Expression of ATP-binding cassette transporter proteins AatA or MdlB facilitates butyric acid production in Clostridium tyrobutyricum

Abstract

ATP-binding cassette (ABC) transporters, one of the largest super families of membrane-associated microbial proteins involved in the transport of a wide range of substrates, play a vital role in bacterial physiology. In this study, we investigated the utility of heterologous expression of the ABC transporter AatA from Acetobacter pasteurianus AC2005 and homologous overexpression of the ABC transporter protein MdlB to improve butyric acid production in Clostridium tyrobutyricum. Compared to the starting wild-type strain C. tyrobutyricum ATCC 25755, recombinant strains of C. tyrobutyricum/aatA and C. tyrobutyricum/mdlB showed increased butyrate yield, productivity, selectivity, and titer during fed-batch fermentation. qRT-PCR revealed that AatA and MdlB upregulated the expression levels of ack, the gene encoding a key enzyme in the acetate synthesis pathway. This result was consistent with the observed increase in acetate titer in the recombinant strains. Moreover, overexpression of AatA or MdlB promoted the synthesis and secretion of acetic acid. An assay of cellular ATPase activities established that the two recombinant strains demonstrated two-folder higher ATPase activities compared to the wild-type strain. This increase in ATPase activity is proposed to accelerate sugar utilization, induce extracellular secretion of acetate for butyrate production, and shorten fermentation periods to improve butyrate productivity.