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Molecules 2014, 19(1), 1004-1022; doi:10.3390/molecules19011004
Article

Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins

1
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1
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2
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1,*  and 2,*
1 Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany 2 Dipartimento di Scienze Biochimiche A. "Rossi Fanelli", Sapienza Università di Roma, Via degli Apuli 9, Roma 00185, Italy
* Authors to whom correspondence should be addressed.
Received: 4 December 2013 / Revised: 7 January 2014 / Accepted: 9 January 2014 / Published: 15 January 2014
(This article belongs to the Special Issue Bioorthogonal Chemistry)
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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.
Keywords: artificial metabolism/metabolic engineering; bioorthogonality; genetic code expansion; posttranslational modifications; L-methionine; L-azidohomoalanine; click chemistry; O-acetyl-L-homoserine sulfhydrylase artificial metabolism/metabolic engineering; bioorthogonality; genetic code expansion; posttranslational modifications; L-methionine; L-azidohomoalanine; click chemistry; O-acetyl-L-homoserine sulfhydrylase
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Ma, Y.; Biava, H.; Contestabile, R.; Budisa, N.; di Salvo, M.L. Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins. Molecules 2014, 19, 1004-1022.

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