CRISPR-Enabled Metabolic Engineering of the Thermotolerant Yeast Kluyveromyces Marxianus

Engineered CRISPR-Cas9 systems have been developed to enable targeted gene knockouts, gene regulation, and genome-wide screens for functional genomics. The widespread adoption of these systems has rapidly increased access to the genomes of many non-conventional microorganisms, and in doing so has increased the number of hosts that can be engineered for chemical biosynthesis. In this talk, we present our work in developing CRISPR-based genome editing tools for the thermotolerant yeast Kluyveromyces marxianus and demonstrate that these tools can rapidly engineer new strains for biochemical production. We focused on K. marxianus because various strains have the natural ability to grow at temperatures upward of 50 °C and because K. marxianus is commonly described as the fastest growing eukaryote, traits that can benefit high temperature, high rate bioprocessing. The ability to metabolize a range of C5, C6, and C12 sugars, as well as organic acids also makes it well-matched with biomass-derived feedstocks. To better exploit these inherent advantages, we have created a set of CRISPR-based metabolic engineering tools for rapid and precise genome editing, and whole-genome screening of novel gene targets for chemical biosynthesis enhancement. Our CRISPR-mediated multigene integration system allowed creating strains that produce high titers of 2-phenylethanol, a valuable flavor and fragrance compound. The genome-wide library of highly active single guide RNAs (sgNRAs) that target every gene in the genome is a promising screening method for functional genomics. It enables rapid genetic mapping of desired phenotypes and guides directed strain revolution. Together, these CRISPR-based genetic tools and examples of their application in metabolic engineering demonstrate that CRISPR systems facilitate strain engineering in non-conventional microbes, and K. marxianus is a viable host for chemical biosynthesis.