Method for enhancing L-phenylalanine exocytosis of escherichia coli

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...

AI Technical Summary

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 metabolic regulation of phenylalanine is limited by multi-level and different levels of feedback control such as gene transcription initiation, transcription process, translation initiation, translation process, enzyme amount, and enzyme activity, and because the size of the plasmid vector is limited, It is difficult to remove all the regulatory sites by overexpressing multiple consecutive genes. In addition, E. coli will accumulate a by-product during the fermentation process - acetic acid, which can limit the growth of the bacteria and the synthesis of the target product, reducing the yield

In conclusion, genetically engineered strains capable of high-yield L-phenylalanine and low fermentation cost have not been reported so far, and it is difficult to form industrialization

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