通过保存和连续传代培养抑制真菌喜树碱(CPT)的生物合成是阻碍真菌工业应用的挑战,所以,这项工作的主要目标是筛选具有稳定的CPT生产能力的新型真菌分离株。具有不同生物活性代谢物含量的长春花可能具有大量具有独特代谢特性的新型内生菌。在C.roseus的内生菌中,油菜链格孢菌EFBL-NVOR131587.1是最高的CPT生产者(96.5μg/L)。通过HPLC验证了推定的CPT的结构特性,FTIR,HNMR和LC-MS/MS,分子量为349m/z,以及通常与真实分子相同的分子片段模式。纯化的十字花菜CPT对UO-31(0.75μM)和MCF7(3.2μM)具有很强的抗增殖活性,选择性指数分别为30.8和7.1,除了抑制TopoII(IC50值0.26nM)的弹性活性之外,Topo1(IC50值3.2nM)。纯化的CPT对UO-31细胞的伤口愈合有约52%的抑制作用,停止它们的基质形成,细胞迁移和转移。纯化的CPT在S期阻止UO-31的细胞分裂,并诱导它们的细胞凋亡约20.4倍,与对照细胞相比。在用表面响应方法进行生物处理后,油菜的CPT产量提高了约3.3倍,与控制相比。随着真菌的储存和传代培养,油菜素合成CPT的代谢能力减弱。到第6个月的存储和第6代,他们的CPT生产力损失了约50%。实际上,通过添加1%表面灭菌的C.roseus叶,可以恢复减毒的A.brassicicola的CPT生产率。确保通过植物微生物组-A引发油菜CPT的隐蔽基因簇。油菜相互作用。所以,第一次,一种新的内生分离株油菜,来自C.Roseus,被探索有一个相对稳定的CPT生物合成机械,具有使用C.roseus微生物组恢复其CPT生产力的负担得起的可行性,除了提取的CPT抑制拓扑异构酶I和II的独特亲和力之外。
Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 μg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC-MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 μM) and MCF7 (3.2 μM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.