Adaptive evolution of engineered Saccharomyces cerevisiae in favored and unusual chemical environments

Engineered microbial cells can produce a wide range of industrially relevant chemicals such as pharmaceuticals, fuels, and material precursors. The use of microbial cells for chemical production from renewable resources could replace oil-based chemistry and contribute to tackling global grand challenges of climate warming and resource insufficiency. However, it is underexplored how the chemical production by engineered microbial cells is affected by them being proliferating catalysts exposed to Darwinian selection. All proliferating cells are unavoidably subjected to Darwinian selection which favors fitness beneficial phenotypes that seldom include engineered chemical production. Here, adaptive laboratory evolution was performed to characterize the effect of Darwinian selection on Saccharomyces cerevisiae strains expressing two different heterologous pigment producing pathways, blue-coloured indigoidine and red-coloured bikaverin. S. cerevisiae haploid S288C based strain had the genes for bikaverin synthesis integrated in the same locus as the genes for indigoidine synthesis in haploid and diploid S. cerevisiae CEN.PK-based strains. The two different pigment producing strains were cultivated in rich and synthetic defined (without amino acids) media with respirative galactose as the sole carbon source for [~]200 and [~]175 generations, respectively. While CEN.PK-based lineages rapidly lost indigoidine pigmentation independent of growth medium or ploidy, bikaverin pigmentation in S288C-based lineages was robust. The adaptive solutions detected in S288C-based bikaverin producing lineages involved mutations in the galactose utilization pathway whereas the heterologous indigoidine pathway was recurrently mutated in the corresponding lineages. When the bikaverin producing S288C-based lineages were adaptively evolved on the favored glucose carbon source instead, pigmentation declined. Thus, the robustness of the engineered traits appears dependent on challenges in production environment and availability and fitness benefits of adaptive solutions. Whether or when engineered traits of microbial cells are robust when they proliferate in industrial use has scarcely been assessed. Here light was shed to the factors affecting the adaptive loss of engineered traits to facilitate the development of strains and biotechnological processes, including chemical environments, for robust long-term production.