Journal of Microbiology

Journal Articles

The Gut Microbiota Mediates the Protective Effects of Spironolactone on Myocardial Infarction.: Lu Li, Jian-Yong Sun, Yu-Lin Li, Shi-Wei Zhu, Sheng-Zhong Duan; J. Microbiol. 2024;62(10):883-895. Published online September 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00164-7

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Abstract: Myocardial infarction (MI) is a type of cardiovascular disease that influences millions of human beings worldwide and has a great rate of mortality and morbidity. Spironolactone has been used as a critical drug for the treatment of cardiac failure and it ameliorates cardiac dysfunction post-MI. Despite these findings, whether there is a relationship between the therapeutic effects of spironolactone and the gut microorganism after MI has not been determined. In our research, we used male C57BL/6 J mice to explore whether the gut microbiota mediates the beneficial function of spironolactone after myocardial infarction. We demonstrated that deletion of the gut microbiota eliminated the beneficial function of spironolactone in MI mice, displaying exacerbated cardiac dysfunction, cardiac infarct size. In addition, the gut microbiota was altered by spironolactone after sham or MI operation in mice. We also used male C57BL/6 J mice to investigate the function of a probiotic in the myocardial infarction. In summary, our findings reveal a precious role of the gut flora in the therapeutic function of spironolactone on MI.

Autotrophy to Heterotrophy: Shift in Bacterial Functions During the Melt Season in Antarctic Cryoconite Holes.: Aritri Sanyal, Runa Antony, Gautami Samui, Meloth Thamban; J. Microbiol. 2024;62(8):591-609. Published online May 30, 2024; DOI: https://doi.org/10.1007/s12275-024-00140-1

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Abstract: Microbes residing in cryoconite holes (debris, water, and nutrient-rich ecosystems) on the glacier surface actively participate in carbon and nutrient cycling. Not much is known about how these communities and their functions change during the summer melt-season when intense ablation and runoff alter the influx and outflux of nutrients and microbes. Here, we use high-throughput-amplicon sequencing, predictive metabolic tools and Phenotype MicroArray techniques to track changes in bacterial communities and functions in cryoconite holes in a coastal Antarctic site and the surrounding fjord, during the summer season. The bacterial diversity in cryoconite hole meltwater was predominantly composed of heterotrophs (Proteobacteria) throughout the season. The associated functional potentials were related to heterotrophic-assimilatory and -dissimilatory pathways. Autotrophic Cyanobacterial lineages dominated the debris community at the beginning and end of summer, while heterotrophic Bacteroidota- and Proteobacteria-related phyla increased during the peak melt period. Predictive functional analyses based on taxonomy show a shift from predominantly phototrophy-related functions to heterotrophic assimilatory pathways as the melt-season progressed. This shift from autotrophic to heterotrophic communities within cryoconite holes can affect carbon drawdown and nutrient liberation from the glacier surface during the summer. In addition, the flushing out and export of cryoconite hole communities to the fjord could influence the biogeochemical dynamics of the fjord ecosystem.

Repeated Exposure of Vancomycin to Vancomycin-Susceptible Staphylococcus aureus (VSSA) Parent Emerged VISA and VRSA Strains with Enhanced Virulence Potentials.: An Nguyen, J Jean Sophy Roy, Ji-Hoon Kim, Kyung-Hee Yun, Wonsik Lee, Kyeong Kyu Kim, Truc Kim, Akhilesh Kumar Chaurasia; J. Microbiol. 2024;62(7):535-553. Published online May 30, 2024; DOI: https://doi.org/10.1007/s12275-024-00139-8

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Abstract: The emergence of resistance against the last-resort antibiotic vancomycin in staphylococcal infections is a serious concern for human health. Although various drug-resistant pathogens of diverse genetic backgrounds show higher virulence potential, the underlying mechanism behind this is not yet clear due to variability in their genetic dispositions. In this study, we investigated the correlation between resistance and virulence in adaptively evolved isogenic strains. The vancomycin-susceptible Staphylococcus aureus USA300 was exposed to various concentrations of vancomycin repeatedly as a mimic of the clinical regimen to obtain mutation(s)-accrued-clonally-selected (MACS) strains. The phenotypic analyses followed by expression of the representative genes responsible for virulence and resistance of MACS strains were investigated. MACS strains obtained under 2 and 8 µg/ml vancomycin, named Van2 and Van8, respectively; showed enhanced vancomycin minimal inhibitory concentrations (MIC) to 4 and 16 µg/ml, respectively. The cell adhesion and invasion of MACS strains increased in proportion to their MICs. The correlation between resistance and virulence potential was partially explained by the differential expression of genes known to be involved in both virulence and resistance in MACS strains compared to parent S. aureus USA300. Repeated treatment of vancomycin against vancomycin-susceptible S. aureus (VSSA) leads to the emergence of vancomycin-resistant strains with variable levels of enhanced virulence potentials.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.: Woo Young Kim, Seung-Jin Kim, Hye-Rin Seo, Yoonyong Yang, Jong Seok Lee, Moonsuk Hur, Byoung-Hee Lee, Jong-Geol Kim, Min-Kyu Oh; J. Microbiol. 2024;62(7):569-579. Published online May 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00136-x

