Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

Abstract

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activation of non-natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 10 5 -fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O-propargyl-l-tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non-natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.[**] We thank Prof. Mohamed A. Marahiel (Philipps Universität Marburg) for supplying strain HM0079 and plasmids pSU18_tycA and pTrc99a_tycB1, and Prof. Hans-Martin Fischer (ETH Zürich) for assistance with radiochemical experiments. We are also grateful to Prof. Chaitan Khosla (Stanford University) for valuable discussions.

Modelling the variable incorporation of aromatic amino acids in the tyrocidines and analogous cyclodecapeptides

“…Our model can easily be adapted to peptides with variability in their Aaa positions, for example the related steptocidines, loloatins and laterocidin, as well as the co‐produced gramicidins. This rapid method of culture manipulation, combined with our mathematical modelling can be used for understanding and manipulation of the production of other nonribosomally produced peptides as well as bioengineered enzyme systems where the synthetase enzymes have been mutated to produce novel analogues (Calcott and Ackerley ; Kries et al ; Kries ; Niquille et al ; Bozhüyük et al ). Moreover, our mathematical modelling can be incorporated into the computational design (Stevens et al ; Jiang et al ; Rothlisberger et al ; Privett et al ) and genome‐mining approach in the design of novel antimicrobial peptides and antibiotics produced by nonribosomal synthesis (Nguyen et al ; Gaudelli et al ; Weber et al ; Kries ; Baltz ).…”

“…Computational algorithms are used to predict mutation of the synthetase enzyme modules (Stevens et al ), however, mutations may also disrupt the interaction of proteins in the synthetase complex resulting in low yields (Stachelhaus et al , ; Schneider et al ; Trauger et al ; Wilkinson and Micklefield ; Winn et al ). Successful modification was reported for the D‐Phe 1 binding module which was induced to accept modified analogues through mutation in the active site (Kries et al ; Niquille et al ). To our knowledge no full‐length tyrocidine has been produced via recombinant technology, although a shortened dipeptide analogue was produced by expression of the first two modules in Escherichia coli (Gruenewald et al ).…”

Biosynthetic engineering of nonribosomal peptide synthetases

“…A. We have introduced a single W239S mutation into the A domain to reprogram specificity of a diketopiperazine synthetase for O‐propargyl‐Tyr . The active site of the A domain is shown with Phe bound (green spheres) .…”

Systematic analysis of the kalimantacin assembly line NRPS module using an adapted targeted mutagenesis approach

“…Recently, the Phe‐incorporating A domain of gramicidin synthetase (GrsA) has been successfully re‐engineered to introduce azide‐ and alkyne‐substituted AA (Kries et al. ). Attempts to drastically change the substrate specificity have resulted in a decrease in the A domain activity (Chen et al.…”