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.

Graphical abstract

中文翻译：

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在单肽酰胺生物合成中，单模块NRPS形成哌嗪环，α-KG依赖性非血红素铁双加氧酶裂解。

摘要

巴西酰胺是巴西青霉产生的一类含哌嗪的生物碱，具有多种药物活性。Brasiliamide生物合成的机制，包括哌嗪环的形成和多种修饰修饰，仍不清楚。在这项研究中，从海洋衍生的巴西霉菌青霉WZXY-M122-9中鉴定了巴西酰胺类生物合成基因brs。使用CRISPR / Cas9基因编辑系统删除组蛋白脱乙酰基酶编码基因导致产生了一种新化合物，即巴西甲酰胺I（1）。该brs-编码的单模块非核糖体肽合成酶（NRPS）BrsA参与了巴西酰胺的哌嗪骨架的形成。纯化了全长的BrsA蛋白（113.6 kDa），体外酶活性的重建证实BrsA立体选择性地接受1-苯丙氨酸作为底物。剪裁基因的多次缺失和体外纯化蛋白的分析使我们能够提出一种Brasiliamide生物合成途径。在剪裁步骤中，已确定了α-酮戊二酸（KG）依赖性非血红素铁双加氧酶BrsJ可以催化巴西来酰胺A（2）的生物合成过程中的哌嗪环裂解。

关键点

• 确定了编码巴西甲酰胺生物合成的基因簇brs。

• 组蛋白脱乙酰基酶编码基因的缺失产生巴西来酰胺I。

• BrsA催化形成巴西来酰胺哌嗪骨架。

• BrsJ催化哌嗪环裂解产生巴西来酰胺A。

图形概要