Construction Of Recombinant Coryitebacterium Glutamicum Producing L-tryptophan And Preliminary Studies On Its Fermentation Conditions

L-tryptophan is an essential amino acid of humans and animals, which has been widelyused in pharmaceutical, food and feed industry. C. glutamicum SPT9（4-FP~r、6-FT~r、Phe~-、Tyr~-）is a phenylalanine-and tyrosine-auxotrophic mutant strain, in which the feedbackregulations of aromatic amino acids were removed. In order to improve the production oftryptophan, The csm gene was deleted, the inducible promoter of the expression plasmidpEC-XK99E was replaced and the trpBA gene was over-expressed. The specific contents ofthis study are as follows:(1) Suicide vector pK18mobsacB-Δcsm was constructed by crossover PCR and then itwas transferred into SPT9by electroporation to obtain csm gene deficient mutant SPT9Δcsm.After72h fermentation, the L-tryptophan yield in broth of the recombinant strain SPT9Δcsmwas0.38g/L, increasing by18.75%comparing with the original strain SPT9.The fluorescence quantitative PCR analysis indicated that the csm gene was not expressed inSPT9Δcsm.(2) The P-cspB promoter sequence was synthesised to replace the inducible promoterPtrc of the expression vector pEC-XK99E, the resulted expression vector containing a strongpromoter was designated pEC-P-cspB.(3) C. glutamicum-E. Coli shuttle expression vectors pEC-XK99E and pEC-P-cspB wereemployed to over-expressed trpBA gene in SPT9Δcsm, the recombinant strains weredesignated as SPT9csm-pEC-trpBA and SPT9csm-P-cspB-trpBA. After72h fermentation,the L-tryptophan yield in broth of the two recombinant strains were0.45g/L,0.49g/L,increasing by18.4%,28.9%respectively comparing with SPT9Δcsm. The fluorescencequantitative PCR analysis indicated that the expressions of trpBA gene inSPT9csm-pEC-trpBA and SPT9csm-P-cspB-trpBA were2.6，3.4times respectively ofthat in strain SPT9Δcsm.