Heme oxygenase-1/carbon monoxide signaling participates in the accumulation of triterpenoids of Ganoderma lucidum

Abstract

Ganoderic triterpenoids (GTs) are the primary bioactive constituents of the Basidiomycotina fungus, Ganoderma lucidum. These compounds exhibit antitumor, anti-hyperlipidemic, and immune-modulatory pharmacological activities. This study focused on GT accumulation in mycelia of G. lucidum mediated by the heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling. Compared with the control, hemin (10 μmol/L) induced an increase of 60.1% in GT content and 57.1% in HO-1 activity. Moreover, carbon monoxide-releasing molecule-2 (CORM-2), CO donor, increased GT content by 56.0% and HO-1 activity by 18.1%. Zn protoporphyrin IX (ZnPPIX), a specific HO-1 inhibitor, significantly reduced GT content by 26.0% and HO-1 activity by 15.8%, while hemin supplementation reversed these effects. Transcriptome sequencing showed that HO-1/CO could function directly as a regulator involved in promoting GT accumulation by regulating gene expression in the mevalonate pathway, and modulating the reactive oxygen species (ROS) and Ca²⁺ pathways. The results of this study may help enhance large-scale GT production and support further exploration of GT metabolic networks and relevant signaling cross-talk.

Keywords: Ganoderma lucidum; Heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling; Transcriptome sequencing; Triterpenoid.

Fig. 1. Effects of different hemin concentrations (0‒80 μmol/L) on the biomass, ganoderic triterpenoid (GT) accumulation, and heme oxygenase-1 (HO-1) activity of Ganoderma lucidum. Compared with the control (CK), mycelial biomass increased by 15.7% (20 μmol/L hemin) and 17.9% (40 μmol/L hemin); the GT content increased by 34.6% (5 μmol/L hemin), 60.1% (10 μmol/L hemin), and 51.2% (20 μmol/L hemin); HO-1 activity increased by 57.1% (10 μmol/L hemin) and 52.5% (20 μmol/L hemin). Data are expressed as mean±standard deviation (SD), n=3. Different letters indicate significant differences among groups at P<0.05.

Fig. 2. Effects of different treatments on the biomass, ganoderic triterpenoid (GT) accumulation, and heme oxygenase-1 (HO-1) activity of Ganoderma lucidum.Hemin, Zn protoporphyrin IX (ZnPPIX), carbon monoxide-releasing molecule-2 (CORM-2), bilirubin (BR), Fe²⁺, and ZnPPIX+hemin were added separately to fermentation cultures and induced for 72 h at a concentration of 10 μmol/L. Compared with the control (CK), the mycelial biomass increased by 37.1% (CORM-2); GT content increased by 60.1% (hemin), 56.0% (CORM-2), and 32.3% (BR), but decreased by 26.2% (ZnPPIX) and then recovered with hemin supplementation; HO-1 activity increased by 57.1% (hemin), 18.1% (CORM-2), and 11.2% (BR), but decreased by 15.8% (ZnPPIX), which was reversed by hemin supplementation. Data are expressed as mean±standard deviation (SD), n=3. Different letters indicate significant differences among groups at P<0.05.

Fig. 3. Effects of different treatments on the heme oxygenase-1 (HO-1) gene expression level of Ganoderma lucidum. Compared with the control (CK), HO-1 gene expression was significantly up-regulated to (1.64±0.10) times by hemin and (1.48±0.15) times by carbon monoxide-releasing molecule-2 (CORM-2), and were down-regulated to (0.71±0.14) times by Zn protoporphyrin IX (ZnPPIX), but hemin supplementation reversed this effect. Data are expressed as mean±standard deviation (SD), n=3. Different letters indicate significant differences among groups at P<0.05. BR: bilirubin.

Fig. 5. Gene Ontology (GO) categories of differentially expressed unigenes identified after hemin (a) and Zn protoporphyrin IX (ZnPPIX) (b) treatments relative to control. CC: cellular component; MF: molecular function; BP: biology process.

Fig. 5. Gene Ontology (GO) categories of differentially expressed unigenes identified after hemin (a) and Zn protoporphyrin IX (ZnPPIX) (b) treatments relative to control. CC: cellular component; MF: molecular function; BP: biology process.

Fig. 5. Gene Ontology (GO) categories of differentially expressed unigenes identified after hemin (a) and Zn protoporphyrin IX (ZnPPIX) (b) treatments relative to control. CC: cellular component; MF: molecular function; BP: biology process.

Fig. 6. Real-time polymerase chain reaction (RT-PCR) validation of transcriptome analysis. Eleven selected gene fragments involved in antioxidant properties and Ca²⁺ signaling after hemin (a) and Zn protoporphyrin IX (ZnPPIX) (b) treatments. After the ZnPPIX treatment, the expression levels of REDX and CAMP were not significantly different in the transcriptome analysis, which is not labeled in the figure. Other RT-PCR results bolstered the credibility of transcriptome experiments. Data are expressed as mean±standard deviation (SD), n=3. POD: peroxidase; GST: glutathione S-transferase; MnSOD: manganese superoxide dismutase; TXN: thioredoxin; REDX: redoxin; CAT: catalase; CAMP: calcium-binding mitochondrial carrier protein; CALM: calmodulin; cch1: calcium-channel protein cch1; CamK 1B: calcium/calmodulin-dependent protein kinase type 1B; ATPase: adenosine-triphosphate enzyme.

Fig. 6. Real-time polymerase chain reaction (RT-PCR) validation of transcriptome analysis. Eleven selected gene fragments involved in antioxidant properties and Ca²⁺ signaling after hemin (a) and Zn protoporphyrin IX (ZnPPIX) (b) treatments. After the ZnPPIX treatment, the expression levels of REDX and CAMP were not significantly different in the transcriptome analysis, which is not labeled in the figure. Other RT-PCR results bolstered the credibility of transcriptome experiments. Data are expressed as mean±standard deviation (SD), n=3. POD: peroxidase; GST: glutathione S-transferase; MnSOD: manganese superoxide dismutase; TXN: thioredoxin; REDX: redoxin; CAT: catalase; CAMP: calcium-binding mitochondrial carrier protein; CALM: calmodulin; cch1: calcium-channel protein cch1; CamK 1B: calcium/calmodulin-dependent protein kinase type 1B; ATPase: adenosine-triphosphate enzyme.

Fig. 7. Schematic representation showing that heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling participates in the regulation of ganoderic triterpenoid (GT) biosynthesis by mediating the gene expression of hmgr, sqs,and ls in Ganoderma lucidum. Transcriptome sequencing showed that HO-1/CO signaling also affects transduction of reactive oxygen species (ROS) and Ca²⁺ signaling. Question marks denote as yet undescribed or unverified pathways. The T-bar indicates inhibition. BV: biliverdin; BR: bilirubin; ZnPPIX: Zn protoporphyrin IX; hmgr: 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) reductase; sqs:squalene synthase; ls:lanosterol synthase.