The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans.

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, resulting in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hypha-inducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hypha-inducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

De Novo Biosynthesis of the Anticancer Compound Euphol in Saccharomyces cerevisiae.

Euphol is a euphane-type tetracyclic triterpene which is primarily found in the Euphorbia genus. Euphol has been renowned because of its great potential as a promising anticancer drug. Surprisingly, despite its diverse antitumor effects, the respective gene for euphol biosynthesis had not been identified until this study. In our experiments with Euphorbia tirucalli, euphol was detected predominantly in latex, the element that is often used for cancer treatments in Brazil. Two latex-specifically expressed oxidosqualene cyclases (OSCs) from E. tirucalli, designated as EtOSC5 and EtOSC6, were functionally characterized by expression in a lanosterol synthase knockout yeast strain GIL77. EtOSC5 produces euphol and its 20S-isomer tirucallol as two of the major products, while EtOSC6 produces taraxasterol and ß-amyrin as the major products. These four compounds were also detected as the major triterpenes in the E. tirucalli latex, suggesting that EtOSC5 and EtOSC6 are the primary catalysts for the formation of E. tirucalli latex triterpene alcohols. Based on a model structure of EtOSC5 followed with site-mutagenesis experiments, the mechanism for the EtOSC5 activity was proposed. By applying state-of-the-art engineering techniques, the expression of EtOSC5 together with three other known precursor genes were chromosomally integrated into Saccharomyces cerevisiae. The resulting engineered yeast strain YS5E-1 produced 1.84 ± 0.17 mg/L of euphol in shake flasks.

De Novo Cholesterol Biosynthesis and Its Trafficking in LAMP-1-Positive Vesicles Are Involved in Replication and Spread of Marek's Disease Virus.

Marek's disease virus (MDV) transforms CD4+ T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens. While MDV-infected chickens have normal serum concentrations of cholesterol, their aortic tissues were found to have elevated concentrations of free and esterified cholesterol. Here, we demonstrate that infection of chicken embryonated fibroblasts (CEFs) with highly pathogenic MDV-RB1B increases the cellular cholesterol content and upregulates the genes involved in cholesterol synthesis and cellular cholesterol homeostasis using comprehensive two-dimensional gas chromatography-mass spectrometry and real-time PCR (RT-PCR), respectively. Using small pharmacological inhibitors and gene silencing, we established an association between MDV-RB1B replication and mevalonic acid, sterol, and cholesterol biosynthesis and trafficking/redistribution. We propose that MDV trafficking is mediated by lysosome-associated membrane protein 1 (LAMP-1)-positive vesicles based on short hairpin RNA (shRNA) gene silencing and the colocalization of LAMP-1, glycoprotein B (gB) of MDV, and cholesterol (filipin III) fluorescence signal intensity peaks. In conclusion, our results demonstrate that MDV hijacks cellular cholesterol biosynthesis and cholesterol trafficking to facilitate cell-to-cell spread in a LAMP-1-dependent mechanism.IMPORTANCE MDV disrupts lipid metabolism and causes atherosclerosis in MDV-infected chickens; however, the role of cholesterol metabolism in the replication and spread of MDV is unknown. MDV-infected cells do not produce infectious cell-free virus in vitro, raising the question about the mechanism involved in the cell-to-cell spread of MDV. In this report, we provide evidence that MDV replication depends on de novo cholesterol biosynthesis and uptake. Interruption of cholesterol trafficking within multivesicular bodies (MVBs) by chemical inhibitors or gene silencing reduced MDV titers and cell-to-cell spread. Finally, we demonstrated that MDV gB colocalizes with cholesterol and LAMP-1, suggesting that viral protein trafficking is mediated by LAMP-1-positive vesicles in association with cholesterol. These results provide new insights into the cholesterol dependence of MDV replication.

Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral.

BACKGROUND: Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood. RESULTS: In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral. CONCLUSION: Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9ß,19-cyclolanost-24-en-3ß-ol) and not lanosterol (lanosta-8,24-dien-3ß-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

IUPHAR-DB: updated database content and new features.

