Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Henderson, Ryan K. (University Medical Center Groningen)
de Valk, Sophie C. (Student TU Delft)
Poolman, Bert (University Medical Center Groningen)
Mans, R. (TU Delft BT/Industriele Microbiologie)

2020

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products.

ATP
Chemostat
Directed evolution
Energy coupling
Membrane transport
Microbial bioenergetics
Substrate uptake
Sucrose

http://resolver.tudelft.nl/uuid:bab59bcc-84df-47d7-9d7c-87f112e43fca

https://doi.org/10.1016/j.ymben.2020.11.014

1096-7176

Metabolic Engineering, 65, 243-254

Institutional Repository

journal article

© 2020 Ryan K. Henderson, Sophie C. de Valk, Bert Poolman, R. Mans