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

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.

An ADAMTS3 missense variant is associated with Norwich Terrier upper airway syndrome.

In flat-faced dog breeds, air resistance caused by skull conformation is believed to be a major determinant of Brachycephalic Obstructive Airway Syndrome (BOAS). The clinical presentation of BOAS is heterogeneous, suggesting determinants independent of skull conformation contribute to airway disease. Norwich Terriers, a mesocephalic breed, are predisposed to Upper Airway Syndrome (UAS), a disease whose pathological features overlap with BOAS. Our health screening clinic examined and scored the airways of 401 Norwich terriers by laryngoscopy. Genome-wide association analyses of UAS-related pathologies revealed a genetic association on canine chromosome 13 (rs9043975, p = 7.79x10-16). Whole genome resequencing was used to identify causal variant(s) within a 414 kb critical interval. This approach highlighted an error in the CanFam3.1 dog assembly, which when resolved, led to the discovery of a c.2786G>A missense variant in exon 20 of the positional candidate gene, ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). In addition to segregating with UAS amongst Norwich Terriers, the ADAMTS3 c.2786G>A risk allele frequency was enriched among the BOAS-susceptible French and (English) Bulldogs. Previous studies indicate that ADAMTS3 loss of function results in lymphoedema. Our results suggest a new paradigm in the understanding of canine upper airway disease aetiology: airway oedema caused by disruption of ADAMTS3 predisposes dogs to respiratory obstruction. These findings will enhance breeding practices and could refine the prognostics of surgical interventions that are often used to treat airway obstruction.