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Genetic approaches to study phenotypic variation in Kluyveromyces marxianus
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Date
2025
Authors
Huff, Franziska M.
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Publisher
University College Cork
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Abstract
Kluyveromyces marxianus is rapidly emerging as a promising candidate for industrial biotechnology, valued for its thermotolerance, fast growth, and ability to thrive on diverse substrates. Yet despite this potential, its full exploitation has been hindered by a lack of classical genetic tools and limited understanding of the molecular basis behind its stress resilience. This thesis addresses these gaps by applying classical genetics to dissect complex traits in K. marxianus—a leap toward making this non-model yeast genetically accessible and industrially robust.
Chapter 2 builds the foundation by equipping K. marxianus with a classical genetic toolkit. Through the construction of heterothallic, auxotrophic derivatives, the work enabled controlled intra- and inter-strain crosses and stable diploid formation. Broadly applicable protocols for sporulation, mating, and strain improvement were established and optimized, creating a flexible system for exploring genetic variation across diverse backgrounds.
Chapter 3 shifts from tools to traits. A diploid hybrid (NBRC 1777 × L04) was generated and sporulated, yielding 112 confirmed haploid F1 segregants To pinpoint phenotypes suitable for QTL analysis, 21 representative segregants, their parental strains, and the hybrid were screened with under five stress conditions: high temperature (46 °C), non-fermentable carbon (2% glycerol), salt (1.5 M KCl), and two forms of cell wall stress (caffeine, 20 mM; Calcofluor White, 1 mM).The data revealed transgressive segregation and continuous trait variation, suggesting a rich underlying genetic architecture. The traits of relative growth and survivability at 46 °C and 1 mM CFW combined broad variation and industrial relevance and were prioritized for QTL mapping.
Chapter 4 delivers the first ever QTL analysis in K. marxianus. Ninety-two segregants were phenotyped under selected conditions and their whole genome sequenced. QTL mapping revealed four significant loci — one for relative growth at 46 °C and three under cell wall stress (1 mM CFW). The biological relevance of these loci was underscored by the discovery of known stress-related genes. The thermotolerance QTL harboured GTT1, involved in glutathione-mediated oxidative stress defence, and ALD4, a mitochondrial aldehyde dehydrogenase previously linked to metabolic rewiring due to temperature stress in K. marxianus. One of the CFW QTLs contained CHS1, encoding a chitin synthase critical for cell wall integrity and previously implicated in CFW sensitivity in S. cerevisiae. Intriguingly, the QTL regions also included uncharacterized genes and medium-chain dehydrogenases/reductases (MDRs), hinting at novel stress tolerance pathways unique to K. marxianus.
The final chapter draws the thesis together by situating these findings in the broader context of yeast genetics and industrial strain development. This work not only establishes K. marxianus as a genetically tractable system for dissecting complex traits but also sets the stage for targeted strain improvement. The combination of classical genetics, trait mapping, and functional gene insights opens the door to precision engineering in this untapped yeast species.
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Keywords
Kluyveromyces , Kluyveromyces marxianus , QTL , Quantitative trait loci , Microbiology , Genetics , Biotechnology , Thermotolerance , Cell wall stress tolerance , Novel genes , Mating , Sporulation , Homothallic , Heterothallic , Yeast
Citation
Huff, A. N. 2025. Genetic approaches to study phenotypic variation in Kluyveromyces marxianus. PhD Thesis, University College Cork.