Evolutionary and reverse engineering of single vitamin requirement in Saccharomyces cerevisiae CEN.PK113-7D

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 requirement could be eliminated through directed evolution, this approach is still limited to biotin requirement. In this study, the CEN.PK113-7D vitamin requirements was 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 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 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 naive background strain. A limited number of mutations were necessary to reverse engineered the parent strain into a single vitamin prototroph revealing new aspect of vitamin metabolism and its regulation. These results pave to way towards design of cheap and entirely mineral media that would have to be complemented with a carbon and nitrogen source only.