WO2019030808A1 - Micro-organisme capable de produire un composé aromatique - Google Patents

Micro-organisme capable de produire un composé aromatique 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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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/fr
Priority to JP2019535461A priority patent/JP7034496B2/ja
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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.

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Abstract

L'invention concerne un micro-organisme qui est exempt de plasmide et qui présente une productivité améliorée. Dans un aspect, la présente invention concerne un micro-organisme transgénique appartenant au genre Escherichia, dans lequel au moins cinq gènes présentés ci-dessous sont introduits sur le chromosome correspondant conjointement avec des promoteurs respectivement capables d'induire l'expression des gènes : (1) aroA, (2) aroB, (3) aroC, (4) aroGfbr ou aroFfbr et (5) pheAfbr ou tyrAfbr. Dans un autre aspect, la présente invention concerne un procédé de production d'un composé aromatique, comprenant la culture du micro-organisme dans un milieu de culture.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08504594A (ja) * 1992-12-21 1996-05-21 パーデュー・リサーチ・ファウンデーション 芳香族アミノ酸合成における共通経路の阻害除去
JPH09121872A (ja) * 1995-08-30 1997-05-13 Ajinomoto Co Inc Ｌ−アミノ酸の製造方法
WO2007088977A1 (fr) * 2006-02-02 2007-08-09 Ajinomoto Co., Inc. Procede destine a la production d'un acide amine l
KR20170055438A (ko) * 2015-11-11 2017-05-19 포항공과대학교 산학협력단 타이로신 고생산 균주 및 이를 이용한 타이로신 생산방법

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US10030112B2 (en) * 2014-05-07 2018-07-24 Solvay Sa Hybrid fluoropolymer composites

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08504594A (ja) * 1992-12-21 1996-05-21 パーデュー・リサーチ・ファウンデーション 芳香族アミノ酸合成における共通経路の阻害除去
JPH09121872A (ja) * 1995-08-30 1997-05-13 Ajinomoto Co Inc Ｌ−アミノ酸の製造方法
WO2007088977A1 (fr) * 2006-02-02 2007-08-09 Ajinomoto Co., Inc. Procede destine a la production d'un acide amine l
KR20170055438A (ko) * 2015-11-11 2017-05-19 포항공과대학교 산학협력단 타이로신 고생산 균주 및 이를 이용한 타이로신 생산방법

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Title
KOMA, D. ET AL.: "Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 78, no. 17, 2012, pages 6203 - 6216, XP009508161, DOI: doi:10.1128/AEM.01148-12 *

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