Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid

Journal article (2021)

Authors

N. Baldi BT/Industriele Microbiologie - AS
S.C. de Valk BT/Industriele Microbiologie - AS
Maria Sousa-Silva University of Minho
Margarida Casal University of Minho
Isabel Soares-Silva University of Minho
R. Mans BT/Industriele Microbiologie - AS
Research Group
BT/Industriele Microbiologie (AS) (TU Delft)
Copyright: © 2021 N. Baldi, S.C. de Valk, Maria Sousa-Silva, Margarida Casal, Isabel Soares-Silva, R. Mans
DOI
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https://doi.org/10.1093/femsyr/foab033
Transport Reverse engineering Evolutionary engineering Carboxylic acids Protein structure
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Published Date

2021

Language

English

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Faculty

Applied Sciences

Department

BT/Biotechnologie

Research Group

BT/Industriele Microbiologie

Abstract

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.