Use of evolutionary engineering for strain development and scale-up studies for carotenoids production in yeast

Genetic determinants outside of the direct biosynthetic pathways have been shown to be important for the production of secondary metabolites such as carotenoids.  Since the identities of these genetic determinants are generally not known, this imposes a limit on the rational engineering of producers.  Evolutionary engineering does not require a prior genetic or mechanistic knowledge of the desired phenotype, and is thus a power tool for strain development if the desired trait is growth-coupled.  However, as the production of most secondary metabolites are generally not coupled with growth, evolutionary engineering has not been used to enhance productivity.  In this work, we successfully applied evolutionary engineering to enhance the heterologous production of carotenoids in Saccharomyces cerevisiae.  Since carotenoids are known antioxidants, we hypothesized that the antioxidant properties of these compounds can be used as a driving force for selection of hyper-producers in the presence of oxidative stress.  This approach allowed the coupling between the biosynthesis of carotenoids with growth-related process.  With this approach, we successfully evolved and isolated mutants with up to a 3-fold increase in carotenoids yield compared to the unevolved parental strain. Transcriptomics and genomics analyses of isolated hyper-producers revealed genotypic and regulatory modifications related with increased productivity.  Subsequent scale-up studies using bench-top bioreactor were used to assess fermentation parameters relevant for productivity using the best producer.