The state of metabolic engineering in the versatile bacterial cell factory Cupriavidus necator H16

Cupriavidus necator strain H16 has emerged as a versatile microbial chassis for sustainable bioproduction due to its metabolic flexibility, enabling growth on a wide range of substrates, including H₂/CO₂, formate, and organic carbon (waste) sources such as volatile fatty acids. This review first provides a comprehensive overview of recent advances in systems-level understanding and metabolic engineering of C. necator. We next discuss recent advances in omics analyses and genome-scale metabolic modeling that are increasingly used to understand the large genome and wide metabolic portfolio of C. necator. We further discuss the native metabolic network, including autotrophic growth via the Calvin Benson Bassham (CBB) cycle, as well as heterotrophic and mixotrophic capabilities. Engineering strategies to enhance substrate utilization and conversion efficiency particularly for H₂/CO₂, formate, and mixed feedstocks are discussed alongside efforts to expand the substrate range in this organism. Other than the already industrialized production of the bioplastic polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates in C. necator, we provide an overview of the wide range of products for which proof-of-principles have been shown in C. necator. We also discuss recent advances in bioprocess design for gas fermentation, electromicrobial production, and H₂-based biocatalytic reduction using C. necator. Finally, we compare C. necator with other hydrogen and formate-utilizing bacteria to identify key knowledge gaps and outline future directions for advancing C. necator as a host for sustainable industrial biotechnology.