The Molecular Breeding Of Tryptophan Industrial Producer Based On Whole Genomics Sequencing

L-Tryptophan is an essential amino acid which has been widely used in pharmaceuticaland food industry. Escherichia coli is used as the dominant industrial producers ofL-tryptophan with its advantage of robust growth and the perfect of genetic operating system.With the application of genetic and metabolic engineering, it is paid attention that theconstruction of high-level L-tryptophan by means of molecular biological approach. Based onthe sequencing of L-tryptophan production strain.We used the molecular biological approachto modificating L-tryptophan pathway to improve L-tryptophan production. The maincontents of this study were as follows:（1）Based on whole-genome sequencing of Escherichia coli try and analysis of thetryptophan synthesis, trpR gene of the tryptophan repression protein, tnaB gene in theL-tryptophan uptake system were deficiency in the genome of Escherichia coli try. The ANTAsynthase encoded by trpE was mutanted by replacing the residue Ala63with Val.（2）Based on whole genome sequencing of Escherichia coli try, we applied theapproach of the SacB homologous recombination system to release the feedback inhibition ofDAHP synthase encoded by aroG and aroF, and ANTA synthase encoded by trpE by themodification of point mutation including the residue Asp146to Asn in aroG, the residue Pro148to Leu in aroF and the residue Met293to Thr in trpE. This construction strain wasnamed as Escherichia coli try SS-02. After24h fermentation, the strain Escherichia coli trySS-02produced0.2g/L L-tryptophan,0.98g/L L-tyrosine and4.3g/L L-phenylalanine.Theproduction ratio of three amino acids were increasing by80%,88%and60%respectivelycomparing with that of the original strain Escherichia coli try.（3）Based on the production strain Escherichia coli try SS-02with feedback inhibitionreleasing of DAHP synthase and ANTA synthase in L-tryptophan pathway, the attention oftryptophan operon was further removed by the means of Red recombination system to knockout the sequences from10to118of trpL.And then the competitive ways of L-tyrosine andL-phenylalanine were cut off by knocking out the tyrA and pheA gene. Finally the high-levelL-tryptophan producing strain was constructed as Escherichia coli try SS-05. After24hfermentation, the strain Escherichia coli try SS-05produced0.395g/L L-tryptophan. Compare with original strain Escherichia coli try, the production of L-tryptophan was increased2.7times.