Advancing Metabolic Engineering through Combination of Systems Biology and Adaptive Evolution

Abstract: Understanding evolutionary strategies of microorganisms may provide opportunities for advanced strain development with the aim to produce valuable bio-products from renewable biomass resources. Through evolutionary processes, microorganisms can attain new traits associated with genetic changes that may be useful for the construction of improved strains. Therefore, the characterization of evolutionary strategies may result in identification of the molecular and genetic changes underlying newly obtained traits, and can hereby become an essential step in strain development. However, so far the depth of analysis has limited the range of comprehension. This thesis applied genome-wide analyses such as transcriptome, metabolome and whole-genome sequencing to investigate the evolutionary strategies of the yeast Saccharomyces cerevisiae. Three evolved mutants were independently generated by adaptive evolution on galactose minimal media to obtain the trait of improved galactose utilization by yeast. Those strains expressed higher galactose utilization rates than a reference strain in terms of both maximum specific growth rate and specific galactose uptake rate. Application of the genome-scale comparative analyses employing engineered strains as controls elucidated unique changes obtained by adaptive evolution. Molecular bases referred from the changes of transcriptome and metabolome were located around galactose metabolism, while genetic bases from whole-genome sequencing showed no mutations in those changes. Common mutations among the evolved mutants were identified in the Ras/PKA signaling pathway. Those mutations were placed on the reference strain background and their effects were evaluated by comparison with the evolved mutants. One of the site-directed mutants showed even higher specific galactose uptake rate than the evolved mutants, and just few number of genetic and molecular changes were enough to recover complete the adaptive phenotype. These results indicate that identification of key mutations provide new strategies for further metabolic engineering of strains. In addition, the pleiotropy of obtained phenotype that is improved galactose availability was tested. When the galactose-evolved mutants were cultured on glucose that is the most favorite carbon source of yeast, those mutants showed reduction of glucose utilization. Genome-wide analyses and sitedirected mutagenesis were applied again to understand underlying molecular and genetic bases of this trade-off in carbon utilization. The results indicated that loosening of tight glucose regulation was likely the reason of increased galactose availability. The implications of evolutionary strategies and the impact of genome-scale analyses on characterization of evolved mutants are discussed.