CRISPR-Cas9 Enabled Metabolic Engineering of Oleaginous and Thermotolerant Yeasts

Identification of organisms which natively possess desirable phenotypes and application of advanced engineering techniques to enhance and understand these properties is a valuable approach for strain engineering in synthetic biology and metabolic engineering. Genome engineering in these non-model organisms has been significantly advanced by the widespread adoption of the type II CRISPR-Cas9 system. We have adapted CRISPR-Cas9 from Streptococcus pyogenes for genome editing in the yeasts Yarrowia lipolytica, of interest due to its ability to synthesize and accumulate lipids, and Kluyveromyces marxianus, which has a native capacity to synthesize volatile esters and ethanol at high temperatures. To overcome low gene disruption efficiencies, we designed synthetic RNA polymerase III promoters for guide RNA expression. These hybrid promoters enhanced CRISPR-Cas9 function in both organisms. In Y. lipolytica, the CRISPR-Cas9 system was applied to markerlessly integrate genes into well-characterized genomic loci for standardized pathway engineering, yielding strains that synthesize the carotenoid lycopene. We further applied the developed system for gene expression regulation via CRISPR interference (CRISPRi), which allowed us to enhance homologous recombination by transiently repressing genes involved in nonhomologous end-joining. In K. marxianus, a knockout library of alcohol dehydrogenases was generated to enable fundamental studies of gene function and allowed the identification of novel ethyl acetate synthesis activity. A CRISPRi system was then developed and used to shift metabolism away from respiration and towards fermentation to improve ester production. Together, these applications show the potential of CRISPR-Cas9 to enable advanced genome editing and transcriptional regulation in organisms with favorable native phenotypes.