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Pichia pastoris mutant strain and application thereof in penicillin acylase production

A penicillin acylase and Pichia pastoris technology, which is applied in the fields of genetic engineering and microbial transformation, can solve the problems of low enzyme production and limited types of microbial penicillin acylase enzyme preparations, so as to reduce production costs and promote popularization and application. Effect

Active Publication Date: 2020-02-28
QINGDAO VLAND BIOTECH GRP
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AI Technical Summary

Problems solved by technology

Compared with foreign countries, the research and development of penicillin acylase in my country started late, and the types of industrialized microbial penicillin acylase enzyme preparations are limited. The use of high-end genetic engineering technology to improve the characteristics of low enzyme production in wild bacteria and the development of new varieties of penicillin acylase still need further research to broaden its application fields and meet the industrial synthesis of β-lactam antibiotics. need

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Synthesis and amplification of penicillin acylase gene

[0015] According to the gene sequence of penicillin acylase AsPGA published by NCBI (GeneBank AY919310), add 8 bases CG before its start codon ATG GAATTC CG (underlined as EcoR I restriction site), add 16 bases ATTT after its stop codon TGA GCGGCCGC TTTA (underlined as not I restriction site), and then this sequence was codon-optimized according to the codon preference of Pichia pastoris, and the optimized gene sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0016] restriction endonuclease EcoR I and not I (Fermentas) digests the penicillin acylase gene; EcoR I and not I digested plasmid pPIC9k. The digested product was purified using a gel purification kit, and the above two digested products were ligated with T4 DNA ligase (Fermentas). The ligation product was transformed into DH5α E. coli and selected with ampicillin. Pick transformants for sequencing verificat...

Embodiment 2

[0018] Example 2 Construction of Pichia pastoris engineering strain

[0019] 1. Transformation and screening

[0020] The recombinant yeast expression plasmid pPIC9k-AsPGA was linearized with Sal I, and the linearized product was purified with a column purification kit, then transformed into Pichia pastoris GS115 by electroporation, and coated on an MD plate. The colony grown on the MD plate is the engineering strain of Pichia pastoris, and then multi-copy transformants are screened on the YPD plate containing different concentrations of geneticin G418.

[0021] 2. Shake flask fermentation verification

[0022] Pick multiple copies of transformants and inoculate them into BMGY medium respectively, shake and culture at 30°C, 220rpm for 24 hours, then transfer to BMMY medium, shake at 30°C, 220rpm, add 0.5% methanol every 24 hours. After 4 days of induced expression, the bacteria were removed by centrifugation, and the supernatant was subjected to SDS-PAGE protein electrophore...

Embodiment 3

[0030] Example 3 Mutagenesis Screening

[0031] The mutations caused by ultraviolet mutagenesis are very random, and the effects of mutations are also random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to carry out multiple rounds of ultraviolet mutagenesis, the workload of screening is relatively large, and there is a possibility that effective positive mutations cannot be obtained. However, because ultraviolet mutagenesis requires simple equipment, low cost, and a large number of mutants can be obtained in a short period of time, it is still a commonly used method of mutagenesis selection.

[0032]The applicant used Pichia pastoris GSpga as the starting strain, and carried out genetic modification on it by means of ultraviolet mutagenesis to further increase the production of its penicillin acylase.

[0033] Inoculate Pichia pastoris GSpga on a YPD plate, culture at 30°C for 2-3 days, wash the bacteria with ...

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Abstract

The invention belongs to the technical field of genetic engineering and microbial engineering transformation, and provides a Pichia pastoris mutant strain with high yield of penicillin acylase and anapplication of the strain. The mutant strain is obtained through ultraviolet mutagenesis screening, with the preservation number being CCTCC NO. M2019924. The strain can be widely applied to the production of the penicillin acylase, thereby being beneficial to reducing the production cost of the penicillin acylase and promoting the popularization and application of the penicillin acylase in the industrial synthesis of beta-lactam antibiotics.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial transformation, and specifically relates to Pichia pastoris with stable and high-yield penicillin acylase and its application in the production of penicillin acylase. Background technique [0002] β-lactam antibiotics mainly include antibiotics such as penicillins and cephalosporins, which are currently the main antibiotics used at home and abroad. This type of antibiotics occupies an important share in the pharmaceutical industry. Compared with developed countries, domestic antibiotic pharmaceutical companies have a large gap in innovation capabilities in high-end varieties and cutting-edge technologies, especially enzymatic ampicillin, amoxicillin, and cephalosporins. It is necessary to accelerate the industrialization of technological innovation. The currently used chemical synthesis method has high production costs, harsh reaction conditions, low temperature reaction...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N13/00C12N9/84C12R1/84
CPCC12N15/815C12N13/00C12N9/84C12Y305/01011
Inventor 宋清清程斯达田延军康丽华李宾黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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