Bioconversion of Ginsenoside Rf into Its Hydrated Derivative with Elevated Anti-inflammatory Effect by a Highly Specific Biocatalytic System of Cordyceps Sinensis.

The presence of 25-OH moiety has been proved to enhance the bioactivity of dammarane saponins in many cases. However, such modification by previous strategies had compromised yield and purity of target products. Herein ginsenoside Rf was specifically transformed into 25-OH-(20S)-Rf with a conversion rate of 88.03 % by a Cordyceps Sinensis-mediated biocatalytic system. The formulation of 25-OH-(20S)-Rf was calculated by HRMS, whilst its structure was validated by 1 H-NMR, 13 C-NMR, HSQC, and HMBC analysis. Time-course experiments unveiled straightforward hydration of the double bond on Rf with undetectable side reactions and maximum production of 25-OH-(20S)-Rf on the 6th day, which collectively suggested the suitable timing of harvesting this target compound. In vitro bioassay of (20S)-Rf and 25-OH-(20S)-Rf against lipopolysaccharide-induced macrophages indicated a significant boost of anti-inflammatory effects after the C24-C25 double bond was hydrated. Therefore, the biocatalytic system in this article could be leveraged to deal with macrophage-mediated inflammation under defined circumstances.

Lowering the Intraocular Pressure in Rats and Rabbits by Cordyceps cicadae Extract and Its Active Compounds.

Cordyceps cicadae (CC), an entomogenous fungus that has been reported to have therapeutic glaucoma, is a major cause of blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) death, mostly due to elevated intraocular pressure (IOP). Here, an ethanolic extract of C. cicadae mycelium (CCME), a traditional medicinal mushroom, was studied for its potential in lowering IOP in rat and rabbit models. Data showed that CCME could significantly (60.5%) reduce the IOP induced by microbead occlusion after 56 days of oral administration. The apoptosis of retinal ganglion cells (RGCs) in rats decreased by 77.2%. CCME was also shown to lower the IOP of normal and dextrose-infusion-induced rabbits within 60 min after oral feeding. There were dose effects, and the effect was repeatable. The active ingredient, N6-(2-hydroxyethyl)-adenosine (HEA), was also shown to alleviate 29.6% IOP at 0.2 mg/kg body weight in this rabbit model. CCME was confirmed with only minor inhibition in the phosphorylated myosin light chain 2 (pMLC2) pathway.

Identification of an Oxidosqualene Cyclase Gene Involved in Steroidal Triterpenoid Biosynthesis in Cordyceps farinosa.

Various fungi including Cordyceps farinosa, an entomopathogenic fungus, can produce steroidal triterpenoids. Protostadienol (protosta-17(20)Z,24-dien-3ß-ol) is a precursor of steroidal triterpenoid compounds. To identify oxidosqualene cyclase (OSC) gene candidates involved in triterpenoid biosynthesis, genome mining was performed using Illumina sequencing platform. In the sequence database, two OSC genes, CfaOSC1 and CfaOSC2, in the genome of C. farinosa were identified. Predicted amino-acid sequences of CfaOSC2 shared 66% similarities with protostadienol synthase (OSPC) of Aspergillus fumigatus. Phylogenetic analysis showed a clear grouping of CfaOSC2 in the OSPC clade. Function of CfaOSC2 was examined using a yeast INVSc1 heterologous expression system to endogenously synthesize 2,3-oxidosqualene. GC-MS analysis indicated that CfaOSC2 produced protosta-13(17),24-dien-3ß-ol and protostadienol at a 5:95 ratio. Our results demonstrate that CfaOSC2 is a multifunctional triterpene synthase yielding a predominant protostadienol together with a minor triterpenoid. These results will facilitate a greater understanding of biosynthetic mechanisms underlying steroidal triterpenoid biosynthesis in C. farinosa and other fungi.

The Enzymatic Decarboxylation Mechanism of 5-Carboxy Uracil: A Comprehensive Quantum Chemical Study.

Dynamic regulation of DNA methylation is an important process for the control of gene expression in mammals. It is believed that in the demethylation pathway of 5-methyl cytosine, the intermediate 5-carboxy cytosine (5caC) can be actively decarboxylated alongside the substitution in the base excision repair. For the active decarboxylation of 5caC, a decarboxylase has not been identified so far. Due to the similar chemistry of the decarboxylation of 5-carboxy uracil (5caU) to uracil (U) in the pyrimidine salvage pathway catalyzed by the iso-orotate decarboxylase (IDCase), the study of this reaction might give valuable insights into the active 5caC decarboxylation process. In this work, we employ quantum chemical and molecular mechanic calculations and find that the catalytic mechanism of IDCase proceeds via a direct decarboxylation mechanism. Detailed investigations on the reaction coordinate reveal that it is a one-step mechanism with concerted proton transfer and C-C bond opening.

Enzymatic characterization and validation of gene expression of phosphoglucomutase from Cordyceps militaris.

