Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies inSaccharomyces cerevisiae

Abstract

<jats:title>Abstract</jats:title><jats:p>Quantitative physiological studies on <jats:italic>Saccharomyces cerevisiae</jats:italic> 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 <jats:italic>S. cerevisiae</jats:italic> CEN.PK113-7D, requirements for biotin could be 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, <jats:italic>para</jats:italic>-aminobenzoic acid (<jats:italic>p</jats:italic>ABA) or thiamine were 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 <jats:italic>myo</jats:italic>-inositol, nicotinic acid or <jats:italic>p</jats:italic>ABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Many strains of <jats:italic>Saccharomyces cerevisiae</jats:italic>, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, <jats:italic>para</jats:italic>-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 phenotype 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 towards development of <jats:italic>S. cerevisiae</jats:italic> strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.</jats:p></jats:sec>

Unlocking the functional potential of polyploid yeasts

“…The RTG approach, coupled with selection based on natural phenotypes generates non-GMO yeast strains that can be unrestrictedly introduced into the market. Compared to other non-GMO approaches such as serial transfer 28,29 , RTG induces a random genome shuffling as it does not select for a specific trait except for the genetic loci regulating the natural phenotype selected. Nevertheless, RTG has several benefits over the previous improvement strategies.…”

Innovative Tools and Strategies for Optimizing Yeast Cell Factories

“…In particular, ALE allows overcoming numerous physiological limitations (i.e. thermotolerance, osmotic stress, low pH, toxicity, etc) [48,[84][85][86][87][88][89][90][91][92] as well as to complement the rational engineering approaches presented in the above sections, and providing new targets for the next round of rational design [64,[83][84][85][86][92][93][94][95][96][97][98][99][100][101][102]. Noteworthily, the use of CRISPR/Cas9 system in combination with ALE approach enabled the generation of evolved YCF with improved thermotolerance, as mentioned in the "Genome editing" section of this review [48].…”

Sexual Dimorphism through the Lens of Genome Manipulation, Forward Genetics, and Spatiotemporal Sequencing

“…These limitations can be avoided by using transgenics tools only after E&R to verify candidate genes. For example, CRISPR (box 2) can replace a haplotype in the ancestral background with the evolved haplotype ( Perli et al. 2020 ).…”