Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

Regulation of a phenylalanine ammonia lyase (BbPAL) by calmodulin in response to environmental changes in the entomopathogenic fungus Beauveria bassiana.

Phenylalanine ammonia lyase (PAL, E.C. 4.3.1.5) catalyses the deamination of L -phenylalanine to trans-cinnamic acid and ammonia, facilitating a critical step in the phenylpropanoid pathway that produces a variety of secondary metabolites. In this study, we isolated BbPAL gene in the entomopathogenic fungus Beauveria bassiana. According to multiple sequence alignment, homology modelling and in vitro PAL activity, we demonstrated that BbPAL acts as a typical PAL enzyme in B. bassiana. BbPAL interacted with calmodulin (CaM) in vitro and in vivo, indicating that BbPAL is a novel CaM-binding protein. The functional role of CaM in BbPAL action was to negatively regulate the BbPAL activity in B. bassiana. High-performance liquid chromatography analysis revealed that L -phenylalanine was reduced and trans-cinnamic acid was increased in response to the CaM inhibitor W-7. Dark conditions suppressed BbPAL activity in B. bassiana, compared with light. In addition, heat and cold stresses inhibited BbPAL activity in B. bassiana. Interestingly, these negative effects of BbPAL activity by dark, heat and cold conditions were recovered by W-7 treatment, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbPAL plays a role in the phenylpropanoid pathway mediated by environmental stimuli via the CaM signalling pathway.

Promotion of Tricholoma matsutake mycelium growth by Penicillium citreonigrum.

Tricholoma matsutake has been the most valuable ectomycorrhizal fungi in Asia because of its unique flavor and taste. However, due to the difficulty of artificial cultivation, the cultivation of T. matsutake has relied on natural growth in forests. To cultivate the T. matsutake artificially, microorganisms in fairy rings were introduced. In this study, we isolated 30 fungal species of microfungi from the soil of fairy rings. Among them, one single fungal strain showed a promoting effect on the growth of T. matsutake. The growth effect was confirmed by measuring the growth area of T. matsutake and enzyme activities including α-amylase, cellulase, and ß-glucosidase. In comparison with control, microfungal metabolite increased the growth area of T. matsutake by 213% and the enzyme activity of T. matsutake by 110-200%. The isolated fungal strain was identified as Penicillium citreonigrum by BLAST on the NCBI database. The Discovery of this microfungal strain is expected to contribute to artificial cultivation of T. matsutake.

Draft Genome Sequence of the Reference Strain of the Korean Medicinal Mushroom Wolfiporia cocos KMCC03342.

Wolfiporia cocos is a wood-decay brown rot fungus belonging to the family Polyporaceae. While the fungus grows, the sclerotium body of the strain, dubbed Bokryeong in Korean, is formed around the roots of conifer trees. The dried sclerotium has been widely used as a key component of many medicinal recipes in East Asia. Wolfiporia cocos strain KMCC03342 is the reference strain registered and maintained by the Korea Seed and Variety Service for commercial uses. Here, we present the first draft genome sequence of W. cocos KMCC03342 using a hybrid assembly technique combining both short- and long-read sequences. The genome has a total length of 55.5 Mb comprised of 343 contigs with N50 of 332 kb and 95.8% BUSCO completeness. The GC ratio was 52.2%. We predicted 14,296 protein-coding gene models based on ab initio gene prediction and evidence-based annotation procedure using RNAseq data. The annotated genome was predicted to have 19 terpene biosynthesis gene clusters, which was the same number as the previously sequenced W. cocos strain MD-104 genome but higher than Chinese W. cocos strains. The genome sequence and the predicted gene clusters allow us to study biosynthetic pathways for the active ingredients of W. cocos.

Mitrula aurea sp. nov., A New Aero-Aquatic Species from the Republic of Korea.

