ID NO: 02) primer pair and AroG-FM-Nde (CGATCC GCA CATGGATA GC, SEQ ID NO: 03) and EcAroG- with the genomic DNA of E. coli MG1655 as a template The two DNA fragments were amplified by Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific, hereinafter the same) using the R (CAAAGCTTTTACCCGCGACGCGCTTTTAC, SEQ ID NO: 04) primer pair. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. Next, using the two amplified fragments as a template, and using the EcAroG-F and EcAroG-R primer pair, overlap extension PCR was performed by Phusion Hot Start II DNA polymerase, and the aroG gene in which the NdeI site in the sequence was eliminated The DNA of was obtained. Furthermore, using the amplified DNA fragment as a template, EcAroG-F and EcAroG-D146N-RM (GGGGTGATCATATTATTGAGAAACTC, SEQ ID NO: 05) and EcAroG-D146N-FM (GAGTTTCTCAATATATGATCACCCC, SEQ ID NO: 06) and EcAroG-R (SEQ ID NO: 6) Two DNA fragments were obtained by Phusion Hot Start II DNA polymerase using the primer pair of 2.). Finally, overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the two amplified fragments as a template and the EcAroG-Nde (CCAACCATATGAATTATCAGAACGACGATTTACG, SEQ ID NO: 08) and EcAroG-R primer pair, and the 146th asparagine was subjected to overlap extension PCR. AroG-encoding DNA (aroG fbr ) in which the acid was substituted with asparagine was obtained.
The amplified DNA and pET21a-FRT vector (Non-patent document 2, Koma et al. Appl. Microbiol. Biotechnol. 93: 815-829, 2012, the same applies hereinafter) are digested with NdeI and XhoI, respectively, and 1% agarose gel electric After separating the DNA by electrophoresis, the corresponding DNA was extracted from the gel and purified using QIAquick Gel Extraction kit (Qiagen, hereinafter the same). The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer (TAATACGACTCACTATAGG, SEQ ID NO: 09, the same applies in the following) and T7 terminator primer (GCTAGTTATTGCTCAGCGG, SEQ ID NO: 10, the same in the following) to select a strain having a desired plasmid. Finally, the strain having the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid is extracted using NucleoSpin Plasmid QuickPure (MACHERY-NAGEL, the same applies hereinafter). Refined. The LB agar medium was adjusted to pH 7.0 with 10 g polypeptone (Nippon Pharmaceutical Co., Ltd.), 5 g dry yeast extract (Nippon Pharmaceutical Co., Ltd.), 10 g sodium chloride, 20 g agar, and sodium hydroxide per liter of pure water. . The LB liquid medium was adjusted to pH 7.0 with 10 g of polypeptone (Nippon Pharmaceutical Co., Ltd.), 5 g of dried yeast extract (Nippon Pharmaceutical Co., Ltd.), 10 g of sodium chloride, and sodium hydroxide per liter of pure water. same as below.
　［ｐＥＴ２１ａ－ＦＲＴ－ｐｈｅＡfbrの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型として、ＥｃＰｈｅＡ－Ｆ（ＣＣＡＡＣＣＡＴＡＴＧＡＣＡＴＣＧＧＡＡＡＡＣＣＣＧＴＴＡＣ，配列番号１１）とＥｃＰｈｅＡ－Ｓ３３０Ｐ－ＲＭ（ＧＡＡＴＣＧＧＧＣＧＴＧＧＴＴＣＣＡＧＡＣ，配列番号１２）プライマーペア及びＥｃＰｈｅＡ－Ｓ３３０Ｐ－ＦＭ（ＧＴＣＴＧＧＡＡＣＣＡＣＧＣＣＣＧＡＴＴＣ，配列番号１３）とＥｃＰｈｅＡ－Ｒ（ＣＡＣＴＣＧＡＧＴＣＡＧＧＴＴＧＧＡＴＣＡＡＣＡＧＧＣＡＣ，配列番号１４）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより２つのＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。つぎに、２つの増幅断片を鋳型として、ＥｃＰｈｅＡ－ＦとＥｃＰｈｅＡ－Ｒのプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、３３０番目のセリンがプロリンに置換されたＰｈｅＡをコードするＤＮＡ（ＰｈｅＡfbr）を得た。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-pheA fbr ]
EcPheA-F (CCAACCATATGACATCGGAAAACCCGTTAC, SEQ ID NO: 11) and EcPheA-S330P-RM (GAATCGGGCGTGTGGTTCCAGAC, SEQ ID NO: 12) primer pair and EcPheA-S330P-FM (GTCTGGAACCACGCCGCGTC, SEQ ID NO: 13) using the genomic DNA of E. coli MG1655 as a template The two DNA fragments were amplified by Phusion Hot Start II DNA polymerase using the R (CACTCGAGTCAGGTTGATCAACAGGCAC, SEQ ID NO: 14) primer pair. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. Next, overlap extension PCR is performed by Phusion Hot Start II DNA polymerase using the two amplified fragments as templates and the EcPheA-F and EcPheA-R primer pairs, and PheA in which the 330th serine is substituted with proline The DNA encoding (PheA fbr ) was obtained.
The amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ｐｐｓＡの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型として、ｐｐｓ－Ｆ（ＣＡＡＣＣＡＴＡＴＧＴＣＣＡＡＣＡＡＴＧＧＣＴＣＧＴＣ，配列番号１５）とｐｐｓ－ＲＭ－Ｎｃｏ（ＧＧＡＴＴＴＴＴＴＴＣＧＡＣＣＣＣＡＴＡＧＴＧＣＧＧＣＧＣ，配列番号１６）プライマーペア及びｐｐｓ－ＦＭ－Ｎｃｏ（ＧＣＧＣＣＧＣＡＣＴＡＴＧＧＧＧＴＣＧＡＡＡＡＡＡＡＴＣＣ，配列番号１７）とｐｐｓ－Ｒ（ＣＡＣＴＣＧＡＧＴＴＡＴＴＴＣＴＴＣＡＧＴＴＣＡＧＣＣＡＧＧ，配列番号１８）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより２つのＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。つぎに、２つの増幅断片を鋳型としてｐｐｓ－Ｆとｐｐｓ－Ｒのプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、配列中のＮｃｏＩサイトを消失させたｐｐｓＡ遺伝子のＤＮＡを得た。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-ppsA]
Using genomic DNA of E. coli MG1655 as a template, pps-F (CAACCATATGTCCACAAATGGCTCGTC, SEQ ID NO: 15) and pps-RM-Nco (GGATTTTTTTCTCACCACCATAGTGCGGCGC, SEQ ID NO: 16) primer pair and pps-FM-Nco (GCGCGCACTACTGGGGTCGAAAAAATCC, SEQ ID NO: 17) and pps- The two DNA fragments were amplified by Phusion Hot Start II DNA polymerase using the R (CACTCGAGTTATTTCTTCAGTTC AGCCAGG, SEQ ID NO: 18) primer pair. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. Next, overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the two amplified fragments as a template and the pps-F and pps-R primer pairs to remove the NcoI site in the sequence of the ppsA gene. DNA was obtained.
The amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ｔｋｔＡの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型としてｔｋｔＡ－Ｎｄｅ（ＣＣＡＡＣＣＡＴＡＴＧＴＣＣＴＣＡＣＧＴＡＡＡＧＡＧＣＴＴＧＣＣ，配列番号１９）とｔｋｔＡ－Ｘｈｏ（ＣＡＣＴＣＧＡＧＴＴＡＣＡＧＣＡＧＴＴＣＴＴＴＴＧＣＴＴＴＣ，配列番号２０）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりｔｋｔＡ遺伝子のＤＮＡを増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-tktA]
The DNA of the tktA gene was amplified by Phusion Hot Start II DNA polymerase using the genomic DNA of E. coli MG1655 as a template and tktA-Nde (CCAACCATATGTCCTCAGTAAAGAGCTTGCC, SEQ ID NO: 19) and tktA-Xho (CACTCGAGTTACAGCAGTTCTTTTTTGCTTTC, SEQ ID NO: 20) primer pair. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual.
The amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－Ｔ７(-8TC)－ｔｋｔＡの作製］
　ｐＥＴＤｕｅｔ－ＦＲＴ（非特許文献２、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．９３：８１５－８２９，２０１２）を鋳型としてＦ－Ｂｇｌ（ＣＡＡＣＡＧＡＴＣＴＡＴＴＣＣＧＧＧＧＡＴＣＣＧＴＣＧＡＣＣ，配列番号２１）と－８ＴＣ－ＲＭ１（ＣＣＣＣＴＡＴＡＧＴＧＧＧＴＣＧＴＡＴＴＡＡＴＴＴＣＧ，配列番号２２）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。一方、ｐＥＴ２１ａ－ＦＲＴ－ｔｋｔＡを鋳型として－８ＴＣ－ＦＭ１（ＣＧＡＡＡＴＴＡＡＴＡＣＧＡＣＣＣＡＣＴＡＴＡＧＧＧＧ，配列番号２３）とＴ７ターミネータープライマーのペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりＤＮＡ断片を増幅した。つぎに、２つの増幅断片を鋳型としてＦ－ＢｇｌとＴ７ターミネータープライマーのペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、Ｔ７プロモーターの転写開始起点から８塩基上流の配列がＴからＣに置換された変異型Ｔ７プロモーター配列を含むＤＮＡ断片を得た。
　このＤＮＡ断片とｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＢｇｌＩＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-T7 (-8TC) -tktA]
pETDuet-FRT (non-patent document 2, Koma et al. Appl. Microbiol. Biotechnol. 93: 815-829, 2012) as a template, F-Bgl (CAACAGATCTATTCCGGGGATCCGTCGACC, SEQ ID NO: 21) and -8TC-RM1 (CCCCTATAGTGGGTCGTATTAATTTCG, SEQ ID NO: 22) The DNA fragment was amplified by Phusion Hot Start II DNA polymerase using a primer pair. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. On the other hand, a DNA fragment was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-tktA as a template and a pair of -8TC-FM1 (CGAAATTAATACGACCCACTATAGGGG, SEQ ID NO: 23) and a T7 terminator primer. Next, overlap extension PCR is performed with Phusion Hot Start II DNA polymerase using the pair of F-Bgl and T7 terminator primer with the two amplified fragments as a template, and the sequence of 8 bases upstream from the transcription start origin of T7 promoter is A DNA fragment containing a mutant T7 promoter sequence substituted for T to C was obtained.
