Abstract

In vitro protein expression has gained considerable attention due to its experimental flexibility and time efficiency. This method employs essential transcriptional components and is particularly advantageous for expressing proteins that are challenging to produce in living systems, such as membrane or toxic proteins. Membrane acetate transporters regulate cellular acetate levels and are important for microbial engineering in industrial fermentation processes. In this study, the succinate–acetate transporter protein SatP from Escherichia coli was functionally expressed utilizing a cell-free system and subsequently characterized through single-channel recordings. Beyond confirming its transport activities, we identified malate and fumarate as additional potential substrates of SatP. The successful in vitro expression of SatP lays the foundation for cell-free screening of acetate transporters. We then further investigated a preliminary screening assay and proposed a potential strategy for the rapid expression and functional characterization of acetate transport proteins. Because acetate accumulation can inhibit microbial growth and reduce fermentation efficiency, a better understanding of SatP-mediated acetate transport may facilitate the engineering of microbial strains with enhanced acetate tolerance and improved product yields. This knowledge provides a foundation for the rational optimization of industrial fermentation processes.

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