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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

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, 1
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, 1,*  and 2,*
Received: 4 December 2013; in revised form: 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 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

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.

AMA Style

Ma Y, Biava H, Contestabile R, Budisa N, di Salvo ML. Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins. Molecules. 2014; 19(1):1004-1022.

Chicago/Turabian Style

Ma, Ying; Biava, Hernán; Contestabile, Roberto; Budisa, Nediljko; di Salvo, Martino L. 2014. "Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins." Molecules 19, no. 1: 1004-1022.



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