The DNA fragment and the pET21a-FRT vector were digested with BglII and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from the gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ｔｙｒＡfbrの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型として、ＥｃＴｙｒＡ－Ｆ（ＣＣＡＡＣＣＡＴＡＴＧＧＴＴＧＣＴＧＡＡＴＴＧＡＣＣＧＣ，配列番号２４）とＥｃＴｙｒＡ－ＲＭ１（ＣＧＣＧＡＧＧＣＣＡＡＧＡＴＡＧＡＴＧＣＣＴＣＧＣＧＣ，配列番号２５）プライマーペア、ＥｃＴｙｒＡ－ＦＭ１（ＧＣＧＣＧＡＧＧＣＡＴＣＴＡＴＣＴＴＧＧＣＣＴＣＧＣＧ，配列番号２６）とＥｃＴｙｒＡ－ＲＭ２（ＣＴＣＴＧＡＡＡＡＣＧＣＴＧＴＡＣＧＴＡＡＴＣＧＣＣＧＡＡＣ，配列番号２７）プライマーペア、及びＥｃＴｙｒＡ－ＦＭ２（ＧＴＴＣＧＧＣＧＡＴＴＡＣＧＴＡＣＡＧＣＧＴＴＴＴＣＡＧＡＧ，配列番号２８）とＥｃＴｙｒＡ－Ｒ（ＣＡＣＴＣＧＡＧＴＴＡＣＴＧＧＣＧＡＴＴＧＴＣＡＴＴＣＧＣＣ，配列番号２９）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより３つのＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。つぎに、３つの増幅断片を鋳型としてＥｃＴｙｒＡ－ＦとＥｃＴｙｒＡ－Ｒのプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、５３番目のメチオニンがイソロイシンに置換され、かつ３５４番目のアラニンがバリンに置換されたＴｙｒＡをコードするＤＮＡ（ｔｙｒＡfbr）を得た。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-tyrA fbr ]
EcTyrA-F (CCAACCATATGGTTGCTGAATTGACGC, SEQ ID NO: 24) and EcTyrA-RM1 (CGCGAGGCGAGAGATAGATGCCTCGGC, SEQ ID NO: 25) and a pair of EcTyrA-F1 (GCGAGGCATCTATCTTGTGCTCGGC) and a GlcNAG as a template, using E. coli MG1655 as a template SEQ ID NO: 27) Primer pair and EcTyrA-FM2 (GTTCGGCGATTACGTACAGCGTTTTCAGAG, SEQ ID NO: 28) with EcTyrA-R (CACTCGAGTTACTGGCGATTGTCATTCGCC, SEQ ID NO: 29) primer pair Used to amplify three DNA fragments by Phusion Hot Start II DNA polymerase. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. Next, overlap extension PCR is performed by Phusion Hot Start II DNA polymerase using the three amplified fragments as a template and a primer pair of EcTyrA-F and EcTyrA-R, and methionine at position 53 is substituted with isoleucine, and 354 A DNA (TyrA fbr ) encoding TyrA in which the second alanine was substituted by valine was obtained.
The amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ｔｙｒＢの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型としてｔｙｒＢ－Ｎｄｅ（ＣＡＡＣＡＣＡＴＡＴＧＴＴＴＣＡＡＡＡＡＧＴＴＧＡＣＧＣ，配列番号３０）とｔｙｒＢ－Ｘｈｏ（ＴＡＣＴＣＧＡＧＴＴＡＣＡＴＣＡＣＣＧＣＡＧＣＡＡＡＣ，配列番号３１）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりｔｙｒＢ遺伝子のＤＮＡ断片を増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。
　増幅されたＤＮＡとｐＥＴ２１ａ－ＦＲＴベクターをそれぞれＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたＤＮＡを常法によりＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-tyrB]
The DNA fragment of the tyrB gene was amplified by Phusion Hot Start II DNA polymerase using the genomic DNA of E. coli MG1655 as a template and tyrB-Nde (CAACACATATGTTTCAAAAGTTGACGC, SEQ ID NO: 30) and tyrB-Xho (TACTCGAGTTACATCACCGCAGCAAAAC, SEQ ID NO: 31) primer pair. . The conditions for the PCR reaction were based on the basic conditions described in the instruction manual.
The amplified DNA and the pET21a-FRT vector were digested with NdeI and XhoI, respectively, and the DNA was separated by 1% agarose gel electrophoresis, and then the corresponding DNA was extracted and purified from gel using QIAquick Gel Extraction kit. The purified DNA was ligated in a conventional manner, transformed into E. coli DH5α, and cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＢの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型としてＡｒｏＢ－ｏｐｔ－ｇｉｂ－Ｆ（ＧＴＴＴＡＡＣＴＴＴＡＡＧＡＡＧＧＡＧＡＴＡＴＡＣＡＴＡＴＧＧＡＧＣＧＴＡＴＴＧＴＣＧＴＴＡＣＴＣ，配列番号３２）とＡｒｏＢ－ｇｉｂ－Ｒ（ＧＴＧＧＴＧＧＴＧＧＴＧＣＴＣＧＡＧＴＴＡＣＧＣＴＧＡＴＴＧＡＣＡＡＴＣＧＧＣ，配列番号３３）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＢ遺伝子のＤＮＡを増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。
　ＤｐｎＩで処理したａｒｏＢ遺伝子のＤＮＡとＮｄｅＩとＸｈｏＩで消化したｐＥＴ２１ａ－ＦＲＴをギブソン・アッセンブリー・システム（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ、以下同じ）を用いて反応させ、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-aroB]
Using AroB-opt-gib-F (GTTTAACTTTAAGAAGGAGATATACATATGATACATATGGAGCCATTATTGTCGTTACTC, SEQ ID NO: 32) and AroB-gib-R (GTGGTGGTGGTGCTCAGATTACGCTGATTGACAATCGGC, SEQ ID NO: 33) primer pair with the genomic DNA of E. coli MG1 655 as a template for using the PhusionA DNA according to Was amplified. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual.
E. coli DH5α is transformed by reacting the DpnI-treated aroB gene DNA with NdeI and XhoI-digested pET21a-FRT using the Gibson assembly system (New England Biolabs, the same shall apply hereinafter), and 20 μg / ml kanamycin It culture | cultivated at 37 degreeC overnight using LB agar medium containing. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬＣの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型としてＥｃＡｒｏＡ－Ｆ（ＣＡＡＣＡＣＣＡＴＧＧＡＡＴＣＣＣＴＧＡＣＧＴＴＡＣＡＡＣ，配列番号３４）とＥｃＡｒｏＡ－Ｒ（ＣＡＡＡＧＣＴＴＴＣＡＧＧＣＴＧＣＣＴＧＧＣＴＡＡＴＣＣ，配列番号３５）プライマーペア、ＥｃＡｒｏＬ－Ｆ（ＣＣＡＡＣＣＡＴＡＴＧＡＣＡＣＡＡＣＣＴＣＴＴＴＴＴＣＴＧＡＴＣ，配列番号３６）とＥｃＡｒｏＬ－Ｒ（ＣＡＣＴＣＧＡＧＴＣＡＡＣＡＡＴＴＧＡＴＣＧＴＣＴＧＴＧＣＣ，配列番号３７）プライマーペア、及びＥｃＡｒｏＣ－Ｆ（ＣＡＡＣＡＣＣＡＴＧＧＣＴＧＧＡＡＡＣＡＣＡＡＴＴＧＧＡＣ，配列番号３８）とＥｃＡｒｏＣ－Ｒ（ＣＡＡＡＧＣＴＴＴＴＡＣＣＡＧＣＧＴＧＧＡＡＴＡＴＣＡＧＴＣ，配列番号３９）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＡ、ａｒｏＬ、及びａｒｏＣ遺伝子のＤＮＡを増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。ａｒｏＡ遺伝子とａｒｏＣ遺伝子のＤＮＡをＮｃｏＩとＨｉｎｄＩＩＩで消化し、一方、ａｒｏＬ遺伝子のＤＮＡをＮｄｅＩとＸｈｏＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いてそれぞれのＤＮＡをゲルから抽出・精製した。ｐＥＴ２１ａ－ＦＲＴベクターをＮｄｅＩとＸｈｏＩで消化し、ｐＥＴ２１ｄ－ＦＲＴベクター（非特許文献２、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．９３：８１５－８２９，２０１２）をＮｃｏＩとＨｉｎｄＩＩＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたａｒｏＡ遺伝子のＤＮＡとｐＥＴ２１ｄ－ＦＲＴ、精製されたａｒｏＣ遺伝子のＤＮＡとｐＥＴ２１ｄ－ＦＲＴ、及び精製されたａｒｏＬ遺伝子のＤＮＡとｐＥＴ２１ａ－ＦＲＴを常法によりそれぞれＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて抽出し精製することで、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＡ、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＣ、及びｐＥＴ２１ａ－ＦＲＴ－ａｒｏＬを得た。
　ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＡを鋳型としてＡＬＣ－Ｆ（ＧＡＡＡＴＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧＧＡＡＴＴＧ，配列番号４０）とＡＬＣ－ａｒｏＡ－Ｒ（ＴＣＡＧＧＣＴＧＣＣＴＧＧＣＴＡＡＴＣ，配列番号４１）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＡ遺伝子を含むＤＮＡ断片を増幅した。また、ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＬを鋳型として、ＡＬＣ－ａｒｏＬ－Ｆ（ＧＡＴＴＡＧＣＣＡＧＧＣＡＧＣＣＴＧＡＣＴＴＴＡＡＧＡＡＧＧＡＧＡＴＡＴＡＣＡＴＡＴＧＡＣＡＣ，配列番号４２）とＡＬＣ－ａｒｏＬ－Ｒ（ＧＧＡＴＧＧＣＣＴＣＣＴＴＴＡＧＡＴＣＣＴＣＡＡＣＡＡＴＴＧＡＴＣＧＴＣＴＧＴＧＣ，配列番号４３）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＬ遺伝子を含むＤＮＡ断片を増幅した。さらに、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＣを鋳型としてＡＬＣ－ａｒｏＣ－Ｆ（ＧＧＡＴＣＴＡＡＡＧＧＡＧＧＣＣＡＴＣＣＡＴＧＧＣＴＧＧＡＡＡＣＡＣＡＡＴＴＧＧ，配列番号４４）とＡＬＣｏｒＥＤＢ－Ｒ（ＧＧＡＴＧＧＣＣＴＣＣＴＴＴＡＧＡＴＣＣＴＣＡＡＣＡＡＴＴＧＡＴＣＧＴＣＴＧＴＧＣ，配列番号４５）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＣ遺伝子を含むＤＮＡ断片を増幅した。つぎに、３つの増幅断片を鋳型としてａｒｏＡＬＣ－Ｆ１（ＡＴＧＧＡＡＴＣＣＣＴＧＡＣＧＴＴＡＣＡＡＣ，配列番号４６）とａｒｏＡＬＣ－Ｒ１（ＴＴＡＣＣＡＧＣＧＴＧＧＡＡＴＡＴＣＡＧＴＣＴＴＣ，配列番号４７）のプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、ａｒｏＡＬＣを含むＤＮＡ断片を得た。得られたＤＮＡ断片をｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ、以下同じ）を含むＴＯＰＯクローニングの試薬と反応させ、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ａｒｏＡＬＣ－Ｆ１とａｒｏＡＬＣ－Ｒ１を用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ａｒｏＡＬＣを抽出し精製した。
　一方、ｐＥＴ２１ａ－ＦＲＴを鋳型として、ｐＥＴ２１ａ－ＡＬＣ－Ｒ（ＧＴＡＡＣＧＴＣＡＧＧＧＡＴＴＣＣＡＴＧＧＴＡＡＴＡＴＣＴＣＣＴＴＣＴＴＡＡＡＧＴＴＡＡＡＣ，配列番号４８）とｐＥＴ２１ａ－ＡＬＣ－Ｆ（ＣＴＧＡＴＡＴＴＣＣＡＣＧＣＴＧＧＴＡＡＣＴＣＧＡＧＣＡＣＣＡＣＣＡＣＣＡＣＣ，配列番号４９）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより、プラスミドバックボーンとなるＤＮＡ断片を増幅した。得られたＤＮＡ断片をｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTMを含むＴＯＰＯクローニングの試薬と反応させ、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ｐＥＴ２１ａ－ＡＬＣ－ＲとｐＥＴ２１ａ－ＡＬＣ－Ｆを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ＢＢ－ａｒｏＡＬＣを抽出し精製した。
　つぎに、ｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ａｒｏＡＬＣを鋳型として、ａｒｏＡＬＣ－Ｆ１とａｒｏＡＬＣ－Ｒ１プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＡＬＣ遺伝子を含むＤＮＡ断片を増幅した。一方、ｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ＢＢ－ａｒｏＡＬＣを鋳型として、ｐＥＴ２１ａ－ＡＬＣ－ＲとｐＥＴ２１ａ－ＡＬＣ－Ｆプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりプラスミドバックボーンを含むＤＮＡ断片を得た。得られた両ＤＮＡ断片をギブソン・アッセンブリー・システムを用いて反応させ、大腸菌ＤＨ５αを形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ａｒｏＡＬＣ－Ｆ１とａｒｏＡＬＣ－Ｒ１プライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-aroALC]
EcAroA-F (CAACAC CATGGA ATCC CTGACGTTACAAC, SEQ ID NO: 34) and EcAroA-R (CAAAGCTTTCAGGCTGCCTGGCTAATCC, SEQ ID NO: 35) primer pair using the genomic DNA of E. coli MG1 655 as a template and EcAroA-F (CAAAAGCTTTAGGCTGCCTGGCTAATCC, SEQ ID NO: 35). No. 37) Using a primer pair and EcAroC-F (CAACACCATGGCTGGAACACACAATTGGAC, SEQ ID NO: 38) and EcAroC-R (CAAAGCTTTTACCAGCGTGGAATATCAGTC, SEQ ID NO: 39) primer pair , AroA the Phusion Hot Start II DNA polymerase was amplified aroL, and the DNA of the aroC gene. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. DNA of aroA gene and aroC gene is digested with NcoI and HindIII, while DNA of aroL gene is digested with NdeI and XhoI, and DNA is separated by 1% agarose gel electrophoresis, respectively using QIAquick Gel Extraction kit DNA was extracted and purified from the gel. pET21a-FRT vector is digested with NdeI and XhoI, and pET21d-FRT vector (Non-patent document 2, Koma et al. Appl. Microbiol. Biotechnol. 93: 815-829, 2012) is digested with NcoI and HindIII, 1% After separating the DNA by agarose gel electrophoresis, the corresponding DNA was extracted and purified from the gel using QIAquick Gel Extraction kit. The DNA of purified aroA gene and pET21d-FRT, the purified DNA of aroC gene and pET21d-FRT, and the purified DNA of aroL gene and pET21a-FRT are respectively transformed into E. coli DH5α The cells were cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, extracted and purified using NucleoSpin Plasmid QuickPure, pET21d-FRT-aroA, pET21d -FRT-aroC and pET21a-FRT-aroL were obtained.
