Method for improving acid production rate of L-phenylalanine gene engineering bacteria

Abstract

The invention relates to a method for improving the acid production rate of L-phenylalanine gene engineering bacteria. The method comprises the following steps of: directionally mutating recombinant expression plasmids of L-phenylalanine gene engineering bacteria constructed in the laboratory, which are taken as a whole; in the process of mutating the expression plasmids with an alkylation agent EMS, controlling the mutation rate by the length of the duration of the reaction of the EMS and the plasmids to obtain the optimal mutation time under the same mutation conditions, and then electrically converting the expression plasmids after the optimal mutation time of the EMS mutation into host bacteria to construct a strain mutation library; and screening to obtain 4 good mutant strains the yields of L-phenylalanine of which are improved in different degrees. The method ensures the reconstruction of acid-producing strains of L-phenylalanine gene engineering bacteria which are easy to be large-scale fermented and industrialized, and facilitates the reconstruction.

Description

【Technical field】 [0001] The invention relates to the technical field of amino acid preparation, in particular to a method for increasing the acid production rate of L-phenylalanine genetically engineered bacteria. 【Background technique】 [0002] L-phenylalanine (L-phenylalanine) is one of the eight essential amino acids in human and animal life activities. It plays an important role in the growth, development and metabolism of human and animals. It is widely used in medicine, food, feed and nutrition. fields of health care. In recent years, with the deepening of the understanding of the physiological functions of L-phenylalanine and the deepening of scientific research, the application of L-phenylalanine has become more and more extensive, and it has become a product with good development prospects and large market demand in the international market. product. [0003] The production methods of L-phenylalanine at home and abroad mainly include hydrolysis extraction method,...

AI Technical Summary

Problems solved by technology

However, the disadvantage of this intracellular gene recombination technology is that it can only be carried out between microorganisms of the same species or close relatives, and the success rate is low.

And these gene products often influence each other and check and balance each other, and the change of a certain gene is often difficult to cause phenotypic changes

[0015] In view of the above shortcomings, the applicant provided a strain of L-phenylalanine in the patent of Chinese Invention Application No. 200910111381. The aroG and pheA genes on the amino acid genetically engineered bacteria were expressed in tandem as a whole, and a combination of error-prone PCR and DNA shuffling was used to carry out in vitro directional co-evolutionary transformation, and a strain of L-phenylalanine was screened Acid production increased by 114% of the mutant strain, although on the basis of constructing genetically engineered bacteria, the expression vector constructed was further improved by directional transformation technology and the acid production of L-phenylalanine engineered bacteria was greatly improved , but because it needs to extract the aroG and pheA genes from the expression plasmid vector and then transform them, the operation is complicated and cumbersome