Ganoderic Acid Production Research Articles

Development of a 2A peptide-based multigene expression system and its application for enhanced production of ganoderic acids in Ganoderma lucidum

Ganoderma has received much attention for its medicinal value, but the manipulation of multiple genes remains a challenge, hindering the genetic engineering of this species for the development of cell factories. Here, we first showed that the presence of an intron is necessary for the efficient expression of the endogenous cDNA of carboxin-resistant gene (cbx) in G. lucidum. Then, the self-cleaving function of 2 A peptide was investigated in G. lucidum by linking cbx cDNA to the codon-optimized hygromycin B-resistant gene (ophph) using the 2A-peptide sequence. The results showed that cbx cDNA and ophph can be successfully expressed in G. lucidum in a bicistronic manner from a single transcript. Moreover, the expression of both genes was not affected by the order within the 2 A cassette. In addition, simultaneous expression of cbx cDNA, ophph, and codon-optimized yellow fluorescent protein gene (opyfp) was conducted for the first time in G. lucidum using the 2 A peptide-based approach. The developed method was successfully applied to express both cDNA of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and squalene epoxidase gene (se) for enhanced production of ganoderic acids (GAs) in G. lucidum. The engineered strain produced the maximum content of GA-Mk, GA-T, GA-S, and GA-Me were 26.56±3.53,39.58±3.75, 16.54±2.16, and 19.1±1.87 μg/100 mg dry weight, respectively. These values were 3.85-, 4.74-, 3.65-, and 3.23-fold higher than those produced by the control strain. The developed method will be useful for the manipulation of complex metabolic or regulatory pathways involving multiple genes in Ganoderma.

Strategies to Increase the Production of Triterpene Acids in Ligzhi or Reishi Medicinal Mushroom (Ganoderma lucidum, Agaricomycetes): A Review.

Ganoderic acids (GAs) are the main active ingredient of Ganoderma lucidum, which has been widely accepted as a medicinal mushroom. Due to the low yield of GAs produced by liquid cultured Ganoderma mycelium and solid cultured fruiting bodies, the commercial production and clinical application of GAs are limited. Therefore, it is important to increase the yield of GA in G. lucidum. A comprehensive literature search was performed with no set data range using the following keywords such as "triterpene," "ganoderic acids," "Ganoderma lucidum," and "Lingzhi" within the main databases including Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI). The data were screened using titles and abstracts and those relevant to the topic were included in the paper and was not limited to studies published in English. Present review focuses on the four aspects: fermentation conditions and substrate, extrinsic elicitor, genetic engineering, and mutagenesis, which play significant roles in increasing triterpene acids production, thus providing an available reference for further research on G. lucidum fermentation.

Effect of Methyl Jasmonate Elicitation on Triterpene Production and Evaluation of Cytotoxic Activity of Mycelial Culture Extracts of Ganoderma applanatum (Pers.) Pat.

Abiotic elicitation, a well-known strategy in mushroom biotechnology, promotes increased accumulation of secondary metabolites in mycelial cultures. The study aimed the effects of methyl jasmonate (MeJA) on the production of triterpenes in submerged cultures of Ganoderma applanatum. Further, the study evaluated the cytotoxic activity of the extract corresponding to the optimal elicitation variant in selected human cancer cell lines as well as the selectivity against normal cells. MeJA was added on days 1, 4, 6, and 8 in the 10-day growth cycle at concentrations of 10, 50, 100, 150, and 200 µM MeJA. The HPLC-DAD was used to analyze the triterpenes. The cytotoxic activity was tested using the MTTFc assay in grouped panels of skin, prostate, and gastrointestinal cancer cells. The results of the quantitative analyses confirmed the stimulating effect of MeJA on the production of ganoderic acid A and ganoderic acid C. The greatest increase in total triterpenes was found on day 6 of the culture cycle compared to the control group-with the concentration of MeJA-150 µM. Compared to the control samples, mycelial culture extract after the most productive elicitation variant showed significant cytotoxic activity against prostate cancer cells and moderate effects on melanoma cells. Ganoderma applanatum mycelial cultures can be proposed as a model to study the dynamics of the accumulation of compounds with therapeutic values through abiotic elicitation.

