The nonconventional yeast Kluyveromyces marxianus is a promising microbial host for chemical biosynthesis due to its thermotolerance, fast growth kinetics, and ability to consume a wide range of carbon sources. A critical limitation thus far has been the lack of genetic engineering tools for strain manipulation. Here, we present our work in creating a metabolic engineering toolbox for K. marxianus. The toolbox includes CRISPR-Cas9 genome editing, multiplexed CRISPR interference (CRISPRi), and a standardized gene integration strategy. In addition, we have characterized a series of 25 promoters native to K. marxianus that vary in expression level. In studying these promoters, we observed significant changes in expression level as culture temperature increased from 30 to 45 °C. In the vast majority of cases expression was significantly reduced at elevated temperature. One solution to this problem was the design of a synthetic promoter that combines high expression elements from the TEF promoter and the high temperature responsiveness of the SSA3 promoter. The result was a promoter that maintains strong expression independent of temperature. Finally, for a subset of six promoters, we also evaluated protein expression and triacetic acid lactone production in xylose at different temperatures. Taken together, the genetic engineering tools presented here demonstrate the capacity to rapidly engineer K. marxianus and advance this host as a viable candidate for metabolic engineering.

Developing a metabolic engineering toolbox for the thermotolerant yeast K. marxianus

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.