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

Author:

Baldi Nicolò1,de Valk Sophie Claire1ORCID,Sousa-Silva Maria23ORCID,Casal Margarida23,Soares-Silva Isabel23ORCID,Mans Robert1

Affiliation:

1. Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands

2. Centre of Molecular and Environmental Biology (CBMA), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

3. Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Abstract

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.

Funder

Research and Development

FCT

European Regional Development Fund

ERDF

Publisher

Oxford University Press (OUP)

Subject

Applied Microbiology and Biotechnology,General Medicine,Microbiology

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