Increased ʟ-glutamate production from gaseous nitrogen using Klebsiella pasteurii NG13 with modified citrate synthase

The fermentation of nitrogen-containing compounds by biological nitrogen fixation is a sustainable strategy that is independent of the Haber-Bosch process. We previously reported that the nitrogen-fixing bacterium Klebsiella pasteurii (formerly K. oxytoca) NG13 synthesized and excreted large amounts of ʟ-glutamate using gaseous nitrogen when citrate synthase (CS) and citrate transporter (CitS) were overproduced; however, the majority of carbon atoms in ʟ-glutamate were derived from citrate, not glucose, in the glucose and citrate-containing medium. To examine biased carbon flux to ʟ-glutamate, K. pasteurii overproducing CS and a 2-oxoglutarate (2-OG) transporter (KgtP) was constructed, and its carbon origin was investigated. This strain produced 2-OG-derived ʟ-glutamate in a culture medium containing glucose and 2-OG as the carbon sources. Since CS was inhibited by 2-OG competitively with oxaloacetate, a cognate substrate of CS, the deviated carbon flux from citrate/2-OG to ʟ-glutamate was attributed to the suppression of CS by 2-OG. Based on the structural model of CS from K. pasteurii (KpCS), H227 and V362 were selected as candidates to detect 2-OG binding, and KpCS variants (KpCS*) with H227L, H227Q, and V362L substitutions were confirmed to have inhibition constants that increased by 2.5- to 12.5-fold. As expected, the strains co-overproducing each of the KpCS variants and CitS generated larger amounts of ʟ-glutamate from glucose than the wild-type KpCS + CitS strain. When the KpCS(H227Q) + CitS strain was cultured under continuous glucose-fed conditions, maximum ʟ-glutamate production reached 2.35 g L^-1. These results suggest the potential of the Haber-Bosch process-independent strategy as a technological basis for the sustainable and eco-friendly utilization of nitrogen. KEY POINTS: • CS was inhibited by 2-OG in K. pasteurii • CS variants with increased K_i^2-OG allowed glucose-derived ʟ-glutamate production • Under glucose-fed culture, ʟ-glutamate production finally reached 2.35 g L^-1.