Multi-Omics Reveals the Regulatory Effects of Chinese Herbal medicine Substrates on Secondary Metabolite Biosynthesis in Inonotus glomeratus

Chengbo Peng^1,2Meng Meng¹Xiaolong Yuan¹Jiaojun Yu³Boyi Wang⁴Li Zhong¹Yi Wang^1*Lu Li¹Tingwen He²Yuan Zheng^2,3*

• ¹Yunnan Academy of Forestry, Kunming, China
• ²Southwest Forestry University, Kunming, China
• ³Huanggang Normal University, Huanggang, China
• ⁴Yunnan Forestry Technological College, Kunming, China

The Inonotus genus, known for its medicinal properties, exhibits antioxidant and anti-tumor activities and is abundant in terpenoid and polyphenolic secondary metabolites. This study explored the impact of different Chinese herbal medicine powders as culture media, including Polygonum multiflorum, Coix lacryma-jobi, Pisum sativum flour, Salvia miltiorrhiza, Panax ginseng, and Astragalus membranaceus, on the secondary metabolite profile of Inonotus glomeratus using a multi-omics approach. Utilizing Illumina and Nanopore sequencing technologies, the genome of I. glomeratus was assembled, resulting in a 38.68 Mb genome consisting of 23 scaffolds with a GC content of 47.98%. The genome annotation process identified 67 transcription factors, four polyketide synthases (PKSs), one non-ribosomal peptide synthase, and 11 terpenoid synthases (TPSs). Multi-omics analysis revealed that terpenoid biosynthesis in I. glomeratus was significantly enhanced in DS and RS media. Betulin and betulinic acid exhibited the most dramatic increases in RS medium, reaching 4,658–9,275-fold and 4–503-fold higher concentrations, respectively. The transcriptome results showed that the expression of enzymes such as IgAACT, IgHMGR, IgSQS, IgSES, and IgTPS9 was significantly higher in the RS medium than in the other treatment groups. Integrated metabolomic and transcriptomic analyses suggested that IgPKS1 participates in orsellinic acid biosynthesis. while IgPKS2 is likely involved in naringenin biosynthesis. Additionally, IgTPS9 was associated with betulinic acid biosynthesis, and IgTPS10 contributed to tetracyclic sesquiterpene B-type triterpene formation. Co-expression network analysis and transcription factor binding site prediction indicated that IgMYB3 may regulates IgPKS1 expression, whereas IgHSF1 may simultaneously modulate IgTPS4 and IgTPS6. This study provides novel insights into the regulatory mechanisms governing secondary metabolite production in I. glomeratus.