(152b) Building a Full Set of Genetic Toolkit to Engineer Scheffersomyces Stipitis

Many of the 1800 other known yeast species have highly unusual metabolic, biosynthetic, physiological and fermentative capacities that are not possessed by model yeasts such as Saccharomyces cerevisiae. These nonconventional yeasts remain largely unexplored due to the lack of efficient genetic engineering tools. In general, stable episomal expression platforms and precise genome-editing tools are the two foundational technologies used for engineering nonconventional species. We recently succeeded in establishing the first stable expression platform for engineering Scheffersomyces stipitis, one of the species with the highest native capability for xylose fermentation. The current expression plasmid is extremely unstable, being lost within 2 days of cultivation. Through the perfect integration of in silico centromere prediction and library-based screening, we were able to rapidly isolate all eight centromere sequences from S. stipitis genome. The identified centromeres significantly enhanced the stability of the episomal vector, resulting in homogenous expression of target protein and significant increase in production of valuable biochemicals. In addition, many nonconventional species rely on non-homologous end joining (NHEJ) mechanism for DNA repairing, which makes targeted gene knockout extremely challenging. We addressed this limitation by adapting CRISPR-Cas9 system for markerless gene knockouts and insertions in S. stipitis. A functional plasmid-based system was created using a codon-optimized Cas9 from Streptococcus pyogenes and single guide RNA (SgRNA). An efficiency of â?¥80% indel was achieved. Ku70 and ku80 genes, which were required for NHEJ, were subsequently disrupted to suppress NHEJ and make homologous recombination (HR) as the primary double strand break repair mechanism. Using Î?ku mutant strain and the CRISPR-Cas9 system, we successfully constructed ura3, trp1, his3, and leu2 auxotroph mutants, and demonstrated that S. stipitis is well suited for producing shikimate pathway derivatives such as shikimate and muconic acid. Our work represents the new explorations in expanding the current collection of microbial factories.