The International Union of Basic and Clinical Pharmacology (IUPHAR) database, IUPHAR-DB (http://www.iuphar-db.org) is an open access, online database providing detailed, expert-driven annotation of the primary literature on human and rodent receptors and other drug targets, together with the substances that act on them. The present release includes information on the products of 646 genes from four major protein classes (G protein-coupled receptors, nuclear hormone receptors, voltage- and ligand-gated ion channels) and ∼3180 bioactive molecules (endogenous ligands, licensed drugs and key pharmacological tools) that interact with them. We have described previously the classification and curation of data for small molecule ligands in the database; in this update we have annotated 366 endogenous peptide ligands with their amino acid sequences, post-translational modifications, links to precursor genes, species differences and relationships with other molecules in the database (e.g. those derived from the same precursor). We have also matched targets with their endogenous ligands (peptides and small molecules), with particular attention paid to identifying bioactive peptide ligands generated by post-translational modification of precursor proteins. Other improvements to the database include enhanced information on the clinical relevance of targets and ligands in the database, more extensive links to other databases and a pilot project for the curation of enzymes as drug targets.

Disorders of sterol synthesis: beyond Smith-Lemli-Opitz syndrome.

Since the discovery in 1993 that Smith-Lemli-Opitz syndrome (SLOS) is a disorder of cholesterol biosynthesis, human disorders associated with additional enzymes involved in the conversion of lanosterol to cholesterol have been identified. This review will focus primarily on the clinical aspects of these disorders, highlighting newly described syndromes, such as SC4MOL deficiency and CK syndrome. We will also provide clinical descriptions of additional cases for extremely rare disorders, such as desmosterolosis. We will compare and contrast the findings with those found in SLOS and briefly discuss possible mechanisms of disease pathogenesis.

Enhanced production of ganoderic acids and cytotoxicity of Ganoderma lucidum using solid-medium culture.

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.

Current progress in the study on biosynthesis and regulation of ganoderic acids.

Ganoderic acids (GAs) isolated from Ganoderma lucidum, which have shown remarkable pharmacological activities and a variety of therapeutic effects on a number of human diseases, have provided an important resource for the development of new medicines. The yield of GAs in field cultivation is still limited, which is mainly due to a scarcity of information regarding its biosynthesis pathway and its regulation. Here, we review the recent publication that has been made in the biosynthesis and regulation of GAs. From these studies, researchers have identified part of the biosynthesis pathway of GAs named mevalonate pathway. They have cloned and characterized the genes involved in the biosynthesis pathway. Additionally, they found that expression of the genes involved in GA biosynthesis is closely related to the impact of environmental factors through transcriptional profiling analysis. Moreover, this review focuses on suggesting new directions for studying GAs and attempts to gain some insights for better understanding of the biosynthesis and regulation of GAs.

Methyl jasmonate induces ganoderic acid biosynthesis in the basidiomycetous fungus Ganoderma lucidum.

This is the first time study to assess the novel use of methyl jasmonate (MeJA) to elicit ganoderic acid (GA) biosynthesis in Ganoderma lucidum and the resulting experiments demonstrated that MeJA was indeed a potent inducer of GA biosynthesis. To maximize GA synthesis, a statistical methodology called uniform design (UD) was used to optimize inducement conditions, which were determined to be 254 microM MeJA solubilized in Tween-20 that was added to the culture on day 6. The resulting GA yield was 4.52 mg/100mg dry weight (DW), which was 45.3% higher than the untreated control sample. To characterize the effect of MeJA on GA biosynthesis, quantitative real-time PCR was used to measure transcription levels of several genes in the synthesis pathway including hydroxy-3-methylglutaryl-Coenzyme A synthase (hmgs), hydroxy-3-methylglutaryl-Coenzyme A reductase (hmgr), mevalonate-5-pyrophosphate decarboxylase (mvd), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs) and oxidosqualene cyclase (osc). Quantification of transcription levels determined that MeJA significantly induced expression of these genes.

Significance of protein elicitor isolated from Tuber melanosporum on the production of ganoderic acid and Ganoderma polysaccharides during the fermentation of Ganoderma lucidum.

