A new Clonostachys Sp. ZBS49 filamentous fungus with high production of betulinic acid and its inhibitory effect on liver cancer cells.

Background: Triterpenoid compounds such as betulinic acid (BA) and oleanolic acid (OA) exhibit considerable pharmacological activities. However, their current production primarily relies on plant extraction and chemical synthesis, methods that are often plagued by low efficiency, complex extraction processes, and environmental concerns. Microbial-based synthesis has emerged as one of the most effective approaches for producing BA and OA.

Results: This study presented the first identification of filamentous fungal strains efficiently synthesizing both BA and OA. The strain ZBS49 is a newly identified species of Clonostachys isolated from Gleditsia japonica Miq. (showing 99.82% sequence identity), produced 47.7 mg/L of BA. The strain XJ1-1, characterized as Colletotrichum gloeosporioides and isolated from Cannabis sativa L., yielded 65.76 mg/L of OA. After optimizing the culture medium and cultivation conditions, the yields of ZBS49 and XJ1-1 increased to 288.97 and 86.14 mg/L, representing improvements of 506% and 31%, respectively. Furthermore, we discovered that the BA extract of the ZBS49 strain significantly inhibited hepatocellular cancer cells (SMMC-7721 and HepG2) in a dose-dependent manner, with a minimum inhibitory concentration of 70 µM. Genomic analysis of Clonostachys sp. ZBS49 elucidated that the presence of 16 putative genes was related to triterpenoid biosynthesis and 6 distinct terpene biosynthetic gene clusters. Among the 145 CYP450, 5 genes involved in C-28 oxidation were predicted.

Conclusions: This research underscores the effectiveness of filamentous fungi as a biotechnological platform for the efficient production of BA and its derivatives, highlighting their potential applications in cancer therapy. Furthermore, these findings provide valuable genetic resources and establish a robust technical and theoretical framework for utilizing ZBS49 as a microbial platform for the biosynthesis of triterpenoids.