WO2019223433A1 - Bactérie de fidaxomicine génétiquement modifiée et procédé de construction et application correspondants - Google Patents

Bactérie de fidaxomicine génétiquement modifiée et procédé de construction et application correspondants Download PDF

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Publication number
WO2019223433A1
WO2019223433A1 PCT/CN2019/081486 CN2019081486W WO2019223433A1 WO 2019223433 A1 WO2019223433 A1 WO 2019223433A1 CN 2019081486 W CN2019081486 W CN 2019081486W WO 2019223433 A1 WO2019223433 A1 WO 2019223433A1
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genetically engineered
fidaxomycin
plateau
seq
actinoplanes
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PCT/CN2019/081486
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English (en)
Chinese (zh)
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李永泉
毛旭明
俞品
郦月萍
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浙江大学
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • the invention belongs to the field of microbial pharmaceuticals, and relates to a high-yield fidamicin genetically engineered bacterium Degan Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) and a method for constructing the same and application thereof.
  • CDI Clostridium difficile infection
  • Vancomycin and metronidazole are the first choice drugs for the treatment of CDI, but with the emergence of drug-resistant strains, finding more effective treatments has become the main research target in recent years.
  • Fidaxomicin also known as Tiacumicin (also known as Lipiramycin A3, OPT-80, PAR-101), is a new macrolide antibiotic that can be administered orally.
  • Original research plant Optimer The company was approved by the FDA in May 2011, but so far no drug has been approved by the CFDA in China.
  • Clinical studies have shown that compared with vancomycin orally, fidaxomycin is more effective than vancomycin, and has a strong inhibitory effect on C. difficile after administration, and can effectively reduce the recurrence rate of C. difficile infection.
  • the object of the present invention is to provide a non-dacmycin engineering bacteria Degan Plateau zooplankton actinomycete YP-2, which is classified and named: Actinoplanes deccanensis YP-2, and has been deposited in the General Microbial Center of the China Microbial Species Collection Management Committee ( (CGMCC for short), deposit number: CGMCC No. 15743, deposit date: May 8, 2018, deposit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • CGMCC General Microbial Center of the China Microbial Species Collection Management Committee
  • Another object of the present invention is to provide a method for constructing the actinomycete engineering bacteria YP-2 (Actinoplanes deccanensis YP-2), which includes the introduction of a gene for positive control of fidaxomycin biosynthesis.
  • the starting strain was screened to obtain a genetically engineered strain (Actinoplanes deccanensis YP-2) of Degan plateau zooplankton with high yield of fidaxomycin.
  • Upstream primer SEQ ID No. 3 aagatctgcccagcatatgaactgttgaaagttgttta
  • Downstream primer SEQ ID NO. 4 ccaagatctgcccagcatatgtcaggcggaatccgccatg;
  • step 2 The fragment recovered in step 1 is digested and inserted into the expression vector pIJ8630 to obtain the plasmid pIJ8630-ermE * -fadR1 and verified;
  • step 2 The plasmid obtained in step 2 is introduced into E. coli ET12567 / pUZ8002 through transformation;
  • steps 2-4 the sequence of SEQ ID NO. 1 is inserted into the plasmid pIJ8630 and introduced into the starting fungus Degan Plateau Actinomyces YP-1.
  • SEQ ID NO.1 in step 1 consists of 3063 nucleotides, positions 1-19 are NdeI recognition sites and protecting bases, positions 20-297 are erythromycin resistance gene promoters, and 297th -3042 is the coding sequence of the gene for positive regulation of fidaxomycin biosynthesis, positions 3043-3063 are the recognition site of NdeI and the protective base, and SEQ ID NO. 2 is the amino acid sequence of the gene for positive regulation of fidaxomycin biosynthesis.
  • step 2 inserts the SEQ ID NO.1 sequence fragment into NdI enzyme digestion and inserts it into the expression vector pIJ8630, the promoter is a erythromycin resistance gene-containing promoter;
  • step 3-4 uses the expression vector Escherichia coli was subjected to parental conjugation to obtain the high-yield fidaxomycin genetically engineered strain Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2).
  • the starting bacteria described in step 4 are Actinoplanes deccanensis YP-1, and the E. coli described in step 3 is E.coil ET12567 (pUZ8002).
  • the yield of the genetically engineered strain of non-dacomycin obtained from Degan Plateau Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) was 400% to 500% higher than the starting bacteria and up to 130mg / L. .
  • Yet another object of the present invention is to provide the application of the fidaxomycin genetically engineered bacteria Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) in the preparation of a medicament for treating diarrhea caused by Clostridium difficile infection.
  • the present inventor purchased a strain of zooxactinomycetes that produces fidaxomycin from the Common Microbial Center of the China Microbial Species Collection Management Committee (CGMCC for short), but this strain has low fidamycin production and is not suitable for industrial applications .
  • CGMCC Common Microbial Center of the China Microbial Species Collection Management Committee
  • the high-yield Fidaxomycin genetically engineered strain Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) can produce a large amount of Fidaxomycin, a drug used to treat diarrhea caused by Clostridium difficile, through fermentation
  • the yield is increased by 400% to 500% compared to the starting bacteria, up to 130mg / L, which has a good application prospect and can promote the industrial development of fidaxomycin.
