Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering.

Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S. stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.