Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase

Abstract

Restriction fragments containing the gene encoding cyanase, cynS, without its transcriptional regulatory sequences were placed downstream of lac and tac promoters in various pUC derivatives to maximize production of cyanase. Plasmid pSJ105, which contains the cynS gene and an upstream open reading frame, gave the highest expression of cyanase. Approximately 50% of the total soluble protein in stationary-phase cultures of a lac-deleted strain containing plasmid pSJ105 was cyanase. The inserted DNA fragment of pSJ105 was transferred into pUC18 derivatives that contain a hybrid tac promoter, instead of the lac promoter, and a strong terminator to generate pSJ124. Stationary-phase cultures of JM101 containing plasmid pSJ124 overexpressed a similar level of cyanase. In JM101(pSJ124), maximum production of cyanase could be obtained either by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h or by growth without IPTG into late stationary phase. The latter conditions resulted in a 10- to 20-fold increase in plasmid content and presumably titration of the lac repressor. The nucleotide sequence of the cloned cynS gene from Escherichia coli K-12 was determined. The predicted amino acid sequence differed from the known amino acid sequence of cyanase isolated from a B strain by four residues. However, overexpressed cyanase was purified to homogeneity, and a comparison of the enzymes from the two sources indicated that they did not differ with respect to physical and kinetic properties. The cynS gene was located next to the lac operon, and the direction of cynS transcription was opposite that of lac.