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Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

ABSTRACT: In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn₂C₆ transcription factors YRR1 and YRM1 (p < 1 × 10^-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

SUBMITTER: Ottilie S

PROVIDER: S-EPMC8837787 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

Ottilie Sabine S Luth Madeline R MR Hellemann Erich E Goldgof Gregory M GM Vigil Eddy E Kumar Prianka P Cheung Andrea L AL Song Miranda M Godinez-Macias Karla P KP Carolino Krypton K Yang Jennifer J Lopez Gisel G Abraham Matthew M Tarsio Maureen M LeBlanc Emmanuelle E Whitesell Luke L Schenken Jake J Gunawan Felicia F Patel Reysha R Smith Joshua J Love Melissa S MS Williams Roy M RM McNamara Case W CW Gerwick William H WH Ideker Trey T Suzuki Yo Y Wirth Dyann F DF Lukens Amanda K AK Kane Patricia M PM Cowen Leah E LE Durrant Jacob D JD Winzeler Elizabeth A EA

Communications biology 20220211 1

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription facto ...[more]

PMID: 35149760

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Project description: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.

Project description:BackgroundLignocellulose is a renewable and sustainable resource used to produce second-generation biofuel ethanol to cope with the resource and energy crisis. Furfural is the most toxic inhibitor of Saccharomyces cerevisiae cells produced during lignocellulose treatment, and can reduce the ability of S. cerevisiae to utilize lignocellulose, resulting in low bioethanol yield. In this study, multiple rounds of progressive ionizing radiation was combined with adaptive laboratory evolution to improve the furfural tolerance of S. cerevisiae and increase the yield of ethanol.ResultsIn this study, the strategy of multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution significantly improved the furfural tolerance of brewing yeast. After four rounds of experiments, four mutant strains resistant to high concentrations of furfural were obtained (SCF-R1, SCF-R2, SCF-R3, and SCF-R4), with furfural tolerance concentrations of 4.0, 4.2, 4.4, and 4.5 g/L, respectively. Among them, the mutant strain SCF-R4 obtained in the fourth round of radiation had a cellular malondialdehyde content of 49.11 nmol/mg after 3 h of furfural stress, a weakening trend in mitochondrial membrane potential collapse, a decrease in accumulated reactive oxygen species, and a cell death rate of 12.60%, showing better cell membrane integrity, stable mitochondrial function, and an improved ability to limit reactive oxygen species production compared to the other mutant strains and the wild-type strain. In a fermentation medium containing 3.5 g/L furfural, the growth lag phase of the SCF-R4 mutant strain was shortened, and its growth ability significantly improved. After 96 h of fermentation, the ethanol production of the mutant strain SCF-R4 was 1.86 times that of the wild-type, indicating that with an increase in the number of irradiation rounds, the furfural tolerance of the mutant strain SCF-R4 was effectively enhanced. In addition, through genome-transcriptome analysis, potential sites related to furfural detoxification were identified, including GAL7, MAE1, PDC6, HXT1, AUS1, and TPK3.ConclusionsThese results indicate that multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution is an effective mutagenic strategy for obtaining furfural-tolerant mutants and that it has the potential to tap genes related to the furfural detoxification mechanism.

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Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

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Adaptive laboratory evolution in S. cerevisiae highlights role of transcription factors in fungal xenobiotic resistance.

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