Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast Kluyveromyces marxianus

Abstract

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 Kluyveromyces marxianus can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in K. marxianus by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, KLMX_70384, is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. KLMX_70384 is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose de novo in K. marxianus after the speciation event that separated K. marxianus and K. lactis contribute to some of its unique traits.

Transcriptional and translational profiling in yeast reveals the use of diverse genome decoding mechanisms to generate functionally distinct proteoforms

“…In a recent comparative omics study, we discovered that genes that were evolutionarily young and / or unique to K. marxianus are overrepresented among genes that display differential expression when growing under stressful conditions (Doughty et al , 2020). We also established that at least one of these genes is required for competitive growth at high temperature (Montini et al , 2022). These findings reinforce the need to thoroughly explore and characterise the K. marxianus genome as many of the unique phenotypic traits in K. marxianus could be due to genes not yet characterised in any species, or indeed to known genes that are regulated differently or encode proteins with alternative functionality.…”

Genotypic and Phenotypic Diversity of Kluyveromyces marxianus Isolates Obtained from the Elaboration Process of Two Traditional Mexican Alcoholic Beverages Derived from Agave: Pulque and Henequen (Agave fourcroydes) Mezcal

“…No conserved thermotolerance mechanism has been found in thermotolerant yeasts [ 78 ]. It has been reported that an evolutionary young gene with an unknown function was required for the competitive growth of K. marxianus at a high temperature [ 79 ]. However, this gene did not confer thermotolerance to Kluyveromyces lactis , indicating that thermotolerance might be linked to the de novo emergence of species-specific genes.…”

Dissecting an ancient stress resistance trait syndrome in the compost yeastKluyveromyces marxianus

“…If adaptive traits in K. marxianus were mediated by hundreds of independent variants, it would conform to the current view of dizzying genetic complexity as the rule rather than the exception for trait variation, across the tree of life 56,57 . In fact, even our large set of loci with signatures of selection could represent an underestimate of the full genetic architecture of K. marxianus traits, given the potential of false negatives in our approach as well as the contribution from species-specific genes 58 not analyzed here. But even as they stand, the derived alleles we have discovered in K. marxianus represent candidate determinants that would be sufficient to reconstitute K. marxianus traits in a stress-sensitive background; as such, they provide an important complement to the few reports focused on genes necessary for thermotolerance in this species 10,13,58 .…”