Characterization of L‐Ascorbate Metabolism in Ralstonia eutropha H16

Abstract

L‐Ascorbate (Vitamin C), a primary antioxidant in plants and animals, is an abundant carbon source highly produced by plants so is an available carbon source for soil bacteria. We previously identified a novel L‐ascorbate metabolic strategy in Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) by eight enzymes up to three transporters; the first enzyme is a membrane bound L‐ascorbate oxidoreductase (LaaO). Which face of LaaO, periplasmic or cytoplasmic, is catalytic was investigated by complementation of an laaO mutant with a cytoplasmic plant L‐ascorbate oxidase. Growth with L‐ascorbate of two of the three transporter deletion mutants was impaired but not abolished. The growth defects correlated with concentrations of L‐ascorbate or spontaneous degradation products in the media. Transcript levels of each transporter gene, fluorescence of mCherry‐transporter fusion mutants, and the L‐ascorbate and dehydroascorbate concentrations were monitored to observe the regulation of transporter genes. This work elaborates on environmental carbon cycling by a novel L‐ascorbate pathway found in hundreds of bacteria while clarifying transporter function in three different protein families.