%0 Journal Article
%T Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins
%A Ying Ma
%A Hern¨¢n Biava
%A Roberto Contestabile
%A Nediljko Budisa
%A Martino Luigi di Salvo
%J Molecules
%D 2014
%I MDPI AG
%R 10.3390/molecules19011004
%X 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 NaN 3, 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.
%K artificial metabolism/metabolic engineering
%K bioorthogonality
%K genetic code expansion
%K posttranslational modifications
%K L-methionine
%K L-azidohomoalanine
%K click chemistry
%K O-acetyl-L-homoserine sulfhydrylase
%U http://www.mdpi.com/1420-3049/19/1/1004