Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter

Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter

Marques, W.L. (TU Delft BT/Industriele Microbiologie; University of Campinas)
van der Woude, L.N. (TU Delft Science Education and Communication)
Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie)
van den Broek, M.A. (TU Delft BT/Industriele Microbiologie)
Nijenhuis, J.M. (TU Delft Applied Sciences)
Pronk, J.T. (TU Delft BT/Biotechnologie)
van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie)
Mans, R. (TU Delft BT/Industriele Microbiologie)
Gombert, Andreas K. (University of Campinas)

Applied Sciences

BT/Biotechnologie

2018

Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (μ = 0.19 h⁻¹ and μ = 0.08 h⁻¹, respectively). All five mutants displayed sucrose-induced proton uptake (13–50 μmol H⁺ (g biomass)⁻¹ min⁻¹). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1^{I209F C265F G326C}) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h⁻¹) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.

laboratory evolution
plant sucrose facilitator
plant transporter expression
sucrose uptake
yeast physiology

http://resolver.tudelft.nl/uuid:48b2d0d8-338c-404b-8e97-47109b8615c7

https://doi.org/10.1002/yea.3357

2019-09-16

0749-503X

Yeast

Institutional Repository

journal article

© 2018 W.L. Marques, L.N. van der Woude, M.A.H. Luttik, M.A. van den Broek, J.M. Nijenhuis, J.T. Pronk, A.J.A. van Maris, R. Mans, Andreas K. Gombert