(466d) Systematic Characterization of a Panel of Strong Promoter/Terminator Pairs to Facilitate Scheffersomyces Stipitis Engineering

Systematic Characterization of a Panel of Strong Promoter/Terminator Pairs to Facilitate Scheffersomyces stipitis Engineering 

Scheffersomyces stipitis is an unconventional yeast that has the highest native capability for xylose fermentation, therefore holding a lot of potentials in lignocellulosic biomass conversion. However, its broad application as a popular host in biorenewables field is limited by the lack of genetic manipulation tools. Here, we report the identification and characterization of a panel of strong constitutive promoters and terminators. Based on RNA-seq analysis of S. stipitis growing in both mixed sugars and single sugar conditions, nine promoters were selected for further comprehensive characterization under conditions with various sugar and oxygen levels. The strengths of majority of these promoters were more than 10-fold higher than that of the commonly used TEF1 promoter, representing a collection of strongest Scheffersomyces promoters reported in literature. In parallel, the transcriptional termination efficiency of the selected terminators was studied and compared to the tight TEF1 terminator. Such a systematic characterization of potent promoters and terminators not only paves the way for manipulating pathways in S. stipitis to produce industry-relevant high-value biochemicals from mixed sugars, but also provides a foundation for future mechanism study of xylose uptake and conversion in the native host, which will benefit the entire biorenewables field.