Microbial optimization through adaptation: How microbes adapt to (variable) environments through regulation and evolution

This thesis revolves around the themes of fitness and adaptation. How well do the microorganisms Escherichia coli and Lactococcus cremoris fit in their environment and how much offspring are they able to produce to ‘achieve evolutionary success’? Here, we study the adaptive properties of these two model microbes, both on short timescales (where optimal expression tuning of protein production plays a major role) and longer timescales (where adaptive evolution through mutation and selection comes into play). The thesis starts with a literature review on optimality of metabolism (Chapter 2). Many topics that are at the core of the other chapters are introduced here, including broader metabolic and evolutionary concepts. Chapter 3 investigates how a relatively novel culturing method (emulsion culturing which selects for cell number yield, rather than growth or metabolic rate) can impose an alternative selection pressure on E. coli. Interestingly, this evolutionary project helped shed new light on the role of overflow metabolism (generally considered as fast, but inefficient). The biphasic growth pattern associated with overflow metabolism not only seems to have the fitness advantage of fast growth during the first growth phase – as previously observed in batch culture experiments; but also the added benefit that during the second growth phase slower growth can occur which coincided with cell size reduction. This led to a significant increase in viable offspring (as the produced biomass can now be divided over a larger amount of smaller “genome packages”). Hence, this project provided - through both experiments and computational data - an alternative evolutionary scenario where overflow metabolism can be favourable even in the absence of nutrient competition. L. cremoris also has two distinct metabolic strategies. Typically, lactic acid metabolism is associated with fast but inefficient growth, and thus selected for in e.g. suspension culturing. Mixed acid metabolism on the other hand leads to slower growth but a higher biomass yield, and could be selected for through emulsion culturing. In Chapter 4 we describe that a trade-off between these could be circumvented, eventhough in earlier research selection for one property (either yield or rate) went at the expense of the other. Culturing these microbes in a fluctuating environment, alternating between batch culture on lactose and emulsion culture on glucose, allowed ‘breaking through the pareto front’. This gives the impression that the suggested trade-off is rather a negative correlation associated with growth in one specific environment, while an alternative evolutionary path can be followed when the appropriate selection pressure (i.e. a combination of environments) is applied. We also wanted to shed more light on short term adaptations to new environments (through protein expression tuning) in Chapter 5. By comparing the fitness level (i.e. maximal specific growth rate) of a wild type E. coli strain, to that of a strain with titratable H+-ATPase – titrated to the optimal level – we could assess how well this microbe was able to tune its expression level upon exposure to a new environment. Throughout a wide variety of nutrient conditions (with typical or more atypical carbon sources), the wildtype turned out to operate surprisingly close to the optimum, indicating robust optimal protein expression tuning in this species, enabling rapid growth rate maximization. In all, integrating data from the genetic (mutation) to the cellular (metabolic and physiological) to the population (growth rate and yield) to the overall evolutionary (fitness) level, has helped gain novel insights. The combination of these projects leads to a more immersive view on how microorganisms optimize their cellular processes in order to adapt to new environments.