Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production

Abstract

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called ‘Chaga’. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.

Introduction

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota, inhabiting primarily the trunks of Betula trees but also occasionally on other angiosperm trees in nature (Ryvarden and Gilbertson 1993). This fungus rarely forms the fertile fruiting body but usually an irregular shape of sclerotial conk with the appearance of burnt charcoal, which is termed ‘Chaga’ in Russia, Northern Europe, and most of Baltic countries. Morphologically, Chaga is shaped like a wedge, bursts through the bark, and appears as large gall-like structures, varying in size from 5 to 40 cm in diameter, with a very irregularly cracked and deeply fissured surface. Inside the burnt charcoal surface is rusty-colored woody texture consisting of interwoven mycelia. Geographically, I. obliquus is distributed in Russia, Korea, Eastern Europe, Northern China, Northern areas of the United States, and the North Carolina mountains (Huang 2002). Early in the sixteenth century, Chaga had been used as an effective folk medicine in Russia and Northern Europe to treat cancer, gastritis, ulcers, and tuberculosis (Kim et al. 2007b) in the absence of any unacceptable toxic side effects (Zheng et al. 2009d). Traditionally, water extract of the crushed Chaga is the frequently used formulation (Koyanma et al. 2008) and is believed to inhibit oxidative DNA damage in human lymphocytes (Park et al. 2004b) and induce G₀/G₁ arrest and apoptosis in human hepatoma HepG2 cells (Youn et al. 2008). Recently, the ethanol extract of Chaga is also found to protect hydrogen peroxide-induced damage of DNA in human lymphocytes (Park et al. 2005a) and to quench free radicals (Cui et al. 2005; Kim et al. 2007a; Lee et al. 2007; Zheng et al. 2009c).

In nature, however, I. obliquus is restricted to very cold habitats (40° N–68° N latitude) and grows very slowly. According to an observation, a Chaga with a diameter of more than 10 cm must undergo at least 10–15 years’ growth (Ham et al. 2009), which suggests that Chaga is not a reliable source for industrial production of these bioactive metabolites (Zheng et al. 2009d). Previous attempts to grow this fungus axenically focused on the accumulation of mycelia biomass (Wang et al. 2006), polysaccharides (Chen and Zhang 2005), and melanins (Babitskaia et al. 2000b) with little attention to the production of other secondary metabolites and their subsequent biological activities. Recently, attempts have been made to grow this fungus in a continuously stirring tank reactor (CSTR) in the presence of hydrogen peroxide (Zheng et al. 2009d) in a shake-flask culture under darkness (Zheng et al. 2009c) or in the presence of a fungal elicitor (Zheng et al. 2009a). CSTR culture stressed by hydrogen peroxide resulted in an increased production of melanins and flavonoid aglycones, but hispidin analogs were only minor components (Zheng et al. 2009c). Moreover, pharmacological activities such as the immuno-stimulating effects only reached about 50% of those from Chaga (Zheng et al. 2008b). Under darkness or in the presence of fungal elicitor, I. obliquus produced more amounts of hispidin analogs, leading to an enhanced capacity for scavenging free radicals. Yet the levels of these phenolic compounds in cultured mycelia were still less than those found in Chaga (Zheng et al. 2009d). Moreover, the accumulation of secondary metabolites other than phenolic compounds has not yet been well documented.

Submerged culture of medicinal fungi is believed to be a promising alternative for the efficient production of mycelia and metabolites and has received increasing attention worldwide. However, in spite of several decades of efforts, the production of secondary metabolites by submerged culture of medicinal fungi including I. obliquus is still encountering many biological, physiological, and engineering limitations. The lack of information on submerged cultures of medicinal fungi in bioreactors is significant when compared to a relatively large body of information about those of streptomycetes and microfungi (Tang et al. 2007).This review focuses on the latest achievements in discovering the chemical diversity of bioactive metabolites produced by I. obliquus grown in its natural habitats and submerged cultures. It highlights the progress during the last couple of years in the findings of new metabolites from Chaga and the submerged culture strategies for diversifying and up-regulating the production of these metabolites, aiming to gain some insights into the solutions to improve and diversify the biosynthesis of these bioactive metabolites in bioreactors.

