Predictive evolution of metabolic phenotypes using model-designed environments.

Adaptive evolution under controlled laboratory conditions has been highly effective in selecting organisms with beneficial phenotypes such as stress tolerance. The evolution route is particularly attractive when the organisms are either difficult to engineer or the genetic basis of the phenotype is complex. However, many desired traits, like metabolite secretion, have been inaccessible to adaptive selection due to their trade-off with cell growth. Here, we utilize genome-scale metabolic models to design nutrient environments for selecting lineages with enhanced metabolite secretion. To overcome the growth-secretion trade-off, we identify environments wherein growth becomes correlated with a secondary trait termed tacking trait. The latter is selected to be coupled with the desired trait in the application environment where the trait manifestation is required. Thus, adaptive evolution in the model-designed selection environment and subsequent return to the application environment is predicted to enhance the desired trait. We experimentally validate this strategy by evolving Saccharomyces cerevisiae for increased secretion of aroma compounds, and confirm the predicted flux-rerouting using genomic, transcriptomic, and proteomic analyses. Overall, model-designed selection environments open new opportunities for predictive evolution.

Adaptive laboratory evolution of microbial co-cultures for improved metabolite secretion.

Adaptive laboratory evolution has proven highly effective for obtaining microorganisms with enhanced capabilities. Yet, this method is inherently restricted to the traits that are positively linked to cell fitness, such as nutrient utilization. Here, we introduce coevolution of obligatory mutualistic communities for improving secretion of fitness-costly metabolites through natural selection. In this strategy, metabolic cross-feeding connects secretion of the target metabolite, despite its cost to the secretor, to the survival and proliferation of the entire community. We thus co-evolved wild-type lactic acid bacteria and engineered auxotrophic Saccharomyces cerevisiae in a synthetic growth medium leading to bacterial isolates with enhanced secretion of two B-group vitamins, viz., riboflavin and folate. The increased production was specific to the targeted vitamin, and evident also in milk, a more complex nutrient environment that naturally contains vitamins. Genomic, proteomic and metabolomic analyses of the evolved lactic acid bacteria, in combination with flux balance analysis, showed altered metabolic regulation towards increased supply of the vitamin precursors. Together, our findings demonstrate how microbial metabolism adapts to mutualistic lifestyle through enhanced metabolite exchange.