Under the elicitation of protein elicitor isolated from the culture mycelia of Tuber melanosporum, the biosynthesis of ganoderic acids (GA) was significantly stimulated during Ganoderma lucidum fermentation. Compared with our previous results that, GA content was inhibited by polysaccharide elicitor isolated from T. melanosporum, while improved by the elicitor of polysaccharide and protein, protein was identified to be the exact component inducing GA biosynthesis in this work. G. lucidum cell growth was significantly inhibited by elicitor of polysaccharide and protein, and polysaccharide elicitor did not inhibit the cell growth. In this work, the remarkable inhibition on the cell growth was considerably eliminated under the elicitation of protein elicitor isolated from T. melanosporum. These suggested maybe the interaction of polysaccharide and protein components existed in the inhibition on the cell growth of G. lucidum. Not only GA content but also total GA accumulation obtained the highest values after the elicitation of protein elicitor. The maximal GA production of 260.5 ± 5.6 mg/L was 31.2% higher than the control. Under the elicitation of protein elicitor, the production of extracellular polysaccharide (EPS) and the content of intracellular polysaccharide (IPS) were also enhanced; however, total IPS accumulation was lower. GA biosynthesis was also significantly affected by the addition time of protein elicitor, whose optimal value was the culture of day 4.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.

Impact of oxygen level in gaseous phase on gene transcription and ganoderic acid biosynthesis in liquid static cultures of Ganoderma lucidum.

Liquid static cultivation of Ganoderma lucidum was previously found to be very efficient for improving the production of its valuable antitumor compound ganoderic acid (GA) (Fang and Zhong in Biotechnol Prog 18:51-54, 2002). In this work, effects of oxygen concentration within the range of 21-100% (v/v) in the gaseous phase on the mycelia growth, GA production, and gene transcription of key enzymes for GA biosynthesis in liquid static cultures of G. lucidum were investigated. A high cell density of 29.8 +/- 1.7 g/l DW and total GA production of 1427.2 +/- 74.2 mg/l were obtained under an optimal gaseous O(2) level of 80%. The expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, squalene synthase and lanosterol synthase genes of GA biosynthetic pathway as detected by quantitative real-time PCR was also affected by the gaseous oxygen concentration in the liquid static culture. H(2)O(2) was generated as reactive oxygen species in response to high oxygen concentrations in the gas phase, and it seemed to be involved in the regulation of GA biosynthesis. The information obtained in this study provided an insight into the role of gaseous O(2) in the GA production and it will be helpful for further enhancing its productivity.

Modified oxidosqualene cyclases in the formation of bioactive secondary metabolites: biosynthesis of the antitumor clavaric acid.

Hypholoma sublateritium is a basidiomycete that produces the antitumor compound clavaric acid. We cloned a gene encoding an oxidosqualene cyclase (occ) that is involved in the conversion of oxidosqualene to clavaric acid. Mutants disrupted in occ by gene replacement were unable to synthesize clavaric acid, but did not require sterols for growth. Amplification of the occ gene produced a 35-67% increase in clavaric acid yield. Northern analysis of occ gene expression confirmed that there is a clear correlation of occ expression and clavaric acid biosynthesis. Analysis of the occ-encoded oxidosqualene cyclase revealed that it has VSDCVGE motif instead of the consensus VSDCTAE sequence of the active center. In summary, there is an oxidosqualene cyclase specific for secondary metabolite biosynthesis; this is in agreement with the finding of two squalene cyclases in the sequenced genomes of basidiomycetes.

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum.

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.

A novel three-stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides.

