Coupling bioorthogonal chemistries with artificial metabolism: intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins

Molecules. 2014 Jan 15;19(1):1004-22. doi: 10.3390/molecules19011004.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / analogs & derivatives*
  • Alanine / biosynthesis
  • Bacterial Proteins / biosynthesis*
  • Click Chemistry
  • Escherichia coli
  • Fermentation
  • Genetic Engineering
  • Metabolic Networks and Pathways
  • Protein Processing, Post-Translational
  • Recombinant Proteins / biosynthesis
  • Spectrometry, Mass, Electrospray Ionization


  • Bacterial Proteins
  • Recombinant Proteins
  • azidohomoalanine
  • barstar protein, Bacillus amyloliquefaciens
  • Alanine