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

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

Perli, T. (TU Delft BT/Industriele Microbiologie)
Moonen, Dewi P.I. (Student TU Delft)
van den Broek, M.A. (TU Delft BT/Industriele Microbiologie)
Pronk, J.T. (TU Delft BT/Biotechnologie)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)

BT/Biotechnologie

2020

Quantitative physiological studies on Saccharomyces cerevisiae 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 S. cerevisiae 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, para-aminobenzoic acid (pABA), 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 myo-inositol, nicotinic acid, or pABA. 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.

adaptive mutations
evolutionary engineering
media
nutritional requirements
prototrophy
reverse genetic analysis
Saccharomyces cerevisiae
vitamin biosynthesis

http://resolver.tudelft.nl/uuid:477d2241-8d3f-4e9a-8882-a41a5aa47bb0

https://doi.org/10.1128/AEM.00388-20

0099-2240

Applied and Environmental Microbiology, 86 (12)

Institutional Repository

journal article

© 2020 T. Perli, Dewi P.I. Moonen, M.A. van den Broek, J.T. Pronk, J.G. Daran