Biologically active metabolites

In nature, I. obliquus grows in very cold habitats and is exposed to regular seasonal environmental stresses including freezing temperatures, UV irradiation (Hoshino et al. 1998; Zucconi et al. 2002), and the invasion of various pathogenic microbes (Bolwell et al. 2001). In response to these, I. obliquus has evolved a complex series of integrated defense systems. These include the massive production of antioxidant melanins that form a thick layer outside the woody texture of Chaga, enhanced expression of antioxidant enzymes to create a reduced environment for mycelial growth, and accumulation of a plethora of metabolites that assists in competing successfully the invasion of pathogenic organisms (Shwab and Keller 2008; Zheng et al. 2009e). Chemical investigations have showed that Chaga produces a diverse variety of bioactive metabolites including triterpenoids (1–17), ergosterol (18) and its peroxide (19), sesquiterpene (20) (Taji et al. 2005), benzoic acid derivatives (21–28) (Nakajima et al. 2007), hispidin analogs (29–52) (Lee and Yun 2007) (Fig. 1), melanins (Babitskaia et al. 2000b), and polysaccharides (Mizuno et al. 1999) including β-glucans and heteroglucans (Rhee et al. 2008). The biological activities of these metabolites were summarized in Table 1 and the active fractions or extracts in Table 2.

Fig. 1

The structures of compounds 1–52

Table 1 Bioactive metabolites from sclerotia of I. obliquus

Table 2 Biologically active parts and fractions

Antitumoral and immunomodulatory

High incidence of human malicious tumors has always been one of the biggest health threats that is responsible for the annual death of about 7.6 million people worldwide. Studies have shown that a tumor microenvironment influences the functional potential of immune cells by secreting immunosuppressive factors to modify the host immune responses (Chouaib et al. 1997; Chen 1998). These studies raised the possibility that tumors of both mice and human origin can evade immune surveillance by delivering apoptotic death signals to lymphocytes (Fishman et al. 2001). Current strategies for treating malicious tumors commonly involve the combination of surgery and chemotherapy (Efferth and Volm 2005). Yet, surgical removal of breast carcinoma, colon carcinoma, and osteogenic sarcoma, for instance, is frequently followed by metastases to the lung, liver, and other organs of the host (Kimnur et al. 2004). On the other hand, chemotherapy used for the control of tumor growth and metastases is always incurred by cytotoxicity, especially those administered by a combination of more than two drugs (Moreira et al. 2001). In contrast to the current treatment of human malicious tumors, Chaga is an ideal natural drug chemically containing a plethora of metabolites that selectively induce the apoptosis of tumor cells. Evidences in the past decades have shown that Chaga possesses substantial capacities for inhibiting tumor growth and metastasis not only by stimulating host immunity via enhancing the proliferation of lymphocytes (Zheng et al. 2008b) but also by inducing tumor cell apoptosis through up-regulating the expression of Bax genes and down-regulating Bcl-2 genes (Chen et al. 2007a) without incurring any host cell damage. The antitumoral compounds produced in Chaga include triterpenoids inotodiol (2) (Nakata et al. 2007), lanosta-8,23E-diene-3β,22R,25-triol (6), lanosta-7,9(11),23E-triene-3β,22R, 25-triol (8), ergosterol peroxide (19), benzoic acid derivatives (22–28) (Nakajima et al. 2009a), and hispolon (30) and hispidin (31) (Table 1). In addition, the aqueous Chaga extracts and the fractions derived from these extracts containing polysaccharides and polyphenols have also shown to be active in inhibiting tumor growth and metastasis (Burczyk et al. 1996; Rzymowska 1998; Chen et al. 2007a). These underpin water extracts of Chaga that have been used as an effective formulation since the sixteenth century in most Baltic countries. Despite the breakthrough in the bioassay of metabolites in Chaga in the past decades, the antitumoral activity of a large number of phenolic compounds and lanostane-type triterpenoids has not yet been clarified.

Antiviral

Virus is another class of important human pathogens that not only produce the common influenza, chickenpox, and cold sores but also trigger the outbreak of pandemic Ebola, AIDS, avian influenza, severe acute respiratory syndrome, type A H1N1 influenza and hepatitis, etc. (Margolis et al. 2007). In recent years, the growing incidence of avian flu and type A H1N1 influenza poses ongoing threats to human health, killing numerous people each year worldwide. The current therapy of virus-induced diseases includes chemotherapy inhibiting the key enzymes for viral replication in host cells (Li et al. 2009), vaccination to enhance host immunity (Yang et al. 2009), and the delivery of T cells to the infected host (Kumar et al. 2008). Antiretroviral therapy has led to a significant improvement in the prognosis for virus-infected individuals. These involve targeting the envelope protein, reverse transcriptase, integrase, and protease to block virus infection at the stage of entry, reverse transcription, integration, and maturation (Coffin 1995). However, growing clinical evidences have shown that long-term chemotherapy leads to serious multi-drug resistance and toxic side effects in patients (Li et al. 2009). In addition, the increasing number of serious adverse events in children by vaccination have been reported (Rosenberg et al. 2009), and the perception of T cell delivery as invading pathogens by host is still a problem that hinders its application (Nayak and Herzog 2010). As an effective traditional folk remedy, I. obliquus also shows its potential for treating virus-induced diseases. For example, lignin-like polyphenols from water extracts of Chaga showed effective inhibition of protease of HIV-1 (Ichimura et al. 1998). The charcoal-like surface layer of Chaga exhibited 100% inhibition against human and horse type A and B influenza virus, and triterpenoids betulinic acid (20) and mycosterols were thought to be the active constituents (Kahlos et al. 1996). Evidences also indicated that phenolic compounds hispolon (30) and hispidin (31) effectively inhibited the replication of type A and B influenza virus in host cells, and hispidin (31) inactivated HIV-1 integrase at an IC₅₀ value of 2 μM (Singh et al. 2003). Collectively, the metabolites synthesized by I. obliquus demonstrated substantial capacity for treating virus-induced diseases. Yet, the number of active compounds needs further screening and the mechanisms for their antiviral activity remain unknown. Thus, bioprospecting antiviral compounds from Chaga is of great importance for antiviral drug discovery.

