Journal
MOLECULES
Volume 19, Issue 1, Pages 1004-1022Publisher
MDPI AG
DOI: 10.3390/molecules19011004
Keywords
artificial metabolism/metabolic engineering; bioorthogonality; genetic code expansion; posttranslational modifications; L-methionine; L-azidohomoalanine; click chemistry; O-acetyl-L-homoserine sulfhydrylase
Funding
- China Scholarship Council
- Alexander von Humboldt Foundation
- Finanziamento Progetti di Ricerca of Sapienza University of Rome
- FP7 EU
- UniCat Excellence Cluster of Berlin Institute of Technology (TU Berlin)
- DFG Forschergruppe [1805]
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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.
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