Exogenous NADH enhances polycyclic aromatic hydrocarbon degradation in Novosphingobium pentaromativorans US6-1 by boosting energy metabolism and reducing oxidative stress

Polycyclic aromatic hydrocarbons (PAHs) are environmentally persistent pollutants that pose threats to both the environment and human health. Recently, bioremediation has emerged as a promising strategy for PAH pollution. NADH plays a crucial role in microbial metabolism and participates in PAH degradation. However, the mechanism underlying the influence of NADH on polycyclic aromatic hydrocarbon degradation remains unclear. The bacterium Novosphingobium pentaromativorans US6-1 degrades PAHs efficiently, and exogenous NADH supplementation promoted both cell growth and phenanthrene degradation. Promoter activity assays revealed that NADH upregulates the expression of PAH-degrading enzyme genes salA and xylE and enhanced the in vivo activity of the XylE enzyme. Since NADH did not act as a direct cofactor of XylE in cell-free assays, this enhancement is likely attributed to transcription upregulation and optimized intracellular conditions. Furthermore, NADH reduced intracellular reactive oxygen species (ROS) levels more effectively than antioxidant glutathione (GSH), suggesting that NADH supports cell fitness through mechanisms that extend beyond simple chemical ROS scavenging. Interestingly, NADH boosted the intracellular electron flux, and enhancing the electron transport system through either gene overexpression or FAD supplementation similarly improved PAH degradation. In conclusion, NADH promotes PAH degradation in N. pentaromativorans US6-1 through a multifaceted mechanism involving upregulating the key catabolic genes, enhancing in vivo critical enzyme activity, reducing intracellular ROS accumulation, and increasing electron flux. Our findings elucidate novel regulatory pathways of NADH in PAH degradation, thus highlighting its potential as a target for optimizing PAH bioremediation strategies. IMPORTANCE The oxidation of polycyclic aromatic hydrocarbons (PAHs) by degrading bacteria generates toxic intermediates. Accumulation of these compounds disrupts intracellular redox balance, exacerbating cellular toxicity. Furthermore, microbial PAH degradation imposes high energy demands and depletes essential redox cofactors. Despite the critical role of NADH as a central energy currency and reductant in cellular metabolism, the specific mechanisms underlying the influence of NADH on PAH biodegradation, particularly its interplay with redox homeostasis, remain poorly understood. This study has elucidated that NADH enhances PAH degradation efficiency by alleviating oxidative stress due to PAH oxidation. This study provides insights critical for advancing engineered bioremediation solutions.