Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis
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In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction. However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated. We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source. Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium. A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated. DNA microarray experiments revealed increased expression of the malic enzyme gene malE. The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis. Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate. The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate. Whereas during growth of C. glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.
KeywordsSole Carbon Source Pyruvate Kinase Malic Enzyme Pyruvate Carboxylase Corynebacterium Glutamicum
The authors would like to thank Christian Lange and Tino Polen for help with DNA microarray experiments. Malgorzata Krause received an ERASMUS fellowship from the European Union. Roman Netzer and Petra Peters-Wendisch acknowledge support from the Deutsche Bundesstiftung Umwelt (grant AZ13037) and Hermann Sahm acknowledges support by the Fonds der Chemischen Industrie. Roman Netzer and Malgorzata Krause contributed equally to this study.
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