Current textbook knowledge holds that the structural scope of ribosomal biosynthesis is based exclusively on α-amino acid backbone topology. Here we report the genome-guided discovery of bacterial pathways that posttranslationally create β-amino acid-containing products. The transformation is widespread in bacteria and is catalyzed by an enzyme belonging to a previously uncharacterized radical -adenosylmethionine family. We show that the β-amino acids result from an unusual protein splicing process involving backbone carbon-carbon bond cleavage and net excision of tyramine. The reaction can be used to incorporate diverse and multiple β-amino acids into genetically encoded precursors in In addition to enlarging the set of basic amino acid components, the excision generates keto functions that are useful as orthogonal reaction sites for chemical diversification.

Radical S-adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d-configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry-based assay is introduced that permits characterization of products generated in E. coli. Applying this to a range of non-natural peptide-epimerase combinations, it is shown that the d-amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme-leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.

Salmonid alphavirus (SAV) causes pancreas disease and sleeping disease in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) and confers a major burden to the aquaculture industry. A commercial inactivated whole virus vaccine propagated in a salmon cell line at low temperature provides effective protection against SAV infections. Alphaviruses (family Togaviridae) are generally transmitted between vertebrate hosts via blood-sucking arthropod vectors, typically mosquitoes. SAV is unique in this respect because it can be transmitted directly from fish to fish and has no known invertebrate vector. Here, we show for the first time that SAV is able to complete a full infectious cycle within arthropod cells derived from the Asian tiger mosquito Aedes albopictus. Progeny virus is produced in C6/36 and U4.4. cells in a temperature-dependent manner (at 15°C but not at 18°C), can be serially passaged and remains infectious to salmonid Chinook salmon embryo cells. This suggests that SAV is not a vertebrate-restricted alphavirus after all and may have the potential to replicate in invertebrates. The current study also shows the ability of SAV to be propagated in mosquito cells, thereby possibly providing an alternative SAV production system for vaccine applications.

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Industrial and Commercial Training Volume 34 . Number 2 . 2002 . pp. 70±75 # MCB UP Limited .