Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

Authors: HOHENSCHUH, WILLIAM - Oregon State University; Hector, Ronald - Ron; Mertens, Jeffrey; MURTHY, GANTI - Oregon State University

Submitted to: Systems Microbiology and Biomanufacturing
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2020
Publication Date: 6/30/2020
Citation: Hohenschuh, W., Hector, R.E., Mertens, J.A., Murthy, G.S. 2020. Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose. Systems Microbiology and Biomanufacturing. 1: 42-57. https://doi.org/10.1007/s43393-020-00002-y.
DOI: https://doi.org/10.1007/s43393-020-00002-y

Interpretive Summary: This research shows that utilization of xylose, the second most abundant sugar in nature, is enhanced in engineered strains of brewer’s yeast when low levels of oxygen are available. A series of strains was created that utilized a new technology to over express certain proteins only when xylose was present. The protein expressed in this study (STB5) was previously demonstrated to regulate multiple other proteins which are essential for xylose utilization. The effect of expressing STB5 on cellular metabolism was analyzed in an effort to identify bottlenecks in xylose metabolism. Expressing STB5 did lead to induction of specific proteins when xylose was present. However, xylose utilization was not improved. Instead, consistent throughout this study, we observed that oxygen availability played a large role in the strains ability to use xylose. Co-factors required for enzymatic reactions to occur are more balanced when low levels of oxygen are available. When these metabolites and co-factors are not properly balanced, xylose utilization and conversion to products such as ethanol slows down, resulting in incomplete utilization of xylose. Complete and efficient xylose utilization is necessary for a successful bioeconomy to replace petro-chemicals. These results highlight areas of metabolism that need to be optimized in order to generate industry-ready strains with high yield and productivity that are capable of converting all available sugars to value added end-products.

Technical Abstract: Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide evolutionarily optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern.