Rational Analysis And Design Of Escherichia Coli For Production Of Tryptophan With High Efficiency

L-tryptophan（L-trp）is a precursor of various bioactive components.It has been widely used in medicine,food,feed additives and environmental monitoring.With the increasing demand of the market,the research of L-trp has become a hot spot.At present,the world market demand for L-trp is over 10000 tons,and it grows at an annual rate of 10%.However,the biosynthetic pathway from glucose to L-trp is long,and the metabolic flux is also weak under normal conditions.Moreover,the synthesis of L-trp requires various precursors.The regulation mechanism of biosynthetic pathway is rather complex,which limits the production of tryptophan.In recent years,with the development of genetic engineering,metabolic engineering and directional evolution technology,people have successfully constructed the engineering strains of high yield L-trp,overexpressing the endogenous genes or introducing heterogenous genes to eliminate the competition branch.Escherichia coli（E.coli）is widely used in metabolic engineering to synthesize various valuable compounds because of its advantages of easy cultivation,clear genetic background and simple genetic operation.In this study,we engineered a genetically stable E.coli strain capable of efficiently producing L-trp,by applying more advanced rational metabolic engineering approaches within the chromosome.First,an E.coli KW strain,derived from E.coli W3110,was chosen as a host strain.It exhibited enhanced tryptophan production（0.28±0.04 g/L）due to multiple instances of random mutagenesis.Then,we constructed a basic strain that produced L-trp by overexpression of trpEDCBA and aroG^fbr in the strain KW.Next,we gradually inactivated trpR,ptsH,pykF/pykA,and the tryptophan attenuator with CRISPR/cas9 to improve the accumulation of intermediate metabolites.Finally,to improve glucose utilization,we modulated galP and glk,and repressed pta to reduce the accumulation of acetic acid.Our results suggested that the resulting strain,KW023,exhibited increased L-trp accumulation compared to the parental strain.KW023 efficiently produced 39.7 g/L of L-trp with a conversion rate of 16.7%and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.This study provides an important theoretical basis for the industrial production of L-trp by rational analysis and construction.