Enzymology and Fungal Biotechnology Lab, Botany and Microbiology Department, Faculty of Science, Zagazig University, 44519, Zagazig, Egypt.
Pharmacognosy Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.
Mol Biol Rep. 2022 Jun;49(6):4349-4364. doi: 10.1007/s11033-022-07271-x. Epub 2022 Mar 21.
Emerging of endophytic fungi as potent camptothecin producers raise the hope for its commercial production, due to their rapid growth and feasibility of metabolic engineering, nevertheless, their loss of productivity with the fungal storage and subculturing is the challenge. Thus, screening for unique fungal isolate with sustainable camptothecin productivity is the objective of this work.
The camptothecin productivity of the fungal endophytes of wild and in vitro cultured Astragalus fruticosus was evaluated. Aspergillus flavus ER, endophyte of A. fruticosus explant, was the potent producer (51.7 µg/l), the chemical identity of putative compound was resolved by UV, HPLC and LC-MS/MS analyses. The purified A. flavus camptothecin displayed a significant activity against HEPG-2 (IC 0.9 mM), MCF7 and HCT29 (IC 1.2-1.35 mM). The productivity of camptothecin by A. flavus was increased by 1.6 fold with methyljasmonate. Upon Plackett-Burman Design optimization, the yield of camptothecin was enhanced by 3 fold (150 µg/l) comparing to control. The camptothecin biosynthetic machinery of A. flavus was noticed to be attenuated with subculturing, nevertheless, this biosynthetic potency was restored upon addition of A. fruticosus methanolic extract (1%), ensuring the incidence of specific signals from plant tissues that triggers the expression of camptothecin encoding genes.
This is the first study deciphering the feasibility of A. flavus for sustainable production of camptothecin upon addition of A. fruticosus extracts, that could be a new platform for camptothecin scaling-up.
由于内生真菌能够快速生长且具有代谢工程可行性，因此作为潜在的喜树碱产生菌，为其商业化生产带来了希望。然而，随着真菌的储存和传代培养，其生产力会下降，这是一个挑战。因此，筛选具有可持续喜树碱生产力的独特真菌分离株是这项工作的目标。
评估了野生和体外培养的黄芪内生真菌的喜树碱生产力。黄曲霉 ER 是黄芪外植体的内生菌，是一种有效的喜树碱产生菌（51.7μg/l），通过 UV、HPLC 和 LC-MS/MS 分析确定了假定化合物的化学结构。纯化的黄曲霉喜树碱对 HEPG-2（IC 0.9 mM）、MCF7 和 HCT29（IC 1.2-1.35 mM）具有显著的活性。茉莉酸甲酯可使黄曲霉喜树碱的产量提高 1.6 倍。通过 Plackett-Burman 设计优化，与对照相比，喜树碱的产量提高了 3 倍（150μg/l）。黄曲霉的喜树碱生物合成机制随着传代培养而减弱，但在添加黄芪甲醇提取物（1%）后，这种生物合成能力得到恢复，这确保了来自植物组织的特定信号的发生，从而触发喜树碱编码基因的表达。
这是首次研究黄曲霉在添加黄芪提取物的情况下可持续生产喜树碱的可行性，这可能是喜树碱规模化生产的一个新平台。
