Method for enhancing L-phenylalanine exocytosis of escherichia coli

A technology of phenylalanine cells and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of mutant strains with nutritional deficiencies, limited amino acid accumulation, and difficult to release regulatory sites, so as to improve fermentation intensity and enhance the level of extracellular secretion , Accelerate the effect of extracellular transport rate

Active Publication Date: 2013-06-12
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] The microbial strains with acid-producing ability screened from nature generally have limited amino acid accumulation; although the mutant strains selected through special modification can improve the production efficiency of L-phenylalanine, they are blind and have a heavy workload , the production cost is high, and the mutant strains generally carry nutritional deficiencies, which limit the further improvement of yield
The release of metabolic regulatory sites by means of gene cloning to promote the synthesis of target products has been successfully applied to the fermentation production of various metabolites such as citric acid, lactic acid, and succinic acid, which is an inevitable trend in the industrial production of L-phenylalanine. However, L -The metab

Method used

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  • Method for enhancing L-phenylalanine exocytosis of escherichia coli
  • Method for enhancing L-phenylalanine exocytosis of escherichia coli
  • Method for enhancing L-phenylalanine exocytosis of escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Construction of the plasmid pR15ABK overexpressing AroG, PheA, AroK and YdiB

[0024] 1.1 Release of metabolic regulation of 3-deoxy-D-arabinoheptulose-7-phosphate synthase AroG

[0025]Using the fitted DNA of Escherichia coli as a template, the upstream primer aroG15_F146 (base sequence shown in SEQ ID NO: 2) and the downstream primer aroG_HindⅢ_RV (base sequence shown in SEQ ID NO: 3) were used for PCR amplification to obtain gene fragments A, using Escherichia coli nucleoid DNA as a template, PCR amplification was performed with upstream primer aroG_Kpn Ⅰ_FW (base sequence shown in SEQ ID NO:4) and downstream primer aroG15_R146 (base sequence shown in SEQ ID NO:5) Obtain gene fragment B.

[0026] Using gene fragments A and B as templates, by overlapping PCR (according to the PCR site-directed mutagenesis technique, the references are as follows: Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen, and L.R. Pease (1989) Site-directed mutagenesis by overlap extens...

Embodiment 2

[0032] Example 2 Release of metabolic regulation of tyrosine transaminase TyrB

[0033] The tyrB coding gene was obtained by PCR amplification using the fitted DNA of Escherichia coli as a template, and the primers were designed as follows: the base sequence of the upstream primer tyrB_EcoRV_SD_SB_FW is shown in SEQ ID NO:14, and the base sequence of the downstream primer tyrB_AflⅡ_EcoRI_RV is shown in SEQ ID NO:15 , wherein, the SD sequence AAGGAGGAACAGAC is the ribosome binding site, and the SB sequence ATGTTCCAGAAGGTCGATG (as shown in SEQ ID NO: 1) is the starting base sequence of the coding frame of the tyrB gene after replacement.

[0034] The amplified TyrB coding gene was connected to the pMD 18T-simple Vector, sequenced and identified correctly by Shanghai Sangon Bioengineering Co., Ltd., and then ligated by enzyme digestion (for specific methods, please refer to the corresponding restriction endonuclease or ligase instructions) and The plasmid pR15ABK constructed in E...

Embodiment 3

[0040] Example 3 Release of metabolic regulation of methyl viologen efflux pump (YddG)

[0041] The yddG coding gene was obtained by PCR amplification using E. coli fitting DNA as a template, and the primers were designed as follows: the base sequence of the upstream primer yddG_EcoRV_SD_FW is shown in SEQ ID NO:16, and the base sequence of the downstream primer yddG_NcoI_KpnI_RV is shown in SEQ ID NO:17 , wherein, the SD sequence AAGGAGGAACAGAC is the ribosome binding site.

[0042] Ligate the amplified yddG-encoding gene to pMD 18T-simple Vector, after sequenced and identified correctly by Shanghai Sangon Bioengineering Co., Ltd., carry out an enzyme digestion ligation reaction (see the corresponding restriction enzyme or ligase instructions for specific methods) Connect with the plasmid constructed in Example 2, place the yddG coding gene in the strong promoter P L Later, a recombinant expression plasmid that can overexpress YddG, TyrB, AroG, PheA, AroK and YdiB at the sam...

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Abstract

The invention discloses a method for enhancing the L-phenylalanine exocytosis of escherichia coli. The method comprises the steps of transforming host escherichia coli through recombinant plasmids capable of simultaneously overexpressing tyrosine aminotransferase TyrB, methyl viologen exo transport protein YddG, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase AroG, chorismate mutase and prephenate dehydratase PheA, shikimate kinase AroK and shikimate dehydrogenase YdiB and producing L-phenylalanine through liquid fermentation, wherein artificial modification is carried out on TyrB, YddG, AroG, PheA, AroK and YdiB encoding genes from the escherichia coli resource so as to get release from the metabolic regulation and vectors are inserted, thus constructing the recombinant plasmids. The method disclosed by the invention has the advantages that the L-phenylalanine exocytosis level of the escherichia coli can be obviously enhanced, the L-phenylalanine yield is high, the concentration of harmful by-product acetic acid is low, the fermentation cost is low and the process is simple; and therefore, the method is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for enhancing the extracellular secretion of E. coli L-phenylalanine by means of genetic engineering technology and fermentation. Background technique [0002] L-phenylalanine (L-phenylalanine) is a physiologically active aromatic amino acid that is necessary for the human body but cannot be synthesized by itself. It is also an important pharmaceutical and food chemical intermediate. It is mainly used in the pharmaceutical industry to produce Tumor drugs and amino acid infusion preparations are mainly used in the food industry to synthesize aspartame, a dipeptide sweetener, and have good application prospects. There are mainly four methods for producing L-phenylalanine: hydrolysis extraction, chemical synthesis, enzymatic method and fermentation method. Among them, the raw materials used in the microbial fermentation method are cheap and easy to obtain...

Claims

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Application Information

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IPC IPC(8): C12P13/22C12N1/21C12R1/19
Inventor 刘双平石贵阳张梁丁重阳何冬旭李赢
Owner JIANGNAN UNIV
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