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Transcriptome reshaping of the evolved thermotolerant Saccharomyces cerevisiae strain compared with the parental strain by RNA-Seq

ABSTRACT: Elevated thermotolerance is an important desired property of Saccharomyces cerevisiae for its industrial applications. Here, adaptive laboratory evolution experiments were employed to further improve the thermotolerance of an industrial strain ScY. The resulting evolved strain showing enhanced thermotolerance was named ScY01. We sequenced mRNA from the cultures of the evolved strain ScY01 and the parental strain ScY grown on YP medium containing 200 g/l glucose at 40ºC at 200 rpm for 14 h ~ 16 h to the early-log phase. Differences in gene expression in ScY01 versus ScY revealed by RNA deep sequencing revealled that genes involved into glycolysis, amino acid biosynthesis and translation showed increased gene expressions, whereas mitochondrial translation and respiration associated genes showed decreased gene expressions. This suggested that the evolved strain might suppress its mitochondrial respiratory activity but boost its fermentation capacity, thereby providing enough ATP required for its more active energy-consuming pathways including amino acid and protein biosynthetic pathways.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE96829 | GEO | 2024/12/18

REPOSITORIES: GEO

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			Action	DRS
		Other
	GSE96829_Differential_gene_expression_ScY01_vs._ScY.xlsx	Xlsx
	GSE96829_Transcriptome_profiles_of_ScY_and_ScY01.xlsx	Xlsx

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Project description:The growth of E. coli is inhibited by millimolar concentrations of L-homoserine, which may be problematic for the industrial production of this compound or for products derived from it. In this work, an adapted laboratory evolution (ALE) was applied, which resulted in the isolation of an E. coli strain at least 10-fold more tolerant to L-homoserine than the original (MG1655) strain. Only four genomic modifications were identified after genome sequencing of this evolved strain (designed 4E), including a 49 bp truncation starting from the codon stop of thrL and leading to a modified thrL locus carrying a thrL* allele encoding a 30 amino acids polypeptide, which is 9 amino acids more than the leader peptide encoded by thrL. Replacement of thrL with thrL* enabled the initial strain to be as or slightly more tolerant to L-homoserine than the evolved 4E strain, which is explained by the rapid metabolization of L-homoserine to threonine resulting from ThrL*-dependent transcriptional activation of the threonine thrABC operon. Interestingly, in 4E strain, L-homoserine degradation went beyond threonine degradation, as tdh and kbl encoding threonine degradation pathway II and genes of the glycine cleavage system were strongly upregulated. To infer about the toxicity of L-homoserine, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which identified a potent repression of locomotion-motility-chemotaxis process. Since the magnitude of this repression was reduced in a ΔthrL mutant concomitantly with a twofold lower sensitivity of this mutant to L-homoserine, one could argue that the repression of this biological process contributed to growth inhibition by L-homoserine. Furthermore, in both wild type and thrl mutant, a strong repression of the branched-chain amino acids synthesis and transport, as well as activation of the sulphate assimilation pathway process to cysteine synthesis were observed in the presence of L-homoserine, which may also contribute to toxic effect of this compound. How this non-canonical amino acid triggers these transcriptomic changes is discussed.

Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.

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Transcriptome reshaping of the evolved thermotolerant Saccharomyces cerevisiae strain compared with the parental strain by RNA-Seq

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