The genus Mitrula (Mitrulaceae, Helotiales), as also known as swamp beacons, inhabits submerged, decaying vegetation in standing or decaying needles, twigs, leaves, and shallow water. They play an important role in carbon cycling in some freshwater ecosystems. In the herbarium of the Korea National Arboretum (KH), seven Mitrula specimens were collected during mushroom forays in the period from 2019 to 2021. The Korean collections were found to be macromorphologically closely related to M. paludosa and M. elegans, but micromorphologically they could be distinguished by characteristics of slightly narrower asci and aseptate ascospores. Our molecular phylogenetic analyses of the internal transcribed spacer (ITS) and 28S rDNA regions also revealed that our specimens were related to M. paludosa and M. elegans, but formed a distinct clade. Based on these results, we reported our specimens as new to science and discussed the phylogeny and diversity of Mitrula species.

Improved accuracy of geographical origin identification of shiitake grown in sawdust medium: A compound-specific isotope model-based pilot study.

In countries like South Korea and the USA, origin labeling of shiitake grown using imported Chinese-inoculated medium is an issue. Therefore, we evaluated the use of compound-specific isotope analysis (CSIA) for the accurate identification of the geographical origin of shiitake (Korean, Chinese-inoculated medium, and Chinese); Chinese-inoculated medium shiitake were cultivated in Korea using inoculated sawdust medium from China. The CSIA-discriminant model showed an overall accuracy of 100% in the geographical classification of the original set and 96.4% for the cross-validated set. Glutamate and aspartate δ15N values were the most important variables for differentiating shiitake based on their origins. Compared to that observed upon using the bulk stable isotope analysis, the CSIA model was associated with significantly improved predictability of origin identification. Our findings elucidate the importance of isotope signatures in developing a reliable origin labeling method for shiitake cultured on the sawdust medium for the global market.

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.

An origin identification model for labeling of shiitake (Lentinula edodes).

With the increasing globalization of the food trade across countries and continents, reliable identification of the geographical origin of products is critical. In this study, we describe the limitations of the current origin labeling system for non-soil-based agricultural products and suggest alternative strategies for the identification of the geographical origin of such products. An origin identification model based on stable isotope ratio analysis combined with discriminant analysis is used to evaluate the similarities and dissimilarities between domestic and foreign shiitake mushrooms, including Chinese inoculated sawdust blocks and Chinese origin. The results show a classification sensitivity of 92.0%, classification specificity of 91.5%, and overall accuracy of 93.5%. In particular, δ15N was the most important isotope marker for the identification of the origin of shiitake mushrooms. Hence, the current origin labeling system for mushroom species has to be revised to establish fair trade and avoid improper origin labeling in the global shiitake market.

An unattended HS-SPME-GC-MS/MS combined with a novel sample preparation strategy for the reliable quantitation of C8 volatiles in mushrooms: A sample preparation strategy to fully control the volatile emission.

Eight carbon (C8) compounds are the key characteristic flavors of mushrooms. The quantitative analysis of the volatiles in mushrooms is challenging especially with the unattended HS-SPME-GC-MS. An unattended HS-SPME-GC-MS/MS in combination with novel sample preparation of the complete control of volatile emissions was developed for the quantitation of the C8 volatiles in mushrooms. The sample preparation strategy was composed of freeze-drying, rehydration, and the addition of a 15% citric acid solution. With this strategy, the volatile emission from mushroom was fully controlled at a certain time point. This method was found to be highly reliable, sensitive, precise, and accurate. This method was successfully applied to measure the contents of the C8 volatiles in the beech, button, and shiitake mushrooms. 1-Octene-3-ol was the most predominant compound in the mushrooms, representing 62.4, 69.0, and 89.2% of the total C8 volatiles in the beech, button, and shiitake mushrooms, respectively.

Fatty Acids and Stable Isotope Ratios in Shiitake Mushrooms (Lentinula edodes) Indicate the Origin of the Cultivation Substrate Used: A Preliminary Case Study in Korea.