DNA fragment containing aroA gene by Phusion Hot Start II DNA polymerase using pET21d-FRT-aroA as a template and ALC-F (GAAATTAATACGACTCACTATAGGGGAATTG, SEQ ID NO: 40) and ALC-aroA-R (TCAGGCTGCCTGCGTACTAATC, SEQ ID NO: 41) primer pair Was amplified. In addition, using PET21a-FRT-aroL as a template, ALC-aroL-F (GATTAGCCAGGCACTCACTACTTTGAAAGGAGATACATATATGACAC, SEQ ID NO: 42) and ALC-aroL-R (GGATGGCCTCCTTTAGATCCTCAACAATTGATCGTCTGTGC, SEQ ID NO: 43) using a primer pair by using the Phuaroh Hot Start DNA polymerase The DNA fragment containing the gene was amplified. In addition, pET21d-FRT-aroC is used as a template and contains ALC-aroC-F (GGATCTAAAGAGGGCATCCATGGCTGGAACACAATTGG, SEQ ID NO: 44) and ALCorEDB-R (GGATGGCGTCCTTTAGATCCTC AACAATTGATCGTCTGTGC, SEQ ID NO: 45) primer pair containing Phusion Hot Start II DNA aroC by a DNA polymerase The DNA fragment was amplified. Next, overlap extension PCR was performed by Phusion Hot Start II DNA polymerase using the primer pair of aroALC-F1 (ATGGAATCCCTGACGTTACAAAC, SEQ ID NO: 46) and aroALC-R1 (TTACCAGCGTGGAATATCAGTCTTC, SEQ ID NO: 47) using the three amplified fragments as a template. Conducted to obtain a DNA fragment containing aroALC. The resulting DNA fragment pCR TM II-Blunt-TOPO TM (Thermo Fisher Scientific, hereinafter the same) is reacted with a reagent of TOPO cloning including, E. coli DH5α was transformed, the LB agar medium containing 20 [mu] g / ml of kanamycin It was cultured at 37 ° C. overnight. Colony direct PCR using aroALC-F1 and aroALC-R1 was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and pCR TM II-Blunt-TOPO TM -aroALC is extracted and purified using NucleoSpin Plasmid QuickPure did.
On the other hand, using pET21a-FRT as a template, and using pET21a-ALC-R (GTAACGTCAGGGATTCCATGGTAATATCTCCTCTTCTAAAGTTAAAC, SEQ ID NO: 48) and pET21a-ALC-F (CTGATATTCACGCTGGTAACTCGAGCACCACCACC, SEQ ID NO: 49) primer pair, using Phuthion Hot Startia DNA polymerase, by using The amplified DNA fragment was amplified. The resulting DNA fragment is reacted with a reagent of TOPO cloning including pCR TM II-Blunt-TOPO TM , E. coli DH5α was transformed, overnight at 37 ° C. using a LB agar medium containing 20 [mu] g / ml kanamycin culture did. Colony direct PCR using pET21a-ALC-R and pET21a-ALC-F was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and pCR TM II-Blunt-TOPO TM -BB-aroALC is extracted using NucleoSpin Plasmid QuickPure And refined.
Next, the pCR TM II-Blunt-TOPO TM -aroALC as a template, using aroALC-F1 and aroALC-R1 primer pair to amplify a DNA fragment containing the AroALC gene by Phusion Hot Start II DNA polymerase. Resulting Meanwhile, the pCR TM II-Blunt-TOPO TM -BB-aroALC as a template, using the pET21a-ALC-R and pET21a-ALC-F primer pair, a DNA fragment containing the plasmid backbone by Phusion Hot Start II DNA polymerase The Both DNA fragments obtained were reacted using the Gibson assembly system, transformed into E. coli DH5α, and cultured overnight at 37 ° C. on LB agar medium containing 50 μg / ml of ampicillin. Colony direct PCR using aroALC-F1 and aroALC-R1 primer pairs was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 50 μg / ml of ampicillin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤＢの作製］
　大腸菌ＭＧ１６５５のゲノムＤＮＡを鋳型としてＥｃＡｒｏＥ－Ｆ（ＣＡＡＣＣＡＴＡＴＧＧＡＡＡＣＣＴＡＴＧＣＴＧＴＴＴＴＴＧＧ，配列番号５０）とＥｃＡｒｏＥ－Ｒ（ＣＡＣＴＣＧＡＧＴＣＡＣＧＣＧＧＡＣＡＡＴＴＣＣＴＣＣ，配列番号５１）プライマーペア、ＥｃＡｒｏＤ－Ｆ（ＣＣＡＡＣＣＡＴＡＴＧＡＡＡＡＣＣＧＴＡＡＣＴＧＴＡＡＡＡＧＡＴＣ，配列番号５２）とＥｃＡｒｏＤ－Ｒ（ＣＡＣＴＣＧＡＧＴＴＡＴＧＣＣＴＧＧＴＧＴＡＡＡＡＴＡＧＴＴＡＡＴＡＣ，配列番号５３）プライマーペア、及びＥｃＡｒｏＢ－Ｆ（ＣＡＡＣＡＣＣＡＴＧＧＡＧＡＧＧＡＴＴＧＴＣＧＴＴＡＣＴＣ，配列番号５４）とＥｃＡｒｏＢ－Ｒ（ＣＡＡＡＧＣＴＴＴＴＡＣＧＣＴＧＡＴＴＧＡＣＡＡＴＣＧＧＣ，配列番号５５）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＥ、ａｒｏＤ、及びａｒｏＢ遺伝子のＤＮＡを増幅した。なお、ＰＣＲ反応の条件は、説明書に記載の基本的な条件に基づいて行った。ａｒｏＥ遺伝子とａｒｏＤ遺伝子のＤＮＡをＮｄｅＩとＸｈｏＩで消化し、一方、ａｒｏＢ遺伝子のＤＮＡをＮｃｏＩとＨｉｎｄＩＩＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いてそれぞれのＤＮＡをゲルから抽出・精製した。ｐＥＴ２１ａ－ＦＲＴベクターをＮｄｅＩとＸｈｏＩで消化し、ｐＥＴ２１ｄ－ＦＲＴベクターをＮｃｏＩとＨｉｎｄＩＩＩで消化し、１％アガロースゲル電気泳動でＤＮＡを分離した後、ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いて該当のＤＮＡをゲルから抽出・精製した。精製されたａｒｏＥ遺伝子のＤＮＡとｐＥＴ２１ａ－ＦＲＴ、精製されたａｒｏＤ遺伝子のＤＮＡとｐＥＴ２１ａ－ＦＲＴ、及び精製されたａｒｏＢ遺伝子のＤＮＡとｐＥＴ２１ｄ－ＦＲＴを常法によりそれぞれＬｉｇａｔｉｏｎし、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて抽出し精製することで、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＥ、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＤ、及びｐＥＴ２１ａ－ＦＲＴ－ａｒｏＢを得た。
　ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＥを鋳型としてＥＤＢ－Ｆ（ＧＡＡＡＴＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧＧＡＡＴＴＧ，配列番号５６）とＥＤＢ－ａｒｏＥ－Ｒ（ＣＡＴＡＴＧＴＡＴＡＴＣＴＣＣＴＴＣＴＴＡＡＡＧＴＣＡＣＧＣＧＧＡＣＡＡＴＴＣＣＴＣＣ，配列番号５７）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＥ遺伝子を含むＤＮＡ断片を増幅した。また、ｐＥＴ２１ｄ－ＦＲＴ－ａｒｏＤを鋳型として、ＥＤＢ－ａｒｏＤ－Ｆ（ＣＧＴＧＡＣＴＴＴＡＡＧＡＡＧＧＡＧＡＴＡＴＡＣＡＴＡＴＧＡＡＡＡＣＣＧＴＡＡＣＴＧＴＡＡＡＡＧＡＣＣ，配列番号５８）とＥＤＢ－ａｒｏＤ－Ｒ（ＧＧＡＴＧＧＣＣＴＣＣＴＴＴＡＧＡＴＣＣＴＴＡＴＧＣＣＴＧＧＴＧＴＡＡＡＡＴＡＧＴＴＡＡＴＡＣ，配列番号５９）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＤ遺伝子を含むＤＮＡ断片を増幅した。さらに、ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＢを鋳型としてＥＤＢ－ａｒｏＢ－Ｆ２（ＧＧＡＴＣＴＡＡＡＧＧＡＧＧＣＣＡＴＣＣＡＴＧＧＡＧＣＧＴＡＴＴＧＴＣＧＴＴＡＣＴＣ，配列番号６０）とＡＬＣｏｒＥＤＢ－Ｒプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＢ遺伝子を含むＤＮＡ断片を増幅した。つぎに、３つの増幅断片を鋳型としてａｒｏＥＤＢ－Ｆ１（ＡＴＧＧＡＡＡＣＣＴＡＴＧＣＴＧＴＴＴＴＴＧＧ，配列番号６１）とａｒｏＥＤＢ－Ｒ１（ＴＴＡＣＧＣＴＧＡＴＴＧＡＣＡＡＴＣＧＧＣ，配列番号６２）のプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりオーバーラップエクステンションＰＣＲを行い、ａｒｏＥＤＢを含むＤＮＡ断片を得た。得られたＤＮＡ断片をｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTMを含むＴＯＰＯクローニングの試薬と反応させ、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ａｒｏＥＤＢ－Ｆ１とａｒｏＥＤＢ－Ｒ１を用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ａｒｏＥＤＢを抽出し精製した。
　一方、ｐＥＴ２１ａ－ＦＲＴを鋳型として、ｐＥＴ２１ａ－ＥＤＢ－Ｒ（ＣＡＡＡＡＡＣＡＧＣＡＴＡＧＧＴＴＴＣＣＡＴＡＴＧＴＡＴＡＴＣＴＣＣＴＴＣＴＴＡＡＡＧＴＴＡＡＡＣ，配列番号６３）とｐＥＴ２１ａ－ＥＤＢ－Ｆ（ＣＧＡＴＴＧＴＣＡＡＴＣＡＧＣＧＴＡＡＣＴＣＧＡＧＣＡＣＣＡＣＣＡＣＣＡＣＣ，配列番号６４）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼにより、プラスミドバックボーンとなるＤＮＡ断片を増幅した。得られたＤＮＡ断片をｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTMを含むＴＯＰＯクローニングの試薬と反応させ、大腸菌ＤＨ５αを形質転換し、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ｐＥＴ２１ａ－ＥＤＢ－ＲとｐＥＴ２１ａ－ＥＤＢ－Ｆを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ＢＢ－ａｒｏＥＤＢを抽出し精製した。
　つぎに、ｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ａｒｏＥＤＢを鋳型として、ａｒｏＥＤＢ－Ｆ１とａｒｏＥＤＢ－Ｒ１プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＥＤＢ遺伝子を含むＤＮＡ断片を増幅した。一方、ｐＣＲTMＩＩ－Ｂｌｕｎｔ－ＴＯＰＯTM－ＢＢ－ａｒｏＥＤＢを鋳型として、ｐＥＴ２１ａ－ＥＤＢ－ＲとｐＥＴ２１ａ－ＥＤＢ－Ｆプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりプラスミドバックボーンを含むＤＮＡ断片を得た。得られた両ＤＮＡ断片をギブソン・アッセンブリー・システムを用いて反応させ、大腸菌ＤＨ５αを形質転換し、５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。ａｒｏＥＤＢ－Ｆ１とａｒｏＥＤＢ－Ｒ１プライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を５０μｇ／ｍｌのアンピシリンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いて目的のプラスミドを抽出し精製した。 [Preparation of pET21a-FRT-aroEDB]
EcAroE-F (CAACCATATGGAAACC TATACTGTCAGTCAAGCGACCGCGCGCGAGACAAGCC (SEQ ID NO: 50)) EcAroE-F (CAACCATATGGAAACCACTGCTGTTTTTTTG (SEQ ID NO: 50)); No. 53) Primer pair, and EcAroB-F (CAACACCATGGAGAGGTATTGTCGTTACTC, SEQ ID NO: 54) and EcAroB-R (CAAAGCTTTACGCTGATTGACAATCGGC, SEQ ID NO: 55) primer With A, aroE by Phusion Hot Start II DNA polymerase was amplified aroD, and the DNA of the aroB gene. The conditions for the PCR reaction were based on the basic conditions described in the instruction manual. DNA of aroE gene and aroD gene is digested with NdeI and XhoI, while DNA of aroB gene is digested with NcoI and HindIII, and DNA is separated by 1% agarose gel electrophoresis, respectively using QIAquick Gel Extraction kit DNA was extracted and purified from the gel. The pET21a-FRT vector is digested with NdeI and XhoI, the pET21d-FRT vector is digested with NcoI and HindIII, DNA is separated by 1% agarose gel electrophoresis, and then the corresponding DNA is gelled using the QIAquick Gel Extraction kit. It extracted and refined. The DNA of purified aroE gene and pET21a-FRT, the purified DNA of aroD gene and pET21a-FRT, and the purified DNA of aroB gene and pET21d-FRT are respectively transformed into E. coli DH5α by ligation. The cells were cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using T7 promoter primer and T7 terminator primer to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, extracted and purified using NucleoSpin Plasmid QuickPure, pET21d-FRT-aroE, pET21d -FRT-aroD and pET21a-FRT-aroB were obtained.
