Mitigating protocatechuic acid toxicity in Vibrio natriegens enables poly-3-hydroxybutyrate production from an aromatic carbon source

Abstract 3,4-Dihydroxybenzoic acid (protocatechuic acid, PCA) is a central intermediate in bacterial aromatic catabolism - a key node for processing aromatic waste streams such as lignin and polyethylene terephthalate (PET) and a compound of growing biotechnological interest for valorising complex aromatic feedstocks. Here, we present a proof-of-concept for upcycling of PCA as a sole carbon source into a non-aromatic, higher-value bioproduct. We show that Vibrio natriegens , an emerging microbial chassis, can use PCA to produce poly-3-hydroxybutyrate (PHB), with no additional carbon source such as yeast extract or peptone. Efficient growth on PCA required careful optimisation of substrate concentration, cultivation conditions, and medium composition, e.g. replacing Fe 2 ⁺ with Fe 3 ⁺. These strategies reduced PCA autoxidation and limited reactive oxygen species (ROS) toxicity. Adaptive laboratory evolution over ~ 1000 generations identified convergent mutations that reveal insights into the architecture of the oxidative stress response in V. natriegens , including catalytic enzymes such as a Dyp-type peroxidase, and multiple regulatory proteins including OxyR, MarR, RpoS, ToxS, and RseB. Further mutations implicate cell wall repair and protein quality control in the PCA stress response. Functionally, the adapted strains produced 2.5-fold more PHB in 80% of the time required by the parental strain. Overall, we demonstrate the potential and current limitations of V. natriegens for the sustainable upcycling of aromatic, waste-derived carbon into value-added bioproducts.