Unleashing the Potential of Ribosomal and Nonribosomal Peptide

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Unleashing the Potential of Ribosomal and Nonribosomal Peptide Biosynthesis Hsin-Mei Huang and Hajo Kries* Leibniz Institute for Natural Product Research and Infection Biology (HKI), Beutenbergstrasse 11a, 07745 Jena, Germany

Biochemistry Downloaded from pubs.acs.org by 46.148.124.155 on 10/18/18. For personal use only.

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eptide natural products and their derivatives provide potent pharmaceuticals and antibiotics. Several structural features underlie their privileged role in pharmaceutical chemistry: the rigidity of macrocycles facilitates target binding, backbone N-methylations enhance membrane permeability, and D- or β-amino acids contribute proteolytic resistance. Biosynthetically, most peptide natural products belong to the classes of nonribosomal peptides (NRPs) or ribosomally synthesized and post-translationally modified peptides (RiPPs). Powerful strategies not only for bioengineering RiPPs and NRPs but also for creating entirely novel, natural product-like peptides have been pioneered and will be highlighted here. Several library construction and screening methods can identify cyclic ribosomal peptides strongly binding pharmaceutically relevant targets (Figure 1A−C).1−3 Lanthipeptides, for instance, a subgroup of RiPPs, feature networks of macrocycles cross-linked through thioether bonds. Like most RiPPs, lanthipeptides are produced as precursor peptides composed of an N-terminal leader and a C-terminal core. The leader peptide recruits modifying enzymes sequence-specifically to the core peptide, where they perform extensive modifications with a high sequence tolerance. Because of the sequence plasticity of the core peptide, large numbers of variants can be conveniently prepared by mutating the gene of the precursor peptide, potentially with an expanded genetic code. To enable high-throughput screening of lanthipeptide libraries with ∼106 members, cell surface display methods have recently been developed in both eukaryotic and prokaryotic systems hijacking cellular translocation pathways.1 In a yeast display system (Figure 1A), a precursor peptide fused to the C-terminus of surface protein Aga2 translocates into the lumen of the endoplasmatic reticulum, where an independently exported enzyme installs the thio-bridges before exocytosis. For peptides sensitive to N-terminal extensions, phage display has been applied alternatively (Figure 1B). Here, modification occurs in the cytoplasm, and the resulting bulky lanthipeptide is translocated across the inner membrane through the Tat secretion pathway. In an analogous approach, synthetic “double bridges” have been integrated into ribosomal peptides to build bicyclic structures that, in this case, are not modeled after peptide natural products.2 Each phage-displayed peptide contains four cysteine residues that can be bridged in three combinations through two molecules of a thio-reactive cross-linking reagent. Compared to lanthipeptide libraries, the choice of cross-linking reagents contributes additional diversity. RaPID (Random Nonstandard Peptides Integrated Discovery), another platform for biosynthesizing cyclic peptides de © XXXX American Chemical Society

Figure 1. Peptide bioengineering strategies. Libraries of ribosomal peptides can be cyclized by various methods and displayed on (A) yeast, (B) phage, or (C) ribosome for panning. From the pool of peptides, panning retrieves binders to a desired biological target along with the attached genetic information.1−3 (D) High-throughput mutant screening on yeast surface and novel module shuffling strategies have been developed for the re-engineering of nonribosomal peptides.4,5

novo, combines bio-orthogonal tRNA acylation izyme”, in vitro translation, and mRNA display (Figure 1C).3 At the core, flexizyme conjugates broad range of amino acids with little selectivity

with “flextechnology tRNA to a toward the

Special Issue: Future of Biochemistry: The International Issue Received: September 2, 2018

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DOI: 10.1021/acs.biochem.8b00930 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

generates several dozen variants at best,4,5 the size of synthetic DNA template libraries for in vitro translation reaches 1013−14 and strong binders are routinely generated.3 Limiting for in vitro translation in the RaPID system is the scale-up that is prohibited by the price of the reagents. Furthermore, binding assays with displayed peptides cannot directly detect important pharmaceutical properties such as antibiotic activity and membrane permeation. Perhaps the remarkable achievements made with different biosynthetic methods can be combined into a “Swiss army knife” for peptide bioengineering in the future. This tool kit would offer facile library generation and peptide screening, the option for scale-up, and structural variability of side chains, backbone, and cyclization, all in one. Engineers working on both ribosomal and nonribosomal peptides are racing toward this formidable goal, and the new display, screening, and engineering tools showcased in this Viewpoint will certainly accelerate the race.

