Data underlying the research of Evolutionary and reverse engineering of single vitamin requirement in Saccharomyces cerevisiae.

The popular Saccharomyces cerevisiae laboratory strain CEN.PK113-7D is regularly grown in chemically defined also referred to synthetic media (SM) that contain a set of organic compounds known as class B vitamins to support fast growth. While previous work showed how specific vitamin requirements could be eliminated through directed evolution, this approach is still limited to the biotin requirement. In this study, the CEN.PK113-7D vitamin requirements were evaluated and showed that growth was suboptimal upon omission of thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, inositol, or nicotinic acid. The S. cerevisiae strain was then evolved for fast growth in the absence of one of the vitamins. In all evolution lines, strains reached growth rates comparable to those of the strain grown in the presence of the vitamin. After few generations on medium without myo-inositol, nicotinic acid or pABA, CEN.PK113-7D exhibited fast prototrophic growth, conversely evolution for thiamine, pyridoxine and pantothenate had to be prolonged for over 300 generations. The genome of evolved single-colony isolates was re-sequenced and non-synonymous mutations identified. A subset of the mutations was selected and reintroduced using CRISPR/Cas9 in the naïve background strain. A limited number of mutations were necessary to reverse-engineer the parent strain into a single vitamin prototroph revealing a new aspect of vitamin metabolism and its regulation. These results pave the way towards the design of a cheap and entirely mineral media that would have to be complemented with a carbon source only. Measurement data collected and plotted in Figures 2, 3 and 7 are provided

目前广泛使用的酿酒酵母（Saccharomyces cerevisiae）实验室菌株CEN.PK113-7D，通常在添加了一类被称为B族维生素的有机复合物的化学限定培养基（chemically defined media），即合成培养基（synthetic media，缩写SM）中培养，以实现快速生长。此前的研究虽已证明可通过定向进化消除菌株对特定维生素的需求，但该方法目前仅适用于生物素依赖性状的改造。本研究对酿酒酵母CEN.PK113-7D的维生素需求进行了系统评估，结果显示，当培养基中分别缺失硫胺素（thiamine）、吡哆醇（pyridoxine）、对氨基苯甲酸（para-aminobenzoic acid）、泛酸（pantothenic acid）、肌醇（inositol）或烟酸（nicotinic acid）时，菌株的生长状态均未达到最优。随后，研究团队针对上述每一种维生素缺失的培养条件，对该菌株开展定向进化实验，以使其可在无对应维生素的培养基中实现快速生长。所有进化谱系的菌株最终均达到了与在完整维生素培养基中生长的亲本菌株相当的生长速率。其中，在不含肌醇、烟酸或对氨基苯甲酸的培养基中传代数代后，CEN.PK113-7D即可快速实现原养型生长；而针对硫胺素、吡哆醇和泛酸的定向进化则需延长至300代以上才能达成目标。研究团队对进化获得的单菌落分离株进行了全基因组重测序，成功鉴定出所有非同义突变位点。随后筛选出部分突变位点，通过CRISPR/Cas9基因编辑技术将其重新引入原始背景菌株中。实验结果表明，仅需少量突变即可将亲本菌株改造为单维生素原养型菌株，该发现揭示了维生素代谢及其调控机制的全新维度。本研究结果为开发低成本、完全无机的培养基奠定了基础，此类培养基仅需额外补充碳源即可使用。本文还提供了图2、图3及图7中所展示的实验测量数据。