Genetic identification of acetyl-CoA synthetases involved in acetate activation in <i>Haloferax mediterranei</i>

Owing to the high demand and supply challenge of glucose, acetate might be considered a potential alternative carbon source for microbial growth and fermentation. <i>Haloferax mediterranei</i> is capable of utilizing acetate as a carbon source for growth and subsequent value-added product synthesis. Thus, it is essential to identify the genes responsible for acetate utilization in <i>H. mediterranei</i>. As per available literature, haloarchaeal ADP-forming acetyl-CoA synthetase (APD-ACS) catalyzes the reversible conversion of acetate to acetyl-CoA <i>in vitro</i>. However, <i>in vivo</i>, acetate activation and acetate formation are catalyzed by AMP-forming acetyl-CoA synthetase (AMP-ACS) and ADP-ACS, respectively. In this study, we have identified six AMP-ACS enzymes that catalyzed acetate activation in <i>H. mediterrane</i>i. Deletion of these six genes abolished the growth of the resulting mutant (Δ6AMP-ACS) in acetate medium. The natively expressed ADP-ACS was unable to mediate its acetate activation <i>in vivo</i>. Interestingly, an artificial system based on plasmid overexpression of ADP-ACS in Δ6AMP-ACS restored its growth on acetate. This finding suggested that native ADP-ACS was unable to catalyze acetate activation in <i>H. mediterranei</i> due to its low expression level. Together, our study explored the acetate activation in <i>H. mediterranei,</i> and the obtained results would enrich the knowledge of acetate metabolism in archaea. Furthermore, the information offered in this study would benefit the improvement of acetate utilization in haloarchaea for value-added product synthesis.