Advancing Metabolic Engineering through Combination of Systems Biology and Adaptive Evolution
Year: 2012
Type: dissertation
Abstract: Understanding evolutionary strategies of microorganisms may provide opportunities for\nadvanced strain development with the aim to produce valuable bio-products from renewable\nbiomass resources. Through evolutionary processes, microorganisms can attain new traits\nassociated with genetic changes that may be useful for the construction of improved strains.\nTherefore, the characterization of evolutionary strategies may result in identification of the\nmolecular and genetic changes underlying newly obtained traits, and can hereby become an\nessential step in strain development. However, so far the depth of analysis has limited the range\nof comprehension. This thesis applied genome-wide analyses such as transcriptome, metabolome\nand whole-genome sequencing to investigate the evolutionary strategies of the yeast\nSaccharomyces cerevisiae. Three evolved mutants were independently generated by adaptive\nevolution on galactose minimal media to obtain the trait of improved galactose utilization by\nyeast. Those strains expressed higher galactose utilization rates than a reference strain in terms of\nboth maximum specific growth rate and specific galactose uptake rate. Application of the\ngenome-scale comparative analyses employing engineered strains as controls elucidated unique\nchanges obtained by adaptive evolution. Molecular bases referred from the changes of\ntranscriptome and metabolome were located around galactose metabolism, while genetic bases\nfrom whole-genome sequencing showed no mutations in those changes. Common mutations\namong the evolved mutants were identified in the Ras/PKA signaling pathway. Those mutations\nwere placed on the reference strain background and their effects were evaluated by comparison\nwith the evolved mutants. One of the site-directed mutants showed even higher specific galactose\nuptake rate than the evolved mutants, and just few number of genetic and molecular changes were\nenough to recover complete the adaptive phenotype. These results indicate that identification of\nkey mutations provide new strategies for further metabolic engineering of strains. In addition, the\npleiotropy of obtained phenotype that is improved galactose availability was tested. When the\ngalactose-evolved mutants were cultured on glucose that is the most favorite carbon source of\nyeast, those mutants showed reduction of glucose utilization. Genome-wide analyses and sitedirected\nmutagenesis were applied again to understand underlying molecular and genetic bases of\nthis trade-off in carbon utilization. The results indicated that loosening of tight glucose regulation\nwas likely the reason of increased galactose availability. The implications of evolutionary\nstrategies and the impact of genome-scale analyses on characterization of evolved mutants are\ndiscussed.
Author: Kuk-Ki Hong
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W99818487