Biotechnological potential of Paenarthrobacter nicotinovorans ATCC 49919 for the microbial conversion of nicotine and tobacco extracts into 6-hydroxynicotine
TL;DR
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.
Abstract
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 ZnSO4 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.