DNA fragment containing aroE gene by Phusion Hot Start II DNA polymerase using pET21d-FRT-aroE as template and EDB-F (GAAATTAATACGACTCACTATAGGGGAATTG, SEQ ID NO: 56) and EDB-aroE-R (CATATGTATATCTCCTCTTCTTAAGTCACGCGGACAATTCCTCC, SEQ ID NO: 57) primer pair Was amplified. In addition, using EDB-aroD-F (CGTGACTTTAAGAAGGAGATATACATATGAAACCGTAACTGTAAAAGACC, SEQ ID NO: 58) and EDB-aroD-R (GGATGGCCTCCTTTAGATCCTTTATGCCTGTGTAAAATAGTTAATAC, SEQ ID NO: 59) using pET21d-FRT-aroD as a template, using the primer pair as a template. The DNA fragment containing the gene was amplified. Furthermore, a DNA fragment containing the aroB gene was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroB as a template and EDB-aroB-F2 (GGATCTAAAGGAGGCCATCATGGAGCGTATTGTCGTTACTC, SEQ ID NO: 60) and ALCorEDB-R primer pair. Next, overlap extension PCR was performed with Phusion Hot Start II DNA polymerase using the primer pair of aroEDB-F1 (ATGGAAACCTATGCTGTTTTTGG, SEQ ID NO: 61) and aroEDB-R1 (TTACGCTGATTGACAATCGGC, SEQ ID NO: 62) using the three amplified fragments as templates. Conducted to obtain a DNA fragment containing aroEDB. The resulting DNA fragment is reacted with a reagent of TOPO cloning including pCR TM II-Blunt-TOPO TM , E. coli DH5α was transformed, overnight at 37 ° C. using a LB agar medium containing 20 [mu] g / ml kanamycin culture did. Colony direct PCR using aroEDB-F1 and aroEDB-R1 was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and pCR TM II-Blunt-TOPO TM -aroEDB is extracted and purified using NucleoSpin Plasmid QuickPure did.
On the other hand, using pET21a-FRT as a template, and using pET21a-EDB-R (CAAAAAAGACATAGGTTTCCATATGTATATCTCCTTCTTAAGTTAAAC, SEQ ID NO: 63) and pET21a-EDB-F (CGATTGTCAATCAGCGTCAACTCGAGCACCACCACC, SEQ ID NO: 64) primer pair, using Phuthion Hot Startia DNA polymerase, The amplified DNA fragment was amplified. The resulting DNA fragment is reacted with a reagent of TOPO cloning including pCR TM II-Blunt-TOPO TM , E. coli DH5α was transformed, overnight at 37 ° C. using a LB agar medium containing 20 [mu] g / ml kanamycin culture did. Colony direct PCR using pET21a-EDB-R and pET21a-EDB-F was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and pCR TM II-Blunt-TOPO TM -BB-aroEDB is extracted using NucleoSpin Plasmid QuickPure And refined.
Next, the pCR TM II-Blunt-TOPO TM -aroEDB as a template, using aroEDB-F1 and aroEDB-R1 primer pair to amplify a DNA fragment containing the AroEDB gene by Phusion Hot Start II DNA polymerase. Resulting Meanwhile, the pCR TM II-Blunt-TOPO TM -BB-aroEDB as a template, using the pET21a-EDB-R and pET21a-EDB-F primer pair, a DNA fragment containing the plasmid backbone by Phusion Hot Start II DNA polymerase The Both DNA fragments obtained were reacted using the Gibson assembly system, transformed into E. coli DH5α, and cultured overnight at 37 ° C. on LB agar medium containing 50 μg / ml of ampicillin. Colony direct PCR was performed using aroEDB-F1 and aroEDB-R1 primer pairs to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid was cultured overnight at 37 ° C. using LB liquid medium containing 50 μg / ml of ampicillin, and the target plasmid was extracted and purified using NucleoSpin Plasmid QuickPure.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＬＣの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬＣを鋳型としてａｒｏＬＣ－ＤＢ－Ｆ（ＣＴＴＡＡＡＧＴＴＡＡＡＣＡＡＡＡＴＴＡＴＴＴＣＴＡＧＡＧＧ，配列番号６５）とａｒｏＬＣ－Ｒ（ＡＡＧＧＡＧＡＴＡＴＡＣＡＴＡＴＧＡＣＡＣＡＡＣＣＴＣ，配列番号６６）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＬＣ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ、以下同じ）で精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキット（Ｔａｋａｒａ、以下同じ）を用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＬＣを得た。 [Preparation of pET21a-FRT-aroLC]
DNA fragment containing aroLC gene by Phusion Hot Start II DNA polymerase, using pET21a-FRT-aroALC as template with aroLC-DB-F (CTTAAAGTTAAACAAATTATTTCTAGAGG, SEQ ID NO: 65) and aroLC-R (AAGGAGATATACATATGACACACACTC, SEQ ID NO: 66) primer pair Was amplified. The resulting DNA fragment was purified with QIAquick PCR Purification Kit (Qiagen, hereinafter the same), digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, then self-ligated using DNA ligation kit (Takara, the same in the following), transformed into E. coli DH5α, and LB agar medium containing 20 μg / ml kanamycin. And incubated overnight at 37 ° C. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroLC. Obtained.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＣの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬＣを鋳型としてａｒｏＡＣ－Ｆ（ＣＴＴＡＡＡＧＴＣＡＧＧＣＴＧＣＣＴＧＧＣ，配列番号６７）とａｒｏＡＣ－Ｒ（ＡＡＧＧＡＧＧＣＣＡＴＣＣＡＴＧＧＣＴＧＧＡＡＡＣ，配列番号６８）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＡＣ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキットを用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＣを得た。 [Preparation of pET21a-FRT-aroAC]
A DNA fragment containing aroAC gene is amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroALC as a template and aroAC-F (CTTAAAGTCAGGCTGCCTGCGC, SEQ ID NO: 67) and aroAC-R (AAGGAGGCCATCATCATGCTGGAAAC, SEQ ID NO: 68) did. The resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5α, and transformed at 37 ° C using LB agar medium containing 20 μg / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroAC. Obtained.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬＣを鋳型としてａｒｏＡＬ－Ｆ（ＴＣＡＡＣＡＡＴＴＧＡＴＣＧＴＣＴＧＴＧＣＣ，配列番号６９）とａｒｏＡＬ－ＥＤ－Ｒ（ＣＴＣＧＡＧＣＡＣＣＡＣＣＡＣＣＡＣ，配列番号７０）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＡＬ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキットを用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＡＬを得た。 [Preparation of pET21a-FRT-aroAL]
A DNA fragment containing aroAL gene by Phusion Hot Start II DNA polymerase, using pET21a-FRT-aroALC as a template and aroAL-F (TCAACAATTGATCGTCTTGCC, SEQ ID NO: 69) and aroAL-ED-R (CTCGAGCACCACCACCAC, SEQ ID NO: 70) primer pair Was amplified. The resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5α, and transformed at 37 ° C using LB agar medium containing 20 μg / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroAL. Obtained.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＤＢの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤＢを鋳型としてａｒｏＬＣ－ＤＢ－ＦとａｒｏＤＢ－Ｒ（ＡＡＧＧＡＧＡＴＡＴＡＣＡＴＡＴＧＡＡＡＡＣＣＧＴＡＡＣ，配列番号７１）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＤＢ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキットを用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＤＢを得た。 [Preparation of pET21a-FRT-aroDB]
A DNA fragment containing the aroDB gene was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroEDB as a template and aroLC-DB-F and aroDB-R (AAGGAGATATACATATAAAAAGTCAAC, SEQ ID NO: 71) primer pair. The resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5α, and transformed at 37 ° C using LB agar medium containing 20 μg / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using an LB liquid medium containing 20 μg / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroDB. Obtained.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＢの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤＢを鋳型としてａｒｏＥＢ－Ｆ（ＴＣＡＣＧＣＧＧＡＣＡＡＴＴＣＣＴＣＣ，配列番号７２）とａｒｏＥＢ－Ｒ（ＧＧＡＴＣＴＡＡＡＧＧＡＧＧＣＣＡＴＣＣＡＴＧ，配列番号７３）プライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＥＢ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキットを用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＢを得た。 [Preparation of pET21a-FRT-aroEB]
A DNA fragment containing aroEB gene is amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroEDB as a template using aroEB-F (TCACGCGGACAAATTCCTCC, SEQ ID NO: 72) and aroEB-R (GGATCTAAAGAGGGCATCCATG, SEQ ID NO: 73) primer pair did. The resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5α, and transformed at 37 ° C using LB agar medium containing 20 μg / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroEB. Obtained.
