金黑小单孢菌中新颖多烯大环内酰胺类化合物的发现及产量提高

Discovery and yield enhancement of novel polyene macrolactams in Micromonospora auratinigra DSM 44815

摘要:

【背景】 多烯大环内酰胺类化合物具有独特的化学结构和显著的生物活性，如抗菌、抗寄生虫和抗癌等，展现出较大的药物开发潜力，因此受到了广泛关注。【目的】 通过生物信息学分析，结合微生物遗传学和天然产物化学的方法，从微生物资源中发掘新的多烯大环内酰胺类化合物及其生物合成基因簇。【方法】 利用antiSMASH对金黑小单孢菌(Micromonospora auratinigra) DSM 44815基因组中的次级代谢产物生物合成基因簇进行分析，发现可能合成多烯内酰胺类化合物的基因簇。随后，采用同源重组技术敲除基因簇中的核心合成酶基因，并对菌株进行发酵，使用液相色谱法检测代谢物，确认该基因簇的产物。接着，通过天然产物分离纯化、高分辨率质谱和核磁共振等技术确定化合物的结构。最后，筛选培养基以提高该化合物在摇瓶发酵中的产量。【结果】 通过antiSMASH分析，在金黑小单孢菌DSM 44815基因组中发现一个新的I型聚酮合酶基因簇(mms cluster)，预测其可合成多烯大环内酰胺类化合物。通过高分辨率质谱分析，在该菌株发酵液中检测到一个分子量为427.272的化合物，可能为该基因簇产物。经分离纯化与核磁共振分析，确定该化合物(命名为MMS-A)为一种新型多烯大环内酰胺类天然产物。通过同源重组敲除基因簇中的聚酮合酶基因mmsB后，敲除株不再产生MMS-A，证实其负责MMS-A的生物合成。根据化合物结构及基因簇编码蛋白的功能，推测了MMS-A的生物合成途径。进一步优化发酵条件，将MMS-A产量提高至624.5 mg/L。【结论】 在金黑小单孢菌DSM 44815中发现了一个新的生物合成基因簇，并鉴定其合成的产物MMS-A为一种新结构的多烯大环内酰胺类天然产物。通过优化培养基成功提高了该产物的产量。本研究不仅丰富了多烯大环内酰胺类天然产物的化学和生物合成多样性，还为该类化合物的开发应用提供了新的分子、菌株和生物合成元件资源与工具。

Abstract:

[Background] Polyene macrolactams possess unique chemical structures and significant biological activities, such as antibacterial, antiparasitic, and anticancer effects, showing great potential for drug development. Accordingly, they have attracted considerable attention. [Objective] To discover new polyene macrolactams and their biosynthetic gene clusters from microbial resources through bioinformatics analysis combined with microbial genetics and natural product chemistry. [Methods] Firstly, antiSMASH was used to analyze the biosynthetic gene clusters for secondary metabolites in the genome of Micromonospora auratinigra DSM 44815, and a gene cluster likely involved in the synthesis of polyene macrolactams was identified. Next, homologous recombination was employed to knock out the core synthase gene within the gene cluster, and the obtained mutant strain was used for fermentation. Metabolites were analyzed by liquid chromatography to confirm the product of the gene cluster. Subsequently, the compound was isolated and purified, and its structure was determined by high-resolution mass spectrometry and nuclear magnetic resonance (NMR). Finally, the fermentation medium was optimized to increase the yield of the compound in shake-flask fermentation. [Results] A novel type I polyketide synthase gene cluster (mms cluster) was identified in the genome of M. auratinigra DSM 44815 through antiSMASH analysis, and it was predicted to be capable of synthesizing polyene macrolactams. High-resolution mass spectrometry of the fermentation broth detected a compound with a molecular weight of 427.272, which was suspected to be the product of this gene cluster. Subsequent isolation, purification, and NMR analysis confirmed this compound (named MMS-A) as a novel polyene macrolactam. The knockout of the type I PKS gene mmsB in the mms cluster by homologous recombination resulted in the loss of MMS-A production in the mutant strain, which confirmed that the biosynthesis of MMS-A depended on the mms cluster. On the basis of the compound structure and the predicted functions of the enzymes encoded by the gene cluster, a biosynthetic pathway for MMS-A was proposed. Finally, after optimization of the fermentation conditions, the yield of MMS-A was increased to 624.5 mg/L in the M4 medium. [Conclusion] We identified a new biosynthetic gene cluster in M. auratinigra DSM 44815 and preliminarily determined that the synthesized product, MMS-A, was a novel polyene macrolactam. The yield of MMS-A was successfully increased by optimizing the fermentation medium. This study enriches the chemical and biosynthetic diversity of polyene macrolactams and provides new molecular, strain, and biosynthetic component resources and tools for the future development of such compounds.