Breeding Of High-yielding L-isoleucine Strains By ARTP Mutagenesis Combined With λ-Red Recombination Technology

L-Isoleucine is one of the eight essential amino acids in the human body.It is widely used in food,pharmaceutical,chemical and feed industries.Currently,it is mainly produced by direct microbial fermentation.Obtaining good strains is the key to efficient production of L-isoleucine.In the study of the fine regulation of L-isoleucine metabolic pathway in Escherichia coli,it was found that blocking the synthesis pathway of m ethionine,lysine and leucine and the degradation of L-threonine to L-2-amino-3-oxo The metabolic pathway of butyrate may play an important role in enhancing the synthesis of L-isoleucine.In this study,the wild-type Escherichia coli MG1655 was used as the starting strain,and the methionine and lysine auxotrophic strains were selected by using atmospheric room temperature plasma mutagenesis technology(Atmospheric Room Temperature,ARTP)for multiple rounds of mutagenesis.Analyze the key metabolic pathways of L-isoleucine synthesis and determine the target genes;use λ-Red recombination technology to knock out the key enzyme genes LeuA and L-2-amino-3-oxobutyric acid synthesis pathway of Escherichia coli leucine synthesis pathway The key enzyme gene tdh was used to determine the L-isoleucine production of the recombinant strain by fermentation;the fermentation medium components of the recombinant strain were optimized by single factor experimental design and response surface methodology to obtain the best fermentation parameters.The main research contents and results are as follows:(1)ARTP mutagenesis combined with MMC to breed methionine and lysine auxotrophic strains.Taking the E.coli MG 1655 wild type as the starting strain,using ARTP for stepwise mutagenesis,and using methionine and lysine as the selection markers,the methionine was screened by the Microbial Microdroplet Culture system(MMC).Deficient strain E.coli NXA1 and methionine and lysine double-deficient strain E.coli NXA2 were fermented with E.coli NXA1 and E.coli NXA2 to measure L-isoleucine production.The results showed that the growth rate of E.coli NXA1 was consistent with that of E.coli MG 1655 wild type after 40 h of fermentation.The concentration of L-isoleucine in the fermentation broth was 1.16 g/L,and the genetic characters were stable after continuous passage;E.After 40 hours of fermentation,the growth rate of.coli NXA2 was consistent with that of E.coli NXA1.The L-isoleucine concentration in the fermentation broth was 4.34 g/L,which was 3.74 times that of E.coli NXA1,and increased by 274.14%.The genetic characters were stable after serial passage.(2)Transcriptomic analysis of E.coli transformed strains.Transcriptional sequencing of E.coli MG 1655 wild-type and E.coli NXA2 by RNA-Seq technology showed that there were 643 differentially expressed genes,406 genes were up-regulated and 237 genes were down-regulated.KEGG Pathway analysis of differentially expressed genes showed that the increase of L-isoleucine production was mainly closely related to glycolytic pathway,oxidative phosphorylation,TCA cycle,apoplexy,amino acid metabolism and threonine metabolism.This indicates that the recombinant bacteria can redistribute the metabolic flow.In order to increase the production of L-isoleucine,it is determined that the key enzyme gene LeuA of the leucine synthesis pathway and the key enzyme gene tdh of the L-2-amino-3-oxobutyric acid synthesis pathway are subjected to transformation to provide a theoretical basis for follow-up research.(3)Knockout of LeuA gene and tdh gene in the modified E.coli strain.The gene knockout experiment was performed with E.coli NXA2,and the key enzyme gene Leu A in the leucine synthesis pathway was knocked out by using λ-Red recombination technology to obtain a recombinant strain E.coli NXA3;L-2-amino-3-oxo was knocked out The key enzyme gene tdh in the butyrate synthesis pathway was obtained as a recombinant strain E.coli NXA4.The results showed that the growth rate of E.coli NXA4 had no effect after 40 h of fermentation,and the measured L-isoleucine concentration reached 8.91 g/L,which was 2.05 times that of E.coli NXA2 and increased by 105.30%,and the concentration of L-isoleucine reached 8.91 g/L.The genetic traits were stable after passage.(4)The components of the L-isoleucine fermentation medium of the modified Escherichia coli strain were optimized.Through single factor experiment and response surface optimization,it was concluded that the optimal fermentation and culture conditions for E.coli NXA4 to produce L-isoleucine were:glucose 127.41 g/L,ammonium sulfate 32.97 g/L,corn steep liquor 15.97 g/L,phosphoric acid Potassium dihydrogen 3.0 g/L,magnesium sulfate 2.5 g/L,ferrous sulfate 0.01 g/L,manganese sulfate 0.01 g/L.After the optimization of medium components,E.coli NXA4 was fermented and cultured for 40 hours,and the L-isoleucine concentration was determined to reach 10.26g/L,which was15.15%higher than that before optimization,which proved that the model of the response surface was selected correctly.The optimized medium was more favorable for E.coli NXA4 to accumulate L-isoleucine.