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.

Coupling bioorthogonal chemistries with artificial metabolism: Intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins / Ma, Y; Biava, H; Contestabile, Roberto; Budisa, N.; DI SALVO, Martino Luigi. - In: MOLECULES. - ISSN 1420-3049. - 19:(2014), pp. 1004-1022. [10.3390/molecules19011004]

Coupling bioorthogonal chemistries with artificial metabolism: Intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins

CONTESTABILE, Roberto;DI SALVO, Martino Luigi
2014

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.
Artificial metabolism/metabolic engineering, Bioorthogonality, Click chemistry, Genetic code expansion, L-Azidohomoalanine, L-methionine, O-Acetyl-L-homoserine sulfhydrylase, Posttranslational modifications
01 Pubblicazione su rivista::01a Articolo in rivista
Coupling bioorthogonal chemistries with artificial metabolism: Intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins / Ma, Y; Biava, H; Contestabile, Roberto; Budisa, N.; DI SALVO, Martino Luigi. - In: MOLECULES. - ISSN 1420-3049. - 19:(2014), pp. 1004-1022. [10.3390/molecules19011004]
File allegati a questo prodotto
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/557368
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 15
  • Scopus 43
  • ???jsp.display-item.citation.isi??? 44
social impact