Engineering the activity and specificity of Saccharomyces cerevisiae Acetate Transporter Ady2/Ato1

Organic acids are industrially relevant chemicals with application in polymer, food, agricultural and pharmaceutical sectors. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Since organic acid export over the plasma membrane represents one of the key steps in microbial production of these compounds, organic acid transporters started receiving greater attention in metabolic engineering strategies. Ato1 is the main transporter responsible for uptake of acetic acid into the cytosol in S. cerevisiae, while also being able to mediate organic acid transport in the opposite direction, as it was shown to be involved in the export of lactic acid from S. cerevisiae cells engineered for lactic acid production. Ato1 is a member of the Acetate Uptake Transporter Family (AceTR), with several functionally characterized homologues in yeast, fungi, and bacteria. Recently solved crystal structure of its bacterial homologue, SatP, depicts a hexameric anion channel. In this work, we studied the relationship between structure and function of Ato1 via rational mutagenesis and identified residues critical for Ato1 substrate specificity and transport activity. By utilizing computer-assisted three-dimensional modelling tools, we provide possible explanations of acquired features. Our final goal is to test applicability of these transporters in yeast cell factories that produce organic acids.Supported by strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and TransAcids (PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood (NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement. TC acknowledges Yeastdoc European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 764927

Engineering of AceTr membrane transporters to improve organic acid production in yeast

Organic acids are industrially relevant chemicals obtainable from renewable feedstocks via microbial cell factories. Microbially produced organic acids have a wide variety of applications, including bioplastic synthesis. Thus, they possess the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for microbial production of organic acids, namely due to their tolerance of low pH environments. Such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in low pH conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Our current objective is to deepen the knowledge on organic acid transporters from the AceTR family (1,2,3). We performed functional characterization by studying transporter kinetics, energetics and specificity as well as site-directed mutagenesis to acquire insight into the structural features of transporters. Finally, we aim to improve organic acid production in S. cerevisiae cell factories via expression of engineered AceTR transporters with altered activity and substrate specificity.UID/BIA/04050/2013(POCI-01-0145-FEDER-007569) and TransAcids(PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood(NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement.TCacknowledgesYeastdocEuropean Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curie grant agreement No 76492

Exploring plasma membrane transporters to improve organic acid production in yeast – Characterization and engineering

Organic acids are industrially relevant building-block chemicals obtainable from renewable feedstocks by utilization of microbial cell factories. With a wide variety of applications, including bioplastics synthesis, microbially produced organic acids have the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments, since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in such conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Here, we are characterizing and engineering plasma membrane transporters with the final aim to improve production of dicarboxylic acids, namely succinic acid, in yeast. This includes transporters that have already been described as efficient dicarboxylate transporters, as well as promising transporters from the AceTr family. First, we perform functional characterization by studying transporter kinetics, energetics and specificity, as well as site-directed mutagenesis, to acquire insight into functional-structural relationship of transporters. This insight further uncovers engineering targets that can lead to improved transporter activity as well as altered substrate specificity. Finally, the performance of these transporters can be assessed via their expression in S. cerevisiae that is engineered for succinic acid production.This work was supported by the strategic programme UID/BIA/ 04050 2019 funded by Portuguese funds through the FCT IP, the project TransAcids (PTDC/ 5184 2014 funded by FCT IP and ERDF by COMPETE 2020 POCI and the project EcoAgriFood (NORTE 01 0145 FEDER 000009 supported by NORTE 2020 under the PORTUGAL 2020 Partnership Agreemen

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