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- Title
Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins.
- Authors
Ying Ma; Biava, Hernán; Contestabile, Roberto; Budisa, Nediljko; di Salvo, Martino Luigi
- Abstract
In this paper, we present a novel, "single experiment" methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-azidohomoalanine from O-acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host's methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.
- Subjects
BIOSYNTHESIS; RECOMBINANT proteins; SULFUR amino acids; METHIONINE; RECOMBINANT molecules; ESCHERICHIA coli
- Publication
Molecules, 2014, Vol 19, Issue 1, p1004
- ISSN
1420-3049
- Publication type
Article
- DOI
10.3390/molecules19011004