Laboratory evolution and characterization of nitrate-resistant phosphite dehydrogenase (PtxD) for enhanced cyanobacterial cultivation

Phosphite dehydrogenase (PtxD) catalyzes NAD⁺-dependent oxidation of phosphite (Pt) to phosphate (Pi), offering various biotechnological applications, such as the creation of Pt-dependency for the biological containment of genetically modified organisms. Previously, we established a Pt-dependent cyanobacterial strain (RH714) by expressing PtxD and a reduced phosphorous compound-specific transporter (HtxBCDE) in Synechococcus elongatus PCC 7942 devoid of its endogenous Pi transporters. This strain demonstrated strict Pt dependency but failed to grow in unbuffered BG-11 medium supplemented with 2 % CO₂ owing to medium acidification below approximately pH 6.5. The present study aimed to overcome this limitation by passaging the RH714 strain in an unbuffered growth medium, resulting in mutants capable of growing without buffering. The mutant strains carried a Gly157Ser mutation in the Rossmann fold domain of PtxD, leading to approximately five- and eight-fold higher K_m values for NAD⁺ and Pt, respectively, compared with the wild-type enzyme. Interestingly, PtxD^G157S exhibited enhanced resistance to nitrate, a major component of BG-11, suggesting that reduced substrate affinity mitigates nitrate inhibition at lower pH levels. Further kinetic analysis revealed that nitrate inhibits wild-type PtxD through an uncompetitive mechanism, targeting the enzyme-substrate complex at an allosteric site. Consequently, the PtxD^G157S mutation reduces nitrate binding, facilitating sustained growth of Pt-dependent strains under conditions without pH buffering. These findings imply that PtxD^G157S could significantly enhance the applicability of Pt-dependent cyanobacterial strain.