anticodon and side chain. The genetic code can thus be freely reprogrammed by assigning novel amino acids to certain codons. Cyclization events are triggered by incorporating pairs of reactive residues, and orthogonal cross-linking chemistry can create defined multicyclic structures. Although the flexizyme system enables flexible amino acid recruitment, including various non-natural building blocks, ribosome-dependent peptide bond formation sets limits on the backbone structure. Elongation with D-amino acids suffers from poor efficiency, and successive incorporation of β-amino acids is prohibited. Besides, the requirement for cell-free settings severely limits the synthetic scale. Nonribosomal peptide synthetases (NRPSs) generate peptides with valuable bioactivities and highly modified side chains or backbone structures, and some NRPSs are workhorses for industrial fermentation of natural products. Unfortunately, reprogramming NRPSs is more complicated than tinkering with ribosomal peptide genes because genetic changes do not translate directly into new peptide sequences. An NRPS is a long string of large enzyme modules that extend the peptide by one residue per module through the coordinated action of several domains. Adenylation (A) domains specifically activate amino acids and load them onto peptidyl carrier proteins (PCPs), and condensation (C) domains catalyze peptide bond formation. Modules are often equipped with additional tailoring domains to reshape the peptide backbone at specific residues during synthesis. Two recent papers mark significant progress toward targeted manipulation of NRPSs (Figure 1D).4,5 Niquille et al. have switched the substrate specificity of an initiation module from α- to β-Phe through fluorescence-activated cell sorting (FACS) of ∼106 mutants displayed on the yeast surface.4 In this assay, loading of propargylated substrates onto a displayed NRPS module is detected with clickable fluorophores. Similar crosslinking residues could be more generally useful for allowing chemical diversification of NRPs, but here, they served as stepping stones on a substrate walk from an α- to a β-amino acid. A domain specificity was first changed from α-Phe to Opropargyl-α-Tyr with an accessory mutation, then to Opropargyl-β-Tyr via large scale mutant screening on yeast, and finally to β-Phe by reverting the accessory mutation. The engineered module in combination with natural elongation modules yielded 120 mg of the pentapeptide with N-terminal β-Phe per litre culture. Helge Bode and co-workers have generated novel peptides with unprecedented efficiency by shuffling native NRPS modules from the vast pool of natural sequences.5 An innovative choice of the exchange unit was key to their success. On the basis of multidomain crystal structures, they identified a specific position within the C−A domain linker as a suitable split site. The exchange unit is hence “A−PCP−C” instead of the common module definition “C−A−PCP”. NRPS engineering might have struggled before because C domains are selective for the amino acid and the C-terminal peptide residue that they are condensing together. When these residues were kept constant during module exchange the resulting artificial NRPSs were remarkably efficient. In principle, NRPSs could accommodate more diverse backbone structures compared to the ribosome because each bond formation step is catalyzed by a dedicated C domain. However, both A domain engineering and module shuffling are still hindered by the obscure nature of C domain substrate selectivity. While diversification of nonribosomal peptides



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hajo Kries: 0000-0002-4919-2811 Funding

This work was supported by a postdoctoral scholarship of the Daimler und Benz Stiftung (H.K.). Notes

The authors declare no competing financial interest.



REFERENCES

(1) Hetrick, K. J., Walker, M. C., and van der Donk, W. A. (2018) Development and application of yeast and phage display of diverse lanthipeptides. ACS Cent. Sci. 4, 458−467. (2) Kale, S. S., Villequey, C., Kong, X.-D., Zorzi, A., Deyle, K., and Heinis, C. (2018) Cyclization of peptides with two chemical bridges affords large scaffold diversities. Nat. Chem. 10, 715−723. (3) Obexer, R., Walport, L. J., and Suga, H. (2017) Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads. Curr. Opin. Chem. Biol. 38, 52−61. (4) Niquille, D. L., Hansen, D. A., Mori, T., Fercher, D., Kries, H., and Hilvert, D. (2017) Nonribosomal biosynthesis of backbonemodified peptides. Nat. Chem. 10, 282−287. (5) Bozhüyük, K. A. J., Fleischhacker, F., Linck, A., Wesche, F., Tietze, A., Niesert, C.-P., and Bode, H. B. (2017) De novo design and engineering of non-ribosomal peptide synthetases. Nat. Chem. 10, 275−281.

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DOI: 10.1021/acs.biochem.8b00930 Biochemistry XXXX, XXX, XXX−XXX