Understanding Fermentative Glycerol Metabolism and its Application for the Production of Fuels and Chemicals
Clomburg, James M.
Doctor of Philosophy thesis
Due to its availability, low-price, and higher degree of reduction than lignocellulosic sugars, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. However, this high degree of reduction of carbon atoms in glycerol also results in significant challenges in regard to its utilization under fermentative conditions. Therefore, in order to unlock the full potential of microorganisms for the fermentative conversion of glycerol into fuels and chemicals, a detailed understanding of the anaerobic fermentation of glycerol is required. The work presented here highlights a comprehensive experimental investigation into fermentative glycerol metabolism in Escherichia coli, which has elucidated several key pathways and mechanisms. The activity of both the fermentative and respiratory glycerol dissimilation pathways was found to be important for maximum glycerol utilization, a consequence of the metabolic cycle and downstream effects created by the essential involvement of PEP-dependent dihydroxyacetone kinase (DHAK) in the fermentative glycerol dissimilation pathway. The decoupling of this cycle is of central importance during fermentative glycerol metabolism, and while multiple decoupling mechanisms were identified, their relative inefficiencies dictated not only their level of involvement, but also implicated the activity of other pathways/enzymes, including fumarate reductase and pyruvate kinase. The central role of the PEP-dependent DHAK, an enzyme whose transcription was found to be regulated by the cyclic adenosine monophosphate (cAMP) receptor protein (CRP)-cAMP complex, was also tied to the importance of multiple fructose 1,6-bisphosphotases (FBPases) encoded by fbp, glpX, and yggF. The activity of these FBPases, and as a result the levels of fructose 1,6-bisphosphate, a key regulatory compound, appear to also play a role in the involvement of several other enzymes during fermentative glycerol metabolism including PEP carboxykinase. Using this improved understanding of fermentative glycerol metabolism as a platform, E. coli has been engineered to produce high yields and titers of ethanol (19.8 g/L, 0.46 g/g), co-produced along with hydrogen, and 1,2-propanediol (5.6 g/L, 0.21 g/g) from glycerol, demonstrating its potential as a carbon source for the production of fuels and reduced chemicals.
Glycerol fermentation; Escherichia coli; Metabolic engineering; Biofuels; 1,2-propanediol