Adaptive Evolution for the Study of Complex Phenotypes in Microbial Systems
Reyes Barrios, Luis Humberto
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Microbial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.