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

Andrew Yiakoumetti; Charlotte Green; Mark Reynolds; John Ward; Gill Stephens; Alex Conradie

doi:10.1016/j.isci.2025.111787

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

iScience. 2025 Jan 30;28(3):111787. doi: 10.1016/j.isci.2025.111787. eCollection 2025 Mar 21.

Authors

Andrew Yiakoumetti¹, Charlotte Green¹, Mark Reynolds², John Ward³, Gill Stephens¹, Alex Conradie¹

Affiliations

¹ Sustainable Process Technologies Research Group, Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD, UK.
² Mitsubishi Chemical Methacrylates, Wilton Centre, Wilton, Redcar TS10 4RF, UK.
³ Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, UK.

Abstract

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.

Keywords: Biotechnology; Chemical engineering.