Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae.

Quantitative physiological studies on commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, -aminobenzoic acid (ABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without -inositol, nicotinic acid, or ABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. Many strains of , a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, -aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.