An Oxygen-Tolerant Archaeal Dehydratase Enables a Synthetic Pathway for 4-Hydroxybutyrate-Containing Polyesters from Glucose and CO2 in Cupriavidus necator.

4-Hydroxybutyrate (4HB) and the related compounds are important in the chemical industry, and microbial metabolic engineering for 4HB production has been pioneered via the TCA cycle route employing clostridial enzymes. This study focused on an alternative, energy-efficient route for 4HB-CoA production using a unique enzyme from a different domain of life. The [4Fe-4S] cluster-containing 4HB-CoA dehydratase (4HcD), catalyzing radical-based atypical dehydration/hydration, is one of the enzymes difficult to apply in biotechnological applications due to its high sensitivity to reactive oxygen species. The present results demonstrated that an oxygen-tolerant 4HcD derived from an aerobic archaeon enabled conversion of crotonyl-CoA to 4HB-CoA in polyhydroxyalkanoate (PHA)-producing Cupriavidus necator. This archaeal enzyme contributed to establishing an energetically favorable ATP-generating biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) under low-aeration conditions. By rewiring C4-acyl-CoA metabolism in the host and modifying the N-terminus of 4HcD to potentially enhance its translation efficiency, the 4HB molar fraction of up to 12.6 mol % was obtained while inherent 3-hydroxyhexanoate incorporation was suppressed. This pathway was further extended to chemolithoautotrophic metabolism via gas fermentation, which enabled the direct production of the copolyester from CO2 and H2. Together, these findings highlight the potential of integrating archaeal catalytic functions within hydrogen-oxidizing bacteria as a promising strategy for advancing carbon capture and the sustainable biosynthesis of functional biopolymers.