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Abstract: Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Correlation between fat accumulation and fecal microbiota in crossbred pigs: Xin Li , Mengyu Li , Jinyi Han , Chuang Liu , Xuelei Han , Kejun Wang , Ruimin Qiao , Xiu-Ling Li , Xin-Jian Li; J. Microbiol. 2022;60(11):1077-1085. Published online September 9, 2022; DOI: https://doi.org/10.1007/s12275-022-2218-5

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Abstract: Backfat thickness (BF) is an important indicator of fat deposition capacity and lean meat rate in pigs and is very important in porcine genetics and breeding. Intestinal microbiota plays a key role in nutrient digestion and utilization with a profound impact on fat deposition of livestock animals. To investigate the relationship between the pig gut microbiome and BF, 20 low-BF (L-BF) and 20 high-BF (H-BF) pigs were selected as two groups from Yunong Black pigs in the present study. Fecal samples from pigs were analyzed for microbial diversity, composition, and predicted functionality using 16S rRNA gene sequencing. The results showed that there were significant differences in microbial β diversity between the two groups. LEfSe analysis revealed a number of bacterial features being differentially enriched in either L-BF or H-BF pigs. Spearman correlation analysis identified the abundance of Oscillospira, Peptococcus, and Bulleidia were significantly positive correlations with BF (P < 0.05), while Sutterella and Bifidobacterium were significantly negatively correlated with BF (P < 0.05). Importantly, the bacteria significantly positively correlated with BF mainly belong to Clostridium, which can ferment host-indigestible plant polysaccharides into shortchain fatty acid (SCFA) and promote fat synthesis and deposition. Predictive functional analysis indicated that the pathway abundance of cell motility and glycan biosynthesis were significantly widespread in the microbiota of the H-BF group. The results of this study will be useful for the development of microbial biomarkers for predicting and improving porcine BF, as well as for the investigation of targets for dietary strategies.

Phenotypic and genomic characteristics of Brevibacterium zhoupengii sp. nov., a novel halotolerant actinomycete isolated from bat feces: Yuyuan Huang , Lingzhi Dong , Jian Gong , Jing Yang , Shan Lu , Xin-He Lai , Dong Jin , Qianni Huang , Ji Pu , Liyun Liu , Jianguo Xu; J. Microbiol. 2022;60(10):977-985. Published online August 19, 2022; DOI: https://doi.org/10.1007/s12275-022-2134-8

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Abstract: Two strictly aerobic, Gram-staining-positive, non-spore-forming, regular rod-shaped (approximately 0.7 × 1.9 mm) bacteria (HY170T and HY001) were isolated from bat feces collected from Chongzuo city, Guangxi province (22°20􍿁54􍿂N, 106°49􍿁20􍿂E, July 2011) and Chuxiong Yi Autonomous Prefecture, Yunnan province (25°09􍿁10􍿂N, 102°04􍿁39􍿂E, October 2013) of South China, respectively. Optimal growth is obtained at 25–28°C (range, 4–32°C) on BHI-5% sheep blood plate with pH 7.5 (range, 5.0–10.0) in the presence of 0.5– 1.0% NaCl (w/v) (range, 0–15% NaCl [w/v]). The phylogenetic and phylogenomic trees based respectively on the 16S rRNA gene and 845 core gene sequences revealed that the two strains formed a distinct lineage within the genus Brevibacterium, most closely related to B. aurantiacum NCDO 739T (16S rRNA similarity, both 98.5%; dDDH, 46.7–46.8%; ANI, 91.9–92.1%). Strain HY170T contained MK-8(H2), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), galactose and ribose as the predominant menaquinone, major polar lipids, and main sugars in the cell wall teichoic acids, respectively. The meso-diaminopimelic acid (meso-DAP) was the diagnostic diamino acid of the peptidoglycan found in strain HY170T. Anteiso-C15:0 and anteiso-C17:0 were the major fatty acids (> 10%) of strains HY170T and HY001, with anteiso-C17:1A predominant in strain HY170T but absent in strain HY001. Mining the genomes revealed the presence of secondary metabolite biosynthesis gene clusters encoding for non-alpha poly-amino acids (NAPAA), ectoine, siderophore, and terpene. Based on results from the phylogenetic, chemotaxonomic and phenotypic analyses, the two strains could be classified as a novel species of the genus Brevibacterium, for which the name Brevibacterium zhoupengii sp. nov. is proposed (type strain HY170T = CGMCC 1.18600T = JCM 34230T).