Antimutagenic

Mutations in the DNA sequence of a cell’s genome lead primarily to harmful effects to individuals owing to the genetic disorder (Sawyer et al. 2007). Genetic disorder is the origin of severe genetic diseases such as sickle cell anemia, mental retardation, and Down syndrome. These mutagenic diseases occur in high morbidity in most of the developing countries (Verma 2000), resulting in a substantial reduction in the population’s quality of health. The treatment of mutagenic disorders is currently a big challenge and depends on the development of ‘genetic medicine’ therapies using the transfer of DNA and /or RNA to modify gene expression to correct or compensate for an abnormal phenotype. Despite the efficacy of these technologies in treating experimental models of hereditary disorders, their successful application in the clinic still encounters many obstacles (O'Connor and Crystal 2006). Thus, antimutagenic agents have significant application in preventing mutagenic diseases. Metabolites produced by I. obliquus showed remarkable antimutagenic activity. Bioassay-guided fractionation of Chaga methanol extracts led to the isolation of two antimutagenic compounds inotodiol (2) and 3β-hydroxylanosta-8,24-diene-21-al (4). These two compounds inhibit mutations induced by mutagen MNNG by 80.0% and 77.3%, respectively, in Salmonella typhimurium TA100. They also inhibited 4NQO-induced mutagenesis in S. typhimurium TA98 and TA100 by 52.6–62.0% (Ham et al. 2009). The above example suggests that there is a potential in isolating natural compounds produced by I. obliquus in effectively reducing the incidence of mutagenic diseases. However, a thorough evaluation of antimutagenic activity of I. obliquus is necessary to further elucidate the active components and mechanisms of action.

Antioxidant

Oxidative stress stems from the continuous production of H₂O₂ primarily in the mitochondrion of all aerobic organisms during oxidative phosphorylation for energy production (Turrens 2003). In normal physiological conditions, production of reactive oxygen species (ROS) can be quenched by antioxidant enzymes. However, severe oxidative stress will lead to DNA damage in cells and induce apoptosis (Evans and Cooke 2004). ROS, such as hydroxyl and superoxide anion radicals, demonstrates a wide variety of pathological effects on cellular processes (Marx 1987). Superoxide anion radical is one of the most reactive free radicals easily combined with nitric oxide (NO) produced by the endothelium, macrophages, neutrophils, and brain synaptosomes, leading to the production of the stable peroxynitrite anion (ONOO⁻) (Beckman et al. 1990), the highly toxic substance responsible for the subsequent cell injuries (Harman 1997). In addition, hydroxyl radical is also a far more reactive free radical that directly interacts with unsaturated fatty acids in the cell membrane, resulting in lipid peroxidation and thereby dysfunctions of cell membranes (Shen and Qian 2004). Long-term severe oxidative stress also increases substantially the incidence of neurodegenerative (Gilgun-Sherki et al. 2001) and cardiovascular diseases (Madamanchi et al. 2005), cataract (Spector 2000), and autoimmune diseases (Shunichi et al. 2003). I. obliquus produces quite an impressive array of metabolites capable of scavenging free radicals including superoxide anion (SOA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2.2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS). These include lanostane-type triterpenoids 3β-hydroxylanosta-8,24-diene-21-al (4) (Ham et al. 2009), lanosta-24-ene-3β,21-diol (5) (Taji et al. 2005), benzoic acid derivatives (22–28) (Nakajima et al. 2007, 2009b), hispolon (30), hispidin (31), hispidin analogs inonobilins A, B, and C (32–34), phelligridins D–G (36–39) (Lee et al. 2006), interfunginns A–C (42–44) (Lee and Yun 2007), inoscavins A–C (45, 47, 49), methyl inoscavins A–C (46, 48, 50), davallialactone (51), and methyl davallialactone (52) (Lee and Yun 2006). Of these, compounds 42–44 and 51–52 were more effective in quenching SOA with IC₅₀ values of less than 7 μM (Table 1). These metabolites represent antioxidant principles in the extracts of ethanol (Park et al. 2004b; Cui et al. 2005; Hu et al. 2009), ethyl acetate, and n-butanol (Liang et al. 2009). Studies also showed that polysaccharides fractionated from water extracts of Chaga demonstrate remarkable antioxidant activity (Hu et al. 2009). Evidences showed that Chaga polysaccharides inhibit 80% of Fe²⁺–Cys-induced lipid peroxidation of mitochondria at a minimum concentration of 200 μg/ml (Song et al. 2008). Treatment with Chaga aqueous extract reduced 40% of DNA damage of human lymphocytes induced by H₂O₂ (100 μg/ml) at a minimum concentration of 100 μg/ml (Park et al. 2004b). The polyphenolic extract of Chaga demonstrated its scavenging for DPPH and SOA for up to 90% (Cui et al. 2005). Melanins, with chemical moieties of electron donors and acceptors, are powerful antioxidants (Babitskaia et al. 2000a; Shcherba et al. 2000) and have received extensive attention in the past decades owing to their effective protection of human skin against UV irradiation (Paramonov et al. 2004) and low-intensity ionizing radiation (Druzhyna et al. 2001). The combination of metabolites produced by I. obliquus might have applications in reducing the incidence of oxidative stress-induced diseases including cataract, hypertension (Kwon et al. 2007), cancer (Orzechowski 2007), neurodegenerative (Heo and Lee 2005) and autoimmune diseases (Galli et al. 2005). Chemical diversity and effective scavenging for free radicals potentialize I. obliquus to be a promising source for screening lead compounds for antioxidant drug discovery.

