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

Molecules

Ying Ma¹  Hernán Biava¹  Roberto Contestabile²  Nediljko Budisa¹ 

[1] Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany; E-Mails:;Dipartimento di Scienze Biochimiche A. “Rossi Fanelli”, Sapienza Università di Roma, Via degli Apuli 9, Roma 00185, Italy; E-Mail:

关键词: artificial metabolism/metabolic engineering;    bioorthogonality;    genetic code expansion;    posttranslational modifications;    l-methionine;    l-azidohomoalanine;    click chemistry;    O-acetyl-l-homoserine sulfhydrylase;   

DOI  :  10.3390/molecules19011004

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.