Sala cibi gen. nov., sp. nov., an extremely halophilic archaeon isolated from solar salt: Hye Seon Song , Juseok Kim , Yeon Bee Kim , Se Hee Lee , Tae Woong Whon , Seong Woon Roh; J. Microbiol. 2022;60(9):899-904. Published online July 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2137-5

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Abstract: Two novel halophilic archaeal strains, CBA1133T and CBA- 1134, were isolated from solar salt in South Korea. The 16S rRNA gene sequences of the isolates were identical to each other and were closely related to the genera Natronomonas (92.3–93.5%), Salinirubellus (92.2%), Halomarina (91.3– 92.0%), and Haloglomus (91.4%). The isolated strains were coccoid, Gram-stain-negative, aerobic, oxidase-positive, and catalase-negative. Growth occurred under temperatures of 25–50°C (optimum, 45°C), NaCl levels of 10–30% (optimum, 15%), pH levels of 6.0–8.5 (optimum, 7.0), and MgCl2 concentrations of 0–500 mM (optimum, 100 mM). Digital DNADNA hybridization values between the strains and related genera ranged from 18.3% to 22.7%. The major polar lipids of the strains were phosphatidyl glycerol, phosphatidyl glycerol phosphate methyl ester, and phosphatidyl glycerol sulfate. Genomic, phenotypic, physiological, and biochemical analyses of the isolates revealed that they represent a novel genus and species in the family Halobacteriaceae. The type strain is CBA1133T (= KACC 22148T = JCM 34265T), for which the name Sala cibi gen. nov., sp. nov. is proposed.

Improved tolerance of recombinant Chlamydomonas rainhardtii with putative 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase from Pyropia yezoensis to nitrogen starvation: Seo-jeong Park , Joon Woo Ahn , Jong-il Choi; J. Microbiol. 2022;60(1):63-69. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1491-7

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Abstract: In a previous study, a putative 2-amino-3-carboxymuconate- 6-semialdehyde decarboxylase (ACMSD) was highly expressed in a mutant strain of Pyropia yezoensis, which exhibited an improved growth rate compared to its wild strain. To investigate the functional role of the putative ACMSD (Pyacmsd) of P. yezoensis, the putative Pyacmsd was cloned and expressed in Chlamydomonas reinhardtii. Recombinant C. reinhardtii cells with Pyacmsd (Cr_Pyacmsd) exhibited enhanced tolerance compared to control C. reinhardtii cells (Cr_control) under nitrogen starvation. Notably, Cr_Pyacmsd cells showed accumulation of lipids in nitrogen-enriched conditions. These
results
demonstrate the role of Pyacmsd in the generation of acetyl-coenzyme A. Thus, it can be used to enhance the production of biofuel using microalgae such as C. reinhardtii and increase the tolerance of other biological systems to nitrogendeficient conditions.

Characterization of staphylococcal endolysin LysSAP33 possessing untypical domain composition: Jun-Hyeok Yu , Do-Won Park , Jeong-A Lim , Jong-Hyun Park; J. Microbiol. 2021;59(9):840-847. Published online August 12, 2021; DOI: https://doi.org/10.1007/s12275-021-1242-1

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Abstract: Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for Lys- SAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.

