Abstract:[Background] β-glucosidases can hydrolyze various glycosidic substrates into aglycones with industrial significance and have applications in food, medicine, and industry. In medicine, β-glucosidases can produce rare ginsenosides CK and 20(S)-Rh1 by transforming glycosides in natural products. Decaying wood in forests contains abundant lignocellulose, which is the degradation substrate of β-glucosidase. However, the discovery of β-glucosidase-producing bacteria in the study area of this study has not been reported. [Objective] To explore the microbial (gene) resources of β-glucosidases by the pure culture method and metagenomic analysis and compare the microbial diversity of decaying wood among different forest regions. [Methods] Bacteria were isolated by the pure culture method, and β-glucosidase-producing strains were screened by the medium containing aesculin and transformation of ginsenosides Rb1 and Rg1. Metagenomic sequencing was performed to supplement the pure culture and screening work and explore the structures and functions of microbial communities in different decaying wood samples. [Results] A total of 267 bacterial strains were isolated from decaying wood samples collected from five different regions in Yunnan Province, including Junzi Mountain in Shizong County, a primitive forest in Tuanjie Township of Kunming City, Wansong Mountain in Wuding County, Dawei Mountain in Pingbian County, and Fenshui Ridge in Jinping County. Among these strains, 154 β-glucosidase-producing strains were screened out, belonging to 37 genera, 19 families, 13 orders, 8 classes of 4 phyla. Proteobacteria was the dominant phylum among the enzyme-producing strains, and Chryseobacterium was the dominant genus. The primitive forest in Tuanjie Township of Kunming City had the highest diversity of β-glucosidase-producing bacteria. The strain YIM B11765, isolated from Wansong Mountain in Wuding County, exhibited the highest β-glucosidase activity of 0.34 U/mL. This strain and strain YIM B11728 can transform ginsenosides Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1, respectively. The results of the metagenomic analysis of decaying wood samples indicated that bacteria were dominant, with Proteobacteria and Actinomycetota showing the largest relative abundance. Streptomyces was the predominant genus. Among the samples from the five regions, glycoside hydrolases were the most abundant enzymes annotated in the CAZy database. Furthermore, four β-glucosidases with the potential to catalyze the transformation of ginsenosides Rb1 and Rg1 were screened from these enzymes. The microbial diversity was the highest in the samples from the primary forest in Tuanjie Township of Kunming City. Carbohydrate-active enzymes were the richest in the samples from Junzi Mountain in Shizong County. Streptomyces and Bradyrhizobium contributed the most to the main COG functions and KEGG metabolic pathways, as well as the functions of carbohydrate-active enzymes. [Conclusion] A batch of β-glucosidase-producing bacteria was obtained by pure culture isolation and screening, among which two strains could transform ginsenosides Rb1 and Rg1 into rare ginsenosides CK and 20(S)-Rh1, respectively. After metagenomic information mining, AlphaFold 3 prediction of protein tertiary structure, and virtual screening, four β-glucosidases with the potential to transform ginsenosides Rb1 and Rg1 were obtained, which supplemented the pure culture isolation and screening work. Junzi Mountain in Shizong County demonstrates great potential for the mining of functional microbial resources, which provides a theoretical basis for further development and utilization.