Anti-inflammatory

Inflammatory responses are triggered by cytokines that are produced primarily by leucocytes. Excessive production of cytokines IL-1β, IL-6, TNF-α, and NO leads to serious cell damage (Lin et al. 2004). Water extract, 80% ethanol, and ethyl acetate extracts of Chaga inhibited the production of these cytokines by murine macrophages (Van et al. 2009). Chaga extracts by ethanol at 80°C showed 81.2% inhibition of platelet aggregation. Systematic isolation and purification resulted in the identification of anti-inflammatory peptide with a molecular mass of 365 Da and a sequence of Trp-Gly-Cys (Hyun et al. 2006). However, other metabolites with anti-inflammatory activity in the Chaga extracts remain unknown.

Anti-complementary

Metabolites from I. obliquus also showed anti-complementary activity. Ergosterol (18) and ergosterol peroxide (19) showed strong anti-complementary activity on classical pathway. Ergosterol $\left( {{\hbox{I}}{{\hbox{C}}_{{5}0}} = {1}.0 \times {1}{0^{ - {6}}}{\hbox{M}}} \right)$ was more active than ergosterol peroxide $\left( {{\hbox{I}}{{\hbox{C}}_{{5}0}} = { 5}.0 \times {1}{0^{ - {6}}}{\hbox{M}}} \right)$ (Kim et al. 1997).

Other biological activities

Studies also disclosed other than the above-stated biological activities of the metabolites or fractions from I. obliquus. Chloroform extract of Chaga showed remarkable antiproliferative effect against mouse leukemia P388 cells, and inotodiol (2) is the active principle (Nomura et al. 2008). Hispolon (30) showed cell proliferation inhibitory effect against human epidermoid KB cell at an IC₅₀ value of 4.62 μg/ml (Chen et al. 2006). Trametenolic acid (3) suggested inhibition to the growth of Bacillus subtilis at a minimum concentration of 2.5 μg/ml (Keller et al. 1995). Oral administration by methanol extract of Chaga at doses of 100 and 200 mg/kg substantially reduced the frequencies of acetic acid-induced stretching episodes and the subsequent abnormal constriction in mice (Park et al. 2005b). Oral treatment of β-glucan, heteroglucan, and their protein complexes abated blood glucose level for 3–48 h in type I diabetic mice (Mizuno et al. 1999) and decreased the serum glucose level in streptozotocin-induced diabetic rats (Cha et al. 2005). Oral administration of ethyl acetate extract of Chaga evidently suppressed the blood glucose level in alloxan-induced diabetic mice. Bioassay-guided fractionation resulted in the finding of active principles inotodiol (2) and 3β-hydroxylanosta-8,24-diene-21-al (4) able to inhibit amylase activity and scavenge free radicals (Lu et al. 2009). I. obliquus also demonstrated immunomodulatory effects. Oral administration of Chaga water extract at a dosage of 100 mg/kg prevented body weight reduction induced by cyclophosphamide and stimulated the proliferation of peripheral lymphocytes (Zheng et al. 2008b). It has also been reported that lanosterol analogs showed regulatory effects on the biosynthesis of cholesterol (Frye and Leonard 1999). Chemical diversity of lanostane-type triterpenoids produced by I. obliquus makes it possible to be a source for the discovery of drugs reducing cholesterol-induced human hyperlipidemia.