Integrated Transcriptomics and Nontargeted Metabolomics Analysis Reveal Key Metabolic Pathways in Ganoderma lucidum in Response to Ethylene.

Ganoderic acid (GA) is an important secondary metabolite of Ganoderma lucidum with a diverse array of pharmacological properties. In this study, we found that exogenous ethylene increased the production of endogenous ethylene and ganoderic acid in G. lucidum. However, the mechanism by which ethylene is regulated remains unclear. As a result, we performed a combined transcriptomics and nontargeted metabolomics analysis to evaluate the regulatory mechanism of ethylene. A total of 4070 differentially expressed genes (1835 up-regulated and 2235 down-regulated) and 378 differentially accumulated metabolites (289 up-regulated and 89 down-regulated) were identified in all groups. The transcriptomics and nontargeted metabolomics data revealed that genes involved in the tricarboxylic acid (TCA) cycle, polyamine metabolic pathway, acetyl-CoA carboxylase (ACC) pathway, and triterpenoid metabolism were up-regulated, whereas the metabolic intermediates involved in these metabolic pathways were down-regulated. These findings imply that ethylene potentially accelerates normal glucose metabolism, hence increasing the number of intermediates available for downstream biological processes, including polyamine metabolism, ethylene synthesis pathway, and ganoderic acid biosynthesis. The findings will contribute significantly to our understanding of secondary metabolites biosynthesis in fungi.

Biosynthesis, Gene Expression, and Pharmacological Properties of Triterpenoids of Ganoderma Species (Agaricomycetes): A Review.

For more than 6 millennia, Ganoderma species have been used in traditional Asian medicine due to their health benefits. Ganoderma synthesizes several compounds with biological activity, including lanostane-type triterpenoids like ganoderic acids (GAs), lucidones, and colosolactones. These triterpenoids have been investigated for their antiviral, hypoglycemic, and anticancer effects. GAs are highly oxygenated triterpenoids with different functional groups attached to lanostane skeleton. Their great chemical diversity makes GAs prospects for the development of new drugs to treat multiple illnesses such as cancer. The effect of GAs against cancer cells has been associated with their capability to inhibit specific targets such as STAT3, to induce apoptosis and cell cycle blockage, and to increase natural killer cell activity. Due to the biological activity of these molecules, novel strategies are being developed for Ganoderma production mainly by liquid cultivation, gene overexpression (HMGR, SQS, LS) by elicitors, and modified growing conditions (carbon and nitrogen sources, pH, temperature), which induce reactive oxygen species production, key compounds for secondary metabolism. In addition, some transcription factors are mainly expressed under stress conditions, such as cytochrome P450 genes, which participate in the regulation of triterpenoid synthesis. The fermentation process has been scaled up to a 300-L bioreactor, which shows good GA production. This article reviews current knowledge on bioactive triterpenoids of Ganoderma and their production, biosynthesis, and pharmacological properties, emphasizing gene expression in liquid culture. It also discusses the lack of information regarding other species with high potential.

Biosynthesis of a novel ganoderic acid by expressing CYP genes from Ganoderma lucidum in Saccharomyces cerevisiae.

