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Dual phenazine gene clusters enable diversification during biosynthesis

Abstract

Biosynthetic gene clusters (BGCs) bridging genotype and phenotype continuously evolve through gene mutations and recombinations to generate chemical diversity. Phenazine BGCs are widespread in bacteria, and the biosynthetic mechanisms of the formation of the phenazine structural core have been illuminated in the last decade. However, little is known about the complex phenazine core-modification machinery. Here, we report the diversity-oriented modifications of the phenazine core through two distinct BGCs in the entomopathogenic bacterium Xenorhabdus szentirmaii, which lives in symbiosis with nematodes. A previously unidentified aldehyde intermediate, which can be modified by multiple enzymatic and non-enzymatic reactions, is a common intermediate bridging the pathways encoded by these BGCs. Evaluation of the antibiotic activity of the resulting phenazine derivatives suggests a highly effective strategy to convert Gram-positive specific phenazines into broad-spectrum antibiotics, which might help the bacteria–nematode complex to maintain its special environmental niche.

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Acknowledgements

The authors are grateful to T. A. Wichelhaus from Universitätsklinikum Frankfurt for the help with antibiotic susceptibility testing and the following colleagues from Goethe Universität Frankfurt: K. A. J. Bozhüyük for constructive discussion, K. M. Pos for providing the E. coli efflux pump mutant strains, and Y. Kopp and S. Mauer for the initial analysis of the phenazine biosynthesis in X. szentirmaii. This work was supported by the LOEWE program of the state of Hesse (LOEWE Schwerpunkt MegaSyn and LOEWE Zentrum TBG). Y.-M.S. is supported by a Postdoctoral Research Fellowship from the Alexander von Humboldt Foundation.

Author information

Y.-M.S. and H.B.B. conceived the project and wrote the paper. All experiments were performed by Y.-M.S., except initial analysis and heterologous expression of the gene clusters performed by A.O.B. RNA sequencing was performed by M.W. and N.J.T. and G. mellonella injection was performed by N.N. Y.-M.S., N.J.T., and H.B.B. discussed the results and commented on the manuscript.

Correspondence to Helge B. Bode.

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Supplementary Tables 1–6, Supplementary Figures 1–27

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Further reading

Fig. 1: Comparison of the phenazine BGCs in Pseudomonas aeruginosa PAO1 (phz), X. szentirmaii (xpz), and Pantoea agglomerans Eh1087 (ehp).
Fig. 2: Structures and biosynthetic pathway of phenazines from X. szentirmaii.
Fig. 3: Activation of the silent xpz biosynthetic gene cluster.
Fig. 4: In vitro characterization of iodinin and phenazine–polyketide conversions.
Fig. 5: Characterization of XpzPQS as free-standing A, T, and FabH family condensation (KS) enzymes catalyzing phenazine–peptide antibiotics formation via ester and amide bonds.