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Method for Recombining and Regulating Biological Components to Enhance Biosynthesis of Secondary Metabolites

A technology for secondary metabolites and biosynthesis, which can be applied to other methods of inserting foreign genetic materials, recombinant DNA technology, and the introduction of foreign genetic materials using vectors. It can solve problems such as failure to make substantial progress and achieve a large application space. , enhance the effect of synthesis

Active Publication Date: 2016-05-04
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no substantive progress has been made in the research of Micromonospora

Method used

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  • Method for Recombining and Regulating Biological Components to Enhance Biosynthesis of Secondary Metabolites
  • Method for Recombining and Regulating Biological Components to Enhance Biosynthesis of Secondary Metabolites
  • Method for Recombining and Regulating Biological Components to Enhance Biosynthesis of Secondary Metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of recombinant plasmids for the integration of biological components T7promoter

[0036] 1. Primer design and recombinant plasmid construction

[0037] According to the published sisomicin biosynthetic gene cluster sequence (GenBankAccessionNumberJF431003), the 202bp sequence between CDS28 and CDS29 downstream of the gene cluster was used as the replacement target site of T7promoter, and two pairs of exchange arm amplifiers were designed at the upstream and downstream of the site, respectively. Add primers. Primers P1 / P2 amplify the HB1 sequence; primers P3 / P4 amplify the HB2 sequence. Since the T7promoter is only 20bp, and considering that the direction of the T7promoter should be consistent with the transcription direction of the biosynthetic gene, the T7promoter should be added to the 5′ end of the downstream primer (P2) of HB1 to synthesize the T7promoter by amplifying HB1.

[0038]PCR was carried out using the genome of Micromonas ineudene...

Embodiment 2

[0040] Embodiment 2: Recombinant plasmid pHB202 transforms Micromonospora eneurica

[0041] 1. Preparation of Donor Bacteria

[0042] through CaCl 2 The shuttle plasmid pHB202 was transformed into Escherichia coli ET12567 (pUZ8002) to obtain donor bacteria (ET12567 / pUZ8002 / pHB202). The overnight cultured donor bacteria were transferred to 30mL LB liquid medium containing corresponding antibiotics (kanamycin 25ug / ml, chloramphenicol 25ug / ml and apramycin 50ug / ml) at an inoculum of 1%. Incubate at 37°C for 2-3h, control OD 600 Between 0.4-0.6, the cells were collected by centrifugation. After washing the cells twice with fresh LB liquid medium, resuspend with 1 mL of fresh LB, and set aside.

[0043] 2. Preparation of Micromonospora eneurica spore suspension

[0044] Scrape fresh spores from a well-growing slope, suspend them with 5mL of 0.05mol / L TES buffer (pH8.0), shake vigorously on a vortex mixer, and break up the spores; place the spore suspension in a water bath at 5...

Embodiment 3

[0047] Example 3: Screening of Biological Components T7promoter Insertion Gene Cluster Engineering Bacteria

[0048] Micromonospora innuuuatus DTS202 was continuously passed on for 3 generations on a non-resistant slant, and then isolated and purified, and single colonies were picked and placed on plates containing apramycin (50 μg / mL) and plates without apramycin resistance. Apramycin-susceptible strains were screened on a sex-resistant plate. Select one of them, extract its chromosomal DNA as a template, design a pair of primers (P5 / P6) for PCR verification, and sequence it to prove that the biological component T7promoter is inserted between CDS28 and CDS29 downstream of the sisomicin biosynthesis gene cluster . The strain is a double-exchange engineered strain named Micromonospora ineudeneus TS202.

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Abstract

The invention provides a method for reinforcing the biosynthesis of a secondary metabolite by recombining and controlling biological components. The method is mainly used for recombining biological components, namely T7RNApolymase gene and T7 promoter, of T7 bacteriophage and components, namely lacI, lacP and lacO, of lactose operon; the recombined products are respectively integrated to specific sites on upstream and downstream of a biosynthetic gene cluster of the secondary metabolite to enforcedly start the biosynthetic gene cluster so as to achieve high-efficiency transcription and expression. The method comprises the following steps of: constructing a shuttle vector required by biological component integration; introducing recombinant plasmids into industrial microbes; screening engineering bacteria; and controlling biosynthesis of the engineering bacteria. According to the method, the yield of secondary metabolites can be greatly improved.

Description

technical field [0001] The invention belongs to the field of genome improvement and recombination, and more specifically relates to a method for recombining and regulating biological components to strengthen the biosynthesis of secondary metabolites. Background technique [0002] Improving the production of industrial microorganisms, reducing consumption, reducing pollution, and improving economic benefits are the research focus of microbial engineering and metabolic engineering. [0003] With the rapid development of molecular biology technology, genetic engineering, protein engineering, and microbial molecular genetics engineering have emerged one after another; with the development of computer science, bioinformatics has emerged as the times require, and soon integrated into molecular biology. The intermingling of disciplines has made genetic engineering even more powerful, promoting the development of industrial microbial genome improvement at a rapid speed, and graduall...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/02C12N15/87
Inventor 洪文荣肖海鹏
Owner FUZHOU UNIV