Efficient expression of cyclodextrin glycosyltransferase from Geobacillus stearothermophilus in Escherichia coli by promoter engineering and downstream box evolution

Cyclodextrin glycosyltransferase (CGTase) catalyzes hydrolysis, cyclization, coupling, and disproportionation reactions and is widely used in the starch processing industry. In this work, the expression of CGTase from Geobacillus stearothermophilus in Escherichia coli BL21 (DE3) was significantly improved by promoter engineering and downstream box evolution. Firstly, the effects of the promoter type (PT7, Ptrp, PlacUV5, and the hybrid promoters PtacI and PtacII) and spacer sequence on the expression of CGTase were examined. PtacI demonstrated the highest rate of transcriptional activity, which was 4.4-, 7.1-, 3.3-, and 1.5-fold greater than that of PT7, Ptrp, PlacUV5, and PtacII, respectively. The spacer sequence of the promoter was optimized using a degenerate base library, and the GC content of the spacer was found to be inversely proportional to CGTase expression. In addition, CGTase expression was higher when TG:CA and TA:TA dimers were present in the spacer sequence. Under the control of the PtacI promoter with an optimized spacer sequence, extracellular CGTase activity reached 170.6 U/mL, which was seven times higher than that of the original strain (25.2 U/mL). Directed evolution of the downstream box sequence was then performed by randomization of the sequence using degenerate codons, similarly as for the optimization of the spacer sequence. After optimizing the downstream box sequence, CGTase activity increased from 170.6 to 214 U/mL. The results obtained here indicate that in addition to promoter type, the spacer sequence of the promoter and the downstream box are important target elements for improved gene expression.