A Robust Bioprocess for the Global Incorporation of Noncanonical Amino Acids in Auxotrophic Hosts Produces Labeled Proteins at the Gram Scale.

ABSTRACT: 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<

SUBMITTER: Fladischer P 

PROVIDER: S-EPMC12781146 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature