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.
Highlights
The main source of chemical diversity in the majority of mature proteins and peptides is due to posttranslational modifications (PTMs), as only a few polypeptide structures have a final covalent structure deriving from the translation of their genes [1]
It has been previously shown that O-acetylhomoserine sulfhydrylase (OAHSS), the sulfide-utilizing enzyme involved in the direct sulfhydrylation pathway of L-methionine (Met) biosynthesis in various bacteria and yeasts (Figure 1a) [14,15,16], displays a somehow relaxed substrate specificity [15,17,18]
Recent biochemical studies have suggested that the likely sulfur source utilized by OAHSS in the methionine biosynthetic pathway may differ from organism to organism
Summary
The main source of chemical diversity in the majority of mature proteins and peptides is due to posttranslational modifications (PTMs), as only a few polypeptide structures have a final covalent structure deriving from the translation of their genes [1]. The most straightforward way to tackle this problem is to insert natural or synthetic non-canonical amino acids (ncAAs) of interest directly during translation, expanding the scope of ribosomal protein synthesis beyond the 20 canonical amino acids–in other words, to re-engineer the genetic code [5] This process is simpler than PTM itself, as the feature for that particular modification can be based on the unique chemical functionality delivered by the incorporated ncAA, and no longer requires complex signals on the protein scaffold. We repeat the N-terminal addition of a dansyl fluorescent label to the recombinantly expressed Aha-modified ψB*
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