The generation game: Toward the generational genetic stability of continuous culture

Fed-batch bioprocesses are typically deployed to convert renewable feedstocks to bio-based products using metabolically engineered microorganisms. However, for low-value chemicals, fed-batch cultures provide insufficient volumetric productivity to yield commercially viable products. The greater overall volumetric productivity of continuous culture holds techno-economic promise, but the genetic instability of engineered strains has prevented commercial deployment. This study demonstrated the continuous bioproduction of citramalate (CMA) for over 1,000 h at a productivity of 0.32 g_CMA g_DCW ^-1 h^-1. Plasmid segregational stability was ensured via infA-complementation, and structural stability was obtained under phosphate limitation in the chemostat. By contrast, glucose limitation promoted structural plasmid instability. Cost-prohibitive inducers were also avoided by using a constitutive promoter for gene expression. Plasmid-borne expression of CMA synthase delivered enhanced productivity compared to a chromosomal integrant strain also developed in this study. This work advances the techno-economic feasibility of sustainable chemicals manufacturing from renewable feedstocks by engineered strains in microbial cell culture.