Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

将木糖有效转化为具有成本效益的生物燃料的瓶颈限制了植物木质纤维素作为可再生原料的广泛使用。酵母酿酒酵母将葡萄糖发酵成乙醇，代谢通量如此之高，以至于它在有氧条件下发酵高浓度的葡萄糖，这种特性称为 Crabtree/Warburg 效应。与葡萄糖相比，大多数工程化酿酒酵母菌株不会以经济可行的速率和产量发酵木糖，并且它们需要呼吸来实现足够的木糖代谢通量和有氧生长所需的能量返回。在这里，我们进化出了呼吸缺陷型酿酒酵母可以在有氧条件下生长并发酵木糖为乙醇的菌株，这种特性类似于葡萄糖的 Crabtree/Warburg 效应。通过基因组序列比较和定向工程，我们确定编码工程木糖代谢酶的基因以及TKL1（一种编码戊糖磷酸途径中转酮醇酶的基因）的重复是进化表型的致病基因变化。重新设计这些酶的重复，结合HOG1、ISU1、GRE3和IRA2 中的缺失突变，提高了需氧和厌氧木糖发酵的速率。重要的是，我们发现这些基因修饰在另一个遗传背景中起作用，并提高了工业相关柳枝稷水解物中木糖转化为乙醇的速率和产量，表明这些特定的基因修饰可能使酵母可持续生产工业生物燃料成为可能。我们提出了一个模型，说明关键调控突变如何通过降低溢出代谢的阈值来引导酵母进行有氧木糖发酵，允许突变增加木糖通量并将其重定向到发酵产物中。