Generation of bacterial platform strains for raw materials utilization using adaptive laboratory evolution

Abstract

Rising global temperatures and limited fossil resources make it increasingly urgent to find alternative ways of producing fuels and chemicals. Metabolic engineering offers promising solutions to this problem by utilizing biological systems—microbes—as cell factories for manufacturing a diverse set of products from renewable resources. However, rationally developing a microbial cell factory requires an extensive amount of knowledge of cellular processes as well as expensive and time-consuming molecular biology to design strains with desired characteristics. Adaptive laboratory revolution (ALE) is an alternative approach that can accelerate the development of microbial cell factories by harnessing the power of evolutionary processes. An important aspect of sustainable bioproduction is the utilization of renewable raw substrates, as they are abundant and have the potential to be a low-cost feedstock. However, utilization of raw materials can be limited by two issues: a) when deconstructed, they often contain toxic materials that affect cell fitness, and b) most microbial strains are limited in their consumption of such materials. It has been shown that ALE can be used to overcome both issues and, further, that understanding the genetic basis of the desired phenotypes is possible, given the availability of whole-genome sequencing. This thesis addresses the generation of platform strains optimized for the utilization and tolerance of raw material feedstocks, including lignocellulosic hydrolysates and raw sugar sources, focusing on an ALE approach. The main focus for raw materials was on lignocellulosic hydrolysate and raw sugarcane juice. For lignocellulosic hydrolysate, two studies were performed; the first was to overcome the toxicity of ionic liquid (a promising solvent for lignocellulosic biomass), and the second was to increase bacterial tolerance and utilization of the aromatic acids typically found in lignocellulosic hydrolysates, especially from solubilized lignin. A third study was also conducted to establish efficient utilization of sucrose (the main carbon of sugarcane juice) in industrial-relevant strains with improved overall fitness. This thesis contributes to our understanding of how microbial cells adapt to specific stress and growth conditions and provides tangible platform strains and a set of mutations that can be used as engineering tools to generate production strains for biomaterials based on renewable feedstocks.