Shiitake mushroom (Lentinula edodes) is commonly consumed worldwide and is cultivated in many farms in Korea using Chinese substrates owing to a lack of knowledge on how to prepare sawdust-based substrate blocks (bag cultivation). Consequently, issues related to the origin of the Korean or Chinese substrate used in shiitake mushrooms produced using bag cultivation have been reported. Here, we investigated differences in fatty acids (FAs) and stable isotope ratios (SIRs) in shiitake mushrooms cultivated using Korean and Chinese substrates under similar conditions (strain, temperature, humidity, etc.) and depending on the harvesting cycle. The total FA level decreased significantly by 5.49 mg∙g-1 as the harvesting cycle increased (p < 0.0001); however, no differences were found in FAs between shiitake mushrooms cultivated using Korean and Chinese substrates. Linoleic acid was the most abundant FA, accounting for 77-81% of the total FAs during four harvesting cycles. Moreover, the SIRs differed significantly between the Korean and Chinese substrates, and the harvesting cycles resulted in smaller maximum differences in SIR values compared to those of the cultivation substrate origins. Our findings contribute to the identification of the geographical origin of shiitake mushrooms and may have potential applications in international shiitake-mushroom markets.

Ganoderma lucidum Extract Reduces Insulin Resistance by Enhancing AMPK Activation in High-Fat Diet-Induced Obese Mice.

Ganoderma lucidum is used widely in oriental medicine to treat obesity and metabolic diseases. Bioactive substances extracted from G. lucidum have been shown to ameliorate dyslipidemia, insulin resistance, and type 2 diabetes in mice via multiple 5' AMP-activated protein kinase (AMPK)-mediated mechanisms; however, further studies are required to elucidate the anti-obesity effects of G. lucidum in vivo. In this study, we demonstrated that 3% G. lucidum extract powder (GEP) can be used to prevent obesity and insulin resistance in a mouse model. C57BL/6 mice were provided with a normal diet (ND) or a high-fat diet (HFD) supplemented with 1, 3, or 5% GEP for 12 weeks and the effect of GEP on body weight, liver, adipose tissue, adipokines, insulin and glucose tolerance (ITT and GTT), glucose uptake, glucose-metabolism related proteins, and lipogenesis related genes was examined. GEP administration was found to reduce weight gain in the liver and fat tissues of the mice. In addition, serum parameters were significantly lower in the 3% and 5% GEP mice groups than in those fed a HFD alone, whereas adiponectin levels were significantly higher. We also observed that GEP improved glucose metabolism, reduced lipid accumulation in the liver, and reduced adipocyte size. These effects may have been mediated by enhanced AMPK activation, which attenuated the transcription and translation of lipogenic genes such as fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and sterol regulatory element-binding protein-1c (SREBP1c). Moreover, AMP-activated protein kinase (AMPK) activation increased acetyl-CoA carboxylase (ACC), insulin receptor (IR), IR substrate 1 (IRS1), and Akt protein expression and activation, as well as glucose transporter type 1/4 (GLUT1/4) protein production, thereby improving insulin sensitivity and glucose metabolism. Together, these findings demonstrate that G. lucidum may effectively prevent obesity and suppress obesity-induced insulin resistance via AMPK activation.

Characterization of Volatile Profiles of Six Popular Edible Mushrooms Using Headspace-Solid-Phase Microextraction Coupled with Gas Chromatography Combined with Chemometric Analysis.

The classification of six mushroom species (white beech, brown beech, button, oyster, king oyster, and enoki mushrooms) was successfully achieved using canonical discriminant analysis (CDA) on volatile metabolite data sets obtained by headspace-solid-phase microextraction gas chromatography (HS-SPME-GC). Twenty-seven major volatile compounds in six edible mushrooms were positively identified by HS-SPME-GC mass spectroscopy. The total volatile content was highest in brown beech mushroom (P < 0.05). Significant difference in volatile profile was observed between brown beach and white beech mushrooms. Button mushroom contained significantly higher contents of benzaldehyde and benzyl alcohol than the other mushrooms (P < 0.05). Oyster mushroom contained 1-octen-3-ol as the most prevalent volatile, representing 67% out of total volatiles. Hexanal (35.0%) and 1-octen-3-ol (22.5%) were the most abundant volatiles found in king oyster. Hexanal (29.1%) was the most prevalent volatile in enoki mushroom only. Several volatile pairs with very high positive correlation in their levels were identified, representing the highest correlation coefficient (r = 0.970) for the pair of t-2-octenal and 2,4-octandienal. CDA was much more efficient than principal component analysis for the differentiation of mushroom species. PRACTICAL APPLICATION: The present study provided the important information on the volatile metabolite profiles of popular six commercial mushroom species. The present data will be useful for the quality control of mushrooms cultivated in farms and mushroom products processed in food industry. The strategy of canonical discriminant analysis in combination with HS-SPME-GC could be expanded for the determining the authentication of mushroom species.

