Abstract
Microbial metabolism offers a powerful lens through which to understand evolutionary adaptation and a promising toolkit for converting plant-derived compounds into useful biochemicals. Among the most challenging of these compounds is lignin, a major component of plant biomass that breaks down into a mixture of aromatic compounds, many of which are difficult to metabolize, due to toxic metabolic intermediates like formaldehyde. This dissertation investigates how Methylobacterium extorquens, a facultative methylotroph with robust formaldehyde detoxification systems, can be engineered and evolved to expand its capacity to process aromatic compounds derived from lignin. Using a combination of genetic engineering, experimental evolution, and high-throughput fitness profiling, we explore the physiological, genetic, and ecological factors that shape microbial adaptation to lignin-derived aromatics. First, we show that M. extorquens can be equipped with the metabolic pathways needed to degrade aromatics like vanillate and protocatechuate, enabling growth on these compounds while maintaining resistance to formaldehyde stress. Next, we apply evolution to improve growth performance and uncover the genetic trade-offs that emerge—particularly in central metabolism, redox balance, and carbon storage. We find that mutations enhancing substrate use can come at the expense of bioproduction traits, such as accumulation of biopolymers like polyhydroxybutyrate (PHB), highlighting a tension between growth optimization and product yield. Finally, we demonstrate that environmental structure—whether constant, mixed, or fluctuating—plays a critical role in shaping evolutionary outcomes. While stable environments promote specialization, fluctuating conditions do not necessarily favor generalists, and switching between carbon sources can incur asymmetric fitness costs.
Altogether, this work reveals how metabolic capacity, stress tolerance, and environmental history interact to guide microbial evolution in complex chemical landscapes. It offers new strategies for improving microbial bioconversion of lignin-derived aromatics and contributes to a broader understanding of how adaptation is shaped by both biochemical constraints and ecological dynamics.