Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 1;8(11):7834-7843.
doi: 10.1039/c7sc03093b. Epub 2017 Sep 25.

Harnessing Fungal Nonribosomal Cyclodepsipeptide Synthetases for Mechanistic Insights and Tailored Engineering

Affiliations
Free PMC article

Harnessing Fungal Nonribosomal Cyclodepsipeptide Synthetases for Mechanistic Insights and Tailored Engineering

Charlotte Steiniger et al. Chem Sci. .
Free PMC article

Abstract

Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation, in trans expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their in vivo biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using Aspergillus niger as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L-1) and octa-beauvericin (10.8 mg L-1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L-1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against Leishmania donovani and Trypanosoma cruzi compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines.

Figures

Fig. 1
Fig. 1. Alternative CDP biosynthesis models and structures. (a) Models of CDP biosynthesis. Fungal CDP synthetases produce six- and eight-membered CDPs, with module 1 incorporating d-HAs and module 2 activating l-AAs. The building blocks are coupled until the chain length is long enough to be cyclized by the C3 domain. In the looping model (top), the depsipeptide chain grows by attachment of a single building block (HA or AA), shuttling between T1 and T2a/b. The role of T2b is still unclear. In the parallel model (bottom), the depsipeptide chain grows by addition of dipeptidols. a: acceptor site, d: donor site. (b) Cognate, non-methylated and hybrid CDP products. Iterative units are colored.
Fig. 2
Fig. 2. Mechanistic studies on fungal CDP synthetases. In vivo CDP production of (a) wild-type, mutated (EnSYNΔPpant2a/b) and truncated synthetases (SYNΔC1, SYNΔMt) as well as (b) in trans combinations synthesized in E. coli and monitored by MALDI-TOF-MS. (c) CDP yields in E. coli strains containing wild-type, ΔC1- and ΔPpant2a/b-synthetases determined by LC-ESI-MS (n = 3 cultures, standard deviation).
Fig. 3
Fig. 3. Structural diversification of fungal CDPs. In vivo CDP production of wild-type and hybrid synthetases with swapped C-terminal part (see color code) based on EnSYN (a), BaSYN (b) and BeSYN (c) synthesized in E. coli and monitored by MALDI-TOF-MS.
Fig. 4
Fig. 4. Swapping of C3 subdomains and elements thereof. In vivo CDP production of wild-type and hybrid synthetases based on EnSYN (a) and BeSYN (b) with swapped C3NTD, C3CTD, bridging loop region (small green circle) and five distinct AAs of BaSYN (orange line) in E. coli, respectively, as monitored by MALDI-TOF-MS.
Fig. 5
Fig. 5. Multiple swapping of fungal CDP synthetases. (a) In vivo CDP production of the hybrid synthetases (see color code) monitored by MALDI-TOF-MS. (b) CDP titers of wild-type and hybrid NRPSs producing bassianolide and hexa-bassianolide in E. coli compared by LC-ESI-MS (n = 3 cultures, standard deviation). n. d.: not detected.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Süssmuth R. D., Müller J., von Döhren H., Molnár I. Nat. Prod. Rep. 2011;28:99–124. - PubMed
    1. Feifel S. C., Schmiederer T., Hornbogen T., Berg H., Süssmuth R. D., Zocher R. ChemBioChem. 2007;8:1767–1770. - PubMed
    1. Müller J., Feifel S. C., Schmiederer T., Zocher R., Süssmuth R. D. ChemBioChem. 2009;10:323–328. - PubMed
    1. Matthes D., Richter L., Müller J., Denisiuk A., Feifel S. C., Xu Y., Espinosa-Artiles P., Süssmuth R. D., Molnár I. Chem. Commun. 2012;48:5674. - PubMed
    1. Krause M., Lindemann A., Glinski M., Hornbogen T., Bonse G., Jeschke P., Thielking G., Gau W., Kleinkauf H., Zocher R. J. Antibiot. 2001;54:797–804. - PubMed
Feedback