Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter. Saccharomyces cerevisiae strain:IMZ630

Alteration of the energy coupling of sugar transporters can improve product yields in microbial biotechnology. Substitution of the native pathway for sucrose metabolism in Saccharomyces cerevisiae by uptake via a plant sucrose uniporter (Phaseolus vulgaris sucrose facilitator 1) combined with intracellular phosphorolysis (by Leuconostoc mesenteroides sucrose phosphorylase) could increase the ATP yield of anaerobic sucrose fermentation by 25 %. However, recent attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, such PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately two-fold higher anaerobic growth rate in 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 into an unevolved strain enabled it to grow in sucrose at the same rate (0.19 h-1) as the corresponding evolved strain. This study shows how laboratory evolution can be used to improve the uptake of sucrose via heterologous plant transporters and reveal relevant residues for their efficient heterologous expression.