Method for improving acid production rate of L-phenylalanine genetic engineering bacterial

A technology of genetically engineered bacteria and phenylalanine, applied in the field of bioengineering, can solve problems such as enhancing the ideal metabolic pathway, and achieve the effects of enhancing the accumulation of PEP, improving the ability, and achieving great benefits

Inactive Publication Date: 2011-05-04
MAIDAN BIOLOGICAL GROUP FUJIAN
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Problems solved by technology

Moreover, the reactions and metabolic pathways regulated by global metabolic regulators may perform different or even opposite effects, which makes it possible to both enhance the desired metabolic pathway and inhibit the competing metabolic pathway

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  • Method for improving acid production rate of L-phenylalanine genetic engineering bacterial
  • Method for improving acid production rate of L-phenylalanine genetic engineering bacterial
  • Method for improving acid production rate of L-phenylalanine genetic engineering bacterial

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Embodiment Construction

[0042] A method of improving the acid production rate of L-phenylalanine genetically engineered bacteria of the present invention, it specifically comprises the following steps:

[0043] Step 1: Construction of a new generation of L-phenylalanine engineering plasmid MDphe-3, specifically including the following construction steps (see figure 1 ):

[0044] 1. PCR amplification of the pckA gene with restriction sites

[0045] 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:

[0046] pckA-F: 5-ATGCGCGTTAACAATGGTTTG-3

[0047] pckA-R: 5- GCTCAGC TTACAGTTTCGGACCAGCC-3

[0048] 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 production rate of L-phenylalanine genetic engineering bacterial. The method comprises the steps of: constructing the original engineering plasmid MDphe-2 of L-phenylalanine into a new generation of engineering plasmid MDphe-3 by adopting a PCR (polymerase chain reaction)technology; carrying out deletion constructing on a whole metabolism network regulatory gene csrA of the engineering host bacterial of the engineering plasmid MDphe-3 through a Red system; then implanting the engineering plasmid MDphe-3 in the engineering host bacterial which is in deletion of the whole metabolism network regulatory gene csrA to construct a new L-phenylalanine genetic engineering bacterial; and finally verifying and reserving the L-phenylalanine gene engineering bacterial with high hereditary stability and high acid production rate. The method has the advantages that the PEP accumulation of one of precursors of aromatic amino acid is strengthened, simultaneously the carbon metabolism flux can be converted heavily to a shikimic acid way, the large flowing of the metabolism flux of the aromatic amino acid to the synthesis of the L-phenylalanine is ensured, and the ability of the engineering bacterial to ferment the L- phenylalanine is further improved.

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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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12N1/21C12N15/09C12R1/19
CPCC12N15/67C07K14/245C12N9/88C12P13/222C12Y401/01032
Inventor 黄钦耿吴伟斌蔡爱金施巧琴施碧红黄祥峰陈炳生吴松刚
Owner MAIDAN BIOLOGICAL GROUP FUJIAN
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