Ganoderic acids (GAs), a group of highly oxygenated lanostane-type triterpenoids from the traditional Chinese medicinal mushroom Ganoderma lucidum, possessed significant pharmacological activities. Due to the difficulty in its genetic manipulation, low yield, and slow growth of G. lucidum, biosynthesis of GAs in a heterologous host is a promising alternative for their efficient production. Heterologous production of a GA, 3-hydroxy-lanosta-8,24-dien-26-oic acid (HLDOA), was recently achieved by expressing CYP5150L8 from Ganoderma lucidum in Saccharomyces cerevisiae, but post-modification of HLDOA to biosynthesize other GAs remains unclear. In this study, another P450 from G. lucidum, CYP5139G1, was identified to be responsible for C-28 oxidation of HLDOA, resulting in the formation of a new GA 3,28-dihydroxy-lanosta-8,24-dien-26-oic acid (DHLDOA) by the engineered yeast, whose chemical structure was confirmed by UPLC-APCI-HRMS and NMR. In vitro enzymatic experiments confirmed the oxidation of HLDOA to DHLDOA by CYP5139G1. As the DHLDOA production was low (0.27mg/L), to improve it, the strategy of adjusting the dosage of hygromycin and geneticin G418 to respectively manipulate the copy number of plasmids pRS425-Hyg-CYP5150L8-iGLCPR (harboring CYP5150L8, iGLCPR, and hygromycin-resistant gene hygR) and pRS426-KanMx-CYP5139G1 (harboring CYP5139G1 and G418-resistant gene KanMx) was adopted. Finally, 2.2mg/L of DHLDOA was obtained, which was 8.2 fold of the control (without antibiotics addition). The work enriches the GA biosynthetic enzyme library, and is helpful to construct heterologous cell factories for other GA production as well as to elucidate the authentic GA biosynthetic pathway in G. lucidum. KEY POINTS: • Another P450 gene responsible for GA's post-modification was discovered and identified. • One new GA, DHLDOA, was identified and produced via engineered yeast. • With the balance of the two CYP genes expression, DHLDOA production was significantly improved.

Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum.

Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at −80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase (Erk1/2, p38, and JNK) and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts.

Heat stress promotes the conversion of putrescine to spermidine and plays an important role in regulating ganoderic acid biosynthesis in Ganoderma lucidum.

Heat stress (HS) is inescapable environmental stress that can induce the production of ganoderic acids (GAs) in Ganoderma lucidum. Our previous studies found that putrescine (Put) played an inhibitory role in GAs biosynthesis, which appeared to be inconsistent with the upregulated transcription of the Put biosynthetic gene GlOdc under HS. To uncover the mechanism underlying this phenomenon, two spermidine (Spd) biosynthetic genes, GlSpds1 and GlSpds2, were identified and upregulated under HS. Put and Spd increased by 94% and 160% under HS, respectively, suggesting that HS induces polyamine biosynthesis and promotes the conversion of Put to Spd. By using GlSpds knockdown mutants, it is confirmed that Spd played a stimulatory role in GAs biosynthesis. In GlOdc-kd mutants, Put decreased by 62-67%, Spd decreased by approximately 34%, and GAs increased by 15-22% but sharply increased by 75-89% after supplementation with Spd. In GlSpds-kd mutants, Put increased by 31-41%, Spd decreased by approximately 63%, and GAs decreased by 24-32% and were restored to slightly higher levels than a wild type after supplementation with Spd. This result suggested that Spd, rather than Put, is a crucial factor that leads to the accumulation of GAs under HS. Spd plays a more predominant and stimulative role than Put under HS, possibly because the absolute content of Spd is 10 times greater than that of Put. GABA and H2O2, two major catabolites of Spd, had little effect on GAs biosynthesis. This study provides new insight into the mechanism by which environmental stimuli regulate secondary metabolism via polyamines in fungi. KEY POINTS: • HS induces polyamine biosynthesis and promotes the conversion of Put to Spd in G. lucidum. • Put and Spd played the inhibitory and stimulatory roles in regulating GAs biosynthesis, respectively. • The stimulatory role of Spd was more predominant than the inhibitory role of Put in GAs biosynthesis.