Production of bioactive metabolites by submerged cultures

The presence of metabolites with chemical and pharmacological diversities in Chaga manifests I. obliquus to be an ideal medicinal fungus for drug discovery. However, the very slow formation of Chaga in natural habitats shows that these pharmacologically important metabolites cannot be produced for therapeutic purposes using naturally growing I. obliquus (Zheng et al. 2009d, e). Thus, culturing I. obliquus is the only solution for large-scale production of the metabolites similar to those from Chaga. Early submerged culture of I. obliquus focused only on the accumulation of mycelial biomass in which suitable growth conditions were probed in carbon/nitrogen ratio, pH, and light intensity by shake-flask platform (Jiang et al. 2004). Later, sucrose and corn powder and pH between 5 and 7 were found to be beneficial for mycelial growth, whereas light irradiation at an intensity of 500 lx reduced the production of mycelial biomass (Chen and Zhang 2005). Recently, growth conditions were further optimized by duality quadratic regress composite intersect design and orthogonal design for the maximum production of mycelial biomass, which resulted in 19.8 and 13.57 g/l dried mycelia in the media containing reduced sugar plus yeast powder and corn juice at a pH value of 6.0 and in those containing glucose and yeast powder at a pH value of 6.0, respectively (Wang et al. 2006).

Targeting the production of melanins

Melanins are the most conspicuous metabolites produced by I. obliquus. Previous attempts for producing melanins in submerged cultures disclosed that the presence of copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20 mM) stimulated melanogenesis (Babitskaia et al. 2000b) and found that melanins produced in submerged cultures of I. obliquus belonged to eumelanins that physiochemically differed from allomelanins in Chaga (Kukulianskaia et al. 2002). Recently, Zheng et al. (2009e) showed that the presence of hydrogen peroxide in submerged cultures of I. obliquus resulted in a substantial enhancement in melanin production, and melanogenesis was inhibited by supplementing arbutin, which suggests a DOPA pathway for melanin synthesis (Zheng et al. 2009d). It is believed that melanins produced by I. obliquus in submerged cultures are antioxidant and genoprotective (Babitskaia et al. 2000a, b), yet further experiments are needed to elucidate the differences between eumelanins and allomelanins in biological activities.

Targeting the production of polysaccharides

Production of polysaccharides was another early target for the submerged culture of I. obliquus. It has been reported that polysaccharides from submerged cultures showed similar hypoglycemic (Mizuno et al. 1999) and antitumoral potential with those obtained from Chaga, and even more effective in antioxidant activity (Song et al. 2008). Kim et al. (2005) showed that intracellular polysaccharides (IPS) from cultured mycelia of I. obliquus suggested a higher potential for stimulating the proliferation of lymphocytes than that of exopolysaccharides (EPS) (Kim et al. 2005). IPS from submerged culture of the fungus showed no direct cytotoxicity against a variety of tumor cell lines in vitro but a remarkable inhibition in vivo on the growth of B16F melanoma-bearing mice (Kim et al. 2007b). Following these findings, culture conditions for the maximum production of EPS were screened. These included the carbon and nitrogen sources and pH value that favored EPS accumulation by which to increase EPS up to 7.8 g/l in the media containing maltose (2%) and peptone (0.4%) at a pH of 5.5 (Chen et al. 2007b) and up to 3.85 g/l in the media containing corn syrup (2.11%), soluble starch (2.23%), yeast powder (0.51%), and peptone (0.41%) with a pH value of 5.0 (Zhang et al. 2009). Recent endeavor was also made to optimize the conditions for the maximum production of IPS. Zhang (2008) showed that UV irradiation (45 W, 30 cm) for 3 min increased IPS accumulation up to 2.39%.The above studies have improved the culture conditions for polysaccharide production. Further experiments are still needed to investigate the chemical structure and pharmacological activities of polysaccharides, and the links between monosaccharides, and to identify whether increased production of polysaccharides correlated with enhanced potential of pharmacological activities.