  • the present invention obtains high-yield fidamycin genetically engineered bacteria Degan Plateau Actinomycete YP-2 (Actinoplanes deccanensis YP-2) by over-expressing fidaxomycin biosynthesis positive regulatory genes in vivo, without the need for Perform in vitro expression, thereby overcoming the problems of difficult expression of some proteins in vitro, inactivity or low activity after expression, and fast and convenient operation; no need to prepare substrates for in vitro reactions, avoiding the difficulty of preparing or purchasing some substrates; The conditions of in vitro reactions need to be explored to simplify the process.
  • the high-yield non-dacmycin genetically engineered strain Depth plateau zooplankton actinomycete YP-2 (Actinoplanes deccanensis YP-2) produced by the present invention has an increase in yield of 400% to 500%, and up to 130mg / L compared with the starting bacteria. It greatly reduces the production cost of fidaxomycin and provides technical support for industrial production to increase the yield of fidaxomycin.
  • the high-yield fidaxomycin genetic engineering strain Degan Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) is obtained, and the method is highly efficient It is accurate, convenient and easy to operate, and provides a new research method for the construction of an efficient biosynthetic pathway for actinomycetes.
  • the genetically engineered strain of Fidaxomycin Degan Plateau Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) can be used in the preparation of medicines for treating diarrhea caused by Clostridium difficile infection.
  • Figure 1 is a diagram of the construction of a high expression plasmid.
  • Fig. 2 is the biomass curve of the Dekan Plateau Actinomyces YP-1 and the Dekan Plateau Actinomyces YP-2 during fermentation.
  • Figure 3 shows the yield of fidaxomycin during the fermentation of Dekang Plateau S. actinomycetes YP-1 and Dekang Plateau S. actinomycetes YP-2.
  • Figure 4 shows the growth phenotype of the actinomycete YP-2 on the Degan Plateau.
  • ISP4 solid medium soluble starch 1%, MgSO 4 .7H 2 O 0.1% K 2 HPO 4 0.1%, NaCl 0.1%, (NH 4 ) 2 SO 4 0.2%, CaCO 3 0.2%, FeSO 4 . 7H 2 O 0.0001%, MnCl 0.0001% agar 2%, pH 7.0.
  • Seed medium glucose 1.75%, peptone 1.5%, NaCl 1.0%, and the rest is water. The percentages are all mass percentages, and the pH is natural.
  • Fermentation medium B yeast extract 0.3%, peptone 0.5%, malt extract 0.3%, glucose 1%, and the rest is water. The percentages are all mass percentages and the pH is natural.
  • MS solid medium 2% mannitol, 2% soybean powder, 2% agarose, and the rest is water. The percentages are all mass percentages, 10 mM MgCl 2 , and the pH is natural.
  • Example 1 Construction of a genetically engineered bacterium with high yield of Fidaxomycin Actinoplanes Deccanensis YP-2 (Actinoplanes deccanensis YP-2)
  • the expression vector for the positive regulation gene of fidaxomycin biosynthesis constructed in this embodiment is named pIJ8630-ermE * -fadR1, and this vector contains the positive regulation gene fadR1 of fidaxomycin biosynthesis.
  • the positive regulation gene of fidaxomycin biosynthesis The sequence of fadR1 is shown in SEQ ID NO.1.
  • SEQ ID NO.1 consists of 3063 nucleotides, positions 1-19 are NdeI recognition sites and protective bases, positions 20-297 are erythromycin resistance gene promoters, and fate 297-3042 are The coding sequence of the gene for positive control of biomycin biosynthesis. Positions 3043-3063 are NdeI recognition sites and protective bases, and SEQ ID NO. 2 is the amino acid sequence of the gene for positive control of fidaxomycin biosynthesis.
  • Upstream primer SEQ ID No. 3 aagatctgcccagcatatgaactgttgaaagttgttta
  • Downstream primer SEQ ID No. 4 ccaagatctgcccagcatatgtcaggcggaatccgccatg.
  • step (1) The SEQ ID No.1 fragment with NdeI digestion sites at both ends recovered in step (1) was digested with NdeI and the expression vector pIJ8630, which was cut by the same enzyme, was ligated to obtain the recombinant expression vector pIJ8630- ermE * -fadR1 ( Figure 1).
  • Actinoplanes deccanensis YP-1 2.Introduce the pIJ8630-ermE * -fadR1 expression vector into the starting bacteria Actinoplanes deccanensis YP-1 to obtain genetically engineered bacteria with high yields of fidaxomycin. (Actinoplanes deccanensis YP-2), the specific method is as follows.
  • the pIJ8630-ermE * -fadR1 vector was transformed into the demethylated starting E. coli ET1256 / (pUZ8002 by heat shock method to obtain recombinant E.coil ET12567 / pUZ8002 / pIJ8630-ermE * -fadR1 .
  • Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) provided by the present invention has been deposited in the General Microbiology Center of the China Microbial Species Collection Management Committee (CGMCC), and is named and named: Actinoplanes deccanensis YP-2, deposit number: CGMCC No. 15743, deposit date: May 8, 2018, deposit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. [0034] Example 2 Synthesis of Fidaxomycin fermentation by starting bacteria and genetically engineered strains.
  • CGMCC General Microbiology Center of the China Microbial Species Collection Management Committee
  • Bacterial biomass Take 1ml of bacterial solution fermented by fermentation medium B, collect the bacterial cells after centrifugation, wash with 1ml of sterile water, and then collect the bacterial cells after centrifugation. Dry at 50 ° C for 3 days and weigh. 2 is the biomass curve of zooplankton YP-1 and zooplankton YP-2.
  • Fidaxomycin standard curve Fidaxomycin standard was prepared with methanol to a certain concentration gradient solution and measured by HPLC.
  • HPLC conditions Chromatographic column: C18 column (Aglient, Eclipse Plus XDB, 5um, 4.6mm * 250mm); detection wavelength: 254nm; flow rate: 1.00mL / min; injection volume: 10ul; experimental mobile phase: mobile phase A phase is 10% acetonitrile, containing 0.08% trifluoroacetic acid (TFA), mobile phase B phase is 90% acetonitrile; HPLC sampling procedure: 0-20min, 30% -100% B phase; 20-25min, 100% B phase; 25- 30min, 100% -30% B phase.
  • TFA trifluoroacetic acid
  • the strain Deinoplankton actinomycete YP-2 was cultured in ISP4 agar medium for 10 days, and the basal hyphae and aerial hyphae developed well (Figure 4). Apparent characteristics in various different media, the results are shown in Table 1; physiological and biochemical characteristics of YP-2 in Degan Plateau, see Table 2.

