Laboratory Evolution and Reverse Engineering of E. coli to Enhance Lignocellulose Bioconversion

Lignocellulosic biomass represents a sustainable and abundant renewable resource with enormous potential to support large-scale bioproduction of fuels and value-added chemicals. However, efficiently converting the diverse array of substrates present in lignocellulosic hydrolysates into desired products using microbial cell factories remains challenging. Inherent regulatory mechanisms found in bacteria, coupled with metabolic constraints related to redox balance, energy production, and substrate transport, pose significant hurdles to efficient bioproduction from lignocellulosic sugars. Attempts to alleviate these constraints through purely rational engineering have been limited by the intrinsic complexity of biological systems. Adaptive laboratory evolution has emerged as a powerful complement to rational engineering efforts to develop bacterial phenotypes of industrial relevance. In this work, I explore the use of adaptive laboratory evolution and reverse engineering to improve sugar co-fermentation and the production of ethanol and succinate in E. coli. Specifically, I demonstrate that point mutations in the xylose-specific transcriptional regulator, XylR, relieve arabinose-induced repression on xylose fermentation and improves co-utilization of glucose-xylose-arabinose sugar mixtures in an ethanologenic E. coli strain. Furthermore, in Chapter 3 of this dissertation, I present the identification of novel mutations in pdhR, lpd, and agaR that contribute to enhanced anaerobic succinate production. The value of these mutations is demonstrated by their ability to significantly improve succinate yields in engineered E. coli succinate strains, highlighting the potential for ALE to uncover beneficial genetic changes that can be harnessed to optimize microbial production processes for industrial applications.