Targeting the production of triterpenoids and steroids

Attention for metabolite accumulation has also been directed to the production of triterpenoids and steroids. Shin et al. (2001, 2002) isolated lanosterol, ergosterol, and ergosterol peroxide, 3β-22-dihydroxy-lanosta-7,9(11),24-triene, from the mycelia grown in the media containing glucose (2%), malt extract (0.5%), and yeast (0.5%) without modifying the pH after autoclaving. Xiang et al. (2006) determined lanosterol, inotodiol, 3β-hydroxyl-lanosta-8-en-24-methylene, 9(11)-dihydroergosta-benzoate methyl ester, ergosterol, and ξ-ergosterol from the mycelia grown in the media containing glucose (2.5%), peptone (0.3%), and yeast extract (0.1%) without modifying the pH after autoclaving and showed that lanosterol content was considerably less than ergosterol. Recently, I. obliquus from the same medium was also evaluated for the bioconversion from lanosterol to ergosterol, which determined the intermediates of ergosterol biosynthesis including 24-methylene dihydrolanosterol, 4,4-dimethylfecosterol, 4-methylfecosterol, and ergosta-7,22-dien-3-ol in the presence of AgNO₃. Following the presence of these intermediates, lanosterol accumulation, which was very low in the absence of AgNO₃, was obviously increased together with a reduced activity of 3-hydroxyl-3-methylglutaryl coenzyme A reductase (HMGCoAR) and the subsequent slowing down in sterol metabolism. The above observations suggested that a higher amount of Ag⁺ inhibits the activity of enzymes responsible for sterol metabolism (Zheng et al. 2007b, 2008a). These findings highlighted the mechanisms differing in chemical profiles between lanosterol analogs biosynthesized in submerged culture and natural habitats. However, mechanisms in up-regulating lanosterol analogs, particularly those with pharmaceutical importance, remain unclear.

Targeting the production of phenolic compounds

Production of phenolic compounds by I. obliquus has recently received much attention due to their prominent antioxidant activities. Zheng et al. (2007a) showed that supplementation of l-tyrosine and water extracts of Aspergillus flavus and Mucor racemosus to the culture of I. obliquus resulted in an increased production of glycosides of quercetin, naringenin, kaempferol, and isorhamnetin together with enhanced capacities for scavenging DPPH, SOA, and hydroxyl radicals. Yang and Zheng (2007) optimized culture medium for producing hydrolyzable tannins and showed that supplementing Cu²⁺, Co²⁺, Zn²⁺, and Mn²⁺ at a concentration of 0.8, 1.6, 1.6, and 1 mM, respectively, up-regulated the accumulation of hydrolyzable tannins in the medium containing glucose and peptone with a pH value of 5.5. To clarify the difference in phenolic profiles between Chaga and cultured mycelia, phenolic compounds were extracted and compared. It showed that those in Chaga comprised mainly of hispidin analogs concomitant with the presence of minor benzoic acid derivatives, whereas those in cultured mycelia were featured by the dominant presence of glycosylated flavonoids and benzoic acid derivatives; hispidin analogs were determined as the minor components. Further, the capacity for resisting body weight reduction in immuno-suppressed mice by total phenolic compounds from cultured mycelia reached only half of that by total phenolic compounds from Chaga (Zheng et al. 2008b). Recently, the production of phenolic compounds by I. obliquus was conducted in CSTR in the presence of hydrogen peroxide only or in the presence of hydrogen peroxide and arbutin simultaneously, which resulted in the accumulation primarily of flavonoid aglycones and benzoic acid derivatives. Hispidin analogs were still the minor components (Zheng et al. 2009d). Following oxidative stress, I. obliquus enhanced the expression of superoxide dismutases and catalase together with a reduced accumulation of phenolic compounds due to their consumption for free radical scavenging (Zheng et al. 2009e).