First Report of Buchwaldoboletus lignicola (Boletaceae), a Potentially Endangered Basidiomycete Species, in South Korea.

During the 2014 survey of the mushroom flora of Gwangneung forest in South Korea, we collected two specimens of boletoid mushroom growing on a felled tree of Pinus koraiensis. These specimens were characterized by a light brown to reddish-brown pileus with appressed tomentum, pore surface bluing instantly when bruised, golden-yellow mycelium at the base of stipe, and lignicolous habitat. Both specimens were identified as Buchwaldoboletus lignicola, a rare basidiomycete, based on morphological characteristics and sequences of internal transcribed spacer (ITS; fungal barcode). Here, we describe these specimens and provide the first report of this genus in South Korea.

Potential geo-discriminative tools to trace the origins of the dried slices of shiitake (Lentinula edodes) using stable isotope ratios and OPLS-DA.

The Lentinus edodes species, known as shiitake, has a history of extensive use in many cuisines in several East Asian countries owing to its unique and pleasant flavor and texture. As international food trade increases, reliable discrimination of geographical origin is becoming increasingly crucial in Korea to identify cheaper imported Chinese shiitake. Herein, stable isotope ratios (i.e., δ13C, δ15N, δ18O, and δ34S) were measured with a stable isotope ratio mass spectrometer, and a geographical discrimination method using orthogonal projection to latent structure-discriminant analysis was developed. The externally validated discrimination method showed excellent predictability (Q2cum = 0.881) and illustrated that δ18O and δ15N were important isotope markers for the geographical discrimination of dried shiitake slices. This study extends the knowledge of geographical differences between China and Korea evidenced by the shiitake isotope signatures, thereby contributing to potential geographical authentication with broader applications for international shiitake markets.

1H NMR based metabolite profiling for optimizing the ethanol extraction of Wolfiporia cocos.

Metabolite profiling of Wolfiporia cocos (family: Polyporaceae) had been much advancement in recent days, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly important trait of W. cocos still needs advanced protocols despite some standardization. Partial least squares discriminant analysis (PLS-DA) was used as the multivariate statistical analysis of the 1H NMR data set. The PLS-DA model was validated, and the key metabolites contributing to the separation in the score plots of different ethanol W. cocos extract. 1H NMR spectroscopy of W. cocos identified 33 chemically diverse metabolites in D2O, consisting of 13 amino acids, 11 organic acids 2 sugars, 3 sugar alcohols, 1 nucleoside, and 3 others. Among these metabolites, the levels of tyrosine, proline, methionine, sarcosine, choline, acetoacetate, citrate, 4-aminobutyrate, aspartate, maltose, malate, lysine, xylitol, lactate threonine, leucine, valine, isoleucine, uridine, guanidoacetate, arabitol, mannitol, glucose, and betaine were increased in the 95% ethanol extraction sample compared with the levels in other samples, whereas level of acetate, phenylalanine, alanine, succinate, and fumarate were significantly increased in the 0% ethanol extraction sample. A biological triterpenoid, namely pachymic acid, was detected from different ethanol P. cocos extract using 1H-NMR spectra were found in CDCl3. This is the first report to perform the metabolomics profiling of different ethanol W. cocos extract. These researches suggest that W. cocos can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.

The complete mitochondrial genome of Sanghuangporus sanghuang (Hymenochaetaceae, Basidiomycota).

Sanghuang is a polypore mushroom, which has been widely used in oriental medicine. Since recent molecular phylogenetic studies elucidated its species delimitation, Sanghaungporus sanghuang became the official name of this fungus. In this study, the complete sequence of the mitochondrial DNA of S. sanghuang was determined. The whole genome was 112,060 bp containing 14 proteins, 2 ribosomal RNA subunits, and 45 transfer RNAs. The overall GC content of the genome was 23.21%. A neighbour-joining tree based on atp6 sequence data showed its close relationship with the species of Ganoderma and Trametes.

