Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA.
Metab Eng. 2012 Jul;14(4):401-11. doi: 10.1016/j.ymben.2012.03.004. Epub 2012 Mar 18.
Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved V(max) values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.
传统的代谢途径工程很少考虑分子运输的影响。在这里，我们描述了两种异源转运蛋白 Candida intermedia GXS1 和 Scheffersomyces stipitis XUT3 的定向进化。通过突变体转运蛋白的表达，木糖的生长速率提高了 70%。大多数突变体被发现表现出大大提高的 V(max) 值，并显示出高细胞密度糖消耗率的增加。混合葡萄糖和木糖发酵表明，突变体转运蛋白可以改变酵母的双相转换动力学和宿主菌株的同时糖利用能力。突变分析突出了几个影响转运蛋白功能的重要残基，包括 Candida intermedia GXS1 的 F40 点突变和 S. stipitis XUT3 的 E538 点突变。这项工作首次证明，通过在酵母中的定向进化，可以改善分子转运蛋白，用于生物技术应用。
