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

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. The

SUBMITTER: Ma Y 

PROVIDER: S-EPMC6271081 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature