Enzyme expression in Cupriavidus necator H16 for whole-cell biocatalysis

Climate change is an urgent and collective challenge, and new processes to synthesize complex molecules in a more sustainable way are highly desirable. Biocatalysis can be a strong player in this field, due to the specificity of enzymes and their ability to catalyze complex reactions at mild conditions. However, these reactions often require the regeneration of expensive cofactors in order to obtain relevant amounts of product. In vivo biocatalysis offers a solution to this problem by plugging the reaction in the microbial metabolism, which supplies the necessary energy. In particular, Cupriavidus necator H16 (C. necator H16) is an attractive microbial chassis due to its versatility and its lithoautotrophic metabolism. Its O₂-tolerant soluble hydrogenase (SH) can be used to regenerate nicotinamide cofactors in an atom-efficient manner, without the creation of undesired side products. This hydrogenase has already been used as a cofactor regeneration system in vitro, but examples of in vivo biocatalysis are scarce due to the time-consuming genetic engineering process of C. necator H16. In this book chapter, we present a strategy for the engineering of C. necator from plasmid cloning (using a recently developed expression plasmid) to protein expression of a model oxidoreductase. This pipeline allows for rapid and streamlined strain engineering, which can aid the discovery and development of future in vivo biocatalytic processes using C. necator H16.