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

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.Doctor of Philosophy (PhD) in Biomolecular Science

Enhanced extracellular chitinase production in <em>Pseudomonas fluorescens</em>: biotechnological implications

Chitin is an important renewable biomass of immense commercial interest. The processing of this biopolymer into value-added products in an environmentally-friendly manner necessitates its conversion into N-acetyl glucosamine (NAG), a reaction mediated by the enzyme chitinase. Here we report on the ability of the soil microbe Pseudomonas fluorescens to secrete copious amounts of chitinase in the spent fluid when cultured in mineral medium with chitin as the sole source of carbon and nitrogen. Although chitinase was detected in various cellular fractions, the enzyme was predominantly localized in the extracellular component that was also rich in NAG and glucosamine. Maximal amounts of chitinase with a specific activity of 80 µmol NAG produced mg–1 protein min–1 was obtained at pH 8 after 6 days of growth in medium with 0.5 g of chitin. In-gel activity assays and Western blot studies revealed three isoenzymes. The enzyme had an optimal activity at pH 10 and a temperature range of 22–38 ℃. It was stable for up to 3 months. Although it showed optimal specificity toward chitin, the enzyme did readily degrade shrimp shells. When these shells (0.1 g) were treated with the extracellular chitinase preparation, NAG [3 mmoles (0.003 g-mol)] was generated in 6 h. The extracellular nature of the enzyme coupled with its physico-chemical properties make this chitinase an excellent candidate for biotechnological applications

Manganese orchestrates a metabolic shift leading to the increased bioconversion of glycerol into α-ketoglutarate.

Glycerol is a major by-product of the biodiesel industry and its transformation into value-added products is an ongoing technological challenge. Here we report on the ability of the nutritionally-versatile Pseudomonas fluorescens to synthesize copious amount of α-ketoglutarate (KG) in a glycerol medium supplemented with manganese (Mn). The enhanced production of this keto-acid was mediated by the increased activities of isocitrate dehydrogenase (ICDH)-(NAD)P dependent and aminotransaminases. At stationary phase of growth when the optimal quantity of KG was recorded, these enzymes exhibited maximal activities. Two isoforms of pyruvate carboxylase (PC) that were identified in the Mn-treated cells provided an effective route for the synthesis of oxaloacetate, a metabolite critical in the production of KG. Furthermore, the increased activities of phosphoenol pyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) ensured the efficacy of this KG-generating metabolic system by supplying pyruvate and ATP from the oxaloacetate synthesized by PC. Mn-exposed whole cells converted 90% of industrial glycerol into KG. This Mn-evoked metabolic network can be optimized into the economic transformation of glycerol into KG

Deciphering metabolic networks by blue native polyacrylamide gel electrophoresis: A functional proteomic exploration

Metabolism is the consortium of reactions within a cell which directs a variety of processes including energy synthesis, signalling and the behaviour of a biological system. Metabolic networks, and more specifically the activity of enzymes within them, provide an accurate status of how cellular information is being executed. The performance of these networks and their ability to siphon metabolites in a number of directions may be the difference between a healthy and diseased state. Blue native polyacrylamide gel electrophoresis (BN-PAGE), owing to its simplicity and wide-ranging applications, permits the inspection of these nodules. The separation of proteins and enzyme complexes in their native format enables the exploration of enzymatic activity in metabolic networks via in-gel assays. These are quick, specific, and amenable to further studies. This electrophoretic technology not only enables the visualization of enzymatic efficacy but reveals the crosstalk among enzymes and their interactions with other organellar partners

Glycine metabolism and anti-oxidative defence mechanisms in Pseudomonas fluorescens

a b s t r a c t The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show that the nutritionally-versatile microbe, Pseudomonas fluorescens, reconfigures its metabolism in an effort to generate NADPH, ATP and glyoxylate in order to fend off oxidative stress. Glyoxylate was produced predominantly via the enhanced activities of glycine dehydrogenase-NADP + (GDH), glycine transaminase (GTA) and isocitrate lyase (ICL) in a medium exposed to hydrogen peroxide (H 2 O 2 ). This ketoacid was utilized to produce ATP by substrate-level phosphorylation and to neutralize reactive oxygen species with the concomitant formation of formate. The latter was also a source of NADPH, a process mediated by formate dehydrogenase-NADP + (FDH). The increased activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) worked in tandem to synthesize ATP in the H 2 O 2 -challenged cells that had markedly diminished capacity for oxidative phosphorylation. These metabolic networks provide an effective means of combating ROS and reveal therapeutic targets against microbes resistant to oxidative stress