Mushrooms produce a large number of medicinal bioactive metabolites with antioxidant, anticancer, antiaging, and other biological activities. However, whether they produce flavonoids and, if so, how they synthesize them remains a matter of some debate. In the present study, we combined flavonoid-targeted metabolomics and transcriptome analysis to explore the flavonoid synthesis in the medicinal mushroom Sanghuangporus baumii. The S. baumii synthesized 81 flavonoids on a chemically defined medium. The multiple classes of flavonoids present were consistent with the biosynthetic routes in plants. However, paradoxically, most of the genes that encode enzymes involved in the flavonoid biosynthetic pathway are missing from S. baumii. Only four genes related to flavonoid synthesis were found in S. baumii, among which phenylalanine ammonia lyase gene (PAL) is a key gene regulating flavonoid synthesis, and overexpression of SbPAL increases the accumulation of flavonoids. These results suggest that the flavonoid synthesis pathway in S. baumii is different from that in known plants, and the missing genes may be replaced by genes from the same superfamilies but are only distantly related. Thus, this study provides a novel method to produce flavonoids by metabolic engineering using mushrooms.

Sanghuangporus baumii is a traditional medicinal fungus that produces pharmacological terpenoids, but natural resources are insufficient for applications, and its growth and development mechanisms are poorly understood. Combining metabolomic and transcriptomic analyses, we found four terpenoid hormones and a central gene, isopentenyl diphosphate isomerase (IDI), involved in growth and development. Additionally, an exogenous hormone test was used to further confirm the importance of the four terpenoid hormones. Finally, hormone content determination and qRT−PCR were performed to explore the growth and development mechanism; we found thatcis-zeatin (CZ) plays a major role in the mycelia stage, trans-zeatin (TZ) and gibberellin A4 (GA4) are important in the primordia stage, GA4 is crucial for the fruiting bodies stage, and abscisic acid (ABA) may be a marker of maturity. The IDI gene was also found to affectterpenoid hormone content by regulating the relative gene transcript levels, thereby controlling morphological changes in S. baumii. Our results revealthe growth and development mechanisms of S. baumii and may promote the breeding and utilisation of high-quality varieties.

Triterpenoids are secondary metabolites produced by the fungus Sanghuangporus baumii that have important pharmacological activities. However, the yield of triterpenoids is low and cannot meet market demand. Here, we treated S. baumii with several concentrations of MeJA (methyl jasmonate) and found that the total triterpenoid content was highest (23.31 mg/g) when the MeJA concentration was 250 μmol/L. qRT-PCR was used to quantify the transcription of five key genes involved in triterpenoid biosynthesis. The results showed that the relative transcription of most genes increased with increasing MeJA concentration, indicating that MeJA is a potent inducer of triterpenoid biosynthesis in S. baumii. To further explore whether other terpenoid biosynthesis pathways are also involved in the accumulation of triterpenoids induced by MeJA, we measured the contents of cis-Zeatin (cZ), gibberellins (GAs), and the transcript levels of related biosynthesis genes. We found that MeJA significantly inhibited the biosynthesis of cZ, GAs, and the transcription of related genes. The repressive effects of MeJA on cZ and GA accumulation were further confirmed by growth rate and biomass assays. In conclusion, our study provides an effective method to enhance the triterpenoid content of S. baumii, and also provides novel insights into the mechanism of MeJA-induced triterpenoid biosynthesis.

Background: Basic helix–loop–helix (bHLH) transcription factors (TFs) widely exist in eukaryotic organisms and play a key role in plant growth and development in response to environmental stresses. Sanghuangporus baumii, an important medicinal mushroom known for its anticancer properties, has limited research on the bHLH gene family. Methods: This research utilized the genomic data from S. baumii to identify bHLH family members, and their gene structure, conserved motifs, and phylogenetic relationship were characterized. Additionally, we conducted an analysis of promoter cis-elements and predicted protein interaction networks. We also examined the expression profiles of bHLH genes during different developmental stages and in response to four abiotic stresses: heat, cold, oxidative stress, and heavy metal exposure. Finally, we overexpressed the candidate gene SbbHLH3 in yeast to assess its tolerance to these different stress conditions. Results: A total of 12 SbbHLH genes were identified in S. baumii, and the members of the bHLH gene family displayed a variety of physicochemical characteristics, reflecting their diverse array of functions. Based on homology, the SbbHLH proteins are more closely related to those found in Lentinula edodes and Pleurotus ostreatus. The analysis of promoter cis-elements showed that SbbHLHs contain several elements associated with abiotic stress response, and a network prediction identified 28 bHLH-interacting proteins. Expression pattern analysis revealed that most SbbHLH genes exhibited a positive response to different developmental stages and abiotic stresses. Notably, the overexpression of SbbHLH3 significantly enhanced stress tolerance in yeast. Conclusions: This study provides a comprehensive assessment of the bHLH family in S. baumii, delivering new genetic resources for breeding resistant varieties.

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.

Triterpenoids derived from Sanghuangporus baumii exhibit potent antitumor activity, but their yields under natural conditions are relatively low due to their status as secondary metabolites. In this study, we investigated the effects of Zn²⁺ induction on the growth and triterpenoid biosynthesis of S. baumii. The results showed that 0.5 mM Zn²⁺ significantly enhanced the mycelial growth rate (0.43 ± 0.004 cm/d) and biomass (4.8 ± 0.024 g/L), representing increases of 8.71% and 16.95%, respectively, compared with the Zn0 group. This result was mainly caused by an increase in the soluble sugar content. Furthermore, 5 mM Zn²⁺ induced upregulation of genes in the mevalonate (MVA) pathway, thereby promoting triterpenoid accumulation by 167.86% compared with the Zn0 group. Transcriptome analysis identified SbHMGS as the key gene involved in triterpenoid biosynthesis under Zn²⁺ induction. Heterologous expression of SbHMGS in Saccharomyces cerevisiae confirmed its critical role in triterpenoid production. The triterpenoid (squalene) content of the engineered strain (Sc-HMGS) reached 0.88 mg/g under Zn²⁺ induction, which was 208.6% higher than in the non-induced control strain (Sc-NTC). These findings provide a foundation for optimizing the industrial fermentation condition of S. baumii and S. cerevisiae to enhance triterpenoid yields.