(640e) Development of Genetic Toolkits for Engineering Scheffersomyces Stipitis to Produce Biorenewables

Scheffersomyces stipitis has the highest native capability for xylose fermentation, and is considered as a promising microorganism to simultaneously convert both hexose and pentose in lignocellulosic hydrolysates for industrial applications.  Currently, its broader application is largely limited by the lack of genetic manipulation tools.  Consisting of a S. stipitis automatic replication sequence (ARS) and an ura3 selection marker, the expression vector currently available in literature, has an unstable copy number, making heterologous pathway engineering very challenging.  Here we report the successful identification of a functional centromere sequence (CEN) that stabilizes the episomal vector.  The redesigned pMC-CEN vector was evaluated for enhancing the production of L-lactic acid and muconic acid via expressing exogenous lactate dehydrogenase gene (ldhL) and muconic acid pathway genes.  Furthermore, a set of promoters was characterized under various carbon sources and oxygen conditions via real-time PCR analysis and fluorescence reporter evaluation.  Development of stable episomal expression vector and identification of strong constitutive promoters paved the way for further exploration of Scheffersomyces stipitis to produce biorenewable chemicals based on co-utilizing hexose/pentose in the lignocellulosic hydrolysates.