Engineering Escherichia Coli For Production Of Tryptophan From Glucose Via Rational Genetic Manipulations

Tryptophan is one of aromatic amino acids.Due to it with specific physiological function and chemical structure,tryptophan is widely applied to food,pharmaceuticals,cosmetics and other fields.Consequently,the demand for tryptophan is increasing.According to a report of market analysis and investment value of Chinese L-tryptophan from LIMU Information Consultant Company,the world market needs more 2000 t tryptophan and the quantity demanded for tryptophan increases 10 % every year.The engineered Escherichia coli KW001 served as an original strain.A strain which was able to effectively produce tryptophan was successfully constructed via a series of rational genetic manipulations.Firstly,to optimize of glutamine synthesis,heterologous glutamine synthetases from Bacillus subtilis and Bacillus megaterium was respectively expressed in engineered E.coli KW001.The best candidate,strain TS-1 which expressed Bacillus subtilis’ glutamine synthetase,could produce 0.810 g/L tryptophan.Subsequently,the overexpression of native icd and gdh A was to enhance nitrogen flux and strengthen glutamine synthesis,resulting in TS-5 strain which produced 1.060 g/L tryptophan.Secondly,prs was overexpressed by a ptrc promoter to increase PRPP amounts,and mutated ser A and thr A were expressed to optimize the supplement of serine in strain TS-5.The resulting strain,TS-8,could produce 1.380 g/L tryptophan.The expression of DHQ-SDH enzyme from Populus trichocarpa in strain TS-8 was to short shikimate pathway.But the manipulation did not make no different for the improvement of tryptophan production.To further increase tryptophan production,the overexpression of sth A and pnt AB in strain TS-8 balanced cofactor metabolism,which resulted in strain TS-10.It could produce 1.710 g/L tryptophan.Finally,more phenylalanine was accumulated in the fermentation broth deriving from above mentioned engineered strains.As a result,the expression of phe A was weakened by a feeble promoter(J23119)and RBS to decrease phenylalanine synthesis in strain TS-10.The resulting strain,TS-11,could produce 1.820 g/L tryptophan.Compared with the original strain KW001,tryptophan production increased by 2.93 times.Taken together,our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan.Subsequently,weakening the biosynthesis of phenylalanine positively worked for further improving tryptophan production as well.