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Piperazine ring formation by a single-module NRPS and cleavage by an α-KG-dependent nonheme iron dioxygenase in brasiliamide biosynthesis

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Abstract

Brasiliamides are a class of piperazine-containing alkaloids produced by Penicillium brasilianum with a range of pharmaceutical activities. The mechanism of brasiliamide biosynthesis, including piperazine ring formation and multiple tailoring modifications, still remains unclear. In this study, the biosynthetic gene cluster of brasiliamides, brs, was identified from the marine-derived fungal strain Penicillium brasilianum WZXY-M122-9. Deletion of a histone deacetylase–encoding gene using a CRISPR/Cas9 gene editing system led to the production of a new compound, namely brasiliamide I (1). The brs-encoded single-module nonribosomal peptide synthetase (NRPS) BrsA is involved in the formation of the piperazine skeleton of brasiliamides. Full-length BrsA protein (113.6 kDa) was purified, and reconstitution of enzymatic activity in vitro confirmed that BrsA stereoselectively accepts l-phenylalanine as the substrate. Multiple deletion of tailoring genes and analysis of purified proteins in vitro enabled us to propose a brasiliamide biosynthetic pathway. In the tailoring steps, an α-ketoglutarate (KG)-dependent nonheme iron dioxygenase, BrsJ, was identified to catalyze piperazine ring cleavage during biosynthesis of brasiliamide A (2).

Key Points

  • The gene cluster encoding brasiliamide biosynthesis, brs, is identified.

  • Deletion of a histone deacetylase–encoding gene produces brasiliamide I.

  • BrsA catalyzes brasiliamide piperazine skeleton formation.

  • BrsJ catalyzes piperazine ring cleavage to produce brasiliamide A.

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Acknowledgments

The authors thank Dr. Jinwei Ren for the measurements of NMR spectroscopic data.

Author contribution statement

BY conducted genetic manipulation. DL performed the fungus fermentation. XG and YY analyzed the bioinformatics. JZ helped to record the spectroscopic data. DY and YZ assisted with gene deletion steps. MM and WL conceived and designed the research protocol and wrote the manuscript. All authors read and approved the manuscript.

Funding

This work has been funded by the National Natural Science Foundation of China (81991525, 21861142006, 81872793, 81630089, 81673332, and 81573326), COMRA (DY135-B-05), and MOST (2018ZX09711001-001-008).

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Correspondence to Ming Ma or Wenhan Lin.

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This article does not contain any studies with animals performed by any of the authors.

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The authors declare that they have no conflict of interests.

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Yuan, B., Liu, D., Guan, X. et al. Piperazine ring formation by a single-module NRPS and cleavage by an α-KG-dependent nonheme iron dioxygenase in brasiliamide biosynthesis. Appl Microbiol Biotechnol 104, 6149–6159 (2020). https://doi.org/10.1007/s00253-020-10678-w

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  • DOI: https://doi.org/10.1007/s00253-020-10678-w

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