　［ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤの作製］
　ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤＢを鋳型としてａｒｏＥＤ－Ｆ（ＴＴＡＴＧＣＣＴＧＧＴＧＴＡＡＡＡＴＡＧＴＴＡＡＴＡＣＣ，配列番号７４）とａｒｏＡＬ－ＥＤ－Ｒプライマーペアを用いて、Ｐｈｕｓｉｏｎ Ｈｏｔ Ｓｔａｒｔ ＩＩ ＤＮＡポリメラーゼによりａｒｏＥＤ遺伝子を含むＤＮＡ断片を増幅した。得られたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、Ｔ４キナーゼ（Ｔａｋａｒａ）を用いてリン酸化した。リン酸化されたＤＮＡ断片をＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後に、ＤＮＡライゲーションキットを用いてセルフライゲーションさせ、大腸菌ＤＨ５αを形質転換して、２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｔ７プロモータープライマーとＴ７ターミネータープライマーペアを用いたコロニーダイレクトＰＣＲを行い、目的のプラスミドを持つ菌株を選別した。最終的に目的プラスミドを有する菌株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地を用いて３７℃で一晩培養し、ＮｕｃｌｅｏＳｐｉｎ Ｐｌａｓｍｉｄ ＱｕｉｃｋＰｕｒｅを用いてプラスミドを抽出し精製することでｐＥＴ２１ａ－ＦＲＴ－ａｒｏＥＤを得た。 [Preparation of pET21a-FRT-aroED]
A DNA fragment containing the aroED gene was amplified by Phusion Hot Start II DNA polymerase using pET21a-FRT-aroEDB as a template and aroED-F (TTATGCCTGGTGTAAAATAGTTAATACC, SEQ ID NO: 74) and aroAL-ED-R primer pair. The resulting DNA fragment was purified with QIAquick PCR Purification Kit, then digested with DpnI, purified again with QIAquick PCR Purification Kit, and then phosphorylated using T4 kinase (Takara). The phosphorylated DNA fragment is purified with QIAquick PCR Purification Kit, self-ligated with DNA ligation kit, transformed into E. coli DH5α, and transformed at 37 ° C using LB agar medium containing 20 μg / ml kanamycin. It was cultured overnight. Colony direct PCR using T7 promoter primer and T7 terminator primer pair was performed to select a strain having a desired plasmid. Finally, the strain carrying the target plasmid is cultured overnight at 37 ° C. using LB liquid medium containing 20 μg / ml kanamycin, and the plasmid is extracted and purified using NucleoSpin Plasmid QuickPure to obtain pET21a-FRT-aroED. Obtained.
　［染色体に１遺伝子を導入した菌株の作製］
　表１に示す鋳型ＤＮＡとプライマーペアを用いて、ＰｒｉｍｅＳＴＡＲ ＧＸＬ ＤＮＡポリメラーゼ（タカラバイオ）により、表１に示すように、大腸菌の染色体に導入するためのＤＮＡカセットを増幅した。プライマーの配列は表２に示す。 [Preparation of a strain in which one gene has been introduced into the chromosome]
Using the template DNA and primer pair shown in Table 1, the DNA cassette for introduction into the chromosome of E. coli was amplified by PrimeSTAR GXL DNA polymerase (Takara Bio) as shown in Table 1. The sequences of the primers are shown in Table 2.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　大腸菌ＭＧ１６５５（ＤＥ３）／ｐＫＤ４６を１０ｍＭのアラビノースと１００μｇ／ｍｌのアンピシリンを含むＬＢ培地でＯＤ660の値が０．５程度になるまで培養した。培養液を氷冷した後、３ｍｌの培養液を１０，０００ｒｐｍ、４℃で３分間遠心分離し、上清を捨てた。菌体を氷冷した１ｍｌの滅菌水に懸濁し、１０，０００ｒｐｍ、４℃で３分間遠心分離し、上清を捨てた。この操作をもう一度繰り返した後、菌体を５０μｌの滅菌水に懸濁し、約１００ｎｇのＤＮＡカセットを含む溶液を加えた後に、エレクトロポレーションを行った。１ｍｌのＬＢ培地を用いてエレクトロポレーションキュベットから菌体を回収し、３７℃で２．５時間振とうした後に２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｋ２（ＣＧＧＴＧＣＣＣＴＧＡＡＴＧＡＡＣＴＧＣ，配列番号８７、以下同じ）と表１に示した染色体上の挿入座位から約５００ｂｐ下流に設計したプライマーのペアを用いたコロニーダイレクトＰＣＲを行い、染色体上の目的座位に目的のＤＮＡカセットが挿入された菌株を選別した。下流に設計したプライマーの配列は、ｌｄｈＡ座位はＤ－ｌｄｈＡ（ＣＡＧＣＧＴＴＡＡＣＴＧＧＴＴＣＧＣＧＧＴＣ，配列番号８８）、ａｄｈＥ座位はＤ－ａｄｈＥ（ＴＧＣＡＧＧＣＣＧＴＧＣＣＡＧＴＣＡＴＣＣ，配列番号８９）、ｐｆｌＤＣ座位はＤ－ｐｆｌＤＣ（ＣＧＴＴＡＴＴＧＡＡＡＧＧＣＴＡＴＧＡＣＣＴＧＡＡＧ，配列番号９０）、ｐｙｋＦ座位はＤ－ｐｙｋＦ（ＧＡＧＣＴＧＣＧＴＣＡＴＣＴＴＴＡＧ，配列番号９１）、ａｓｃＦ座位はＤ－ａｓｃＦ（ＣＧＴＡＧＣＧＧＣＴＧＡＡＡＡＡＣＴＣＣＡＣＣ，配列番号９２）、ａｃｋＡ－ｐｔａ座位はＤ－ａｃｋＡ－ｐｔａ（ＣＣＴＴＣＡＡＡＣＧＧＧＡＡＧＴＴＣＡＴＣＡＧ，配列番号９３）とした。なお、作製した菌株（染色体に１遺伝子が導入された菌株）の関連する遺伝子型を表３に示す。 E. coli MG1655 (DE3) / pKD46 was cultured in LB medium containing 10 mM arabinose and 100 μg / ml ampicillin until the OD 660 value reached about 0.5. After cooling the culture solution on ice, 3 ml of the culture solution was centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. The cells were suspended in 1 ml of ice-cold sterile water, centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. After repeating this operation again, the cells were suspended in 50 μl of sterile water, and a solution containing about 100 ng of DNA cassette was added, followed by electroporation. The cells were recovered from the electroporation cuvette using 1 ml of LB medium, shaken at 37 ° C. for 2.5 hours, and then cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR was performed using a pair of primers designed approximately 500 bp downstream from the insertion locus on the chromosome shown in Table 1 with K2 (CGGTGCCCTGAATGA ACTGC, SEQ ID NO: 87, and so forth), and the target DNA on the target locus The strain in which the cassette was inserted was selected. The sequence of the primer designed downstream is: ldhA locus: D-ldhA (CAGCGTTAACTGGTTCGCGGTC, SEQ ID NO: 88) adhE locus: D-adhE (TGCAGGCCGTGCCAGTCATCC, SEQ ID NO: 89) pflDC locus: D-pflDC The pykF locus was D-pykF (GAGCTGCGTCATCTTTAG, SEQ ID NO: 91), the ascF locus was D-ascF (CGTAGCGGCTGAAAAACTCCACC, SEQ ID NO: 92), and the ackA-pta locus was D-ackA-pta (CCTTCAAACGGGAAGTTCATCAG, SEQ ID NO: 93). The related genotypes of the prepared strains (strains in which one gene has been introduced into the chromosome) are shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　［Ｐ１ファージライセートの調製］
　染色体に１遺伝子を導入した菌株を２０μｇ／ｍｌのカナマイシンを含む５ｍｌのＬＢ液体培地に植菌し、３７℃でＯＤ660が０．１程度になるまで培養した。１Ｍの塩化カルシウムを５０μｌ添加し、さらに１００μｌのＰ１ファージ溶液（＞１０8ｐｆｕ／ｍｌ）を添加して３７℃で３～４時間程度培養した。１０，０００ｒｐｍで５分間遠心分離し、上澄みを０．２μｍのポアサイズの滅菌フィルターでろ過することで、Ｐ１ファージライセートを得た。 [Preparation of P1 phage lysate]
The strain in which 1 gene was introduced into the chromosome was inoculated into 5 ml of LB liquid medium containing 20 μg / ml kanamycin, and cultured at 37 ° C. until the OD 660 became about 0.1. 50 μl of 1 M calcium chloride was added, and 100 μl of P1 phage solution (> 10 8 pfu / ml) was further added, followed by culturing at 37 ° C. for about 3 to 4 hours. P1 phage lysate was obtained by centrifuging at 10,000 rpm for 5 minutes and filtering the supernatant through a sterile filter of 0.2 μm pore size.
　［Ｐｈｅ１株の作製］
ｐＥＴ２１ａ－ＦＲＴ－ａｒｏＧfbrを鋳型としてｄｅｌｔａ－ｔｙｒＲ（ＧＴＧＴＣＡＴＡＴＣＡＴＣＡＴＡＴＴＡＡＴＴＧＴＴＣＴＴＴＴＴＴＣＡＧＧＴＧＡＡＧＧＴＴＣＣＣＡＴＧＡＴＴＣＣＧＧＧＧＡＴＣＣＧＴＣＧＡＣＣ，配列番号９４）とｄｅｌｔａ－ｔｙｒＲ－ＦＲＴ－Ｒ（ＴＧＧＴＧＴＴＧＣＡＣＣＡＴＣＡＧＧＣＡＴＡＴＴＣＧＣＧＣＴＴＡＣＴＣＴＴＣＧＴＴＣＴＴＣＴＴＣＴＧＴＧＴＡＧＧＣＴＧＧＡＧＣＴＧＣＴＴＣＧ，配列番号９５）プライマーペアを用いて、ＰｒｉｍｅＳＴＡＲ ＧＸＬ ＤＮＡポリメラーゼ（タカラバイオ）によりＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＧfbrのＤＮＡを増幅した。得られたＤＮＡをＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した後にＤｐｎＩで消化し、再びＱＩＡｑｕｉｃｋ ＰＣＲ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔで精製した。
　大腸菌ＭＧ１６５５（ＤＥ３）／ｐＫＤ４６を１０ｍＭのアラビノースと１００μｇ／ｍｌのアンピシリンを含むＬＢ培地でＯＤ660の値が０．５程度になるまで培養した。培養液を氷冷した後、３ｍｌの培養液を１０，０００ｒｐｍ、４℃で３分間遠心分離し、上清を捨てた。菌体を氷冷した１ｍｌの滅菌水に懸濁し、１０，０００ｒｐｍ、４℃で３分間遠心分離し、上清を捨てた。この操作をもう一度繰り返した後、菌体を５０μｌの滅菌水に懸濁し、約１００ｎｇのＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＧfbrを含むＤＮＡ溶液を加えた後に、エレクトロポレーションを行った。１ｍｌのＬＢ培地を用いてエレクトロポレーションキュベットから菌体を回収し、３７℃で２．５時間振とうした後に２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地を用いて３７℃で一晩培養した。Ｋ２とＤ－ｔｙｒＲ（ＣＧＴＡＡＧＴＴＴＡＡＣＣＡＡＣＴＧＧＣＡＡＣＴＧ，配列番号９６）プライマーペアを用いたコロニーダイレクトＰＣＲを行い、ｔｙｒＲ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＧfbrが挿入された菌株を選別した。
　つぎにこの株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地で培養し、ＯＤ660の値が０．５程度になるまで培養した。培養液を氷冷した後、１ｍｌの培養液を１０，０００ｒｐｍ、４℃で３分間遠心分離し、上清を捨てた。菌体を１００μｌのＴＳＳ溶液（１０％ポリエチレングリコール３３５０、５％ジメチルスルホキシド、７０ｍＭ塩化マグネシウム、０．１Ｍ塩化カリウム、３０ｍＭ塩化カルシウム）に懸濁し、５０ｎｇ／μｌのｐＣＰ２０を１μｌ添加し、氷中で３０分間静置した。４２℃で９０秒間ヒートショックした後に氷中で２分間静置し、９００μｌのＬＢ培地を加え、１００μｌを５０μｇ／ｍｌのアンピシリンを含むＬＢ寒天培地に塗布した。３０℃で一晩培養し、出現したコロニーをＬＢ寒天培地にストリークして、４２℃で一晩培養した。つぎに、出現したコロニーを新たなＬＢ寒天培地にストリークして、３７℃で一晩培養した。Ｕ－ｔｙｒＲ（ＧＴＴＴＡＡＴＴＡＡＴＣＧＣＡＴＣＧＣＣＡＣＧＣ，配列番号９７）とＤ－ｔｙｒＲプライマーペアを用いて、ｔｙｒＲ座位に挿入されたＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＧfbrからカナマイシン耐性遺伝子が除去されたことを確認し、ＭＧ１６５５（ＤＥ３）ｔｙｒＲ：：ＰT7－ａｒｏＧfbr株を得た。
　つぎにこの株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｌｄｈＡのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｌｄｈＡ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｐｈｅＡfbrが挿入されていることを確認した。
　つぎにこの株を２０μｇ／ｍｌのカナマイシンを含むＬＢ液体培地で培養し、ＯＤ660の値が０．５程度になるまで培養し、前述のｐＣＰ２０を用いた方法によりカナマイシン耐性遺伝子を除去することにより、ＭＧ１６５５（ＤＥ３）ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr株を得た。この株をＰｈｅ１株と命名した。 [Preparation of Phe1 strain]
The GIV -TGs can be used as a pair of G. When a GIV tag is used as a pair, we can use the GPTs to: G. TIGGTG TCC TCC TCC TCC. The DNA of -Km-FRT-aroG fbr was amplified. The obtained DNA was purified with QIAquick PCR Purification Kit, then digested with DpnI, and purified again with QIAquick PCR Purification Kit.
