Genetically engineered bacteria for producing echinocandin B as well as construction method and application thereof

A technology of genetically engineered bacteria and echinocandin, which is applied to the field of genetically engineered bacteria producing echinocandin B and its construction, can solve the problems of decreased echinocandin B yield and low echinocandin B yield, and the like, Achieve the effect of maintaining stable fermentation traits and increasing yield

Active Publication Date: 2019-04-09
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to produce a large number of strong carcinogens—Varitocin and echinocandin B in the existing transformation process in the existing fermentation process in the existing method for producing echinocandin B. The defect of low yield is to provide a genetically engine...

Method used

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  • Genetically engineered bacteria for producing echinocandin B as well as construction method and application thereof
  • Genetically engineered bacteria for producing echinocandin B as well as construction method and application thereof
  • Genetically engineered bacteria for producing echinocandin B as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning of Aspergillus versicolor synthesis-related polyketide synthase gene and left and right homology arms

[0039] First, the total genome of Aspergillus delacroxii NRRL 3860 (purchased from NRRL, the American Agricultural Research Culture Collection) was used as a template to clone the Aspergillus versicolor polyketide synthase gene and its polyketide synthase gene. For nearby genes, the high-fidelity polymerase KOD-FX (TOYOBO company) was used to clone the fragments. Upstream primer S1: 5'-cagaatatcaaacaccgcccc-3' (SEQ ID NO.4), downstream primer S2: 5'-tccgtcaccgatcgtcctag-3' (SEQ ID NO.5), for PCR, PCR conditions: 98°C for 30s, 55 ℃ 30s, 68 ℃ 8min, 30 cycles. The obtained fragment was sequenced, and the homology between the sequence (see SEQ ID NO.6) and the online sequence (Genbank Accession Number: U34740.1) was 92%.

[0040] Primers were designed according to the obtained sequence of the Aspergillus delacroxii NRRL 3860 polyketide synthase gene an...

Embodiment 2

[0043] Construction of Example 2 Knockout Plasmid and Agrobacterium-mediated Fungal Transformation

[0044] A total of 4 fragments of the left and right homology arms, the hygromycin resistance gene cassette, and the linearized plasmid backbone fragment obtained in Example 1 were obtained by using Novizan’s The MultiS One Step Cloning Kit performs homologous recombination connection (see the manual for the method), and obtains the knockout plasmid pDHt-sk-stcA-hyg-stcAB, transforms E. coli DH5a, picks the transformant and cultures it in LB, and extracts the plasmid for enzyme Cutting and PCR verification, finally constructed into a knockout plasmid pDHt-sk-stcA-hyg-stcAB (15090bp, see figure 1 ). For the flow chart of the above plasmid construction, see figure 2 .

[0045] Transform the plasmid pDHt-sk-stcA-hyg-stcAB into Agrobacterium tumefaciens AGL-1 (purchased from Beijing Huayueyang Biological Co., Ltd.) by chemical transformation. cells, mix well, and ice-bath for ...

Embodiment 3

[0047] Example 3 Screening of Genetically Engineered Bacteria for Knockout of Polyketide Synthase Related to Aspergillus versicolor Synthesis

[0048] Pick the transformant and carry out secondary culture on the slant medium (potato 200g, glucose 20g, agar 18g, water 1L, natural pH) containing hygromycin (75μg / ml). The homologous recombination method of the transformant is shown in the illustration image 3 . Pick transformants, extract total DNA, and use primer A: 5'-gattccggagcgatatagc-3' (SEQ ID NO.13) and primer B: 5'-gatcatcccagccatcaatg-3' (SEQ ID NO.14) from the genome of the starting strain Amplified band 2488bp ( image 3 ), and a 2102bp fragment can be amplified from the transformant genome ( image 3 ), thus verifying that the transformed transformants obtained by amplifying the 2102bp fragment are knockout genetically engineered strains ( image 3 ).

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Abstract

The invention discloses genetically engineered bacteria for producing echinocandin B as well as a construction method and application thereof. The genetically engineered bacteria are characterized inthat a polyketide synthase gene associated with the synthesis of sterigmatomycin in a genome of the genetically engineered bacteria is inactivated or is genetically modified so as not to express the sterigmatomycin. The genetically engineered bacteria for producing echinocandin B as well as the construction method and the application thereof disclosed by the invention have the beneficial effects that the mononuclear treatment is not required to be performed on a strain in the construction process of the genetically engineered bacteria, so that the problems such as single exchange occurring through homologous recombination, poor genetic stability and easy reversion mutation are solved; the genetically engineered bacteria does not produce the sterigmatomycin, and the yield of the echinocandin B can be improved by 44 percent compared with that of a wild strain; through multiple passages, the fermentation property still remains stable.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a genetic engineering bacterium producing echinocandin B and its construction method and application. Background technique [0002] Echinocandin B (ECB), as a natural cyclolipopeptide compound, has a strong antifungal effect. Anifungin obtained by modifying the side chain on its basis was approved by the FDA for the treatment of fungal infections in 2006. It has the advantages of low toxicity, high safety, and broad spectrum, and has a good market prospect. [0003] Echinocandin B can pass through Aspergillus delacroxii (Aspergillus deacroxii) NRRL3860 (=Asp. Voser W, Nyfeler R, Keller-Schierlein, W. Stoffwechselproduktevon Mikroorganismen 143. Mitteilung. Echinocandin B, ein neuartiges Polypeptid-Antibioticum aus Aspergillus nidulans var. echinulatus: Isolierung und Bausteine. Helv Chim. 1987: 7–4987; ;CIBA GEIGY AG.Antifungalantibiotic A32204 production by aerobic cult...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80C12P21/04C12R1/66
CPCC12N9/1029C12P21/02
Inventor 胡海峰闵涛玲
Owner SHANGHAI INST OF PHARMA IND
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