Biotechnological potential of Paenarthrobacter nicotinovorans ATCC 49919 for the microbial conversion of nicotine and tobacco extracts into 6-hydroxynicotine

Nicotine-rich wastes from the tobacco industry represent an environmental liability but also a potential feedstock for value-added bioconversion. In this study, the biotechnological potential of Paenarthrobacter nicotinovorans ATCC 49919 for selective accumulation of 6-hydroxy-(L)-nicotine (6HLN), the first intermediate of the pyridine pathway, was evaluated under flask and bioreactor conditions. Two metabolic engineering strategies were explored: transcriptional silencing of the 6-hydroxynicotine oxidase gene ( 6hlnO ) using a CRISPR/dCas9 system and overexpression of the nicotine dehydrogenase large subunit ( ndhL ) to enhance entry flux into the pathway. Among the tested strains, the ndhL -overexpressing derivative exhibited the highest 6HLN accumulation during growth (up to 1.78 g L⁻ 1 ; space–time yield 104.4 mg L⁻ 1 h⁻ 1 ). Resting-cell bioconversion experiments identified 100 g wet cell weight L⁻ 1 as an optimal biomass loading. Chemical inhibition of 6-hydroxy-L-nicotine oxidase with 0.8 mM ZnSO 4 further expanded the 6HLN accumulation window without substantially impairing nicotine depletion. Scale-up experiments in a stirred bioreactor demonstrated effective conversion of both pure nicotine and tobacco-derived extracts, with matrix-dependent differences in conversion kinetics. Dissolved oxygen profiles correlated reproducibly with substrate depletion and product turnover, providing a potential online process control signature. Although not achieving near-stoichiometric yields reported for knockout-based systems, the described platform offers a controllable and feedstock-flexible approach for nicotine waste valorization.