Enhancement of ganoderic acid production by promoting sporulation in a liquid static culture of Ganoderma species

Ganoderic acids (GAs) produced by Ganoderma are a type of lanostane-type triterpenoids with anticancer and antimetastatic activities; however, low production of GAs limits its wide application. In this study, a novel strategy by promoting sporulation of Ganoderma was developed to increase GA production. First, a high-spore producing Ganoderma strain G. 260125 was obtained from dikaryotic strain CGMCC 5.0026, and the sporulation-specific gene of this strain exhibits a higher transcription level than CGMCC 5.0026. Then, the effect of promoting sporulation on GA content was investigated. The maximum ganoderic acid (GA)-T, GA-Mk, and GA-Me contents in G. 260125 in shake flasks were 358.97, 78.32, and 12.75 μg/100 mg dry weight, respectively, which were 3.42, 2.91, and 1.73 times higher than those obtained in CGMCC 5.0026. Moreover, total and individual GA contents in spores were significantly higher than those in liquid static culture. Both concentrations of intermediates and transcription levels of GA biosynthetic genes also improved in G. 260125 during fermentation compared with those in CGMCC 5.0026. For scaling-up experiments, GA-T, GA-Me, and GA-Mk production in G. 260125 improved by 2.2-, 2.6-, and 2.1-fold compared with those in CGMCC 5.0026. In addition, the effectiveness of the developed strategy was also confirmed in three different Ganoderma strains. This work illustrated that promoting sporulation efficiently improves GA production in liquid static cultures of Ganoderma.

Effects of different graphene-based nanomaterials as elicitors on growth and ganoderic acid production by Ganoderma lucidum.

Graphene-based nanomaterials (GBNs) have attracted considerable interest nowadays due to their wide range of applications. However, very little attention has been paid to the application of nanomaterials as potential elicitors for production of valuable metabolites. Herein, aiming to earn insight into effects of nanomaterials on secondary metabolite biosynthesis by medicinal fungi, we evaluated the influence of GBNs on growth and production of ganoderic acid (GA) by Ganoderma lucidum in submerged culture. Graphene oxide (GO), reduced graphene oxide (rGO), and rGO/Fe3 O4 nanocomposite were synthesized successfully and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy analysis. The prepared nanomaterials were added to the culture of G. lucidum at final concentrations of 50, 100, and 150 mg/L on Day 5. The results showed that the elicitation of G. lucidum with GO and rGO decreased the cell dry weight and GA production slightly, especially in higher concentrations. However, rGO/Fe3 O4 nanocomposite not negatively affected cell growth and improved GA production. G. lucidum growth rate responded to elicitation experiments differently and depended on the type of nanomaterials and their concentrations, but almost all GBNs caused an increase in GA content (mg/100 mg dry weight). Also, field emission scanning electron microscopy morphological study showed that under elicitation, mycelia were more condensed and tightly stacked together. The findings from this study may suggest that GBNs in low concentrations could be applied as elicitors to secondary metabolites production from higher fungus, but further environmental, physiological, and biological studies required.

Role of calcineurin-responsive transcription factor CRZ1 in ganoderic acid biosynthesis by Ganoderma lucidum

Roles of functional genes in secondary metabolism of mushroom-forming basidiomycetes are rarely clarified due to the difficulty of gene knock-out in them. In Ganoderma lucidum, an antitumor ganoderic acid (GA) producing basidiomycete, two homologs of a Cys2-His2 (C2H2)-type zinc finger transcription factor (CRZ1) - GlCRZ1 and GlCRZ2 were found by bioinformatic analysis. Using a recently developed CRISPR/Cas9 genome editing platform, each gene of the two CRZ1 orthologs (glcrz1 and glcrz2) was disrupted to reveal their function in calcium signaling of GA biosynthesis. Compared to the wild type strain (WT), in which Ca2+ addition enhanced the GA production, disruption of glcrz1 did not hamper the cell growth but abolished the calcium signaling effect on stimulating GA biosynthesis. For the glcrz2 knocked-out strain, both the mycelial growth and GA synthesis were severely attenuated and its GA production declined even with Ca2+ treatment. These results indicated that the calcium signaling via GlCRZ1 and GlCRZ2 were critical in the calcineurin-mediated regulation of GA biosynthesis by G. lucidum. The work may be also helpful to understand the function of CRZ1 in secondary metabolism regulation in other basidiomycetes.

