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're interested in 2-phenylethanol (2-PE), an aromatic flavor and fragrance compound that can be produced by biotransformation of L-phenylalanine or from sugar metabolisms via the Shikimate and Ehrlich pathways. We focused on K. marxianus as a production host for food or pharmaceutical grade biochemicals because it is a generally recognized as safe (GRAS) microbial species. It also has many other industrially favorable traits, including the natural ability to grow at temperatures upward of 48 °C, the fastest growth rate in eukaryotes, and its capacity to metabolize a range of C5, C6, and C12 sugars, as well as organic acids. To better exploit these 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-PE of over 0.8 g/L out of 20 g/L glucose and over 0.5 g/L out of 12 g/L Sigmacell after P. indianae hydrolysis. The genome-wide library of highly active single guide RNAs (sgRNAs) that target every gene in the genome is a powerful screening tool for functional genomics. It enables rapid genetic mapping of the L-phenylalanine biosynthesis pathways under different carbon sources in K. marxianus as well as rapid strain evolution experiments. Together, these CRISPR-based genetic tools and examples of their application in metabolic engineering demonstrate that CRISPR systems facilitate non-conventional microbe engineering both in a single-pathway manner and at a whole-cell level, and K. marxianus is a viable industrial host for chemical biosynthesis.

Ethan Hillman

Appearances