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.

质子耦合通过 Mal11 将 α-葡萄糖苷转运到酿酒酵母中每个进口分子需要一个 ATP。活性位点中所有三个酸性残基的靶向突变导致糖单转运，但这些突变转运蛋白在酵母中的表达不能在蔗糖上生长。然后，我们通过酵母的定向进化分离出这些突变体的六种独特的转运蛋白变体，以在蔗糖上生长。在三个变体中，新的酸性残基出现在恢复质子耦合蔗糖转运的活性位点附近，而其他进化的转运蛋白仍然催化蔗糖单转运。突变的定位和突变体的转运特性使我们能够提出 Mal11 的质子耦合糖转运机制模型。在厌氧条件下培养表达蔗糖单向转运蛋白之一的酵母菌株，当考虑到菌株生理学的其他变化时，蔗糖限制的恒化器培养表明蔗糖异化效率提高了 21%。因此，我们表明，定向工程和进化工程的结合可以产生更节能的蔗糖运输，作为设计酵母菌株以提高工业相关产品产量的起点。