Abstract
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. Cupriavidus necator 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 C. necator 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.
Published Version
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