E. coli MG1655 (DE3) / pKD46 was cultured in LB medium containing 10 mM arabinose and 100 μg / ml ampicillin until the OD 660 value reached about 0.5. After cooling the culture solution on ice, 3 ml of the culture solution was centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. The cells were suspended in 1 ml of ice-cold sterile water, centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. After this operation was repeated once, the cells were suspended in 50 μl of sterile water, and a DNA solution containing about 100 ng of FRT-Km-FRT-aroG fbr was added, followed by electroporation. The cells were recovered from the electroporation cuvette using 1 ml of LB medium, shaken at 37 ° C. for 2.5 hours, and then cultured overnight at 37 ° C. using LB agar medium containing 20 μg / ml kanamycin. Colony direct PCR using K2 and D-tyrR (CGTAAGTTTAACCAACTGGCAACTG, SEQ ID NO: 96) primer pair was performed to select strains in which FRT-Km-FRT-aroG fbr was inserted at the tyrR locus .
Next, this strain was cultured in LB liquid medium containing 20 μg / ml kanamycin, and cultured until the OD 660 value reached about 0.5. After cooling the culture solution on ice, 1 ml of the culture solution was centrifuged at 10,000 rpm for 3 minutes at 4 ° C., and the supernatant was discarded. Suspend the cells in 100 μl of TSS solution (10% polyethylene glycol 3350, 5% dimethyl sulfoxide, 70 mM magnesium chloride, 0.1 M potassium chloride, 30 mM calcium chloride), add 1 μl of 50 ng / μl pCP20, and in ice Let stand for 30 minutes. After heat shock for 90 seconds at 42 ° C., the plate was left in ice for 2 minutes, 900 μl of LB medium was added, and 100 μl was applied to LB agar medium containing 50 μg / ml of ampicillin. After overnight culture at 30 ° C., emerging colonies were streaked on LB agar and cultured overnight at 42 ° C. Next, the emerged colonies were streaked on fresh LB agar and cultured overnight at 37 ° C. Using U-tyrR (GTTTAATTAATCGCATCGCCACGC, SEQ ID NO: 97) and D-tyrR primer pair, it was confirmed that the kanamycin resistance gene was removed from FRT-Km-FRT-aroG fbr inserted at the tyrR locus, MG1655 (DE3) ) was obtained tyrR :: P T7 -aroG fbr stock.
Next, this strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S1 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ldhA, FRT-Km- FRT-P T7 -pheA fbr the ldhA locus of strains obtained was confirmed that it is inserted.
Next, this strain is cultured in LB liquid medium containing 20 μg / ml kanamycin, cultured until the OD 660 value becomes about 0.5, and the kanamycin resistance gene is removed by the method using pCP20 described above. to obtain a MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr stock. This strain was designated as Phe1 strain.
　［Ｐｈｅ２株の作製］
　Ｐｈｅ１株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ２株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｄｈＥのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｄｈＥ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｐｐｓＡが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ株を得た。この株をＰｈｅ２株と命名した。 [Preparation of Phe2 strain]
The Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S2 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-adhE, FRT-Km- FRT-P T7 -ppsA the adhE locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA strain I got This strain was designated Phe2 strain.
　［Ｐｈｅ３株の作製］
　Ｐｈｅ２株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ３株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｆｌＤＣのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｆｌＤＣ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｋｔＡが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7－ｔｋｔＡ株を得た。この株をＰｈｅ３株と命名した。 [Preparation of Phe3 strain]
The Phe2 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S3 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pflDC, FRT-Km- FRT-P T7 -tktA to PflDC locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 -tktA strain was obtained. This strain was designated as Phe3 strain.
　［Ｐｈｅ４株の作製］
　Ｐｈｅ２株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ４株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｆｌＤＣのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｆｌＤＣ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7(-8TC)－ｔｋｔＡが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ株を得た。この株をＰｈｅ４株と命名した。 [Preparation of Phe4 strain]
The Phe2 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S4 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Colony direct PCR was performed using a primer pair of K2 and D-pflDC, and it was confirmed that FRT-Km-FRT-PT7 (-8TC) -tktA was inserted into the pflDC locus of the obtained strain. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: PT7 (-8TC) -tktA strain was obtained. This strain was named Phe4 strain.
　［Ｐｈｅ５株の作製］
　Ｐｈｅ１株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ８株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＤＢが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ株を得た。この株をＰｈｅ５株と命名した。 [Preparation of Phe5 strain]
The Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S8 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroEDB the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr ascF :: P T7 -aroEDB strain I got This strain was designated Phe5.
　［Ｐｈｅ６株の作製］
　Ｐｈｅ３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ８株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＤＢが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ株を得た。この株をＰｈｅ６株と命名した。 [Preparation of Phe6 strain]
The Phe3 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S8 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroEDB the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 -tktA ascF :: P T7 -aroEDB strain was obtained. This strain was designated as Phe6 strain.
　［Ｐｈｅ７株の作製］
　Ｐｈｅ４株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ８株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＤＢが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ株を得た。この株をＰｈｅ７株と命名した。 [Preparation of Phe7 strain]
The Phe4 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S8 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroEDB the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB stock. This strain was designated as Phe7 strain.
　［Ｐｈｅ８株の作製］
　Ｐｈｅ１株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ８株と命名した。 [Preparation of Phe8 strain]
The Phe1 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr pykF :: P T7 -aroALC strain I got This strain was designated as Phe8 strain.
　［Ｐｈｅ９株の作製］
　Ｐｈｅ３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ９株と命名した。 [Preparation of Phe 9 strain]
The Phe3 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 -tktA pykF :: P T7 -aroALC stock. This strain was named Phe9 strain.
　［Ｐｈｅ１０株の作製］
　Ｐｈｅ４株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ１０株と命名した。 [Preparation of Phe10 strain]
The Phe4 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA pykF :: P T7 -aroALC stock. This strain was named Phe10 strain.
　［Ｐｈｅ１１株の作製］
　Ｐｈｅ５株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ１１株と命名した。 [Preparation of Phe11 strain]
The Phe5 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr ascF :: P T7 -aroEDB pykF :: P T7 -aroALC strain was obtained. This strain was designated as Phe11 strain.
　［Ｐｈｅ１２株の作製］
　Ｐｈｅ６株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ１２株と命名した。 [Preparation of Phe12 strain]
The Phe6 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroALC stock. This strain was designated Phe12 strain.
　［Ｐｈｅ１３株の作製］
　Ｐｈｅ７株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ９株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ１３と命名した。Ｐｈｅ１３は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（＃１２２，２－５－８　Ｋａｚｕｓａｋａｍａｔａｒｉ，Ｋｉｓａｒａｚｕ－ｓｈｉ，Ｃｈｉｂａ　２９２－０８１８，Ｊａｐａｎ）に寄託した（受託日２０１７年７月２０日、受託番号：ＮＩＴＥ　ＢＰ－０２５１４）。 [Preparation of Phe13 strain]
The Phe7 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S9 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroALC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroALC stock. This strain was named Phe13. Phe13 has been deposited at the Patent Organism Depositary Center for Patent Products and Technologies, Japan Patent Organism Depositary (# 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) (Accession date July 20, 2017, Accession number: NITE BP-02514).
　［Ｐｈｅ１４株の作製］
　Ｐｈｅ１０株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ６株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＢが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ　ａｓｃＦ：：ＰT7－ＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＢ株をＰｈｅ１４株と命名した。 [Preparation of Phe14 strain]
The Phe10 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S6 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroB the ASCF locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroALC ascF :: the P T7 -FRT-Km-FRT- aroB strain was named Phe14 strain.
　［Ｐｈｅ１５株の作製］
　Ｐｈｅ１９株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１４株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＢが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＣ　ａｓｃＦ：：ＰT7－ＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＥＢ株をＰｈｅ１５株と命名した。 [Preparation of Phe15 strain]
The Phe19 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S14 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroEB the ASCF locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroAC ascF :: the P T7 -FRT-Km-FRT- aroEB strain was named Phe15 strain.
　［Ｐｈｅ１６株の作製］
　Ｐｈｅ１９株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１５株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＤＢが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＣ　ａｓｃＦ：：ＰT7－ＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＤＢ株をＰｈｅ１６株と命名した。 [Preparation of Phe 16 strain]
The Phe19 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S15 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroDB the ASCF locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroAC ascF :: the P T7 -FRT-Km-FRT- aroDB strain was named Phe16 strain.
　［Ｐｈｅ１７株の作製］
　Ｐｈｅ１９株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ６株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＢが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＣ　ａｓｃＦ：：ＰT7－ＦＲＴ－Ｋｍ－ＦＲＴ－ａｒｏＢ株をＰｈｅ１７株と命名した。 [Preparation of Phe17 strain]
The Phe19 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S6 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroB the ASCF locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroAC ascF :: the P T7 -FRT-Km-FRT- aroB strain was named Phe17 strain.
　［Ｐｈｅ１８株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１０株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＬが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬ株を得た。この株をＰｈｅ１８株と命名した。 [Preparation of Phe 18 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S10 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroAL the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroAL stock. This strain was designated Phe18 strain.
　［Ｐｈｅ１９株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１１株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＡＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＣ株を得た。この株をＰｈｅ１９株と命名した。 [Preparation of Phe19 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S11 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroAC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroAC stock. This strain was designated Phe19 strain.
　［Ｐｈｅ２０株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１２株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｐｙｋＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｐｙｋＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＬＣが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＬＣ株を得た。この株をＰｈｅ２０株と命名した。 [Preparation of Phe20 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S12 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-pykF, FRT-Km- FRT-P T7 -aroLC the pykF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroLC stock. This strain was designated as Phe20 strain.
　［Ｐｈｅ２１株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１３株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＤが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ２１株と命名した。 [Preparation of Phe21 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S13 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroED the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin-resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroED pykF :: P T7 -aroALC stock. This strain was designated as Phe21 strain.
