9H0Z

Crystal structure of TTL[Nle], thermophilic lipase TTL from Thermoanaerobacter thermohydrosulfuricus containing non-canonical amino acid Nle at the position of Met

Summary for 9H0Z

• Entry DOI: 10.2210/pdb9h0z/pdb
• Descriptor: Serine aminopeptidase S33 domain-containing protein, SULFATE ION (3 entities in total)
• Functional Keywords: lipase, thermophilic, non-canonical amino acid, nle, ncaas, norleucine, lipid binding protein
• Biological source: Thermoanaerobacter thermohydrosulfuricus
• Total number of polymer chains: 6
• Total formula weight: 180445.20

Authors

Hromic-Jahjefendic, A.,Pavkov-Keller, T.,Wiltschi, B.,Gruber, K. (deposition date: 2024-10-09, release date: 2025-10-29, Last modification date: 2026-05-13)

Primary citation

Fladischer, P.,Anderhuber, N.,Murgu, O.,Hromic-Jahjefendic, A.,Pavkov-Keller, T.,Gruber, K.,Mairhofer, J.,Striedner, G.,Wiltschi, B.
A Robust Bioprocess for the Global Incorporation of Noncanonical Amino Acids in Auxotrophic Hosts Produces Labeled Proteins at the Gram Scale.
Chembiochem, 27:e202500669-e202500669, 2026

PubMed 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 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.

PubMed: 41376136
DOI: 10.1002/cbic.202500669

Experimental method

X-RAY DIFFRACTION (2.79 Å)