The Pharmacological Effects of Triterpenoids from <i>Ganoderma lucidum</i> and the Regulation of Its Biosynthesis

Ganoderma lucidum is a wildly used medicinal mushroom and has been used in East Asia because of its health-benefit effects. G. lucidum contains various compounds with different biological activities, which include anti-tumour, anti-viral, and anti-malaria effect. Ganoderic acid (GA) is a triterpenoid from G. lucidum. The biosynthesis of GA in G. lucidum is induced by stressors including various elicitors or signaling molecules, and artificially placing elicitors would enhance GA production. In this paper, bioactivities and potential applications of GAs would be focused, and the elicitation strategies for GA production are also highlighted based on the fundamental role of ROS, JA, Ca2+ and NO, which would shed light to developing a novel approach to manipulating the biosynthesis of GA in the future.

Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum

BackgroundGanoderic acids (GAs), derived from the medicinal mushroom Ganoderma lucidum, possess anticancer and other important pharmacological activities. To improve production of GAs, a homologous farnesyl diphosphate synthase (FPS) gene was overexpressed in G. lucidum. Moreover, the influence of FPS gene overexpression on GA production was investigated by developing the corresponding mathematical models.ResultsThe maximum levels of total GAs and individual GAs (GA-T, GA-S, and GA-Me) in the transgenic strain were 2.76 mg/100 mg dry weight (DW), 41 ± 2, 21 ± 5, and 28 ± 1 μg/100 mg DW, respectively, which were increased by 2.28-, 2.27-, 2.62-, and 2.80-folds compared with those in the control. Transcription levels of squalene synthase (SQS) and lanosterol synthase (LS) genes during GA biosynthesis were upregulated by 2.28- and 1.73-folds, respectively, in the transgenic G. lucidum. In addition, the developed unstructured models had a satisfactory fit for the process of GA production in submerged cultures of G. lucidum. Analysis of the kinetic process showed that FPS gene overexpression had a stronger positive impact on GA production compared with its influence on cell growth. Also, FPS gene overexpression led to a higher non-growth-associated-constant β (1.151) over the growth-associated-constant α (0.026) in the developed models.ConclusionsFPS gene overexpression is an effective strategy to improve the production of GAs in G. lucidum. The developed mathematical models are useful for developing a better GA production process in future large-scale bioreactors.

Enhanced Ganoderic Acids Accumulation and Transcriptional Responses of Biosynthetic Genes in Ganoderma lucidum Fruiting Bodies by Elicitation Supplementation.

Ganoderic acids (GAs) are a type of highly oxygenated lanostane-type triterpenoids that are responsible for the pharmacological activities of Ganoderma lucidum. They have been investigated for their biological activities, including antibacterial, antiviral, antitumor, anti-HIV-1, antioxidation, and cholesterol reduction functions. Inducer supplementation is viewed as a promising technology for the production of GAs. This study found that supplementation with sodium acetate (4 mM) significantly increased the GAs content of fruiting bodies by 28.63% compared to the control. In order to explore the mechanism of ganoderic acid accumulation, the transcriptional responses of key GAs biosynthetic genes, including the acetyl coenzyme A synthase gene, and the expression levels of genes involved in calcineurin signaling and acetyl-CoA content have been analyzed. The results showed that the expression of three key GAs biosynthetic genes (hmgs, fps, and sqs) were significantly up-regulated. Analysis indicated that the acetate ion increased the expression of genes related to acetic acid assimilation and increased GAs biosynthesis, thereby resulting in the accumulation of GAs. Further investigation of the expression levels of genes involved in calcineurin signaling revealed that Na+ supplementation and the consequent exchange of Na+/Ca2+ induced GAs biosynthesis. Overall, this study indicates a feasible new approach of utilizing sodium acetate elicitation for the enhanced production of valuable GAs content in G. lucidum, and also provided the primary mechanism of GAs accumulation.

