Irrational Screening And Rational Construction Of Escherichia Coli L-Tryptophan High-Yield Strain

L-tryptophan(L-trp),as the human body 8 essential amino acids essential amino acid the second largest,is a precursor to many important biologically active substances,is widely used in pharmaceuticals,food and feed and other industry.With the increasing demand for L-tryptophan at home and abroad,it has become an important research hotspot.The microbial fermentation method has been widely used in the production of L-tryptophan because of its advantages such as easy availability of raw materials,low cost,high purity of the final product,easy extraction and environmental protection.Among them,Escherichia coli,as a widely used model strain,has been widely used in the metabolic engineering of industrialized commodities due to its clear genetic background,simple genetic modification,rapid propagation and easy cultivation.In terms of strain transformation,rational transformation and irrational screening have certain advantages,but at the same time there are some limitations.Therefore,in this study based on the traditional chemical mutagenesis,and rational design L-tryptophan strains constructed.Firstly,L-tryptophan production chassis cells were constructed using E.coli wild type MG1655 as the starting strain.Secondly,L-tryptophan high-yield strains were obtained by plasma mutagenesis of ARTP,and the genetic background was analyzed by genome-wide sequencing.Finally,rational transformation design is guided by transcriptomics combined with metabolic engineering.The main research contents and results are as follows:(1)Escherichia coli wild type MG1655 was used as a starting strain to overexpress the genomic tyrosine lyase gene tna A,overexpressing the 3-deoxy-D-arabinoheptanoic acid-7-phosphate synthase gene aro G^fbr and the tryptophan operon trp E^fbrDCBA constructs the chassis cell TD0,and its L-tryptophan production in shake flask fermentation reaches 0.3 g/L.(2)The L-tryptophan biosensor p Trp-GFP was constructed,and the strains after ARTP mutagenesis were screened by high-throughput.A strain of L-tryptophan producing strain TD was screened and its yield reached 1.4 g/L in shake flask fermentation.(3)Extract the TD genome of the strain,perform whole genome sequencing,and compare with the E.coli wild type MG1655 genome to determine the location and type of the mutant gene.Through the recovery mutation of some mutant genes,the high-yield mechanism of L-tryptophan was analyzed to provide a basis for the rational transformation of TD strains.At the same time,transcriptomics analysis of strain TD and TD-rpo S(rpo S gene back mutation strain)was carried out,which further provided theoretical basis for rational transformation of TD strain.(4)Based on genomics and transcriptomics data,combined with the fermentation results of TD strains and back-mutant strains,a series of transformations were carried out on the TD strains from the L-tryptophan branch pathway,the chorismate synthesis pathway and the pentose phosphate pathway.Finally,the strain TD020 with extremely significant increase in L-tryptophan production was obtained,and its yield reached 4.2 g/L,and the conversion rate was 9.01%.