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

Using engineered microbial cells for chemical production from renewable resources could replace oil-based chemistry. However, it is underexplored how the chemical production by engineered microbial cells is affected by them being 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 (ALE) 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 ALE was performed as serial batch cultivations in rich and synthetic defined (without amino acids) media with respirative galactose as the sole carbon source for ∼200 and ∼175 generations, respectively. While indigoidine pigmentation was rapidly lost independent of growth medium or ploidy, bikaverin pigmentation was robust. The adaptive solutions detected in poor galactose utilizer 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, clones having lost the pigmentation were detected. Thus, the robustness of the engineered traits appeared dependent on challenges in production environment and availability and fitness benefits of adaptive solutions.