A novel three-stage light irradiation strategy in the submerged fermentation of medicinal mushroom Ganoderma lucidum for the efficient production of bioactive metabolites ganoderic acid (GA) and Ganoderma polysaccharides was developed. Significance of light quality, i.e., blue light (390-500 nm, lambda(max) = 470 nm), red light (560-700 nm, lambda(max) = 625 nm), and white light (400-740 nm, lambda(max) = 550 nm), was studied at first. Interestingly, there was a gradual decrease trend of GA content after the culture of day 2 when the maximal GA content was obtained, while GA content decreased slowly under white light irradiation after day 6. The dark environment was favorable to the specific GA biosynthesis (i.e., GA content) before day 6, and after that the optimum was white light irradiation. A relatively lower irradiation density of white light (i.e., 0.94 and 2.82 W/m(2)) was beneficial for the specific GA biosynthesis before day 6, while GA content was higher under higher irradiation density of white light (i.e., 4.70 and 9.40 W/m(2)) at the later-stage of cultivation. 4.70 W/m(2) white light irradiation culture was the best from the viewpoint of GA accumulation. Therefore, a two-stage light irradiation strategy by combing the first 2 days dark culture with the following 4.70 W/m(2) white light irradiation culture was developed. The highest GA production in the two-stage culture was 276.0 +/- 12.5 mg/L, which was increased by 19% compared to 4.70 W/m(2) white light irradiation culture (i.e., 232.4 +/- 15.8 mg/L) and by 178% compared to the dark culture (i.e., 99.4 +/- 1.0 mg/L). Although there still existed a gradual decrease trend of GA content after day 2 when the maximal GA content was obtained in the two-stage culture. Following three-stage light irradiation strategy was further demonstrated in order to turn around the sharp decrease of GA content after day 2. The first-stage was the 2-day dark culture; the second-stage was the following six-day 0.94 W/m(2) white light irradiation culture, and the third-stage was 4.70 W/m(2) white light irradiation culture until the end of fermentation. During the three-stage culture of G. lucidum, the gradual decrease trend of GA content after day 2 was turned around, which suggested that 0.94 W/m(2) white light irradiation was beneficial for the metabolic flux towards the GA biosynthesis. The maximal GA content of 3.1 +/- 0.1 mg/100 mg DW was obtained, which was higher by 41% compared to the two-stage culture. The maximal GA production (i.e., 466.3 +/- 24.1 mg/L) and productivity (i.e., 38.9 mg/L per day) in the three-stage culture were 69 and 101% higher than those obtained in the two-stage culture. This is the first report investigating the significance of light irradiation on the medicinal mushroom submerged fermentation. Such work is very helpful to other mushroom fermentations for useful metabolite production.

A squalene epoxidase is involved in biosynthesis of both the antitumor compound clavaric acid and sterols in the basidiomycete H. sublateritium.

The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH, and P450 cofactors. Silencing of the erg1 gene in H. sublateritium using constructions expressed from the gdh promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene resulted in up to 32% to 97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.

Sterol composition in field-grown and cultured mycelia of Inonotus obliquus.

Sterols are one of the active classes of compounds in Inonotus obliquus for their effective therapy of many diseases. In field environment, this fungus accumulates large amount of sterols. In cultured mycelia, however, this class of compounds is less accumulated. For analyzing the factors responsible for differing sterol composition, the field-grown and cultured mycelia were extracted with 80% ethanol at room temperature and total sterols were prepared using silicon gel column chromatography followed by identification using either GC-MS or spectroscopic methods. For culturing Inonotus obliquus, the seed culture was grown either in basic medium consisting of glucose (2%), yeast extract (0.5%), KH2PO4 (0.01%), MgSO4.7H20 (0.05%) and distilled water at pH 6.5, or the basic medium supplemented with serial concentrations of AgNO3. The results indicated that field-grown mycelia contained lanosterol and inotodiol (comprised 45. 47% and 25. 36% of the total sterols, respectively) and other 10 sterols (comprising the remaining 30.17%) including ergosterol biosynthetic intermediates such as 24-methylene dihydrolanosterol, 4,4-dimethylfecosterol, 4-methyl fecosterol, fecosterol and episterol. Column chromatography also led to the isolation of lanosterol, Inotodiol, trametenolic acid, foscoparianol B and a new triterpenoid foscoparianol D in field-grown mycelia. In comparison, the cultured mycelia only contained three sterols with ergosterol as the predominant one (82.20%). Lanosterol only accounted for 3.68%. Supplementing Ag+ into the culture at 0.28 micromol x L(-1) greatly enhanced content of lanosterol (accounting for 56.81%) and decreased the content of ergosterol (18.5%) together with the presence of intermediates for ergosterol biosynthesis. These results suggested that the sterol composition in mycelia of the fungus can be diversified by supplementing substances inhibiting enzymatic process towards the synthesis of ergosterol. Harsh growth conditions in field environment (i.e. temperature variation, UV irradiation etc.) can delay the synthesis of ergosterol and hereby diversify the sterol composition in the mycelia of Inonotus obliquus.

Lanosterol biosynthesis in the prokaryote Methylococcus capsulatus: insight into the evolution of sterol biosynthesis.

A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general.

Trametes versatillis, a fungus highly producing eburicoic acid.

Eburicoic acid was produced and isolated from the cultured mycelia of the fungus, Trametes versatillis. The weight rate of eburicoic acid in the dry weight of cultured mycelia of Trametes versatillis was about to 1%. Trametes versatillis can be expected as an important bioresource to supply eburicoic acid for its industrial exploitation.