　［Ｐｈｅ２２株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１４株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＥＢが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ２２株と命名した。 [Preparation of Phe22 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S14 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroEB the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEB pykF :: P T7 -aroALC stock. This strain was designated as Phe22 strain.
　［Ｐｈｅ２３株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ１５株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｓｃＦのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｓｃＦ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ａｒｏＤＢが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＰｈｅ２３株と命名した。 [Preparation of Phe23 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S15 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ascF, FRT-Km- FRT-P T7 -aroDB the ASCF locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroDB pykF :: P T7 -aroALC stock. This strain was designated Phe23 strain.
　［Ｐｈｅ２４株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ７株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｃｋＡ－ｐｔａのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のａｃｋＡ－ｐｔａ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｙｒＢが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＣ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ａｃｋＡ－ｐｔａ：：ＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｙｒＢ株をＰｈｅ２４株と命名した。 [Preparation of Phe24 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S7 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ackA-pta, FRT- Km-FRT-P T7 -tyrB in ackA-pta locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroAC ascF :: the P T7 -aroEDB ackA-pta :: FRT -Km-FRT-P T7 -tyrB strain was designated as Phe24 shares.
　［Ｔｙｒ７株の作製］
　Ｐｈｅ７株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ５株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｌｄｈＡのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｌｄｈＡ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｙｒＡfbrが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｔｙｒＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ株を得た。この株をＴｙｒ７と命名した。 [Preparation of strain Tyr7]
The Phe7 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S5 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ldhA, FRT-Km- FRT-P T7 -tyrA fbr the ldhA locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -tyrA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB stock. This strain was named Tyr7.
　［Ｔｙｒ１０株の作製］
　Ｐｈｅ１０株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ５株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｌｄｈＡのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｌｄｈＡ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｙｒＡfbrが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｔｙｒＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＴｙｒ１０と命名した。 [Preparation of strain Tyr10]
The Phe10 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S5 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ldhA, FRT-Km- FRT-P T7 -tyrA fbr the ldhA locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -tyrA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA pykF :: P T7 -aroALC stock. This strain was designated Tyr10.
　［Ｔｙｒ１３株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＳ５株のＰ１ファージライセートを２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｌｄｈＡのプライマーペアを用いたコロニーダイレクトＰＣＲを行い、得られた株のｌｄｈＡ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｔｙｒＡfbrが挿入されていることを確認した。つぎに、Ｐｈｅ１株の作製で記載した方法に基づいて、カナマイシン耐性遺伝子を除去し、ＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｔｙｒＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ株を得た。この株をＴｙｒ１３と命名した。Ｔｙｒ１３は、独立行政法人製品評価技術基盤機構　特許微生物寄託センター（＃１２２，２－５－８　Ｋａｚｕｓａｋａｍａｔａｒｉ，Ｋｉｓａｒａｚｕ－ｓｈｉ，Ｃｈｉｂａ　２９２－０８１８，Ｊａｐａｎ）に寄託した（受託日２０１７年７月２０日、受託番号：ＮＩＴＥ　ＢＰ－０２５１３）。 [Preparation of strain Tyr13]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of P1 phage lysate of S5 strain was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. Perform colony direct PCR using primers pairs K2 and D-ldhA, FRT-Km- FRT-P T7 -tyrA fbr the ldhA locus of strains obtained was confirmed that it is inserted. Then, based on the method described in Preparation of Phe1 strain to remove the kanamycin resistance gene, MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -tyrA fbr adhE :: P T7 -ppsA pflDC :: give the P T7 (-8TC) -tktA ascF :: P T7 -aroEDB pykF :: P T7 -aroALC stock. This strain was designated Tyr13. Tyr13 was deposited at the Patent Organism Depositary Center for Patent Products and Technologies, Japan Patent Organism Depositary (# 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) (consignment date July 20, 2017, Accession number: NITE BP-02513).
　［２ＰＥ２株の作製］
　Ｐｈｅ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＡＲ－Ｇ８４のＰ１ファージライセート（非特許文献３、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．７８：６２０３－６２１６，２０１２）を２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ｍｔｌＡ（ＧＡＴＣＡＡＣＧＡＣＡＴＣＡＴＣＡＣＣＡＡＴＧＣ，配列番号９８）のプライマーペアを用い、得られた株のｍｔｌＡ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｉｐｄＣが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ｍｔｌＡ：：ＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｉｐｄＣ株を２ＰＥ２株と命名した。なお、２ＰＥ１株は既報（非特許文献３、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ　Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．７８：６２０３－６２１６，２０１２）に記載されているＰＡＲ－６０株に相当する。 [Production of 2PE2 strain]
The Phe13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of AR-G84 P1 phage lysate (Non-patent Document 3, Koma et al. Appl. Environ. Microbiol. 78: 6203-6216, 2012) was added and mixed, and incubated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. K2 a D-mtlA (GATCAACGACATCATCACCAATGC, SEQ ID NO: 98) using primer pairs, FRT-Km-FRT-P T7 -ipdC to MtIA locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroALC ascF :: the P T7 -aroEDB mtlA :: FRT-Km -FRT-P T7 -ipdC strain was designated as 2PE2 shares. The 2PE1 strain corresponds to the PAR-60 strain described in the previously reported (Koma et al. Appl. Environ. Microbiol. 78: 6203-6216, 2012).
　［４ＨＬＡ２株の作製］
　Ｔｙｒ１３株を５ｍｌのＬＢ液体培地を用いて３７℃で一晩培養した。５０μｌの１Ｍ塩化カルシウムを加えて良く撹拌した後、２００μｌを１．５ｍｌチューブに移した。そこにＡＲ－Ｇ３９のＰ１ファージライセート（非特許文献３、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．７８：６２０３－６２１６，２０１２）を２０μｌ加えて混合し、３７℃で２０分間恒温した。１Ｍのクエン酸３ナトリウム溶液を１００μｌとＬＢ液体培地を７００μｌ加えて混合した後に、３７℃でさらに４０分間恒温した。２０μｇ／ｍｌのカナマイシンを含むＬＢ寒天培地に菌液を塗布し、３７℃で一晩培養した。Ｋ２とＤ－ａｃｓ（ＣＴＣＡＴＧＣＡＧＧＡＣＴＴＣＡＴＴＡＴＴＡＡＧＡＣＧＧＴＣ，配列番号９９）のプライマーペアを用い、得られた株のａｃｓ座位にＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｌｄｈＡが挿入されていることを確認した。作製したＭＧ１６５５（ＤＥ３）　ｔｙｒＲ：：ＰT7－ａｒｏＧfbr　ｌｄｈＡ：：ＰT7－ｐｈｅＡfbr　ａｄｈＥ：：ＰT7－ｐｐｓＡ　ｐｆｌＤＣ：：ＰT7(-8TC)－ｔｋｔＡ　ｐｙｋＦ：：ＰT7－ａｒｏＡＬＣ　ａｓｃＦ：：ＰT7－ａｒｏＥＤＢ　ａｃｓ：：ＦＲＴ－Ｋｍ－ＦＲＴ－ＰT7－ｌｄｈＡ株を４ＨＬＡ２株と命名した。なお、４ＨＬＡ１株は既報（非特許文献３、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．７８：６２０３－６２１６，２０１２）に記載されているＰＡＲ－３株に相当する。 [Preparation of 4 HLA 2 strains]
The Tyr13 strain was cultured overnight at 37 ° C. using 5 ml of LB liquid medium. After adding 50 μl of 1 M calcium chloride and stirring well, 200 μl was transferred to a 1.5 ml tube. To this, 20 μl of AR-G39 P1 phage lysate (Non-patent Document 3, Koma et al. Appl. Environ. Microbiol. 78: 6203-6216, 2012) was added, mixed and thermostated at 37 ° C. for 20 minutes. After 100 μl of 1 M trisodium citrate solution and 700 μl of LB liquid medium were added and mixed, the temperature was further kept constant at 37 ° C. for 40 minutes. The bacterial solution was applied to LB agar medium containing 20 μg / ml kanamycin and cultured overnight at 37 ° C. K2 and D-acs (CTCATGCAGGACTTCATTATTAAGACGGTC, SEQ ID NO: 99) using primer pairs, FRT-Km-FRT-P T7 -ldhA the acs locus of strains obtained was confirmed that it is inserted. It was prepared MG1655 (DE3) tyrR :: P T7 -aroG fbr ldhA :: P T7 -pheA fbr adhE :: P T7 -ppsA pflDC :: P T7 (-8TC) -tktA pykF :: P T7 -aroALC ascF :: the P T7 -aroEDB acs :: FRT-Km -FRT-P T7 -ldhA strain was designated as 4HLA2 shares. The 4HLA1 strain corresponds to the PAR-3 strain described in the previously reported (Non-patent Document 3, Koma et al. Appl. Environ. Microbiol. 78: 6203-6216, 2012).
　［菌株の培養と生産物の定量］
　菌株を５ｍｌのＬＢ液体培地または０．８％のグルコースを含むＬＢ液体培地を用いて２７℃で一晩前培養した。５０μｌの前培養液をＭ９Ｍ２液体培地に加えて植菌し、３７℃でＯＤ660の値が０．３程度になるまで振とう培養した。Ｍ９Ｍ２液体培地は、９００ｍｌの蒸留水に１０ｇのグルコース、６ｇのリン酸水素２ナトリウム、３ｇのリン酸２水素カリウム、０．５ｇの塩化ナトリウム、および４ｇの塩化アンモニウムを加え、さらに１Ｍ 塩化マグネシウムを１ｍｌ、１０ｍｇ／ｍｌチアミン塩酸塩を１ｍｌ、１００ｍＭ硫酸鉄（二価）を０．５ｍｌ、１Ｍ塩化カルシウムを１００μｌ、微量金属塩溶液を１０μｌ加え、１Ｌにメスアップしたものを０．２μｍのフィルターを用いてろ過除菌したものを用いた。微量金属塩溶液は１００ｍｌの蒸留水中に０．３７１ｇの（ＮＨ4）6Ｍｏ7Ｏ24・４Ｈ2Ｏ、２．４７３ｇのＨ3ＢＯ3、０．７１４ｇのＣｏＣｌ2・６Ｈ2Ｏ、０．２３９ｇのＣｕＳＯ4・５Ｈ2Ｏ、１．５８３ｇのＭｎＣｌ2・４Ｈ2Ｏ、０．２８８ｇのＺｎＳＯ4・７Ｈ2Ｏを含む。なお、２ＰＥ１株と２ＰＥ２株を培養する場合にはＭ９Ｍ２培地にフェニルアラニンを２ｍＭとなるように加えた。イソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を終濃度が１ｍＭとなるように添加し、２７℃で４４時間振とう培養した。９５０μｌの蒸留水と２００μｌのメタノールと５０μｌの培養液を混合し、１０，０００ｒｐｍで５分間遠心分離し、上清を０．４５μｍのフィルターを用いてろ過したものをＨＰＬＣのサンプルとして用いた。ＨＰＬＣは既報（非特許文献３、Ｋｏｍａ ｅｔ ａｌ．Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．７８：６２０３－６２１６，２０１２）に記載されているとおりに行い、培養液中のフェニルアラニン、チロシン、２－フェニルエタノール、または４－ヒドロキシフェニル乳酸の生成量を定量した。
　その結果を表４～７に示す。これらの表において、Ｐｈｅはフェニルアラニンを、Ｔｙｒはチロシンを、２ＰＥは２－フェニルエタノールを、４ＨＰＬＡは４－ヒドロキシフェニル乳酸、Ｇｌｃはグルコースを表す。 [Strain culture and product determination]
The strain was precultured overnight at 27 ° C. using 5 ml of LB liquid medium or LB liquid medium containing 0.8% glucose. 50 μl of the preculture liquid was added to M9M2 liquid medium to inoculate, and shake culture was performed at 37 ° C. until the value of OD 660 became about 0.3. M9M2 liquid medium is prepared by adding 10 g of glucose, 6 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 0.5 g of sodium chloride and 4 g of ammonium chloride to 900 ml of distilled water, and further adding 1 M magnesium chloride 1 ml, 1 ml of 10 mg / ml thiamine hydrochloride, 0.5 ml of 100 mM iron sulfate (divalent), 100 μl of 1 M calcium chloride, 10 μl of a trace metal salt solution, and the volume up to 1 L is 0.2 μm filter The filter-sterilized one was used. Trace metal salt solution of 0.371g distilled water 100ml (NH 4) 6 Mo 7 O 24 · 4H 2 O, H 3 BO 3 of 2.473g, CoCl of 0.714g 2 · 6H 2 O, 0 . It contains 239 g of CuSO 4 .5H 2 O, 1.583 g of MnCl 2 .4H 2 O, 0.288 g of ZnSO 4 .7H 2 O. In the case of culturing the 2PE1 strain and the 2PE2 strain, phenylalanine was added to the M9M2 medium so as to be 2 mM. Isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and shake culture was performed at 27 ° C. for 44 hours. The culture solution was mixed with 950 μl of distilled water, 200 μl of methanol and 50 μl of culture solution, centrifuged at 10,000 rpm for 5 minutes, and the supernatant was filtered using a 0.45 μm filter and used as an HPLC sample. HPLC is carried out as described in Non-patent document 3 (Koma et al. Appl. Environ. Microbiol. 78: 6203-6216, 2012), and phenylalanine, tyrosine, 2-phenylethanol, or The amount of 4-hydroxyphenyl lactic acid produced was quantified.
