Abstract
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.
Highlights
The ability of microbial systems to transform chemical wastes and renewable biomass into value-added products has been widely exploited industrially
As succinate levels were markedly higher, this prompted us to evaluate how the homeostasis of this dicarboxylic acid was maintained and whether it was utilized in the biogenesis of KG, a metabolite found in elevated amount in the spent fluid
When the isocitrate lyase (ICL) activity band was excised and treated with the isocitrate, peaks attributed to glyoxylate, and succinate were observed by high-performance liquid chromatography (HPLC) (Figure 2B)
Summary
The ability of microbial systems to transform chemical wastes and renewable biomass into value-added products has been widely exploited industrially. Apart from being a pivotal chemical ingredient that participates in a variety of biological processes including protein synthesis, immune response, and antioxidative defense, it is utilized clinically in wound healing, to treat chronic kidney deficiency and to decrease uremia (Tapiero et al, 2002; Bayliak et al, 2015). This keto acid is found in food flavoring, in cosmetics, and is utilized as a nutritional supplement (Wu et al, 2016)
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