Abstact

This study presents a robust bioprocess for the global incorporation of noncanonical amino acids (ncAAs) into proteins, enabling gram-scale production in auxotrophic Escherichia coli strains. The two-phase approach adapts from shake flask to bioreactor cultures and relies on cost-effective synthetic minimal media with glucose as the sole carbon source and yeast extract as an amino acid supply. It supports both external ncAA supplementation and in situ biosynthesis. A versatile E. coli BL21(DE3) auxotroph platform ensures broad ncAA and protein compatibility. Model proteins, such as a thermophilic lipase (TTL) and an oxidoreductase are labeled with biosynthesized norleucine (Nle), synthetic fluoroprolines, and fluorophenylalanine. Under optimal conditions, we achieved titers of up to 2 g L(-) (1) with near-quantitative incorporation. To demonstrate the utility of the bioprocess for applications that require substantial amounts of proteins, the crystal structure of Nle-labeled TTL is solved. Future work should optimize media composition and feeding strategies to improve ncAA bioavailability and integrate biosynthesis pathways into the host genome to reduce metabolic burden and eliminate antibiotic use. These advances will make the process a cost-effective industrial platform for designer protein production.