Enzymology and Fungal Biotechnology Lab, Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt.
Enzymology and Fungal Biotechnology Lab, Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, 44519, Egypt.
Enzyme Microb Technol. 2021 Feb;143:109718. doi: 10.1016/j.enzmictec.2020.109718. Epub 2020 Nov 25.
Epothilones are secondary metabolites produced by Sorangium cellulosum with powerful antiproliferative activity against tumor cells by stabilizing their microtubule arrays, arresting their cellular division at G2-M phase. Unfortunately, the lower yield of epothilone is the challenge for its higher accessibility, thus, searching for alternative sources with promising epothilone producing potency is the prospective. Endophytic fungi are the potential repertoire for bioactive metabolites, thus exploring the epothilone producing potency of endophytic fungi of medicinal plants was objective. Thirty-two fungal isolates were recovered from the tested medicinal plants and their potency to produced epothilone have been assessed using the TLC, HPLC and molecular markers epoA, epoC and epoK. Aspergillus fumigatus EFBL, an endophyte of Catharanthus roseus, was the potent epothilone producer (21.5 μg/g biomass) as revealed from the chromatographic analyses and PCR of molecular markers. The chemical identity of extracted epothilone was verified from the HPLC, NMR, FTIR and LC-MS analyses as epothilone B analogue. The putative epoA gene from A. fumigatus was amplified using RT-PCR with the conservative corresponding primers to the active-sites of S. cellulosum. The amplicons of epoA was 517 bp displayed 98 % similarity with A. fumigatus PKS-NRPS domains, and 40 % similarity with epoA of S. cellulosum. From the in silico analyses, Val, Ala and Ser are the conservative amino acids of epoA protein of A. fumigatus and S. cellulosum. Epothilone B from A. fumigatus displayed a strong antiproliferative activity against HepG-2, MCF-7 and LS174 T as revealed from the IC values 6.4, 8.7 and 10.21 μM, respectively. The productivity of epothilone B from A. fumigatus was optimized by surface response methodology with Plackett-Burman and Faced Centered Central Composite. With the Plackett-Burman design, the yield of epothilone (54.4-60.1 μg/g biomass) by A. fumigatus was increased by about 2.8-3.0 folds comparing to non-optimized cultures (21.5 μg/ g biomass). From the FCCD design, sucrose, tryptone and incubation time being the highest significant variables medium components affecting the epothilone yield of A. fumigatus. This is the first report exploring the feasibility of endophytic fungi for epothilone producing potency, that could be a novel platform for industrial production of epothilone.
表柔比星是由 Sorangium cellulosum 产生的次级代谢产物，通过稳定微管阵列，将细胞分裂阻滞在 G2-M 期，对肿瘤细胞具有强大的抗增殖活性。不幸的是，表柔比星的产量较低，这是其更高可及性的挑战，因此，寻找具有有前途的表柔比星产生潜力的替代来源是有前景的。内生真菌是生物活性代谢物的潜在库，因此探索药用植物内生真菌的表柔比星产生潜力是我们的目标。从测试的药用植物中回收了 32 个真菌分离物，并使用 TLC、HPLC 和分子标记 epoA、epoC 和 epoK 评估它们产生表柔比星的能力。从色谱分析和分子标记物的 PCR 中发现，内生真菌 Aspergillus fumigatus EFBL 是 Catharanthus roseus 的一种有效的表柔比星产生菌（21.5 μg/g 生物质）。从 HPLC、NMR、FTIR 和 LC-MS 分析中验证了提取的表柔比星的化学鉴定为表柔比星 B 类似物。使用 RT-PCR 用与 S. cellulosum 活性位点相对应的保守对应引物从 A. fumigatus 扩增了推定的 epoA 基因。epoA 的扩增子为 517 bp，与 A. fumigatus PKS-NRPS 结构域显示出 98%的相似性，与 S. cellulosum 的 epoA 显示出 40%的相似性。从计算机分析来看，Val、Ala 和 Ser 是 A. fumigatus 和 S. cellulosum epoA 蛋白的保守氨基酸。从 IC 值分别为 6.4、8.7 和 10.21 μM 可知，来自 A. fumigatus 的表柔比星 B 对 HepG-2、MCF-7 和 LS174 T 具有很强的增殖抑制活性。通过 Plackett-Burman 和 Faced Centered Central Composite 方法对面粉比星 B 的产量进行了优化。使用 Plackett-Burman 设计，与非优化培养物（21.5 μg/g 生物质）相比，A. fumigatus 的表柔比星（54.4-60.1 μg/g 生物质）产量增加了约 2.8-3.0 倍。从 FCCD 设计来看，蔗糖、胰蛋白胨和培养时间是影响 A. fumigatus 表柔比星产量的最高显著变量培养基成分。这是探索内生真菌产生表柔比星潜力的可行性的第一个报告，这可能是表柔比星工业生产的新平台。