The results are shown in Tables 4-7. In these tables, Phe represents phenylalanine, Tyr represents tyrosine, 2PE represents 2-phenylethanol, 4HPLA represents 4-hydroxyphenyllactic acid, and Glc represents glucose.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示すとおり、Ｐｈｅ１１株～Ｐｈｅ１７株、Ｐｈｅ１９株、Ｐｈｅ２２株、及びＰｈｅ２３株は、良好なフェニルアラニンの生成量を示した。また、これらの株は、前培養においてグルコースを添加していなくても良好なフェニルアラニンの生成量を示した。
　Ｐｈｅ１３株とＰｈｅ２４株との比較から、ｔｙｒＢの過剰発現は、フェニルアラニン産生にネガティブな影響があることが認められた。
　また、Ｐｈｅ１１株～Ｐｈｅ１７株、Ｐｈｅ１９株、Ｐｈｅ２２株、及びＰｈｅ２３株の結果から、良好なフェニルアラニンの産生には、少なくともａｒｏＡ、ａｒｏＢ、ａｒｏＣ、ａｒｏＧfbr、及びｐｈｅＡfbrの過剰発現が必要であると認められる。
　Ｐｈｅ１１株～Ｐｈｅ１３株の比較から、ｐｐｓＡの過剰発現はフェニルアラニン産生に好ましく、ｔｋｔＡの過剰発現も好ましいが、ｔｋｔＡは誘導が弱まる変異の入ったＴ７プロモーターによって発現されることがより好ましいことが認められる。
　Ｐｈｅ１３株～Ｐｈｅ１７株、Ｐｈｅ１９株、Ｐｈｅ２２株、及びＰｈｅ２３株の結果から、ａｒｏＤ，ａｒｏＥ及びａｒｏＬは、これらのうち少なくとも１つが過剰発現することが好ましいと認められた。
　表４に示すように、ａｒｏＧfbr及びｐｈｅＡfbrを過剰発現し、かつ、ａｒｏＡ、ａｒｏＢ、及びａｒｏＣの３つの遺伝子の少なくとも１つを過剰発現していないフェニルアラニン生産株では、導入した遺伝子の過剰発現による代謝負荷がかかっており、構築されたフェニルアラニンの合成経路が円滑に機能していないと考えられる。したがって、このような菌株では前培養にグルコースを添加してカタボライトリプレッションを引き起こすことにより、基底状態での遺伝子発現レベルを低下させる必要がある（例えば、Ｐｈｅ１、２、５、８、９、１０、１８、２０及び２１株）。一方、ａｒｏＧfbr及びｐｈｅＡfbrに加えてａｒｏＡ、ａｒｏＢ、及びａｒｏＣの３つの遺伝子を過剰発現している菌株では、構築されたフェニルアラニンの合成経路が円滑に機能していると考えられる。そのため、前培養でのグルコース添加の有無にかかわらず、フェニルアラニンの高生産が達成できる（例えば、Ｐｈｅ１１～１７、１９、２２、及び２３株）。 As shown in Table 4, strains Phe11 to Phe17, Phe19, Phe22 and Phe23 showed good production of phenylalanine. In addition, these strains showed good phenylalanine production even without addition of glucose in the preculture.
From the comparison between Phe13 strain and Phe24 strain, it was recognized that overexpression of tyrB had a negative influence on phenylalanine production.
Also, according to the results of strains Phe11 to Phe17, Phe19, Phe22 and Phe23, it is considered that at least aroA, aroB, aroC, aroG fbr , and pheA fbr overexpression is necessary for good phenylalanine production. Is recognized.
From the comparison of Phe11 strain to Phe13 strain, it is recognized that overexpression of ppsA is preferable for phenylalanine production and overexpression of tktA is also preferable, but tktA is more preferably expressed by the T7 promoter containing a mutation that weakens induction .
From the results of the strains Phe13 to Phe17, Phe19, Phe22 and Phe23, it was found preferable that at least one of these aroD, aroE and aroL be overexpressed.
As shown in Table 4, in the phenylalanine producing strain which overexpresses aroG fbr and pheA fbr and does not overexpress at least one of the aroA, aroB and aroC three genes, the overexpression of the introduced gene It is thought that the phenylalanine synthesis pathway constructed is not functioning smoothly. Therefore, in such a strain, it is necessary to reduce the level of gene expression in the ground state by adding glucose to the preculture to cause catabolite repression (eg, Phe 1, 2, 5, 8, 9, 10) , 18, 20 and 21 stocks). On the other hand, in the strain overexpressing three genes of aroA, aroB and aroC in addition to aroG fbr and pheA fbr , it is considered that the synthetic pathway of phenylalanine constructed functions smoothly. Therefore, high production of phenylalanine can be achieved regardless of the addition of glucose in the preculture (eg, strains Phe11 to 17, 19, 22, and 23).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表５の結果から、チロシン産生に関しても表４のフェニルアラニン産生と同様の傾向があると認められた。 From the results of Table 5, it was recognized that tyrosine production also had the same tendency as phenylalanine production of Table 4.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表６及び表７の結果から、Ｐｈｅ１１株～Ｐｈｅ１７株、Ｐｈｅ１９株、Ｐｈｅ２２株、及びＰｈｅ２３株やＴｙｒ１３株に、さらに代謝酵素をコードする遺伝子を導入することで、その他の芳香族化合物の高生産が可能になることが認められた。 From the results in Table 6 and Table 7, high production of other aromatic compounds is achieved by introducing genes encoding metabolic enzymes into strains Phe11 to Phe17, Phe19, Phe22, Phe23 and Tyr13. Was recognized to be possible.
　ＮＩＴＥ　ＢＰ－０２５１３
　ＮＩＴＥ　ＢＰ－０２５１４ NITE BP-02513
NITE BP-02514

Claims (10)

  1. 　形質転換されたエシェリキア（Escherichia）属に属する微生物であって、
    　少なくとも下記５つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されている微生物。
    （１）ａｒｏＡ
    （２）ａｒｏＢ
    （３）ａｒｏＣ
    （４）ａｒｏＧfbr又はａｒｏＦfbr
    （５）ｐｈｅＡfbr又はｔｙｒＡfbr     A microorganism belonging to the transformed Escherichia genus,
    A microorganism in which at least the following five genes are introduced together with an expression inducible promoter on a chromosome.
    (1) aroA
    (2) aroB
    (3) aroC
    (4) aroG fbr or aroF fbr
    (5) pheA fbr or tyrA fbr
  2. 　さらに、下記２つの遺伝子が染色体上に発現誘導可能なプロモーターとともに導入されている、請求項１に記載の微生物。
    （６）ｐｐｓＡ
    （７）ｔｋｔＡ     Furthermore, the microorganism according to claim 1, wherein the following two genes are introduced on a chromosome together with an expression-inducible promoter.
    (6) ppsA
    (7) tktA
  3. 　さらに、下記３つの遺伝子の少なくとも１つが染色体上に発現誘導可能なプロモーターとともに導入されている、請求項１又は２に記載の微生物。
    （８）ａｒｏＤ
    （９）ａｒｏＥ又はｙｄｉＢ
    （１０）ａｒｏＬ又はａｒｏＫ     Furthermore, the microorganism according to claim 1 or 2, wherein at least one of the following three genes is introduced together with an expression inducible promoter on a chromosome.
    (8) aroD
    (9) aroE or ydiB
    (10) aroL or aroK
  4. 　染色体上に発現誘導可能なプロモーターとともに導入される遺伝子にｔｙｒＢが含まれない、請求項１から３のいずれかに記載の微生物。 The microorganism according to any one of claims 1 to 3, wherein the gene introduced together with an expression inducible promoter on a chromosome does not contain tyrB.
  5. 　前記（５）の遺伝子が、ｐｈｅＡfbrであり、フェニルアラニン生産能を有する、請求項１から４のいずれかに記載の微生物。 The microorganism according to any one of claims 1 to 4, wherein the gene of (5) is pheA fbr and has phenylalanine producing ability.
  6. 　前記（５）の遺伝子が、ｔｙｒＡfbrであり、チロシン生産能を有する、請求項１から４のいずれかに記載の微生物。 The microorganism according to any one of claims 1 to 4, wherein the gene of (5) is tyrA fbr and has tyrosine production ability.
  7. 　さらに、フェニルピルビン酸、４－ヒドロキシフェニルピルビン酸、フェニルアラニン、又はチロシンを基質とする酵素をコードする同種又は異種起源の遺伝子が染色体上に発現誘導可能なプロモーターとともに導入された、請求項１から４のいずれかに記載の微生物。 Furthermore, a gene of the same or different origin encoding an enzyme that uses phenylpyruvate, 4-hydroxyphenylpyruvate, phenylalanine or tyrosine as a substrate is introduced together with an expression inducible promoter on a chromosome. The microorganism according to any one of the above.
  8. 　さらに、フェニルピルビン酸デカルボキシラーゼをコードする異種起源の遺伝子ｉｐｄＣが染色体上に発現誘導可能なプロモーターとともに導入され、２－フェニルエタノール又は２－（４－ヒドロキシフェニル）エタノールの生産能を有する、請求項１から４のいずれかに記載の微生物。 Furthermore, a heterologous gene ipdC encoding phenylpyruvate decarboxylase is introduced on a chromosome together with an expression-inducible promoter, and has an ability to produce 2-phenylethanol or 2- (4-hydroxyphenyl) ethanol. The microorganism according to any one of 1 to 4.
  9. 　さらに、乳酸デヒドロゲナーゼをコードする異種起源の遺伝子ｌｄｈＡが染色体上に発現誘導可能なプロモーターとともに導入され、フェニル乳酸又は４－ヒドロキシフェニル乳酸の生産能を有する、請求項１から４のいずれかに記載の微生物。 Furthermore, the heterologous gene ldhA encoding lactate dehydrogenase is introduced on a chromosome together with an expression-inducible promoter, and has the ability to produce phenyllactic acid or 4-hydroxyphenyllactic acid according to any one of claims 1 to 4. Microorganisms.
  10. 　請求項１から９のいずれかに記載の微生物を培地で培養することを含む、芳香族化合物の製造方法。 A method for producing an aromatic compound, comprising culturing the microorganism according to any one of claims 1 to 9 in a culture medium.
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