Comparative analysis of type 2 diabetes-associated gut microbiota between Han and Mongolian people: Shu-chun Li , Yao Xiao , Ri-tu Wu , Dan Xie , Huan-hu Zhao , Gang-yi Shen , En-qi Wu; J. Microbiol. 2021;59(7):693-701. Published online May 15, 2021; DOI: https://doi.org/10.1007/s12275-021-0454-8

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Abstract: Due to the different rates of diabetes in different ethnic groups and the structural differences in intestinal microbiota, this study evaluated the changes in diabetes-related intestinal microbiota in two ethnic groups. Fifty-six stool samples were collected from subjects from the Han and Mongolian ethnic groups in China, including participants without diabetes (non-diabetic, ND) and with type 2 diabetes (T2D). The 16S rDNA gene V3 + V4 area was extracted from microbiota, amplified by PCR, and used to perform high-throughput sequencing and screen differential microbiota associated with ethnicity. The results showed that there were 44 T2D-related bacterial markers in the Han subjects, of which Flavonifractor, Alistipes, Prevotella, Oscillibacter, Clostridium XlVa, and Lachnospiracea_incertae_sedis were most closely related to diabetes. There were 20 T2D-related bacterial markers in the Mongolian subjects, of which Fastidiosipila and Barnesiella were most closely related to diabetes. The common markers of T2D bacteria in the two ethnic groups were Papillibacter and Bifidobacterium. There were 17 metabolic pathways with significant differences between the ND and T2D groups in the Han group, and 29 metabolic pathways in the Mongolian group. The glutamatergic metabolic pathway was the only common metabolic pathway in two ethnic groups. The composition and function of diabetes-related bacteria were significantly different among the different ethnic groups, which suggested that the influence of ethnic differences should be fully considered when studying the association between diabetes and bacteria. In addition, the common bacterial markers found in diabetic patients of different ethnic groups in this study can be used as potential targets to study the pathogenesis and treatment of diabetes.

Salicibibacter cibarius sp. nov. and Salicibibacter cibi sp. nov., two novel species of the family Bacillaceae isolated from kimchi: Young Joon Oh , Joon Yong Kim , Seul Ki Lim , Min-Sung Kwon , Hak-Jong Choi; J. Microbiol. 2021;59(5):460-466. Published online April 28, 2021; DOI: https://doi.org/10.1007/s12275-021-0513-1

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Abstract: To date, all species in the genus Salicibibacter have been isolated in Korean commercial kimchi. We aimed to describe the taxonomic characteristics of two strains, NKC5-3T and NKC21-4T, isolated from commercial kimchi collected from various regions in the Republic of Korea. Cells of these strains were rod-shaped, Gram-positive, aerobic, oxidase- and catalase- positive, non-motile, halophilic, and alkalitolerant. Both strains, unlike other species of the genus Salicibibacter, could not grow without NaCl. Strains NKC5-3T and NKC21-4T could tolerate up to 25.0% (w/v) NaCl (optimum 10%) and grow at pH 7.0–10.0 (optimum 8.5) and 8.0–9.0 (optimum 8.5), respectively; they showed 97.1% 16S rRNA gene sequence similarity to each other and were most closely related to S. kimchii NKC1-1T (97.0% and 96.8% similarity, respectively). The genome of strain NKC5-3T was nearly 4.6 Mb in size, with 4,456 protein-coding sequences (CDSs), whereas NKC21-4T genome was nearly 3.9 Mb in size, with 3,717 CDSs. OrthoANI values between the novel strains and S. kimchii NKC1-1T were far lower than the species demarcation threshold. NKC5-3T and NKC21-4T clustered together to form branches that were distinct from the other Salicibibacter species. The major fatty acids in these strains were anteiso-C15:0 and anteiso-C17:0, and the predominant menaquinone was menaquinone-7. The polar lipids of NKC5-3T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and five unidentified phospholipids (PL), and those of NKC21-4T included DPG, PG, seven unidentified PLs, and an unidentified lipid. Both isolates had DPG, which is the first case in the genus Salicibibacter. The genomic G + C content of strains NKC5-3T and NKC21-4T was 44.7 and 44.9 mol%, respectively. Based on phenotypic, genomic, phylogenetic, and chemotaxonomic analyses, strains NKC5-3T (= KACC 22040T = DSM 111417T) and NKC21-4T (= KACC 22041T = DSM 111418T) represent two novel species of the genus Salicibibacter, for which the names Salicibibacter cibarius sp. nov. and Salicibibacter cibi sp. nov. are proposed.

Reviews

Trans-acting regulators of ribonuclease activity: Jaejin Lee , Minho Lee , Kangseok Lee; J. Microbiol. 2021;59(4):341-359. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0650-6

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Abstract: RNA metabolism needs to be tightly regulated in response to changes in cellular physiology. Ribonucleases (RNases) play an essential role in almost all aspects of RNA metabolism, including processing, degradation, and recycling of RNA molecules. Thus, living systems have evolved to regulate RNase activity at multiple levels, including transcription, post-transcription, post-translation, and cellular localization. In addition, various trans-acting regulators of RNase activity have been discovered in recent years. This review focuses on the physiological roles and underlying mechanisms of trans-acting regulators of RNase activity.

