Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production

Abstract Clostridium tyrobutyricum is an anaerobe known for its ability to produce short‐chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine‐tuning gene expression in C. tyrobutyricum . Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCa thl ) from Clostridium acetobutylicum , with the best one (LacI‐Pto4s) showing a 5.86‐fold dynamic range with isopropyl β‐ d ‐thiogalactoside (IPTG) induction. A LT‐Pt7 system with a dynamic range of 11.6‐fold was then created by combining LacI‐Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7 lac promoter. Furthermore, two inducible expression systems BgaR‐PbgaLA and BgaR‐PbgaLB with a dynamic range of ~40‐fold were developed by optimizing a lactose‐inducible expression system from Clostridium perfringens with modified 5′ untranslated region (5′ UTR) and ribosome‐binding site (RBS). BgaR‐PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adh E2 and butyryl‐CoA/acetate Co‐A transferase encoded by cat 1 in C. tyrobutyricum wild type and Δ cat 1:: adh E2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat 1 expression also confirmed that the Cat1‐catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum . The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.