Enhanced production and anti-inflammatory activity of exopolysaccharides through the overexpression of β-1,3-glucanosyltransferase gene in Ganoderma lingzhi.

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This study aimed to enhance the production and anti-inflammatory activity of exopolysaccharides (EPS) in Ganoderma lingzhi through genetic engineering. The β-1,3-glucanosyltransferase gene of the glycosyl-hydrolase 72 family was cloned and overexpressed in G. lingzhi. The overexpressed strain exhibited a maximum intracellular polysaccharide content of 26.49 ± 0.19 mg/100 mg dry cell weight and an EPS production of 1.16 ± 0.22 g/L, which were 28% and 23% higher, respectively, than those of the wild-type (WT) strain. Furthermore, the structural characteristics of G. lingzhi EPS were analyzed using methylation, one- and two-dimensional nuclear magnetic resonance, and Congo red assays. The major fraction GEL-0.1-1 from the overexpressed strain contained higher levels of →2)-α-D-Manp-(1→, →3)-β-D-Glcp (1→, and →4)-β-D-Glcp (1→ but lower levels of →6)-α-D-Galp (1→ and a more stable triple-helix structure than the corresponding fraction WT-0.1-1 from the WT strain. Both fractions exerted anti-inflammatory activity by suppressing pro-inflammatory cytokines release and M1 polarization in lipopolysaccharide-stimulated RAW264.7 cells. However, GEL-0.1-1 treatment led to a significantly greater reduction in the transcription levels of tumor necrosis factor-α, inducible nitric oxide synthase, interleukin (IL)-6, and IL-1β, decreasing by 38%, 54%, 54%, and 60%, respectively, compared to those of WT-0.1-1 treatment. Additionally, the proportion of M1-polarized cells under GEL-0.1-1 treatment was 61% lower than that observed in the WT-0.1-1 treatment. These findings support the efficient production and application of G. lingzhi EPS and provide insights into the relationship between EPS structure and biological activity.