RegB/A-dependent redox regulation links iron uptake to methanol metabolism in Methylomonas sp. DH-1

Methanotrophs primarily use methane as a carbon and energy source, but they can also grow on methanol. However, methanol assimilation in the absence of methane oxidation results in a redox imbalance due to the accumulation of NADH. In Methylomonas sp. DH-1, a type I methanotroph, we show that this methanol-induced redox stress activates the RegB/RegA two-component system, a global redox sensor. Methanol triggers the RegB-to-RegA phosphotransfer cascade, resulting in RegA-dependent expression of the feo operon and increased ferrous iron uptake. This iron acquisition is essential for methanol growth, as it supports the function of Na⁺-transporting NADH:ubiquinone reductase (Na⁺-NQR), an iron-sulfur cluster-containing enzyme required for NADH oxidation. The operon encoding Na⁺-NQR complex is upregulated independently of RegA. Together, these findings reveal a redox-sensitive regulatory mechanism in which the RegB/RegA system senses redox imbalance and promotes iron uptake to facilitate Na⁺-NQR-mediated NADH reoxidation, thereby restoring redox homeostasis. This study uncovers a critical adaptive strategy for maintaining redox balance during methanol metabolism in methanotrophs.

Methanotrophs are emerging as sustainable platforms for converting methane into value-added chemicals. Many species can also utilize methanol-a readily available and industrially attractive alternative-as a carbon source. However, their broader industrial applications have been limited by an incomplete understanding of the fundamental physiological processes that underlie their metabolic and regulatory responses. In this study, we demonstrate how Methylomonas sp. DH-1, an aerobic methanotroph, couples carbon source sensing with redox regulation through the conserved RegB/RegA two-component system. We show that RegB/RegA controls iron uptake in response to methanol-induced redox stress, which, in turn, supports NADH reoxidation via Na⁺-NQR. These findings reveal a redox-responsive regulatory mechanism for methanol utilization and provide a foundation for engineering methanotrophs optimized for methane and methanol bioconversion.