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Method for knocking out negative regulatory protein genes to improve fermentation level of acarbose

A kind of acarbose, fermentation level technology, applied in the field of biomedicine

Active Publication Date: 2021-03-19
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are still few studies involving the relevant transcriptional regulators and their regulatory mechanisms in the acarbose biosynthetic pathway

Method used

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  • Method for knocking out negative regulatory protein genes to improve fermentation level of acarbose
  • Method for knocking out negative regulatory protein genes to improve fermentation level of acarbose
  • Method for knocking out negative regulatory protein genes to improve fermentation level of acarbose

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Embodiment

[0039] This example is the specific process of obtaining the knockout negative regulatory protein gene ACPL_5445 and ACPL_3989 mutant strains WXM-06 and WXM-07. The specific operation steps are as follows:

[0040] Step 1: Construction of plasmid pLQ1455

[0041] Using the genomic DNA of Actinomycetes SE50 / 110 as a template, use primers ACPL_5445-LF / LR to amplify by PCR to obtain the 1474bp gene fragment on the left side of ACPL_5445, and use primers ACPL_5445-RF / RR to amplify by PCR to obtain the 1646bp gene fragment on the right side of ACPL_5445 Gene fragments, through gene sequencing to confirm the correctness of the target gene. A 1474bp PCR fragment from the left side of the ACPL_5445 gene and a 1646bp PCR fragment from the right side of the ACPL_5445 gene were inserted into the XbaI / HindIII site of plasmid pLQ752 to obtain plasmid PLQ1455. Under the condition of 37°C, two restriction endonucleases XbaI and HindIII were used for enzyme digestion, and a target band of a...

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Abstract

The invention discloses a method for knocking out negative regulatory genes to improve the fermentation level of acarbose. By adopting the method disclosed by the invention, the negative regulatory protein genes ACPL_5445 and ACPL_3989 are knocked out from actinoplanes to obtain acarbose high-yield mutant strains. By knocking out the negative regulatory protein genes, the expression of acarbose biosynthetic genes can be promoted, and finally the acarbose yield is obviously increased. Compared with an original strain, the high-yield strains obtained by the invention have the advantages that thefermentation yields are improved by 13% and 8%, and the laboratory shake flask fermentation levels reach 3.62 g / L and 3.47 g / L.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a method for improving the fermentation level of acarbose by knocking out negative regulatory genes; by knocking out negative regulatory genes ACPL_5445 and ACPL_3989 in actinomycetes to increase the level of acarbose Transcript levels of wave sugar biosynthetic genes, thereby increasing acarbose production. Background technique [0002] Currently, the number of people suffering from diabetes continues to increase worldwide. Type 2 diabetes is the most common type of diabetes, accounting for more than 90% of the total number of diabetes. Many clinical studies have proved that reasonable control of postprandial blood sugar can effectively slow down or reduce the occurrence of some chronic cardiovascular and cerebrovascular complications. Oral hypoglycemic drugs combined with dietary structure control and moderate exercise are effective intervention methods. The classic hypogly...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/74C12P19/26C12R1/045
CPCC07K14/195C12N1/20C12N15/74C12P19/26
Inventor 白林泉汪雪梅
Owner SHANGHAI JIAO TONG UNIV
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