| L-lysine is one of the essential amino acids for human body and plays an important role in food,medicine and cosmetics industry.Most of the studies on L-lysine biosynthesis have focused on Corynebacterium glutamicum as host bacteria,and most of them are random mutation of strains,plasmid-mediated genetic modification and optimization measures in industrialization.There are some problems such as high metabolic pressure,genetic instability and unclear genetic background of strains.However,few studies on genome modification strategies using Escherichia coli(E.coli)as host bacteria have been reported.In this paper,a series of rational modifications of E.coli genome were carried out to improve L-lysine production in E.coli by using the technology and strategy of metabolic engineering.Firstly,two L-lysine decarboxylase coding genes were knocked out,and the pathway of L-lysine transformation to downstream products was blocked.A strain capable of detecting L-lysine accumulation was constructed.Secondly,high-yield strains of L-lysine carrying plasmids were screened by overexpressing key enzyme genes with plasmids as vectors,which provided basis for further genomic integration.Thirdly,the endogenous biosynthetic pathway of L-lysine was modified.For the first time,the promoters of dihydropyridine dicarboxylate synthase and aspartic acid kinase were replaced by strong promoter T7 in E.coli genome.The accumulation of L-lysine in E.coli by genome modification was realized,and the production of L-lysine was increased by 15.6%.Subsequently,the carbon flow ratio of glycolysis pathway and pentose phosphate pathway was adjusted to increase the production of NADPH and L-lysine by 15.02%.Finally,the introduction of heterologous multifunctional enzymes and the integration of them with T7 strong promoter into the genome of E.coli were attempted to replace the original four-step enzymes with a heterologous multifunctional enzymes for catalytic reaction,which greatly increased the production of L-lysine by 2.68 times.Through a series of strategy studies,a genome-modified strain of E.coli with clear genetic background and high L-lysine production was obtained,and the yield could reach 2.07 g/L.This paper is different from the reported methods of producing L-lysine in E.coli.In the construction of L-lysine high-yield platform,random mutation of strains and gene expression through plasmid construction were abandoned.The key enzymes coding genes and strong promoters in recombinant L-lysine synthesis pathway were directly used to make L-lysine high-yield platform.The strain is more stable and the genetic background is clearer.Therefore,the downstream products of high-yield platform through modular co-culture will be more convenient,and the application prospect of this platform will be broader. |
