Abstract:

In Escherichia coli， tnaA gene encodes tryptophanase and it′s inactivation contributes to the L-tryptophan accumulation in E.coli. In the present study, CRISPR/Cas9-mediated gene editing technology was used to knockout the tnaA gene of E.coli. Sequence targeted by CRISPR/Cas9 was designed and gRNA expression plasmid was constructed. Donor DNA for homologous repair was constructed. Donor DNA, gRNA expression plasmid and pCas plasmid co-constituted the CRISPR/Csa9 system. This system was performed on E.coli w3110-Δ. PCR and sequencing results confirmed the successful knockout of tnaA gene with the knockout efficiency of 75%. In conclusion, CRISPR/Cas9-mediated knockout system for E.coli tnaA gene was successfully established, which would provide an effective tool for strain construction for L-tryptophan production.

Key words: CRISPR/Cas9 system； knockout, Escherichia coli； tnaA gene