The antitumor compound psymberin as a model to develop heterologous expression systems for polyketides from uncultivated sponge symbionts
Zusammenfassung der Projektergebnisse
A great challenge in the development of complex, bioactive natural products isolated from marine invertebrates is the supply problem. We have previously provided evidence that symbiotic, as yet uncultivated bacteria are the true producers of many, if not most, complex polyketides in sponges. Many of these compounds seem to be generated by trans-AT polyketide synthases (PKSs), a still poorly understood family of highly complex enzymes. Aim of this project was to provide insights into strategies that provide renewable supplies of sponge-derived natural products. At the time of application, the only available full-length biosynthetic gene cluster from a sponge symbiont was the psymberin cluster. Several plasmids were prepared that carried the complete gene cluster assembled by yeast recombination. Additional plasmids were constructed that contained the misakinolide trans-AT PKS cluster, which was isolated during the funding period. These clusters were introduced into verious test hosts, revealing transcription of mRNA from the native promoters, but no polyketide formation. Further work therefore focused on understanding the function of trans-AT PKS components and their requirement for functional expression in E. coli. KS and ACP domains from the psymberin and additional trans-AT PKS systems were expressed, revealing significant substrate selectivity particularly in the polyketide elongation step. A new domain type, the pyran synthase domain, was identified that catalyzes stereoselective formation of five- and six-membered ether rings, thus bearing interesting potential for chemoenzymatic synthesis. During the funding period we were also able to functionally reconstitute all components of the polytheonamide pathway from a sponge symbiont in bacterial hosts. Rhizobium leguminosarum was identified as a suitable expression host for radical SAM methyltransferase genes of the polytheonamide cluster. These results set the stage for expression of the entire pathway. Finally, as an alternative to heterologous expression for generating renewable production sources, we demonstrated the feasibility of identifying homologous gene clusters in culturable bacteria. As a result, two new compounds were isolated from culturable cyanobacteria that are congeners of psymberin/onnamides and misakinolides, respectively.
Projektbezogene Publikationen (Auswahl)
- "An enzymatic domain for the formation of cyclic ethers in complex polyketides". Angew. Chem. Int. Ed. Eng. (2013) 52, 13215-13218
P. Pöplau, S. Frank. B.I. Morinaka, J. Piel
(Siehe online unter https://doi.org/10.1002/anie.201307406) - "Defensive bacteriome symbiont with a drastically reduced genome". Curr. Biol. (2013) 23, 1478-1484
A. Nakabachi, R. Ueoka, K. Oshima, R. Teta, A. Mangoni, M. Gurgui, N.J. Oldham, G. van Echten-Deckert, K. Okamura, K. Yamamoto, H. Inoue, M. Ohkuma, Y. Hongoh, S.-Y. Miyagishima, M. Hattori, J. Piel, T. Fukatsu
(Siehe online unter https://doi.org/10.1016/j.cub.2013.06.027) - “Substrate specificity in ketosynthase domains from trans-AT polyketide synthases”. Angew. Chem. Int. Ed. Eng. (2013) 52, 1143-1147
M. Jenner, S. Frank, A. Kampa, C. Kohlhaas, P. Pöplau, G.S. Briggs, J. Piel, N.J. Oldham
(Siehe online unter https://doi.org/10.1002/anie.201207690) - "An environmental bacterial taxon with a large and distinct metabolic repertoire". Nature (2014) 506, 58-62
M.C. Wilson, T. Mori, C. Rückert, A.R. Uria, M.J. Helf, K. Takada, C. Gernert, U.A.E. Steffens, N. Heycke, S. Schmitt, C. Rinke, E.J.N. Helfrich, A.O. Brachmann, C. Gurgui, T. Wakimoto, M. Kracht, M. Crüsemann, U. Hentschel, I. Abe, S. Matsunaga, J. Kalinowski, H. Takeyama, J. Piel
(Siehe online unter https://doi.org/10.1038/nature12959) - "Acyl chain elongation drives ketosynthase substrate selectivity in trans-acyl transferase polyketide synthases". Angew. Chem. Int. Ed. Eng. (2015), 54, 1817-1821
M. Jenner, J.P. Afonso, H.R. Bailey, S. Frank, A. Kampa, J. Piel, N.J. Oldham
(Siehe online unter https://doi.org/10.1002/ange.201410219) - "Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms". Nat. Chem. Biol. (2015) 11, 705-712
R. Ueoka, A.R. Uria, S. Reiter, T. Mori, P. Karbaum, E.E. Peters, E.J.N. Helfrich, B.I. Morinaka, M. Gugger, H. Takeyama, S. Matsunaga, J. Piel
(Siehe online unter https://doi.org/10.1038/NCHEMBIO.1870)
