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It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology., Fermentation parameters of industrial processes are often not the ideal growth conditions for industrial microbes. Here, the authors reveal that young genes are more responsive to environmental stress than ancient genes using a new gene age assignment method and provide targeted genes for metabolic engineering.

Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus.