In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3^T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h^-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.

中文翻译：

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进化工程揭示了 Ato2 和 Ato3 中的氨基酸取代，这使得酿酒酵母在乳酸上的生长得到改善

在Saccharomyces cerevisiae 中，尚未确定参与乳酸跨细胞膜转运的完整蛋白质组。在这项研究中，我们旨在通过定向进化、全基因组重测序和逆向工程的组合来鉴定以前未描述过的参与乳酸转运的转运蛋白。缺乏所有已知乳酸转运蛋白的菌株的进化导致发现突变的 Ato2 和 Ato3 作为两种新型乳酸转运蛋白。与先前鉴定的参与乳酸转运的酿酒酵母基因相比，ATO3 ^{T284C 的}表达能够促进最高的生长速率 (0.15 ± 0.01 h ^-1) 在这个碳源上。（进化的）序列与转运蛋白的 3D 模型之间的比较表明，大多数已识别的突变导致阴离子通道最窄的疏水收缩变宽。我们假设这一观察结果，有时与乳酸与该收缩附近位点的结合亲和力增加相结合，是进化蛋白质中乳酸转运改善的原因。