The purification and characterization of PGM (Phosphoglucomutase) from Cordyceps militaris (C. militaris) was investigated. PGM was purified using a combination of ultrafiltration, salting-out and ion exchange chromatography resulting in 4.23-fold enhancement of activity with a recovery of 20.01%. Molecular mass was 50.01 kDa by SDS-PAGE. The optimal activity was achieved at pH 7.5 and 30 °C with NADPH as substrate. The results showed that SDS, DTT Li+, Cu2+, Na+, Mn2+ and Al3+ were effective PGM inhibitors; whereas glycerol, Zn2+, Mg2+, Ca2+, Fe2+ and Fe3+ could enhance the activity of PGM, and the Km and Vmax values were 11.62 mmol/L and 416.67 U/mL, respectively. At the same time, qRT-PCR was used to test the changes of mRNA transcription level of PGM gene encoding under two fermentation conditions: basic medium and optimized medium. The relative quantitative results of PGM target genes resulting in 2.60-fold enhancement than the control group.

Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris.

The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.

Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris.

Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.

Expression, purification and characterization of 5'-nucleotidase from caterpillar fungus by efficient genome-mining.

5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 µmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

Gene cloning, expression and homology modeling of first fibrinolytic enzyme from mushroom (Cordyceps militaris).

Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents.

Structural basis for the specific cleavage of core-fucosylated N-glycans by endo-ß-N-acetylglucosaminidase from the fungus Cordyceps militaris.

N-Linked glycans play important roles in various cellular and immunological events. Endo-ß-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris.

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of Vmax , Km , and kcat were determined to be 4.86 µmol·min-1 ·mg-1 , 0.07 mM, and 0.29 min-1 , respectively. In particular, the relatively low Km value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.

Cloning and protein expression of the sn-1(3) regioselective lipase from Cordyceps militaris.

In this study, the gene of a novel lipase with sn-1(3) regioselectivity (i.e., sn-1 or sn-3 specific) from Cordyceps militaris was successfully expressed by a heterologous expression system. Total RNA was extracted from C. militaris and then single-stranded cDNA was synthesized. The resulting C. militaris lipase (CML) gene was inserted in Escherichia coli expression plasmids [pET-29b(+), pET-26b, and pColdIII] to construct plasmids encoding CML, which were then transformed to E. coli strains BL21 (DE3), C43 (DE), C41 (DE3), and Origami (DE3) for protein expression. Although the recombinant CML expression level was high, it was overproduced in the form of inclusion bodies. Under a specific condition, the soluble form of the recombinant CML was detected using Western blot analysis; however, no enzyme activity was observed. To overcome the lack of post-translational modifications in recombinant CML, a baculovirus-insect expression system was introduced for eukaryotic lipase expression. pDualBac was used as the transfer vector, and the CML gene was fused under the control of the polyhedrin promoter. After generating the recombinant baculovirus, the active form of CML was successfully produced and its kinetic parameters were determined.

Insight into cordycepin biosynthesis of Cordyceps militaris: Comparison between a liquid surface culture and a submerged culture through transcriptomic analysis.

Cordyceps militaris produces cordycepin, which is known to be a bioactive compound. Currently, cordycepin hyperproduction of C. militaris was carried out in a liquid surface culture because of its low productivity in a submerged culture, however the reason was not known. In this study, 4.92 g/L of cordycepin was produced at the 15th day of C. militaris NBRC 103752 liquid surface culture, but only 1 mg/L was produced in the submerged culture. RNA-Seq was used to clarify the gene expression profiles of the cordycepin biosynthetic pathways of the submerged culture and the liquid surface culture. From this analysis, 1036 genes were shown to be upregulated and 557 genes were downregulated in the liquid surface culture compared with the submerged culture. Specifically, adenylosuccinate synthetase and phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthase in purine nucleotide metabolism were significantly upregulated in the liquid surface culture. Thick mycelia formation in the liquid surface culture was found to induce the expression of hypoxia-related genes (GABA shunt, glutamate synthetase precursor, and succinate-semialdehyde dehydrogenase). Cytochrome P450 oxidoreductases containing heme were also found to be significantly enriched, suggesting that a hypoxic condition might be created in the liquid surface culture. These results suggest that hypoxic conditions are more suitable for cordycepin production in the liquid surface culture compared with the submerged culture. Our analysis paves the way for unraveling the cordycepin biosynthesis pathway and for improving cordycepin production in C. militaris.

Isaria fumosorosea KCh J2 Entomopathogenic Strain as an Effective Biocatalyst for Steroid Compound Transformations.