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Abstract

L'invention concerne une bactérie de fidaxomicine génétiquement modifiée et un procédé de construction et une application. La bactérie génétiquement modifiée est Actinoplanes deccanensis YP-2, possédant un numéro d'accès CGMCC n° : 15743. Le procédé de construction associé consiste à : introduire un gène de régulation positive de la biosynthèse de la fidaxomicine dans les Actinoplanes deccanensis YP-1 et cribler pour obtenir une bactérie génétiquement modifiée d'Actinoplanes deccanensis YP-2 qui possède un rendement élevé en fidaxomicine et augmenter le rendement de la bactérie de départ de 400 % à 500 % pour atteindre 130 mg/l. L'invention concerne en outre l'application de la bactérie de fidaxomicine génétiquement modifiée d'Actinoplanes deccanensis YP-2 pour la préparation d'un médicament destiné au traitement de la diarrhée provoquée par une infection par Clostridium difficile.
PCT/CN2019/081486 2018-05-24 2019-04-04 Bactérie de fidaxomicine génétiquement modifiée et procédé de construction et application correspondants WO2019223433A1 (fr)

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CN108841769B (zh) * 2018-05-24 2021-03-02 浙江大学 一种非达霉素基因工程菌及构建方法和应用
CN112430555B (zh) * 2020-12-11 2022-06-24 浙江大学 一种放线菌底盘菌株及其应用

Citations (4)

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Title
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LV , WEIXUN: "Breeding of Fidaxomicin Production Strain and Optimization of Fermentation Conditions", SCIENCE -ENGINEERING (A), CHINA MASTER S THESES FULL-TEXT DATABASE, 15 January 2018 (2018-01-15), ISSN: 1674-0246 *

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