title: Coupling bioorthogonal chemistries with artificial metabolism: intracellular biosynthesis of azidohomoalanine and its incorporation into recombinant proteins.
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.
authors.affiliation: Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany. katrinama2010@gmail.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany. biava@chem.tu-berlin.de.
authors.forename: Hernán
authors.initials: H
authors.lastname: Biava
authors.affiliation: Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany. roberto.contestabile@uniroma1.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Contestabile
authors.affiliation: Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany. nediljko.budisa@tu-berlin.de.
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.affiliation: Department of Chemistry, Biocatalysis Group, Technical University Berlin/Berlin Institute of Technology, Müller-Breslau-Str. 10, Berlin 10623, Germany. martino.disalvo@uniroma1.it.
authors.forename: Martino Luigi
authors.initials: ML
authors.lastname: di Salvo
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000096:biosynthesis*; D057930:Click Chemistry; D004926:Escherichia coli; D005285:Fermentation; D005818:Genetic Engineering; D053858:Metabolic Networks and Pathways; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C506098:azidohomoalanine; C057438:barstar protein, Bacillus amyloliquefaciens; D000409:Alanine