Genetically engineered nifA mutations in Rhodobacter capsulatus to enhance ammonium tolerance and hydrogen production

NifA, the activator of nitrogenase, is sensitive to ammonium concentration, particularly within its N-terminal domain. In this work, genetically engineered mutants with N-terminal deletions of the nifA1 and nifA2 genes were constructed using overlap extension PCR to reduce the inhibitory effect of ammonium on nitrogenase expression in Rhodobacter capsulatus SB1003. Under 3 mM NH₄⁺, the hydrogen production rate of ZX03 (nifA1^-, nifA2^-) reached 0.65 mmol L⁻¹ h⁻¹, with a 31.2 % increase in hydrogen production compared to the wild-type. When 8 mM NH₄⁺ was used as the sole nitrogen source, H₂ production in all strains decreased substantially compared to 5 mM NH₄⁺. However, ZX03 demonstrated a 5.2-fold enhancement in hydrogen production relative to the wild-type under 8 mM NH₄⁺, underscoring its improved ammonium tolerance. During hydrogen production, the gene expression levels of nifA and nifH in all mutant strains were significantly up-regulated under ammonium conditions compared to the wild-type. These findings reveal distinct roles of nifA1 and nifA2 in ammonium tolerance and hydrogen production.