Calcium-Induced Regulation of Sanghuangporus baumii Growth and the Biosynthesis of Its Triterpenoids

Abstract

Sanghuangporus baumii, a fungus used in traditional Chinese medicine, produces important pharmacological compounds such as triterpenoids, but at levels significantly lower than those required for medical use. This study investigated the effects of various concentrations of Ca²⁺ on S. baumii mycelial growth and the heterologous biosynthesis of S. baumii triterpenoids. Under induction by 10 mM Ca²⁺, the growth rate (0.39 cm/d) and biomass (4.48 g/L) of S. baumii mycelia were 1.03% and 10.05% higher than those in the 0 mM Ca²⁺-treatment group, respectively. In contrast, 200 mM Ca²⁺ significantly inhibited the growth rate and biomass of the mycelia. Notably, the total triterpenoid content reached its peak (17.71 mg/g) in the 200 mM Ca²⁺-treatment group, with a significant increase in the Ca²⁺ content (3869.97 µg/g) in the mycelia. Subsequently, the differential metabolic pathways and related genes between the S. baumii groups were examined using transcriptomic analysis. The results indicated that the increase in the growth rate and biomass of S. baumii mycelia was primarily due to elevated soluble sugar content, whereas the growth inhibition was associated with the toxic effects of H₂O₂. The observed differences in triterpenoid content were mainly attributed to the activation of the terpenoid backbone biosynthesis pathway and the AACT gene. Finally, the AACT gene was cloned and transformed into yeast cells, thus creating strain Sc-AA1. Upon treatment at the optimal Ca²⁺ concentration, the squalene content of strain Sc-AA1 reached 0.78 mg/g, 2.89-fold higher than that in the control group. These findings are significant for the heterologous biosynthesis of triterpenoids from S. baumii. Our study demonstrates the feasibility of producing triterpenoids in Saccharomyces cerevisiae and provides a foundation for future optimization toward achieving industrially relevant yields.

Keywords: AACT gene; Saccharomyces cerevisiae; Sanghuangporus baumii; calcium; heterologous biosynthesis; triterpenoid.

Figure 1

Growth characteristics and total triterpenoid content of Sanghuangporus baumii mycelia treated with various concentrations of Ca²⁺. (A) Mycelial growth rate; (B) biomass; (C) total triterpenoid content in S. baumii mycelia; (D) Ca²⁺ content in S. baumii mycelia. Different letters share significant difference, p < 0.05.

Figure 2

Transcriptome analysis to analyze the differences in growth characteristics and metabolite synthesis between Sanghuangporus baumii mycelia treated with various Ca²⁺ concentrations. (A) RNA gel electrophoresis of nine samples (three replicates each of the 0 mM Ca²⁺ [Ca0], 10 mM Ca²⁺ [Ca10], and 200 mM Ca²⁺ treatment groups [Ca200]); (B) PCA score plot of transcript profiles of the Ca0, Ca10, and Ca200 groups; (C) volcano plot of upregulated and downregulated genes identified in pairwise comparisons; (D) qRT-RCR validation of gene expression; (E) KEGG enrichment analysis of DEGs from pairwise comparisons.

Figure 3

Soluble sugar and H₂O₂ content of Sanghuangporus baumii treated with various Ca²⁺ concentrations. (A) Soluble sugar content; (B) H₂O₂ content. Different letters share significant difference, p < 0.05.

Figure 4

Ca²⁺-induced biosynthesis of triterpenoids in Sanghuangporus baumii. acetyl-CoA acyltransferase, AACT; 3-hydroxy-3-methylglutaryl-CoA synthase, HMGS; 3-hydroxy-3-methylglutaryl-CoA reductase, HMGR; mevalonate kinase, MVK; phosphomevalonate kinase, PMK; mevalonate pyrophosphate decarboxylase, MVD; squalene synthase, SQS; lanosterol synthase, LS. (A) Expression levels of genes associated with triterpenoid biosynthesis; (B) results of Pearson correlation analysis among Ca²⁺ content, total triterpenoid content, and expression levels of key genes involved in triterpenoid biosynthesis.

Figure 5

Heterologous biosynthesis of Sanghuangporus baumii triterpenoid in yeast. (A) AACT gene cloning; (B) construction of the AACT gene overexpression vector; (C) detection of positive transformants; (D) homologous alignment analysis of the AACT gene; (E) squalene content in yeast (Ck1, yeast cells transformed with pYES2-NTC; Ca150, cells transformed with pYES2-NTC treated with 150 mM Ca²⁺; Ca150+pYE-AACT, cells expressing the AACT gene and treated with 150 mM Ca²⁺). Different letters share significant difference, p < 0.05.