链霉菌CB02959中四霉素及四烯菌素的鉴定及培养基优化

Identification and medium optimization of tetramycins and tetrins from Streptomyces sp. CB02959

摘要:

[背景] 四霉素（tetramycin）和四烯菌素（tetrin）是具有广谱抗真菌活性的四烯大环内酯类抗生素。链霉菌CB02959是一株雷纳霉素（leinamycin）类化合物的潜在产生菌株，利用antiSMASH分析其基因组发现该菌株含有一个纳他霉素（natamycin）类四烯大环内酯化合物的生物合成基因簇。[目的] 对Streptomyces sp.CB02959中次级代谢产物进行研究，确定其是否可以产生四烯大环内酯化合物，对其发酵产物进行分离和结构鉴定，并进行初步的发酵优化以提高产量。[方法] 基于生物信息学预测和高分辨质谱数据，推测CB02959中多烯化合物的结构；在不同发酵培养基中培养CB02959，确定适合大规模发酵的培养基；敲除tetrA基因以确定目标基因簇和四烯大环内酯化合物产生的相关性；分离和鉴定CB02959产生的主要代谢物的结构；通过改变培养基中葡萄糖、麦芽提取物和胰蛋白胨的含量，提高四烯大环内酯化合物的产量。[结果] 通过对CB02959中纳他霉素类化合物生物合成基因簇的分析及16S rRNA基因序列的进化树分析，推测CB02959可能是一株新的四霉素和四烯菌素产生菌；在YEME发酵培养基中对CB02959进行大规模发酵，分离得到4个化合物，鉴定为四霉素A（1）、四霉素B（2）、四烯菌素A（3）、四烯菌素B（4）；最后通过培养基的初步优化，将化合物1–4的产量分别提高至208.1、100.0、1 315.6、109.9 mg/L。[结论] 通过基因组挖掘策略发现了一株新的四霉素和四烯菌素产生菌链霉菌CB02959，并通过培养基优化提升了其四烯大环内酯化合物的产量，此发现为这类抗真菌天然产物的后续开发奠定了基础。

Abstract:

[Background] Tetramycin and tetrin are tetraene macrolides with broad-spectrum antifungal activity. Streptomyces sp. CB02959 was initially screened as a potential producer of leinamycin-like compounds. We noticed the existence of a putative natamycin biosynthetic gene cluster in CB02959 using antiSMASH analysis. [Objective] In this study, we aim to characterize the secondary metabolites from Streptomyces sp. CB02959, determine whether CB02959 is a tetraene macrolide-producing strain. We also aim to characterize the major products from CB02959 and improve their titers by media optimization.[Methods] Guided by bioinformatic analysis and high-resolution mass spectrum data, we predicted the structures of the compounds to be isolated. Streptomyces sp. CB02959 was then cultivated in different media to determine the right medium for large-scale fermentation. The tetrA gene, which encodes the first polyketide synthase of the polyketide assembly line, was disrupted to correlate the target gene cluster with the production of tetraene macrolides. The structures of major metabolites from CB02959 were elucidated based on extensive spectra analysis. The concentration of glucose, malt extract and tryptone were adjusted to improve the titers of the produced tetraenes. [Results] Based on the analysis of the predicted natamycin biosynthetic gene cluster in CB02959 and phylogenetic analysis of the 16S rRNA gene, CB02959 was proposed as a new tetramycin and tetrin-producing strain. After the large-scale fermentation in YEME medium, we isolated four metabolites from CB02959, which were determined as tetramycin A (1), tetramycin B (2), tetrin A (3), and tetrin B (4). We increased the titers of compounds 1-4 to 208.1, 100.0, 1 315.6, 109.9 mg/L via preliminary optimization of the fermentation medium. [Conclusion] In this study, we identified Streptomyces sp. CB02959 as a new producer for tetramycins and tetrins using the genome mining strategy, the titers of the produced tetraenes were improved by medium optimization. Our findings lay a foundation for the further development of these potent antifungal agents.