초록
<P>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 <SMALL>L</SMALL>-azidohomoalanine from <I>O</I>-acetyl-<SMALL>L</SMALL>-homoserine and NaN<SUB>3</SUB>, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph <I>E. coli</I> 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 <I>O</I>-acetylhomoserine sulfhydrylase from <I>Corynebacterium glutamicum</I>. Then, the expression of the target protein barstar, accompanied with efficient <SMALL>L</SMALL>-azidohomoalanine incorporation in place of <SMALL>L</SMALL>-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.</P>