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In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains

A technology of genetically engineered bacteria and genetically engineered strains, applied in the field of in vitro directional co-evolutionary transformation of L-phenylalanine genetically engineered bacteria, can solve problems such as phenotype changes

Inactive Publication Date: 2010-04-28
MAIDAN BIOLOGICAL GROUP FUJIAN
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Problems solved by technology

These gene products often influence each other and check and balance each other, and a single gene change is often difficult to cause phenotypic changes

Method used

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  • In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains
  • In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains
  • In-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains

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Embodiment Construction

[0039] The present invention provides a method for transforming L-phenylalanine genetically engineered bacteria in vitro through directional cooperative co-evolution, which mainly includes the following steps:

[0040] Step 1: the construction of L-phenylalanine genetically engineered bacteria, specifically including the following construction steps:

[0041] 1. Construction of tyrosine auxotrophic strain

[0042] The wild-type E.coil K12 was treated with UV and NTG compound mutagenesis, the wild-type strain was eliminated with antibiotics, and the auxotrophic strain was enriched; the auxotrophic strain was obtained by spot planting control method, and finally the auxotrophic strain was identified. Screening to obtain tyrosine auxotrophic host bacteria.

[0043] 2. Extraction of E.coil K12 genomic DNA by alkaline lysis

[0044] 3. Acquisition of key enzyme aroG and pheA genes

[0045] Genomic DNA was used as a template, and the designed upstream and downstream primers of ar...

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Abstract

The invention relates to an in-vitro directed coevolution method for modifying L-phenylalanine gene engineering strains, which is realized in a way that: using genes aroG and pheA on an L-phenylalanine gene engineering strain constructed in the room as a whole; carrying out the in-vitro directed coevolution modification by using an error-prone PCR technology and a recombinant DNA technology; and screening to obtain a mutant strain, of which the yield of L-phenylalanine is increased by 114%. In the invention, by modifying the gene formed by coupling and connecting aroG and pheA in series, the key genes aroG and pheA in the metaboly process of the L-phenylalanine are used as a whole to carry out the directed modification, thereby obtaining a new metabolic balance, so that the expressed enzyme has high-efficiency catalytic activity and can resist the feedback inhibition of the L-phenylalanine, thereby obtaining the new modified high-yield L-phenylalanine gene engineering strain by screening. The method can provide an example for modifying the acid production rate of any amino acid gene engineering strain.

Description

【Technical field】 [0001] The invention relates to the technical field of amino acid preparation, and relates to a method for transforming L-phenylalanine genetically engineered bacteria in vitro through directional co-evolution. 【Background technique】 [0002] L-phenylalanine (L-phenylalanine) is a white crystalline powder. It is one of the essential amino acids that cannot be synthesized by humans and animals in the body. It is widely used in food, feed, medicine and cosmetics, especially in low-calorie, Aspartame, a dipeptide sweetener with high sweetness, is widely used, and the market demand for L-phenylalanine, one of the two raw materials for the synthesis of aspartame, is increasing rapidly. In addition, L-phenylalanine is also an essential raw material for antineoplastic drugs and amino acid infusion preparations. With its continuous development in the field of medicine, the demand is also increasing. Therefore, the research on the biosynthetic pathway of L-phenylal...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P19/34C12P13/22C12R1/19C12R1/00
Inventor 黄祥峰施巧琴吴伟斌郑斌施碧红翁雪清刘建明黄维绵林宜云王明兹吴松刚
Owner MAIDAN BIOLOGICAL GROUP FUJIAN
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