Metabolic engineering aimed at the production of keto acids from glycerol : an industrial by-product

Abstract

Worldwide, energy consumption is at an all-time high and projected to increasingly grow in the upcoming years. Thus, it is critical to uncover alternative sources of energy that are independent of fossil fuels and environmentally neutral. The transformation of biomass into various energy-rich chemicals is an important strategy that is being pursued globally. Biodiesel can be an interesting substitute to fossil fuels. However, this process generates excessive amounts of glycerol, a byproduct that needs to be converted into valuable products if the biodiesel industry is to be sustainable. The principle objective of this thesis is to study how glycerol can be used as a raw material by microbial systems to produce valuable products. The soil microbe, Pseudomonas fluorescens widely utilized in the numerous biotechnological applications due to its nutritional versatility is an obvious choice to tailor into a glycerol-transforming nanofactory. The abiotic modulators namely hydrogen peroxide (H2O2) and manganese (Mn) afforded uniquely facile means of triggering metabolic reprogramming aimed at the enhanced formation of pyruvate and α-ketoglutarate (KG). Under the influence of H2O2, P. fluorescens engineers an intricate metabolic network to synthesize ATP and pyruvate. As oxidative phosphorylation is severely impeded, the microbe invokes substrate level phosphorylation to generate energy. This is accomplished via the increased activities of various enzymes including pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) that were analyzed by blue-native polyacrylamide gel electrophoresis (BN-PAGE) and high performance liquid chromatography (HPLC). The high-energy phosphoenolpyruvate (PEP) is then converted into ATP and pyruvate, a process mediated by pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK). Supplementation with a micro-nutrient such as Mn, a divalent metal involved in a variety of enzymes results in the reprogramming of the metabolic networks aimed at the accumulation of KG. The increased activities of isocitrate dehydrogenase (ICDH)- (NAD)P dependent and aminotransaminases aided the exocellular secretion of KG. The overexpression of pyruvate carboxylase (PC) that is evident in the Mn-treated cells provides oxaloacetate, an important precursor to the synthesis of citrate, a key ingredient in the synthesis of KG. Isocitrate lyase (ICL), fumarate reductase (FUMR), succinate semialdehyde dehydrogenase (SSADH), α-ketoglutarate decarboxylase (KDC) and γ-aminobutyric acid transaminases (GABAT) work in concert to produce KG. 13C-NMR helped identify the metabolites participating in the metabolic networks. Immunoblot experiments confirmed the presence of overexpressed enzymes. These disparate metabolic pathways that promote the overproduction of the keto-acids in P. fluorescens have the potential of converting glycerol to value-added products commercially. As the process utilized is devoid of any genetic manipulation, it can be readily implemented in an industrial setting. In conclusion, both H2O2 and Mn can orchestrate metabolic changes in P. fluorescens inducing the production of pyruvate and KG from glycerol respectively. These chemical manipulations may also be applied to other microbial systems.