New insights into the acetate uptake transporter (AceTr) family: Unveiling amino acid residues critical for specificity and activity

Aiming at improving the transport of biotechnologically relevant carboxylic acids in engineered microbial cell factories, the focus of this work was to study plasma membrane transporters belonging to the Acetate Uptake Transporter (AceTr) family. Ato1 and SatP, members of this family from Saccharomyces cerevisiae and Escherichia coli, respectively, are the main acetate transporters in these species. The analysis of conserved amino acid residues within AceTr family members combined with the study of Ato1 3D model based on SatP, was the rationale for selection of site-directed mutagenesis targets. The library of Ato1-GFP mutant alleles was functionally analysed in the S. cerevisiae IMX1000 strain which shows residual growth in all carboxylic acids tested. A gain of function phenotype was found for mutations in the residues F98 and L219 located at the central constrictive site of the pore, enabling cells to grow on lactic and on succinic acid. This phenotype was associated with an increased transport activity for these substrates. A dominant negative acetic acid hypersensitivity was induced in S. cerevisiae cells expressing the E144A mutant, which was associated with an increased acetic acid uptake. By utilizing computer-assisted 3D-modelling tools we highlight structural features that explain the acquired traits found in the analysed Ato1 mutants. Additionally, we achieved the proper expression of the Escherichia coli SatP, a homologue of Ato1, in S. cerevisiae. To our knowledge, this constitutes the first report of a fully functional bacterial plasma membrane transporter protein in yeast cells.This work was supported by the strategic programme UID/BIA/04050/2019 funded by Portuguese funds through the FCT-IP, the project TransAcids (PTDC/BIAMIC/5184/2014) funded by FCT-IP and ERDF by COMPETE 2020-POCI and the project River2Ocean NORTE-01-0145-FEDER-000068, co-financed by the European Regional Development Fund (ERDF) , through Programa Opera-cional Regional do Norte (NORTE 2020) as well as the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Yeastdoc grant agreement No 764927