Glycerol is an important by-product of the biodiesel industry and its transformation into value-added products like keto acids is being actively pursued in order to improve the efficacy of this renewable energy sector. Here, we report that the enhanced production of α-ketoglutarate (KG) effected by Pseudomonas fluorescens in a mineral medium supplemented with manganese (Mn) is propelled by the increased activities of succinate semialdehyde dehydrogenase (SSADH), γ-aminobutyric acid aminotransaminase (GABAT), and isocitrate lyase (ICL). The latter generates glyoxylate and succinate two key metabolites involved in this process. Fumarate reductase (FRD) also aids in augmenting the pool of succinate, a precursor of succinate semialdehyde (SSA). The latter is then carboxylated to KG with the assistance of α-ketoglutarate decarboxylase (KDC). These enzymes work in tandem to ensure copious secretion of the keto acid. When incubated with glycerol in the presence of bicarbonate ( ), cell-free extracts readily produce KG with a metabolite fingerprint attributed to glutamate, γ-aminobutyric acid (GABA), succinate and succinate semialdehyde. Further targeted metabolomic and functional proteomic studies with high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and gel electrophoresis techniques provided molecular insights into this KG-generating machinery. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed the transcripts responsible for ICL and SSADH were elevated in the Mn-supplemented cultures. This hitherto unreported metabolic network where ICL and SSADH orchestrate the enhanced production of KG from glycerol, provides an elegant means of converting an industrial waste into a keto acid with wide-ranging application in the medical, cosmetic, and chemical sectors.
Keywords: biofuel; enzymes; isocitrate; metabolic engineering; succinate semialdehyde; α-Ketoglutarate; γ-Amino butyric acid.