A novel whole system integrated genomics approach to identify key genetic components which facilitate synthetic design of a genetically engineered strain of Escherichia coli K12 with enhanced isobutanol tolerance

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University London.There has been an increased global interest in biofuels which provide a renewable and sustainable alternative to fossil fuels. Isobutanol is an attractive and superior alternative to the currently produced bioethanol possessing several key advantages. Previous work focuses on strategies for metabolic optimisation of carbon utilisation. However, existing solutions reach a stage where the amount of alcohol produced reaches toxic thresholds for bacteria. This inhibits growth and reduces carbohydrate consumption resulting in lower product yields rendering the biofuel production process uneconomical. In this project, a novel strategy has been adopted which uses a whole system integrated genomics approach consisting of expression profiling, selection to create isobutanol-adapted lineages, next generation sequencing, and comparative behavioural genomics to interrogate the system thoroughly and identify critical determinants of resistance to isobutanol. These were used in the highly-defined model species, E. coli K12 to deliver results of the adaptive mechanisms which take place across the entire genome. 41 gene candidates (4 previously identified in literature) were identified to play a role in isobutanol tolerance. These candidates belong to a range of functional groups such as carbohydrate metabolism, oxidative stress response, osmotic stress response; but also identified novel membrane-associated functions such as the Tol-Pal system, BAM complex and colanic acid production. The results also identify critical genes with unknown functions. The results support previous notions that central carbon metabolism shifts from aerobic to anaerobic metabolism in the presence of isobutanol, but also shows there is a transitionary phase where mixed acid fermentation pathways are utilised. This shift was previously thought to be mediated by the ArcA-ArcB two-component system. However, these results suggest the inactive 2Fe-2S core of the anaerobic-regulator Fnr is re-activated by Fe2+ to form the 4Fe-4S core transported by the FeoAB ferrous iron transport system. The strategy also identified the Tol-Pal system and show it is essential to grow in the presence of isobutanol, which is responsible for the maintaining the integrity of the cell envelope structure and increasing the rate of cell division. The BAM complex is responsible for folding and assembly of outer membrane proteins (OMP) and OMP membrane permeability- this system was found to be important for growth in isobutanol, and SurA, which is the primary OMP assembly pathway provided tolerance which was specific to isobutanol. Colanic acid, an extracellular polysaccharide is produced when the cell experiences stress, and provides protection by forming a physical barrier around the cell. The results show that the presence of colanic acid plays a large role in allowing E. coli to grow in presence of isobutanol, and its role becomes essential at critical concentrations. The results also show deletion of the negative regulator of the colanic acid gene cluster improves growth at critical and growth-inhibiting concentrations. When consolidated, these results facilitated knowledge-led based design and subsequently led to the identification of components for a synthetic design schedule, which lists the genetic manipulations proposed to exploit E. coli to enhance isobutanol tolerance