Construction and Optimization of Malonyl-CoA Sensors in Saccharomyces cerevisiae by Combining Promoter Engineering Strategies

Abstract

Biosensors can be used for high-throughput screening, real-time monitoring of metabolites, and dynamic regulation of metabolic processes, which have been a popular research direction in recent years. Here, five promoters from Saccharomyces cerevisiae were selected to construct Malonyl-CoA sensors with the fapO/fapR system derived from Bacillus subtilis, and pCCW12 was finally selected for further optimization. Based on pCCW12, a series of sensors with different response sensitivities were obtained by selecting different fapO insertion sites and combining the best two or three of them. Then, through a combination of promoter hybrid, intron insertion, and transcription factor modification strategies, we obtained sensors with different effects, one of which, the H-pCCW12(TFBS)-Cti6~fapR sensor, had the lowest background noise, doubled response range and higher response sensitivity compared to the original sensor. Sensors with different characteristics constructed in this study, can be applied to Malonyl-CoA related high-throughput screening and finer regulation of metabolism. It also proves that the combined application of different promoter engineering strategies is a feasible idea for the precise construction and regulation of biosensors.