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Method for improving acid yield of L-phenylalanine gene engineering bacteria

A technology of genetically engineered bacteria and phenylalanine, applied in the field of bioengineering, can solve the problems of heavy workload, inability to increase the number of gene copies, and large blindness.

Inactive Publication Date: 2011-04-20
MAIDAN BIOLOGICAL GROUP FUJIAN
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Problems solved by technology

Utilizing the method of conventional mutation breeding, its blindness is large, the workload is heavy, and the copy number of the gene cannot be increased, and the mutant strains generally carry a variety of nutritional deficiencies, which limits further increase in yield

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  • Method for improving acid yield of L-phenylalanine gene engineering bacteria
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  • Method for improving acid yield of L-phenylalanine gene engineering bacteria

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Embodiment Construction

[0023] A method of improving the acid production rate of L-phenylalanine genetically engineered bacteria of the present invention, it specifically comprises the following steps:

[0024] Step 1: Construction of a new generation of L-phenylalanine engineering plasmid MDphe-3, specifically including the following construction steps (see figure 1 ):

[0025] 1. PCR amplification of the pckA gene with restriction sites

[0026] According to the complete pckA gene sequence (GenBank NO.EG10688) published by NCBI, the primers with restriction sites were designed as follows, and were commissioned to be synthesized by Bioengineering (Shanghai) Co., Ltd. using a DNA synthesizer:

[0027] pckA-F: 5-ATGCGCGTTAACAATGGTTTG-3

[0028] pckA-R: 5- GCTCAGC TTACAGTTTCGGACCAGCC-3

[0029] Among them, the downstream primer has the Esp 1 restriction sequence, the length of the PCR amplified product is 1623bp, and the optimal annealing temperature is 57.5°C. The PCR amplification reaction syst...

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Abstract

The invention relates to a method for improving acid yield of L-phenylalanine gene engineering bacteria, comprising the following steps: carrying out deletion construction on a tyrA gene in csrA gene deletion-type engineering host bacteria of new-generation engineering plasmid NDphe-3 and serial trpE / trpsrA genes by a Red system; planting the csrA gene, the tyrA gene and the engineering host bacteria deleted from the serial trpE / trpD gene; constructing the new L-phenylalanine gene engineering bacteria; and finally verifying the genetic stability and acid yield of the new L-phenylalanine gene engineering bacteria. The method provided by the invention has the advantages of breaking two phenylalanine biosynthetic competitive metabolic branches synthesized by phenylalanine biology, such as tyrosine and tryptophan, thus ensuring that the metabolic stream of the aromatic amino acid flows to the synthesis of the L-phenylalanine in a large scale, and further improving the ability of the fermentation of the L-phenylalanine by the engineering bacteria.

Description

【Technical field】 [0001] The invention relates to the field of bioengineering, in particular to a method for improving the acid production rate of L-phenylalanine by transforming engineering plasmids through PCR technology and using the Red system to mediate host bacteria of the transformed plasmids. 【Background technique】 [0002] L-phenylalanine (L-phenylalanine), referred to as L-Phe, 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, cosmetics and other fields. In particular, the low-calorie, high-sweetness dipeptide sweetener Aspartame (Aspartame) 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 th...

Claims

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Application Information

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IPC IPC(8): C12P13/22C12N1/21C12N15/09C12R1/19
Inventor 黄钦耿吴伟斌蔡爱金施碧红施巧琴黄祥峰陈炳生吴松刚
Owner MAIDAN BIOLOGICAL GROUP FUJIAN
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