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Fidaxomicin genetically engineered bacterium, construction method and application

A technology of genetically engineered bacteria and fidaxomicin, which is applied in the field of high-yielding fidaxomycin genetically engineered bacteria Actinomycetes Deccan plateau YP-2 and its construction, and achieves high efficiency, reduced production cost, and quick and convenient operation. Effect

Active Publication Date: 2018-11-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Fidaxomicin genetically engineered bacterium, construction method and application
  • Fidaxomicin genetically engineered bacterium, construction method and application
  • Fidaxomicin genetically engineered bacterium, construction method and application

Examples

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Effect test

Embodiment 1

[0036] Example 1 Construction of a genetically engineered bacterium that produces high-yield fidaxomicin Actinoplanes deccanensis YP-2 (Actinoplanes deccanensis YP-2)

[0037] 1. Construction of pathway-specific gene expression vector pIJ8630-ermE*-fadR1

[0038] 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 297th-3042 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 sequ...

Embodiment 2

[0048] Example 2 Fermentation verification of Fidaxomycin synthesized by starting bacteria and genetically engineered strains.

[0049] (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.

[0050] (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 of 200rpm; inoculated the mycelium in the seed medium into the fermentation medium B to OD 600 0.15, cultured at 30°C for 168 hours, after 12, 24, 48, 72, 96, and 120 hours of culture, samples were taken to measure the biomass of bacteria and the yield of fidaxomicin. Experiments were repeated three times.

Embodiment 3

[0051] Example 3 Genetic engineering strains and starting bacterium biomass, fidaxomicin output comparative verification

[0052] (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.

[0053] (2) Fidaxomycin standard curve: the Fidaxomicin 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), mobile phase B phase is 90% acetonitri...

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Abstract

The invention discloses a high-yield Fidaxomicin genetically engineered bacterium Actinoplanes deccanensis YP-2 and a construction method. A positive regulation gene biosynthesized by Fidaxomicin is guided into starting bacterium Actinoplanes deccanensis YP-1, the high-yield Fidaxomicin genetically engineered bacterium Actinoplanes deccanensis YP-2 is sieved and obtained, the yield is improved by400% to 500% compared with the starting bacterium and reaches up to 130 mg / L, and the Fidaxomicin genetically engineered bacterium has extremely high application prospects. The method is efficient, accurate and convenient in operation, and a new research means is provided for the construction of efficient biosynthetic pathway of an actinomycetes drug. The Fidaxomicin genetically engineered bacterium Actinoplanes deccanensis YP-2 can be applied in the preparation of drugs for curing diarrhea caused by the infection of clostridium difficile.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and relates to a high-yielding fidaxomicin genetically engineered bacterium Actinoplanes deccanensis 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 uni...

Claims

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Application Information

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Patent Type & Authority Applications(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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