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.

Enhanced production of individual ganoderic acids by integrating Vitreoscilla haemoglobin expression and calcium ion induction in liquid static cultures of Ganoderma lingzhi.

SummaryGanoderic acids produced by Ganoderma exhibit anticancer and antimetastatic activities. A novel approach by combining Vitreoscilla haemoglobin (VHb) expression and calcium ion induction was developed to enhance ganoderic acid (GA) production in liquid static cultures of G. lingzhi. The maximum contents of GA‐O, GA‐S and GA‐Me were 1451.33 ± 67.50, 1431.23 ± 79.74 and 1283.81 ± 85.13 μg per 100 mg cell weight, respectively under the integrated approach, which are the highest contents as ever reported in Ganoderma. The contents of squalene and lanosterol were increased by 2.0‐ and 3.0‐fold in this case compared with those in the control. The transcription levels of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, farnesyl‐diphosphate synthase, squalene synthase and cytochrome P450 CYP5150L8 were upregulated by 2.56‐, 3.31‐, 2.59‐ and 6.12‐fold respectively. Additionally, the expression of VHb improved the ratio of type I to type II GA in liquid static cultivation of G. lingzhi. The transcription levels of cyp512a2, cyp512v2 and cyp512a13, candidate cytochrome P450 genes involved in oxidative modification of the lanostane skeleton in GA biosynthesis, were also increased by 2.28‐, 2.65‐ and 3.54‐fold in the VHb‐expressing strain respectively. Our results illustrated that the approach described here efficiently improved GA production in G. lingzhi fermentation.

Optimization of Culture Condition for Ganoderic Acid Production in Ganoderma lucidum Liquid Static Culture and Design of a Suitable Bioreactor.

Ganoderma lucidum, a famous medicinal mushroom used worldwide, is a rich source of triterpenoids which, together with polysaccharides, are believed to be the main effective constituents of G. lucidum. With the increase of market demand, the wild resource is facing serious limitations, and the quality of cultivated fruiting bodies can be seriously affected by the availability of wood resources and by cultivation management practices. In the present study, we aimed to develop an alternative way to produce useful triterpenoids from G. lucidum. We cultured the strain using a two-stage liquid culture strategy and investigated the effects of nitrogen limitation, carbon supply, static culture volume and air supply in the static culture stage on the accumulation of five triterpenoids (GA-P, GA-Q, GA-T, GA-S, GA-R). Our results showed that, under optimized condition, the total yield of the five triterpenoids reached 963 mg/L (as determined by HPLC). Among the five triterpenoids, GA-T accounted for about 75% of the total yield. Besides, a bioreactor suitable for fungal liquid static culture with a 10 L extensible plastic bag shaped culture unit was designed and in which the maximum total yield of the five GAs reached 856.8 mg/L, and the GAs content reached 5.99%. Our results demonstrate the potential of industrial application of G. lucidum culture for the production of triterpenoids, especially GA-T. Air supply significantly improved the accumulation of triterpenoids, and this will provide important clues to understand why more triterpenoids are produced in the mycelia mat under static liquid culture conditions.

Cross Talk between Nitric Oxide and Calcium-Calmodulin Regulates Ganoderic Acid Biosynthesis in Ganoderma lucidum under Heat Stress.

