Microbiome-based screening and co-fermentation of rhizospheric microorganisms for highly ginsenoside Rg₃ production

Ginsenoside Rg₃ has a wide range of pharmacological activities and application value while the content of Rg₃ in Panax plants is extremely low. The interaction between medicinal plants and microorganisms will be beneficial to produce active compounds by biotechnology. In this study, the rhizosphere soil samples from different P. notoginseng producing areas (Asanlong, Huilong, Demonstration garden) were collected to analyze the soil microflora characteristics by Illumina MiSeq sequencing technology. Based on the highest contents of ginsenosides in Huilong, the first, second, and the specificity dominant strains were predicted and the database was established. Besides, a total of 6 strains of bacteria and 3 strains of fungi were isolated from the soil of P. notoginseng. Among them, F24 was identified as Chaetomium sp, which was not only consistent with the second dominant strain of P. notoginseng predicted in the database, but also had a high level of Rg₃. It also indicated that the method of screening the dominant strains in soil was reasonable and the database was reliable. Through the optimization of fermentation conditions, the highest yield of Rg₃ was obtained when F24 was cultured in the medium supplemented with 8 mg/L glucose and 5 mg/L methyl jasmonate (MeJA) for 7 days. On this basis, the yield of ginsenoside Rg₃ in shake flask reached 108.95 mg/L (4.93 mg/g, ~4.2-fold higher than cultivated P. notoginseng plants) by the co-fermentation with A. niger in a concentration ratio of 2:1. The increase of Rg₃ yield mainly came from the conversion of other 20S-protopanaxdiol saponins into Rg₃ by β-glucosidase in A. niger. In addition, the expression levels of ginsenoside biosynthesis genes in different strains were compared, it was found that the expression of key genes was significantly increased after co-fermentation. This research provided certain theoretical and technical support for the large-scale industrial production of ginsenoside Rg₃ by microbiology.