Many yeast species have highly desirable features that are not possessed by model yeasts. Scheffersomyces stipitis is one of the nonconventional yeasts that have the highest native xylose utilization capacity. Recent study suggested its potential as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical and 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 a unique approach to pinpoint the centromeric DNA as the genetic element to guarantee stable plasmid segregation. The method involved the integration of in silico GC3 chromosome scanning and high-throughput functional screening, and efficiently enabled the isolation of all eight S. stipitis centromeres. The identified 500-bp sequence not only stabilized episomal plasmid, enabled homogenous gene expression, increased the titer of a commercially relevant compound by 3-fold, also dramatically increased gene knockout efficiency from &lt; 1% to 83% with the integration of CRISPR components. This study elucidated that rapid establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeasts to expand the current collection of microbial factories. In an in-parallel project, we are also exploring Y. lipolytica in producing long-chain wax esters as one of the major additives in the skin care products due to their structural similarity with the wax esters contained in human skin. Y. lipolytica, which is well known for its superior ability to accumulate triacylglycerol (TAG) under appropriate culturing condition and genetics combinations. To divert the metabolic flux from TAG biosynthesis to wax ester biosynthesis, the genes involved in the wax synthetic pathway from difference sources with unique substrate specificities were tested, and several metabolic engineering strategies have been applied to increase precursor availability and remove substrate competition. The resulting strain produced wax esters at a titer 7-fold higher than the previous record in literature. With a comprehensive optimization on medium recipe, we anticipate a further significant improvement in wax ester titer.

Exploring nonconventional yeasts to produce pharmaceutical precursors and cosmetics additives

253rd National Meeting (2017)

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