Construction of dynamic down-regulation systems in Clostridium tyrobutyricum for butyrate production

Abstract

The acetate biosynthesis pathway is an important component of central carbon metabolism, providing adenosine triphosphate (ATP) and acetate for cell growth. However, enhanced acetate production tends to be toxic to the cell and inhibits butyrate production by Clostridium tyrobutyricum. In this study, we evaluated the expression strength of 22 endogenous promoters, whose expression levels ranged from 4.4 % to 121 % of the strong promoter P_thl. By integrating the endogenous promoters with 8 degradation tag mutants, we obtained the optimal combination of P_sigK-LAA+ 4 after evaluating the constructed dynamic down-regulation systems. P_sigK-LAA+ 4 was used for the dynamic downregulation of the acetate branch pathway to improve butyrate production. Although the titers, yields and selectivity of butyrate production were not improved significantly, productivities of P_sigK-pta-ack-LAA+ 4 and P_sigK-pta-LAA+ 4-ack were increased by 84 % and 121 %, due to the shortened fermentation period and higher tolerance to acetate. Additionally, downregulating the acetate branch pathway in the logarithmic growth phase and overexpressing key enzymes involved in the acetate branch pathway in the stationary phase could effectively mitigate acetate toxicity and improve butyrate production. To our knowledge, this study is the first to report the dynamic downregulation systems in C. tyrobutyricum, providing potential regulation strategies for biofuel production in the future.