Iterative enrichment cultivation and multiomic analysis reveal potential endophytic bacteria affecting the sinomenine synthesis in Sinomenium acutum.

Endophytes play important roles in plant metabolite synthesis, and certain strains were capable of producing bioactive compounds identical to those of their hosts. However, it remains unknown whether culturable endophytes of S. acutum can synthesize intermediate metabolites for the plant principal bioactive compound-sinomenine (SIN) or the compound itself. In this study, we investigated the successions of the culturable bacterial community and the alkaloid profiles within S. acutum endophytes across ten iterative enrichment cultivations using Czapek-Dox and Gause's No. 1 chemically defined media. The results demonstrated significant alterations in the composition and structure of the endophytic consortium and metabolites of the endophytic consortium during iterative cultivation. Priestia aryabhattai dominated the community in the first generation, whereas Microbacterium paraoxydans and Bacillus velezensis became dominant by the tenth generation. SIN was detected at the first and the fifth generation, with declining concentrations, and was absent at the tenth generation. Correlation network analysis revealed a strong positive correlation between the relative abundance of P. aryabhattai and the SIN content. Furthermore, a specific strain, L15, identified as P. aryabhattai, was isolated from the iterative culture. UPLC-MS/MS analysis of P. aryabhattai L15 metabolites confirmed the presence of SIN, alongside other alkaloids including cyclanoline, N-methylhigenamine-7-O-glucopyranoside, and isoquinoline. Further metagenomic analysis also indicated that the relative abundance of P. aryabhattai was significantly (p < 0.05) positively correlated with the SIN content in plant tissues. This study systematically elucidated the role of endophytic bacteria and provides potential strains for the synthesis of bioactive compounds and pharmaceutical research.