A kind of fidaxomicin genetically engineered bacteria and its construction method and application

A technology of genetically engineered bacteria and fidaxomicin, applied in the field of high-yielding fidaxomicin genetically engineered bacteria Actinomycetes Deccanum YP-2 and its construction, to achieve the advantages of convenient operation, simplified process and reduced production cost Effect

Active Publication Date: 2021-03-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Fidaxomicin, also known as Tiacumicin (also known as Lipiarmycin A3, OPT-80, PAR-101), is a new type of macrolide antibiotic that can be administered orally. The original research factory Optimer The company was approved by the FDA for marketing in May 2011, but no such drug has been approved by the CFDA for marketing in China so far

Method used

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  • A kind of fidaxomicin genetically engineered bacteria and its construction method and application
  • A kind of fidaxomicin genetically engineered bacteria and its construction method and application
  • A kind of fidaxomicin genetically engineered bacteria and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of a genetically engineered bacterium that produces high-yield fidaxomicin Actinoplanes deccanensis YP-2 (Actinoplanes deccanensis YP-2)

[0039] 1. Construction of pathway-specific gene expression vector pIJ8630-ermE*-fadR1

[0040] The name of the expression vector of the positive regulatory gene of fidaxomicin biosynthesis constructed in this example is pIJ8630-ermE*-fadR1, which contains the positive regulatory gene fadR1 of fidaxomicin biosynthesis. The positive regulatory gene of fidaxomicin biosynthesis The sequence of fadR1 is shown in SEQ ID NO.1. SEQ ID NO.1 consists of 3063 nucleotides, the 1st-19th is the NdeI recognition site and the protective base, the 20th-297th is the erythromycin resistance gene promoter, and the 298th-3042th is Feida The coding sequence of the positive regulation gene of biosynthesis of Fidaxomycin, the 3043-3063 position is the NdeI recognition site and the protection base, and SEQ ID NO.2 is the amino acid se...

Embodiment 2

[0049] (3) Actinoplanes deccanensis YP-2 (Actinoplanes deccanensis YP-2) provided by the present invention has been preserved in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee, and its classification name is Actinoplanes deccanensis YP-2, deposit number: CGMCC No.15743, deposit date: May 8, 2018, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Example 2 Fermentation verification of Fidaxomycin synthesized by starting bacteria and genetically engineered strains.

[0050] (1) Cultivate the starting bacteria Actinoplanes deccanensis YP-1 and the genetically engineered strain Actinoplanes deccanensis YP-2 (Actinoplanes deccanensis YP-2) on ISP4 solid medium 10 days.

[0051] (2) On the ISP4 solid medium cultured by Actinomycetes YP-1 and Actinomyces YP-2, take 1cm×1cm pieces of bacteria and inoculate them into the seed medium medium, cultivated at 30°C for 16 hours, with a rotation speed...

Embodiment 3

[0052] Example 3 Genetic engineering strains and starting bacterium biomass, fidaxomicin output comparative verification

[0053] (1) Bacterial biomass: Take 1ml of the bacterial liquid fermented by fermentation medium B, collect the bacterial cells after centrifugation, wash with 1ml of sterile water and centrifuge again to collect the bacterial cells, dry at 50°C for 3 days, and then weigh them. figure 2 It is the biomass curve of Actinomycetes YP-1 and Actinomycetes YP-2.

[0054] (2) Fidaxomycin standard curve: the Fidaxomycin standard substance was prepared into a solution with a certain concentration gradient with methanol, and determined 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), the mobile phase B phase is 90% aceton...

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Abstract

The invention discloses a high-yield fidaxomicin genetically engineered bacterium Actinomycetes deccanensis YP‑2 (Actinoplanes deccanensis YP‑2) and a construction method. Among the Actinomycetes Deccanum YP‑1, the genetically engineered Actinomycetes YP‑2 with a high yield of fidaxomicin was screened out, and the yield was 400% to 500% higher than that of the original strain, up to 130mg / L, has a good application prospect. The method of the invention is efficient, accurate and easy to operate, and provides a new research method for the construction of the efficient biosynthesis pathway of the actinomycete drug. The fidaxomicin genetically engineered bacterium Actinomyces deccanum YP‑2 can be used in the preparation of medicines for treating diarrhea caused by Clostridium difficile infection.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and relates to a high-yield fidaxomicin genetic engineering bacterium Deccan Plateau Actinomycetes YP-2 (Actinoplanes deccanensis YP-2) and its construction method and application. Background technique [0002] The extensive use of antibiotics in clinical practice has led to drug resistance of pathogenic bacteria. What is more serious is that bacteria can effectively spread drug resistance gene information. The proportion of pathogenic bacteria resistant to various types of antibiotics is increasing, which has a great negative effect on clinical treatment. influences. Clostridium difficile infection (CDI) is caused by an overgrowth of a Gram-positive bacillus, Clostridium difficile, that releases toxins that can lead to colon inflammation, severe diarrhea, and even death. Previous data have shown that CDI mainly affects the elderly, patients in long-term hospitalization and intensive care units...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/67C12P19/62C12R1/045
CPCC12N15/67C12N15/74C12P19/62
Inventor 李永泉毛旭明郦月萍俞品
Owner ZHEJIANG UNIV
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