Hispidin and flavonoids are biosynthesized via phenylpropanoid pathway, which all start with the precursor phenylalanine and produce the intermediate p-coumaric acid in the presence of phenylalanine ammonia lyase (PAL) (Nambudiri et al. 1973; Dewick 2003; Jiang et al. 2005), and p-coumaric acid can either be transformed to naringenin chalcone (flavonoids) in the presence of chalcone synthase (Moore et al. 2002) or to caffeate in the presence of p-coumaric acid hydroxylase, leading to the biosynthesis of hispidin (Nambudiri et al. 1973). Zhao et al. (2009) showed that p-coumaric acid was predominantly converted into flavonoid aglycones and benzoic acid derivatives under oxidative stress and less transformed to hispidin analogs, which contradicts those growing in natural habitats with phenolic profile featured by the predominant presence of hispidin analogs (Zheng et al. 2008b). In order to search for factors favoring the involvement of p-coumaric acid to the biosynthesis of hispidin analogs, Zheng et al. (2009c) examined the NMR profiles of phenolic compounds from cultured mycelia grown under different light conditions, compared these to those determined in Chaga using NMR-based metabonomic analysis, and found that darkness was beneficial to the biosynthesis of hispidin analogs, particularly davallialactone, methyl davallialactone, and phelligridins, with ¹H NMR spectral features close to those of Chaga. In addition, phenolic compounds from cultured mycelia grown in darkness showed a higher potential in scavenging free radicals than those found in day, blue, and red light and concluded that daylight and darkness tend to be beneficial to the accumulation of phenolic compounds. These findings demonstrate that light regulates the biosynthesis of hispidin analogs in I. obliquus, and the light-induced responses are reasonably mediated by white-collar-protein-like photoreceptors capable of initiating signal transduction pathways propitious to the accumulation of hispidin analogs. More recently, Zheng et al. (2009a) conducted further experiments to analyze the effects of fungal elicitor on the production of polyphenols by I. obliquus under darkness and showed that fungal elicitor enhanced the biosynthesis of hispidin analogs and the subsequent capacity of antioxidant activity of total phenolic compounds. Evidences indicated that this enhancement was mediated by an increased production of nitric oxide able to trigger an enhanced expression of PAL, leading to biosynthesis of hispidin analogs. Recently, studies have also been directed to the bioassay of phenolic compounds from submerged cultures of I. obliquus. Sun et al. (2008) showed that the dried material of culture broth, with the constituents mainly being phenolic compounds, demonstrated remarkable antihyperglycemic and antilipidperoxidative effects against alloxan-induced diabetes in mice. The phenolic compounds from submerged culture demonstrated similar or higher potentials in enhancing the tolerance of lead-treated mice to hypoxia than those by Chaga (Zheng et al. 2009b) and the capacities for scavenging free radicals (Zheng et al. 2007a, 2009a, c, d, e; Zhao et al. 2009). These studies uncovered some of the factors affecting the biosynthesis of phenolic compounds by I. obliquus under submerged culture conditions and elucidated the mechanisms leading to the differences in phenolic profiles and biological activities between cultured mycelia and Chaga. Further experiments should be conducted in clarifying the factors affecting the involvement of p-coumaric acid, particularly the signal transduction leading to the gene expression of p-coumaric acid hydroxylase and the enzymes responsible for hispidin biosynthesis. In addition, it is also important to verify the pharmacological activities of the phenolic compounds from submerged culture for evaluating their pharmaceutical significance.

Problems, strategies, and prospects

Current achievements in the study of I. obliquus provide promising prospects not only in the chemical diversity of bioactive metabolites but also in the up-regulation of these metabolites in its submerged cultures. However, as far as the reactors used are concerned, nearly all of the cultures were performed by shake-flasks, where culture parameters such as dissolved oxygen concentration and dynamic changes of pH value can neither be monitored nor controlled (Zheng et al. 2009d). In addition, the configuration of bioreactor greatly affects broth rhenology and thereby oxygen mixing and transfer, leading to the changes of metabolic profiles (Taticek et al. 2004). Thus, the culture conditions probed by shake-flask cultures cannot be applied directly for the scale-up culture of I. obliquus. Further, other factors including shearing force is also involved in the accumulation of mycelial biomass and metabolites, which have not yet been well documented. Thus, it is essential to optimize the growth conditions of the fungus in CSTR for large-scale fermentation by a combined evaluation of mycelial biomass, metabolites and biological activities. In many groups of bacteria, fungi, and plants, there are dozens of secondary metabolite pathways that are not expressed under standard laboratory growth conditions (Pettit 2009), and the presence or absence of environmental factors is critical to the synthesis of secondary metabolites (Knight et al. 2003). Apparently, potentially interesting gene clusters in I. obliquus, while possibly expressing metabolites that increase competitiveness in its natural habitats, can remain silent in its submerged cultures. Based on the current data on submerged culture of the fungus, the differences in growth conditions with natural habitats are the major obstacles for accumulating bioactive metabolites. In this context, strategies aiming at diversification and up-regulation of these metabolites tend either to vary culture conditions to turn on these pathways or to mimic natural microbial environments by growing I. obliquus in the presence of other microbes. Varying culture conditions, i.e., imposing an oxidative stress or programmatically changing growth temperature, does not prove to be cost-effective in that part of metabolites, particularly phenolic compounds will be consumed for resisting the imposed oxidative stresses (Zheng et al. 2009d). In comparison, co-culture with other microbes seems to be a feasible protocol to up-regulate bioactive metabolites by I. obliquus. Zheng et al. showed that simultaneous inoculation of I. obliquus and medicinal fungus Phellinus punctatus at a ratio of 5:1 (w/w) into submerged culture resulted in a reduced production of mycelial biomass (nearly one third reduced) but a substantial increase in the activity of HMGCoAR, PAL, and TAL, the key enzymes responsible for the biosynthesis of triterpenoids, steroids, phenolic compounds, and melanins, respectively, followed by more complex NMR profiles and enhanced production of extracellular melanins , intra- and extracellular phenolic compounds, and triterpenoids (unpublished data). Moreover, culture broth and ethanol/acetone extracts of mycelia from mixed culture all showed significant increase in scavenging free radicals and selective cytotoxicity to HeLa cells (unpublished data). These data evidenced the possibility of diversifying and up-regulating metabolites in submerged culture of the fungus by co-incubation with other microbes. However, the production of adequate quantities of the active compounds needed for drug discovery may require extensive media optimization and scale-up. These include systematic approaches to manipulate the physiology of I. obliquus to activate its natural product machinery by blocking some pathways expressed in submerged cultures and triggering the pathways expressed in nature under laboratory conditions.

