School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, People's Republic of China.
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, People's Republic of China
Appl Environ Microbiol. 2020 Oct 1;86(20). doi: 10.1128/AEM.01388-20.
Inthomycins belong to a growing family of oxazole-containing polyketides and exhibit a broad spectrum of anti-oomycete and herbicidal activities. In this study, we purified inthomycins A and B from the metabolites of sp. strain SYP-A7193 and determined their chemical structures. Genome sequencing, comparative genomic analysis, and gene disruption of sp. SYP-A7193 showed that the inthomycin biosynthetic gene cluster () belonged to the hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. Functional domain comparison and disruption/complementation experiments of resulted in the complete loss of inthomycins A and B and the subsequent restoration of their production, confirming that encodes a discrete acyltransferase (AT), and hence, was considered to belong to the -AT type I PKS system. Moreover, the disruption/complementation experiments of also resulted in the loss and restoration of inthomycin A and B formation. Further gene cloning, expression, purification, and activity verification of revealed that Itm15 is a cyclodehydratase that catalyzes a straight-chain dehydration reaction to form an oxazole ring for the biosynthesis of inthomycins A and B. Thus, we discovered a novel enzyme that catalyzes oxazole ring formation and elucidated the complete biosynthetic pathway of inthomycins. species produce numerous secondary metabolites with diverse structures and pharmacological activities that are beneficial for human health and have several applications in agriculture. In this study, hybrid nonribosomal peptide synthetase/polyketide synthase metabolites inthomycins A and B were isolated from after fermenting sp. SYP-A7193. Genome sequencing, gene disruption, gene complementation, heterologous expression, and activity assay revealed that the biosynthesis gene assembly line of inthomycins A and B was a 95.3-kb -AT type I PKS system in the strain SYP-A7193. More importantly, Itm15, a cyclodehydratase, was identified to be an oxazole ring formation enzyme required for the biosynthesis of inthomycins A and B; it is significant to discover this catalyzation reaction in the PKS/NRPS system in the field of microbiology. Our findings could provide further insights into the diversity of -AT type I PKS systems and the mechanism of oxazole cyclization involved in the biosynthesis of natural products.
茵霉素属于一类不断增长的含恶唑的聚酮化合物家族，具有广谱的抗卵菌和除草活性。在本研究中，我们从 sp. 菌株 SYP-A7193 的代谢产物中纯化了茵霉素 A 和 B，并确定了它们的化学结构。基因组测序、比较基因组分析和 sp. SYP-A7193 的基因敲除表明，茵霉素生物合成基因簇 () 属于杂合聚酮合酶 (PKS)/非核糖体肽合酶 (NRPS) 系统。通过功能域比较和对 的敲除/互补实验，导致茵霉素 A 和 B 的完全缺失，随后恢复其产生，证实 编码一个离散的酰基转移酶 (AT)，因此，被认为属于 -AT 型 I PKS 系统。此外，对 的敲除/互补实验也导致茵霉素 A 和 B 形成的缺失和恢复。进一步克隆、表达、纯化和活性验证 揭示了 Itm15 是一种环脱水酶，它催化直链脱水反应形成恶唑环，用于茵霉素 A 和 B 的生物合成。因此，我们发现了一种新的酶，它催化恶唑环的形成，并阐明了茵霉素的完整生物合成途径。 sp. 产生了许多具有不同结构和药理活性的次级代谢产物，这些产物对人类健康有益，并在农业中有多种应用。在本研究中，从发酵 sp. 后分离出混合非核糖体肽合酶/聚酮合酶代谢产物茵霉素 A 和 B。SYP-A7193。基因组测序、基因敲除、基因互补、异源表达和活性测定表明，茵霉素 A 和 B 的生物合成基因组装线是 SYP-A7193 菌株中的一个 95.3-kb -AT 型 I PKS 系统。更重要的是，鉴定出 Itm15 是一种环脱水酶，是茵霉素 A 和 B 生物合成所需的恶唑环形成酶；这一催化反应在微生物学领域的 PKS/NRPS 系统中具有重要意义。我们的发现可以为 -AT 型 I PKS 系统的多样性和天然产物生物合成中恶唑环化的机制提供进一步的见解。
