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Recombinant streptomyces of high-expression glucose isomerase

A technology of glucose isomerase and streptomyces, applied in the field of genetic engineering, can solve problems such as unreported and unreported strain genetic engineering

Active Publication Date: 2017-02-22
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the genome sequencing and glucose isomerase gene sequencing of Streptomyces olive chromogenicum have never been reported, nor has there been any report on the genetic engineering of the strain

Method used

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  • Recombinant streptomyces of high-expression glucose isomerase
  • Recombinant streptomyces of high-expression glucose isomerase

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Embodiment 1

[0033] The construction of embodiment 1 recombinant vector

[0034] The gene kasopSF14-AO was synthesized in Nanjing GenScript Biotechnology Co., Ltd., with the upstream and downstream restriction sites Xba I and Bam HI sequences respectively, and then inserted into the EcoR V site of pUC57.

[0035] The pUC57 vector inserted with the kasopSF14-AO gene was double digested with Xba I and Bam HI. At the same time, the pSET152 vector was digested with Xba I and Bam HI.

[0036] After the digested product was recovered, it was ligated with TAKALA DNA ligase I, transformed into Escherichia coli DH5a, and spread on LB plates containing 50ug / ml ampicillin resistance for resistance screening.

[0037] PCR identification was carried out on the obtained plate transformants, and E. coli positive transformants containing the recombinant vector pSET152-kasopSF14-AO were obtained. For the vector structure, see figure 1 .

Embodiment 2

[0038] Embodiment 2 Recombinant vector transforms Escherichia coli ET12567 (pUZ8002)

[0039] Plasmid pSET152-kasopSF14-AO was extracted from DH5a recombinant bacteria, passed through CaCl 2 The pSET152-kasopSF14-AO vector was chemically transformed into Escherichia coli ET12567 (pUZ8002).

[0040] PCR identification was performed on the obtained plate transformants, and a positive transformant of Escherichia coli ET12567 (pUZ8002) containing the recombinant vector pSET152-kasopSF14-AO was obtained.

Embodiment 3

[0041] Example 3 Escherichia coli ET12567 (pUZ8002) positive transformants and Streptomyces olive chromogenes are constructed by conjugative transformation of Streptomyces olive chromogenes

[0042]Inoculate recombinant Escherichia coli ET12567 (pUZ8002) in LB medium containing 50 ug / ml of kanamycin, 25 ug / ml of chloramphenicol, and 50 ug / ml of ampicillin. 37°C, 220rpm overnight. Then take 600ul overnight bacterial liquid in 30ml LB liquid medium containing the above three resistances, at 37°C, 220rpm for 5h. Collect the bacterial liquid, centrifuge at 5000rpm for 10min, remove the supernatant, collect the bacterial cells, resuspend twice with 1ml ice LB liquid medium, centrifuge at 5000rpm at 4°C for 3min, remove the supernatant, resuspend with 500ul ice LB again, put on ice on standby.

[0043] Streptomyces olive chromogenes were inoculated on the YMS plate and cultured at 30°C for 3 days. Add 9ml of sterile water to the plate, scrape the spores on the surface of the cult...

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Abstract

The invention, which belongs to the genetic engineering field, discloses a recombinant streptomyces of high-expression glucose isomerase. The provided streptomyces being named streptomyces olivochromogenes has been preserved in China General Microbiological Culture Collection Center on August 8th, 2016 and assigned the preservation number CGMCC NO. 12831. An AO gene is inserted into a streptomyces genome; and resistance screening and fermental cultivation are carried out to obtain a recombinant streptomyces. The AO gene after codon optimization is integrated into the genome by a phic31 integrase; and then resistance screening and genotype screening are carried out to obtain a recombinant streptomyces strain. Because of integration of the new gene, the expression of the glucose isomerase gene can be improved by 18.7%.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a strain of recombinant streptomyces highly expressing glucose isomerase and its application. Background technique [0002] Glucose Isomerase (GI), No.: EC 5.3.1.5 (Xylose Isomerase). Has the activity of isomerizing aldose to the corresponding ketose (such as converting glucose to fructose). This enzyme is widely used in industry to obtain fructose syrup product after isomerizing glucose into fructose with glucose syrup as substrate. [0003] The production strains of glucose isomerase are diverse. Novozymes reported that Bacillus coagulans and Streptomyces grinotis were used to produce glucose isomerase. constitutive enzymes. The strains that have been put into production in China are based on Streptomyces amylase No. 7 screened by He Jiaming and others from the Food Fermentation Industry Research and Design Institute of Shandong Province. Industrialization of Technology Co....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/92C12N15/61C12N15/76C12R1/465
CPCC12N9/92C12N15/76C12N2800/101C12N2800/22C12Y503/01005
Inventor 刘明明卢嫣红杜华东李峰
Owner NANJING BESTZYME BIO ENG CO LTD
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