Regional discrimination of Agaricus bisporus mushroom using the natural stable isotope ratios.

This study was aimed to verify the regional traceability of Agaricus bisporus mushroom using unique δ13C, δ15N, δ18O, and δ34S features combined with chemometric approaches. Ten cultivars from 15 mushroom farms in six regions of Korea were measured their isotopic signatures by stable isotope ratio mass spectrometer. The δ13C, δ15N, δ18O, and δ34S values were significantly different among mushrooms collected from six cultivation regions (P < 0.05). Multi-dimensional plots including the δ15N and δ34S values demonstrated clear regional discrimination of the Saedo and Saehan cultivars produced from Buyeo, Nonsan, Eumseong, Boryeong, or Gyeongju. Partial least-squares discriminate analysis models showed good discrimination for Saedo (Rx2 = 0.798 and Q2 = 0.563) and Saehan (Rx2 = 0.819 and Q2 = 0.894). These preliminary results can extend knowledge of regional isotope signatures in A. bisporus mushroom produced in Korea, contributing to accurate geographical authenticity with potential broader applications for the international mushroom market.

Studies on seasonal dynamics of soil-higher fungal communities in Mongolian oak-dominant Gwangneung forest in Korea.

We surveyed macrofungi biweekly at defined plots from April to December in 2014, in the Mongolian oak-dominant forest, Gwangneung Forest, Pochen-si, Korea, and analyzed a soilhigher fungal diversity during four seasons (represented by April, August, October, and December). Based on morphological observation of collected specimens, the collected macrofungi were classified into 2 phyla 3 classes 7 orders, 14 families, 21 genera, and 33 species (36 specimens). DNA-based community analyses indicated that soil-higher fungi were classified into 2 phyla, 18 classes, 49 orders, 101 families, and 155 genera (83,360 sequence reads), defined herein as 155 genus-level operational taxonomic units (GOTUs). In the present study, we evaluated and discussed the fungal diversity in seasonal dynamics and soil layers based on collected macrofungi and pyrosequencing data while considering environmental parameters (pH, exchangeable K, T-P, NH 4 (+) , NO 3 (-) , OM, WR, TOC, and T-N). Moreover, principal components analysis (PCA) showed distinct clusters of the GOTU assemblage associated with the seasons.

New Records of Xylaria Species in Korea: X. ripicola sp. nov. and X. tentaculata.

During a Korean mushroom diversity survey from 2011 to 2014, we found one new Xylaria species (X. ripicola sp. nov.) and one Xylaria species that had not been previously observed in Korea (X. tentaculata). To confirm the phylogenetic placement of the new species, we conducted a phylogenetic investigation based on internal transcribed spacer regions of ribosomal DNA sequences. Additionally, the new species, X. ripicola, was subsequently analyzed for RNA polymerase II subunit sequences. We also evaluated the macroscopic and microscopic features of this species. Herein, X. ripicola is described as a new species that was collected from a natural beach habitat and X. tentaculata is formally reported as newly found in Korea.

Species identity of Phellinus linteus (sanghuang) extensively used as a medicinal mushroom in Korea.

Sanghuang is a medicinal mushroom that has gained particular attention in Korea. It has been extensively studied for the past few decades as a natural immune booster and cancer suppressor. Although the scientific name, Phellinus linteus, has been commonly used to refer to the sanghuang mushroom, the species identity of sanghuang has been called into question due to the ambiguity of its circumscription and the inadequacy of morphological distinctions within allied species. Because the species concept of sanghuang has been elucidated by recent molecular phylogenetic studies, it has become necessary to clarify the taxonomic positions of sanghuang strains extensively utilized in Korea. We conducted a phylogenetic analysis of 74 strains belonging to the P. linteus-baumii complex based on ITS nrDNA sequences. Parental stains of sanghuang varieties formally registered in the Korea Seed & Variety Service, including ASI 26046 (Corea sanghuang), 26114 (Boolro), and 26115 (HK 1-ho) were grouped with Sanghuangporus sanghuang instead of P. linteus in the inferred phylogeny.