Department of Botany, MMV, Banaras Hindu University, Varanasi, 221005, India.
Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, 221005, India.
World J Microbiol Biotechnol. 2024 Jul 24;40(9):278. doi: 10.1007/s11274-024-04078-9.
This study investigates the synthesis of vinblastine by endophytic fungi isolated from leaf of C. roseus. A total of 10 endophytic fungi were selected for secretion of vinca alkaloids based on the initial screening by biochemical tests and thin-layer chromatography (TLC). Out of these ten, only four fungal extracts showed positive results for presence of vinblastine at same retention time (10 min.) compared to reference compound on HPLC analysis. The detected concentration of vinblastine was maximum (17 µg/ml) in isolate no. CRL 22 followed by CRL 52, CRL 17 and CRL 28. To validate the presence of vinblastine, ultra-high-performance liquid chromatography coupled with high-resolution accurate mass spectrometry (HRMS) was employed. This analysis confirmed the presence of anhydrovinblastine, a precursor of vinblastine through the detection of molecular ions at m/z 793.4185 in extract of CRL 17. In addition to anhydrovinblastine, the intermediate compounds essential to the biosynthetic pathway of vinblastine were also detected in the extract of CRL 17. These host-origin compounds strongly suggest the presence of a biosynthetic pathway within the endophytic fungus. Based on morphological observation and sequence analysis of the ITS region of rDNA, endophytic fungi were identified as Alternaria alternata (CRL 17), Curvularia lunata (CRL 28), Aspergillus terrus (CRL 52), and Aspergillus clavatonanicus (CRL 22).
本研究调查了从长春花叶片中分离出的内生真菌合成长春碱的情况。基于生化试验和薄层层析（TLC）的初步筛选，共选择了 10 种内生真菌来分泌长春生物碱。在这些真菌中，只有 4 种真菌提取物在 HPLC 分析中与参比化合物相比，在相同保留时间（10 分钟）处显示出长春碱的存在呈阳性结果。在分离物 CRL 22 中检测到的长春碱浓度最高（17µg/ml），其次是 CRL 52、CRL 17 和 CRL 28。为了验证长春碱的存在，采用超高效液相色谱与高分辨率精确质量质谱（HRMS）联用进行分析。该分析通过在 CRL 17 提取物中检测到分子离子 m/z 793.4185，证实了长春碱前体脱水长春碱的存在。除了脱水长春碱外，在 CRL 17 的提取物中还检测到了长春碱生物合成途径中必需的中间化合物。这些宿主来源的化合物强烈表明内生真菌中存在生物合成途径。基于形态观察和 rDNA ITS 区序列分析，内生真菌被鉴定为互隔交链孢菌（CRL 17）、旋孢腔菌（CRL 28）、土曲霉（CRL 52）和棒曲霉（CRL 22）。
