Domestication and engineering of the yeast Kluyveromyces marxianus, a favored industrial host

Author(s): Estrela Curado, Raissa | Advisor(s): Doudna Cate, Jamie H | Abstract: Historically, fermentation has allowed the production of a myriad of food products that acquired central roles in human society such as bread, beer and wine. Through the centuries, fermentation has grown into a billion dollar industry; gaining traction for the production of pharmaceuticals, renewable plastics and new materials. The yeast Saccharomyces cerevisiae (baker's yeast), continues to be used by most industries; however, novel species are necessary to attend the growing and diversifying demand as S. cerevisiae has proven difficult to engineer to expand the carbon sources it can use, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeast species, which frequently contain inherent features addressing many of these issues, remain difficult to manipulate genetically. Here, we engineer one of the most promising candidates - the thermotolerant yeast Kluyveromyces marxianus - to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits - thermotolerance, lipid production, and facile transformation with exogenous DNA - into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals. K. marxianus was also successfully employed as a model organism; providing a new platform for a comprehensive, mechanistic, large-scale study of innate thermotolerance. From a library of mutants to identify genes related to innate thermotolerance, we identified 7 mutants that showed impaired growth at high temperature, indicating a potential role of these genes in the high temperature growth phenotype. This work provides a starting point to elucidate the mechanistic basis underlying the ability to sustain growth at high temperatures, the basis of which is still largely unknown in eukaryotes.