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Acarbose-producing engineered bacterial strain, and preparation method and application thereof

An acarbose and engineering bacteria technology, applied in the field of bioengineering, can solve the problems of complex process, increased production cost, instability, etc., and achieves the effects of simple purification steps, product quality assurance, and cost reduction.

Active Publication Date: 2018-10-09
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to ensure product quality, subsequent purification steps are required, resulting in complex processes and increased production costs
[0004] At present, it is widely used to control the content of impurity components by controlling the fermentation process, but this method is unstable

Method used

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  • Acarbose-producing engineered bacterial strain, and preparation method and application thereof
  • Acarbose-producing engineered bacterial strain, and preparation method and application thereof
  • Acarbose-producing engineered bacterial strain, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-1

[0047] Example 1-1: Construction of recombinant plasmid pBS-BYHS-AMT for inactivating bglY gene

[0048] a) Using primers BY3F53 (SEQ ID NO: 6) / BY3R54 (SEQ ID NO: 7) to amplify from Actinomycetes mobilis 8-22 genomic DNA to obtain a fragment BY334 (SEQ ID NO: 23) of about 3.1 kb, the After the fragment was phosphorylated, it was inserted into the HincII site of the vector pBluKS (Genebank X52331.1) to obtain the plasmid pBS-BY334 (for the insertion direction of the fragment, see figure 2 );

[0049] b) Using HisunHF (SEQ ID NO: 9) / HisunCR (SEQ ID NO: 10) as primers and using the single strand (SEQ ID NO: 8) as a template, amplify the fragment HHCPCR (SEQ ID NO: 24). The plasmid pBS-BY334 was transformed into Escherichia coli JM110 and re-extracted to remove the methylation on base A, so that its ClaI restriction site could be cut by endonuclease ClaI. After the plasmid was digested by HindIII / ClaI, it was ligated with the fragment HHCPCR that had been digested by the same r...

Embodiment 1-2

[0052] Example 1-2: Construction of recombinant plasmid SAT-MGSH for inactivating mpbG gene

[0053] a) Using primers MG3F63 (SEQ ID NO: 14) / MG3R64 (SEQ ID NO: 15) to amplify the 3.5 kb fragment MG334 (SEQ ID NO: 27) from the genomic DNA of Actinomycetes mobilis 8-22. After the fragment was phosphorylated, it was inserted into the HincII site of the vector pBluKS to obtain the plasmid pBS-MG334 (for the insertion direction of the fragment, see Figure 6 );

[0054] b) Using HisunHF (SEQ ID NO: 9) / HisunCR (SEQ ID NO: 10) as primers and single strand (SEQ ID NO: 8) as a template, amplify the fragment HHCPCR. After the fragment was digested by HindIII / ClaI, it was ligated with the plasmid pBS-MG334 which had been digested by the same enzyme, to obtain the plasmid pBS-MG334HS;

[0055]c) Cut out a 1229bp fragment containing pUCori (SEQ ID NO: 28) from the Cosmid plasmid supcos-1 (Stratagene, Inc.) with HincII+DraI; cut out a 1271bp fragment containing aac3 (IV) from pIJ773 with ...

Embodiment 2

[0058] Example 2: Transformation of bglY gene and mpbG gene-deleted recombinant plasmids PBS-BYHS-AMT and SAT-MGSH into host bacterium Actinomyces mobilis 8-22

[0059] a) Transform recombinant plasmids PBS-BYHS-AmT and SAT-MGSH into Escherichia coli ET12567 (pUZ8002) (in literature Gust B, Kieser T and Chater K, F. technology: PCR-targeting system in Streptomyces coelicolor. John Innes Centre.2002 has detailed descriptions): Take 1 μl of the recombinant plasmid and add it to 100 μl Escherichia coli ET12567 (pUZ8002) competent cells (with CaCl 2 (Preparation method), place on ice for 30 minutes, heat shock at 42°C for 90 seconds, then quickly cool on ice for 1 minute, add 900 μl LB for culture, and bathe in water at 37°C for 50 minutes. Take 100 μl and smear it on the solid LB culture containing 25 μg / ml chloramphenicol (Cm), 50 μg / ml kanamycin (Km), 50 μg / ml apramycin (Am), cultivate overnight at 37 ° C, and grow Transformants ET12567(pUZ8002, PBS-BYHS-AmT) and ET12567(pUZ8...

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Abstract

The invention relates to an acarbose-producing engineered bacterial strain, and a preparation method and application thereof, belonging to the field of bioengineering. The acarbose-producing engineered bacterial strain is an Actinoplanes strain for acarbose production or derivative strains thereof. In the strain, one or two selected from the group consisting of the following genes are inactivated:(i) a gene M coding a polypeptide with a sequence as shown in SEQ ID No. 2 or with at least 80%, 90%, 95% or 99% sequence identity to the sequence as shown in the SEQ ID No. 2; and (ii) a gene N coding a polypeptide with a sequence as shown in SEQ ID No. 3 or with at least 80%, 90%, 95% or 99% sequence identity to the sequence as shown in the SEQ ID No. 3. When the engineered bacterial strain provided by the invention is used for acarbose production, the content of an impurity component A and / or an impurity component B in acarbose is significantly reduced, so the quality of the acarbose is guaranteed; and a subsequent purification step is not needed or a purification step is relatively simple, so process steps are decreased, and cost is reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an engineering bacterium for producing acarbose, a preparation method and an application of the bacterium. Background technique [0002] According to statistics, the number of diabetic patients in my country has reached 110 million, and China has become the country with the largest number of diabetic patients in the world. In addition, there are about 150 million people in China with pre-diabetes. Just for diabetes management, China now needs to invest nearly 173.4 billion RMB every year; while the direct medical expenditure for diabetes has accounted for 13% of China's medical expenditure. . [0003] Acarbose, a hypoglycemic drug produced by Actinoplanes sp., is the first choice for the treatment of type II diabetes, with an annual domestic sales of nearly 2 billion yuan. At present, there are mainly impurity components in the production of acarbose, which seriously affects the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/26C12R1/045
CPCC07K14/365C12P19/26
Inventor 黄隽李美红周军余贞张鑫培
Owner ZHEJIANG HISUN PHARMA CO LTD
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