We previously reported that high temperature impacts ganoderic acid (GA) biosynthesis in Ganoderma lucidum via Ca2+ Therefore, to further understand the signal-regulating network of the organism's response to heat stress (HS), we examined the role of nitric oxide (NO) under HS. After HS treatment, the NO level was significantly increased by 120% compared to that under the control conditions. The application of a NO scavenger resulted in a 25% increase in GA compared with that found in the sample treated only with HS. Additionally, the application of a NO donor to increase NO resulted in a 30% lower GA content than that in the sample treated only with HS. These results show that the increase in NO levels alleviates HS-induced GA accumulation. Subsequently, we aimed to detect the effects of the interaction between NO and Ca2+ on GA biosynthesis under HS in G. lucidum Our pharmacological approaches revealed that the NO and Ca2+ signals promoted each other in response to HS. We further constructed the silenced strain of nitrate reductase (NR) and calmodulin (CaM), and the results are in good agreement with the silenced strain and pharmacological experiment. The cross-promotion between NO and Ca2+ signals is involved in the regulation of HS-induced GA biosynthesis in G. lucidum, and this finding is supported by studies with NR-silenced (NRi) and CaM-silenced (CaMi) strains. However, Ca2+ may have a more direct and significant effect on the HS-induced GA increase than NO. These data indicate that NO functions in signaling and has a close relationship with Ca2+ in HS-induced GA biosynthesis.IMPORTANCE HS is an important environmental stress affecting the growth and development of organisms. We previously reported that HS modulates GA biosynthesis in G. lucidum via Ca2+ However, the signal-regulating network of the organism's response to HS has not yet been elucidated. In this study, we found that NO relieved HS-induced GA accumulation, and NO and Ca2+ could exert promoting effects on each other in response to HS. Further research on the effect of NO and Ca2+ on the production of GAs in response to HS indicated that Ca2+ has a notably more direct and significant effect on the HS-induced GA increase than NO. Our results improve our understanding of the mechanism of HS signal transduction in fungi. A greater understanding of the regulation of secondary metabolism in response to environmental stimuli will provide clues regarding the role of these products in fungal biology.

Biosynthesis of a ganoderic acid in Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganoderma lucidum.

Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of cytochrome P450 monooxygenase (CYP450) gene candidates from G. lucidum, which may be responsible for the GA biosynthesis from lanosterol but have not been functionally characterized. As a result, overexpression of a CYP450 gene cyp5150l8 was firstly found to produce an antitumor GA, 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) in S. cerevisiae, as confirmed by HPLC, LC-MS and NMR. A final titer of 14.5 mg/L of HLDOA was obtained at 120 hr of the yeast fermentation. Furthermore, our in vitro enzymatic experiments indicate that CYP5150L8 catalyzes a three-step biotransformation of lanosterol at C-26 to synthesize HLDOA. To our knowledge, this is the first report on the heterologous biosynthesis of GAs. The results will be helpful to the GA biosynthetic pathway elucidation and to future optimization of heterologous cell factories for GA production.

Overexpression of the Squalene Epoxidase Gene Alone and in Combination with the 3-Hydroxy-3-methylglutaryl Coenzyme A Gene Increases Ganoderic Acid Production in Ganoderma lingzhi.

The squalene epoxidase (SE) gene from the biosynthetic pathway of ganoderic acid (GA) was cloned and overexpressed in Ganoderma lingzhi. The strain that overexpressed the SE produced approximately 2 times more GA molecules than the wild-type (WT) strain. Moreover, SE overexpression upregulated lanosterol synthase gene expression in the biosynthetic pathway. These results indicated that SE stimulates GA accumulation. Then, the SE and 3-hydroxy-3-methylglutaryl coenzyme A (HMGR) genes were simultaneously overexpressed in G. lingzhi. Compared with the individual overexpression of SE or HMGR, the combined overexpression of the two genes further enhanced individual GA production. The overexpressing strain produced maximum GA-T, GA-S, GA-Mk, and GA-Me contents of 90.4 ± 7.5, 35.9 ± 5.4, 6.2 ± 0.5, and 61.8 ± 5.8 μg/100 mg dry weight, respectively. These values were 5.9, 4.5, 2.4, and 5.8 times higher than those produced by the WT strain. This is the first example of the successful manipulation of multiple biosynthetic genes to improve GA content in G. lingzhi.