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Acarbose engineering bacterium as well as preparation method and application thereof

A technology of acarbose and engineering bacteria, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve the problems of inapplicability of actinomycetes and Escherichia coli conjugative transfer, and achieve product quality assurance , Reduction of process steps, low content of impurity components

Active Publication Date: 2013-09-18
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in the prior art, heat shock at 50°C for 10 minutes is used for conjugative transfer of Escherichia coli and actinomycetes (Practical Streptomyces Genetics, 2000, Tobias Kieser, etc.), but this temperature and time are not effective for motile actinomyces and Escherichia coli The spliced ​​transfer does not apply

Method used

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  • Acarbose engineering bacterium as well as preparation method and application thereof
  • Acarbose engineering bacterium as well as preparation method and application thereof
  • Acarbose engineering bacterium as well as preparation method and application thereof

Examples

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preparation example Construction

[0038] The method for preparing and screening acarbose engineering bacteria of the present invention can be summarized as the following steps:

[0039] 1. Using PCR, using the genome of Actinomycetes mobilis as a template, amplify the 3057bp fragment upstream of the treY gene (including 785bp at the 5' end of the coding sequence of the treY gene), and add EcoRI and BamHI restriction sites at both ends ; and a downstream 2885bp fragment (including 930bp at the 3' end of the treY gene coding sequence), with BamHI and Hind III restriction sites added to both ends. These two fragments were sequentially inserted into the corresponding restriction sites of the actinomycete suicide plasmid pUAmT14 to obtain the recombinant plasmid pAT-tre12 for inactivating the treY gene. The treY gene of this plasmid loses its original biological activity due to the deletion of 553 internal bases.

[0040] 2. After the recombinant plasmid pAT-tre12 was transformed into Escherichia coli ET12567 (pUZ...

Embodiment 1

[0043] Example 1: Construction of the recombinant plasmid pAT-tre12 for knocking out the treY gene (the schematic diagram of the construction process is shown in figure 1 shown)

[0044] a) Construction of actinomycetes suicide plasmid pUAmT14: cloning vector pUC19 (Fermentas) was digested with Dra I (TaKaRa) and Ssp I (TaKaRa); (TaKaRa) and BstBI (TaKaRa) double digestion, and the BLK kit (TaKaRa) blunt ends. The above two fragments were connected to obtain the actinomycetes suicide plasmid pUAmT14. Plasmid pIJ773 is described in literature Gust B, Kieser T and Chater K, F. Technology: PCR-targeting system in Streptomyces coelicolor. John Innes Centre. 2002 is detailed. The structure and restriction sites of plasmid pUAmT14 are as follows: figure 2 shown. Use Bgl I, Hinc II and Xba I to digest the plasmid pUAmT14 to identify whether the constructed plasmid is correct. image 3 shown.

[0045] b) Isolation of the Actinomycetes genome: Take 200 μl of the frozen Actinom...

Embodiment 2

[0071] Example 2: Transforming the recombinant plasmid pAT-tre12 with treY gene deletion into the host bacterium Actinomyces mobilis 8-22

[0072] a) Transform recombinant plasmid pAT-tre12 into Escherichia coli ET12567 (pUZ8002): Take 1 μl of recombinant plasmid pAT-tre12 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 solid LB culture containing 25 μg / ml chloramphenicol (Cm), 50 μg / ml kanamycin (Km), 50 μg / ml apramycin (Am), culture overnight at 37°C, and grow As for the transformants, one of the transformants was selected and identified as having been transformed into the plasmid pAT-tre12, which was named ET12567 (pUZ8002, pAT-tre12).

[0073] b) Cultivation of Escherichia coli ET12567 (pUZ8002, pAT-tre12): pick a si...

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Abstract

The invention relates to an acarbose engineering bacterium, a method for preparing the engineering bacterium and an application of the engineering bacterium. The acarbose engineering bacterium provided by the invention is prepared by inactivating treY gene of actinoplanes by introducing homologous recombination fragments by using a conjugational transfer method. The content of the generated acarbose C-component impurity of the acarbose engineering bacterium after the acarbose engineering bacterium is fermented is greatly reduced.

Description

technical field [0001] The present invention relates to an acarbose engineering bacterium constructed by a genetic engineering method, more specifically, to an acarbose engineering bacterium with reduced C component impurities, a method for preparing the engineering bacterium, and the engineering bacterium application. Background technique [0002] According to statistics, the number of diabetic patients in my country has reached 92.4 million, including 50.2 million men and 42.2 million women. The number of cases has surpassed that of India, becoming the country with the largest number of diabetics in the world, and the incidence rate (9.7%) is second only to the United States (11%). In addition, there are about 150 million people with pre-diabetes, and the annual treatment cost reaches 174 billion yuan. [0003] Acarbose, a hypoglycemic drug produced by Actinoplanes sp., is the first choice for the treatment of type 2 diabetes, with annual domestic sales of nearly 2 billi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P19/26C12N15/11C12R1/045
Inventor 黄隽李美红余贞
Owner ZHEJIANG HISUN PHARMA CO LTD
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