To date, at least 20 lanostane-type triterpenoids, 15 flavonoids, 9 benzoic acid derivatives, 10 hispidin analogs, melanins, and polysaccharides were identified or isolated from submerged cultures of I. obliquus. More importantly, metabolites from culture broth and mycelial extracts all showed substantial tumor cytotoxic, hypoglycemic (Cha et al. 2005; Sun et al. 2008), antilipidperoxidative (Sun et al. 2008), immunomodulatory (Zheng et al. 2008b), antioxidant (Zheng et al. 2009c, d, e), and tolerance-enhancing activities (Zheng et al. 2009b). These evidenced that some of the pathways can still be expressed in submerged culture conditions, particularly in the presence of elicitors and oxidative stress. However, most of the desired pathways are not expressed in response to the variation of culture conditions, and the factors inactivating or reducing the expression of these pathways remain unclear.

In higher plants, a conspicuous feature of resistance to the attempted pathogen attack is the synthesis of NO, the signal molecule able to drive the expression of a battery of redox-regulated defense genes (Feechan et al. 2005). S-Nitrosylation, the addition of a NO moiety to a specific cysteine thiol, to form an S-notrosothiol, has emerged as a principal mechanism by which NO orchestrates cellular functions in Arabidopsis thaliana. The associated molecular mechanisms by which NO modulates these diverse cellular responses involve S-nitrosylation of salicylic acid binding protein 3 at cysteine (C) 280. The binding of NO suppresses the immune activator salicylic acid and carbonic anhydrase (CA) activity of this protein, leading to a reduced resistance against pathogen infection. Thus, S-nitrosylation could contribute to a negative feedback loop that governs the plant defense responses (Wang et al. 2008). It has been shown that NO also mediates fungal elicitor-enhanced biosynthesis of hispidin analogs by I. obliquus (Zheng et al. 2009a). Thus, it is highly necessary to investigate whether a similar negative feedback loop exists in I. obliquus that modulates the immune response against the invasion of pathogenic microorganisms and subsequently reduces the production of secondary metabolites.

Synthesis of bioactive metabolites by I. obliquus involves the expression of enzymes in a plethora of pathways, leading to the production of a wide variety of compounds. Rapid and accurate qualification and quantification of these compounds are the preconditions for determining the changes of metabolic profiles of I. obliquus grown in different culture conditions (Zheng et al. 2009c). Metabonomics-based approaches, capable of measuring the dynamic multiparametric responses of living systems to the internal and external stimuli (Nicholson et al. 1999), are claimed to evaluate comprehensively the multiparametric metabolic responses to all stimuli and gene modification (Gao et al. 2008). High-performance liquid chromatography combined with mass spectroscopy (HPLC/MS) provides an authentic platform for identifying metabolites, but establishing suitable conditions and subsequent metabolite isolation and identification are time-consuming and challenging processes. As one of the major techniques in metabonomics, NMR spectroscopy has the disadvantage of low detection limits but with many other advantages over HPLC/MS in that NMR measurements are non-destructive and non-selective and it is feasible to acquire profiles of a comprehensive range of organic metabolites (Li et al. 2007). It is encouraging that this approach has been tentatively used in evaluating the effect of light and fungal elicitor on the biosynthesis of phenolic compounds and well described the metabolites leading to the changes in response to different environmental factors (Zheng et al. 2009a, c). Taken together, it is reasonably believed that biosynthesis of bioactive metabolites by I. obliquus will be diversified towards the desired pathways when we successfully combine gene manipulations and signal transductions with NO-mediated feedback loop of immune response and NMR-based metabonomic analysis.

I. obliquus has already played a pivotal role in treating various human diseases. More thorough exploitation of its metabolic potential via the addition of fungal elicitor or co-culture with other fungi is evidencing to be the cost-effective protocol to enlarge its libraries of bioactive metabolites. With countless fungal elicitors or combinations with other microbes and increasingly sophisticated chemical isolation and structure determination methods, the pharmaceutical potential of I. obliquus for drug discovery seems quite promising.