Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands.
Centre of Molecular and Environmental Biology (CBMA), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
FEMS Yeast Res. 2021 Jun 8;21(4). doi: 10.1093/femsyr/foab033.
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 ATO3T284C 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.
在酿酒酵母中，完整的参与乳酸跨膜运输的蛋白质组尚未确定。在这项研究中，我们旨在通过定向进化、全基因组重测序和反向工程的组合，鉴定以前未描述的参与乳酸运输的转运蛋白。在乳酸上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 是两种新的乳酸转运蛋白。与先前鉴定的参与乳酸运输的酿酒酵母基因相比，ATO3T284C 的表达能够在这种碳源上实现最高的生长速率（0.15±0.01 h-1）。转运蛋白的（进化）序列和 3D 模型之间的比较表明，大多数鉴定的突变导致阴离子通道最窄的疏水性收缩变宽。我们假设这种观察结果，有时与乳酸与该收缩相邻的结合部位的结合亲和力增加相结合，是进化后的蛋白质中乳酸转运能力提高的原因。
