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

Abstract

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. IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-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 S. cerevisiae 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.

Improvement of macrolactins production by the genetic adaptation of Bacillus siamensis A72 to saline stress via adaptive laboratory evolution

“…Of the colonies, 7 of 51 (13.7%) showed a flat, rough, and serrated edge, which is similar to A72, and 44 of 51 (86.4%) were smooth, moist and viscous, milky white. Using ALE to engineer an interesting phenotype, bigger-sized colonies on the screening plates were usually selected [ 31 ]. The final concentration of stress factor used for the ALE experiment has usually been used for selecting desired mutants (e.g., caffeine- or ethanol-resistant strains) from the evolved population [ 25 , 32 ], which leads to our belief that it is easier to select the best-performing mutants at a higher saline level.…”

Unlocking the functional potential of polyploid yeasts

“…The RTG, 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 32 , 33 , RTG induces genome shuffling of multiple loci and it does not select for a specific trait except for the genetic loci regulating the selected colony phenotype. The genome shuffling occurs in association with meiotic hotspots and does not select specific parental alleles 20 .…”

Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast

“…Pantothenate and nicotinate, together with the other compounds belonging to the B-group of water-soluble vitamins, are standard ingredients of chemically defined media for aerobic and anaerobic cultivation of yeasts (62). S. cerevisiae strains have been shown to contain the genetic information required for de novo synthesis of these vitamins and can even be experimentally evolved for complete prototrophy for individual vitamins by prolonged cultivation in single-vitamin-depleted media (63,64). In large-scale processes, addition of nutritional supplements increases costs, reduces shelf-life of media, and increases the risk of contamination during their storage (62).…”