The catalytic activity of enzymes produced by an entomopathogenic filamentous fungus (Isaria fumosorosea KCh J2) towards selected steroid compounds (androstenedione, adrenosterone, progesterone, 17α-methyltestosterone and dehydroepiandrosterone) was investigated. All tested substrates were efficiently transformed. The structure of the substrate has a crucial impact on regio- and stereoselectivity of hydroxylation since it affects binding to the active site of the enzyme. Androstenedione was hydroxylated in the 7α-position to give a key intermediate in the synthesis of the diuretic-7α-hydroxyandrost-4-ene-3,17-dione with 82% conversion. Adrenosterone and 17α-methyltestosterone were hydroxylated in the 6ß-position. Hydroxylated derivatives such as 15ß-hydroxy-17α-methyltestosterone and 6ß,12ß-dihydroxy-17α-methyltestosterone were also observed. In the culture of Isaria fumosorosea KCh J2, DHEA was effectively hydroxylated in the C-7 position and then oxidized to give 7-oxo-DHEA, 3ß,7α- and 3ß,7ß-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one. We obtained 7ß-OH-DHEA lactone with 82% yield during 3 days transformation of highly concentrated (5 g/L) DHEA.

Functional Analysis of Ribonucleotide Reductase from Cordyceps militaris Expressed in Escherichia coli.

Cordyceps militaris produces cordycepin (3'-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2'-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2'-deoxyADP. These results indicate that the pathway from ADP to 3'deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.

Biochemical characterization of a novel fibrinolytic enzyme from Cordyceps militaris.

A fibrinolytic enzyme was produced by the medicinal mushroom, Cordyceps militaris using submerged fermentation. The enzyme was purified from culture supernatant by hydrophobic interaction, ion exchange and gel filtration chromatographies. It was purified by 36 fold, with a specific activity of 1,467.4U/mg protein and the final yield was 5.8%. The molecular weight of the enzyme as determined by SDS-PAGE and gel filtration was 28kDa and 24.5kDa, respectively, and its isoelectric point (pI) was 9.0±0.2. It was found to be a glycoprotein with carbohydrate content of 1.67% (w/v). The enzyme was optimally active at 37°C and pH 7.2. The enzyme activity was strongly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin which indicated it to be a serine protease, while other inhibitors like N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK), phenyl methane sulfonyl fluoride (PMSF), pepstatin and metal chelator EDTA did not inhibit its activity. Amino acid sequences of the purified enzyme were determined partially by Q-TOF2 and they were IEDFPYQVDLR; ANCGGTVISEK; YVLTAGHCAEGYTGLNIR; TNYASVTPITADMICAGFPEGK; KDSCSGDSGGPLVTGGK; VVGIVSFGTGCAR; ANKPGVYSSVASAEIR. Sequences of the seven peptides completely matched with those of a trypsin-like serine protease from Cordyceps militaris CM01 (accession no. EGX95217.1). The purified enzyme degraded α chains of fibrinogen first and then ß and γ chains and also activated plasminogen into plasmin. It can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Based on these studies, the purified enzyme has great potential to be developed as a natural agent for prevention and treatment of thrombolytic diseases.

Improved Biological Activities of Isoepoxypteryxin by Biotransformation.

Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant Angelica shikokiana. This research was designed to study the effect of structural changes through fungal biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested microorganisms, only Cordyceps sinensis had enzymes that could catalyze the ester hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to give two more polar metabolites (+)-cis-khellactone (P1) and a new coumarin derivative (+)-cis-3'-[(2-methyl-3-hydroxybutanoyl)oxy]-4'-acetoxy-3',4'-dihydroseselin (P2), respectively. The polar metabolite P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase 8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1 - 42 (Aß1 - 42 )-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more inhibition of the in vitro aggregation of Aß1 - 42 . Both metabolites showed stronger antiplatelet aggregation by increased inhibition of thromboxane-A2 synthase (TXS) activity and thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic, neuroprotective, and antiplatelet aggregation activities of isoepoxypteryxin through its biotransformation by C. sinensis.

Purification and biochemical characterization of a novel fibrinolytic enzyme from culture supernatant of Cordyceps militaris.

A novel fibrinolytic enzyme from Cordyceps militaris was produced by submerged culture fermentation, purified, and biochemically characterized. The enzyme was purified to homogeneity, with an overall yield of 4.0% and a specific activity of 1682 U/mg. The molecular weight and pI of the enzyme were 32 kDa and 9.3 ± 0.2, respectively. The optimal pH and temperature of the enzyme were 7.4 and 37 °C, respectively. The enzyme activity was inhibited by Fe(2+), phenylmethane sulfonyl fluoride (PMSF), aprotinin, and pepstatin but not by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and ethylenediamine tetracetic acid (EDTA). Three internal peptides of the enzyme, APQALTVAAVGATWAR, EKNVGSTVNLLSYDGNK, and TDATSVLLDGYNVSAVNDLVAK, were obtained. The enzyme could hydrolyze fibrin(ogen) directly and cleave the α-chains more efficiently than ß- and γ-chains, suggesting that it is a plasmin like protein. It degraded thrombin, which indicated that it can act as an anticoagulant and prevent thrombosis. Intravascular thrombosis is one of the major reasons of cardiovascular diseases. On the basis of these results, the purified enzyme can be developed as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.