Bridging Adaptive Laboratory Evolution and Metabolic Engineering for Construction of an Improved Glycerol Utilizing Saccharomyces Cerevisiae Strain

Strucko, T., Technical University of Denmark
Forster, J., Technical University of Denmark
Patil, K. R., EMBL
Feist, A., Technical University of Denmark
Jouhten, P., EMBL Heidelberg
Zirngibl, K., EMBL
Mohamed, E. T., DTU, Center for Biosustainability

With increasing interest in biosustainable technologies, the demand for converting available non-saccharide carbon sources most efficiently is increasing. Highly abundant crude glycerol, a major waste residue in biodiesel production, has attracted attention as an alternative carbon source for microbial fermentation processes. The most commonly known microbial cell factory, the yeast Saccharomyces cerevisiae, has been extensively applied for the production of a wide range of scientifically and industrially relevant products using saccharides as carbon source. However, popular wild-type laboratory yeast strains, commonly applied in metabolic engineering studies, cannot utilize glycerol as a sole carbon source. Moreover, previous attempts of rational metabolic engineering approaches did not result in significantly improved glycerol utilization in S. cerevisiae.

In this work, an adaptive laboratory evolution approach to obtain S. cerevisiae strains with an improved ability to grow on glycerol was applied. A broad array of evolved strains, which exhibited a significant increase in the specific growth rate and a much shortened lag phase, were isolated. The best performing strains were further analyzed by using whole genome re-sequencing and classical genetics in order to identify causative mutations. A set of mutations in genes previously not linked to glycerol utilization was identified, which allowed successful re-engineering of the wild-type CEN.PK113-7D. In conclusion, the knowledge acquired in this study may be further applied for rational S. cerevisiae strain improvement for using glycerol as a carbon source in industrial biotechnology processes.

This work is a part of the DeYeastLibrary consortium financed by ERA-IB

DeYeastLibrary - Designer yeast strain library optimized for metabolic engineering applications