Stable Platform for Mevalonate Bioproduction from CO₂

Stable production of value-added products using a microbial chassis is pivotal for determining the industrial suitability of the engineered biocatalyst. Microbial cells often lose the multicopy expression plasmids during long-term cultivations. Owing to the advantages related to titers, yields, and productivities when using a multicopy expression system compared with genomic integrations, plasmid stability is essential for industrially relevant biobased processes. <i>Cupriavidus necator</i> H16, a facultative chemolithoautotrophic bacterium, has been successfully engineered to convert inorganic carbon obtained from CO₂ fixation into value-added products. The application of this unique capability in the biotech industry has been hindered by <i>C</i>. <i>necator</i> H16 inability to stably maintain multicopy plasmids. In this study, we designed and tested plasmid addiction systems based on the complementation of essential genes. Among these, implementation of a plasmid addiction tool based on the complementation of mutants lacking RubisCO, which is essential for CO₂ fixation, successfully stabilized a multicopy plasmid. Expressing the mevalonate pathway operon (MvaES) using this addiction system resulted in the production of ∼10 g/L mevalonate with carbon yields of ∼25%. The mevalonate titers and yields obtained here using CO₂ are the highest achieved to date for the production of C6 compounds from C1 feedstocks.