Ammonia-oxidizing archaea in biological interactions: Jong-Geol Kim , Khaled S. Gazi , Samuel Imisi Awala , Man-Young Jung , Sung-Keun Rhee; J. Microbiol. 2021;59(3):298-310. Published online February 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1005-z

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Abstract: The third domain Archaea was known to thrive in extreme or anoxic environments based on cultivation studies. Recent metagenomics- based approaches revealed a widespread abundance of archaea, including ammonia-oxidizing archaea (AOA) of Thaumarchaeota in non-extreme and oxic environments. AOA alter nitrogen species availability by mediating the first step of chemolithoautotrophic nitrification, ammonia oxidation to nitrite, and are important primary producers in ecosystems, which affects the distribution and activity of other organisms in ecosystems. Thus, information on the interactions of AOA with other cohabiting organisms is a crucial element in understanding nitrogen and carbon cycles in ecosystems as well as the functioning of whole ecosystems. AOA are self-nourishing, and thus interactions of AOA with other organisms can often be indirect and broad. Besides, there are possibilities of specific and obligate interactions. Mechanisms of interaction are often not clearly identified but only inferred due to limited knowledge on the interaction factors analyzed by current technologies. Here, we overviewed different types of AOA interactions with other cohabiting organisms, which contribute to understanding AOA functions in ecosystems.

Journal Articles

Antimicrobial effect and proposed action mechanism of cordycepin against Escherichia coli and Bacillus subtilis: Qi Jiang , Zaixiang Lou , Hongxin Wang , Chen Chen; J. Microbiol. 2019;57(4):288-297. Published online March 30, 2019; DOI: https://doi.org/10.1007/s12275-019-8113-z

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Abstract: The detailed antibacterial mechanism of cordycepin efficacy against food-borne germs remains ambiguous. In this study, the antibacterial activity and action mechanism of cordycepin were assessed. The results showed that cordycepin effectively inhibited the growth of seven bacterial pathogens including both Gram-positive and Gram-negative bacterial pathogens; the minimum inhibitory concentrations (MIC) were 2.5 and 1.25 mg/ml against Escherichia coli and Bacillus subtilis, respectively. Scanning electron microscope and transmission electron microscope examination confirmed that cordycepin caused obvious damages in the cytoplasmatic membranes of both E. coli and B. subtilis. Outer membrane permeability assessment indicated the loss of barrier function and the leakage of cytoplasmic contents. Propidium iodide and carboxyfluorescein diacetate double staining approach coupled with flow cytometry analysis indicated that the integrity of cell membrane was severely damaged during a short time, while the intracellular enzyme system still remained active. This clearly suggested that membrane damage was one of the reasons for cordycepin efficacy against bacteria. Additionally, results from circular dichroism and fluorescence analysis indicated cordycepin could insert to genome DNA base and double strand, which disordered the structure of genomic DNA. Basis on these results, the mode of bactericidal action of cordycepin against E. coli and B. subtilis was found to be a dual mechanism, disrupting bacterial cell membranes and binding to bacterial genomic DNA to interfere in cellular functions, ultimately leading to cell death.

Metabolomic profiling reveals enrichment of cordycepin in senescence process of Cordyceps militaris fruit bodies: Junsang Oh , Deok-Hyo Yoon , Bhushan Shrestha , Hyung-Kyoon Choi , Gi-Ho Sung; J. Microbiol. 2019;57(1):54-63. Published online December 29, 2018; DOI: https://doi.org/10.1007/s12275-019-8486-z

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Abstract: Cordyceps militaris is a species of Cordyceps that is classified in the Cordycipitaceae family and is well known in East Asia as a traditional medicinal mushroom. Its artificial fruit body has been widely cultivated for commercial use in cosmetics, functional food, and medicine. To explore the metabolites associated with fruit body development, we conducted gas chromatography mass spectrometry (GC-MS) analyses based on developmental stage, which was divided into the growth period (stage 1, stage 2, and stage 3) and aging period (stage 4). We detected 39 biochemical metabolites associated with nucleotide, carbohydrate, and amino acid metabolism. Cordycepin, one of the representative bioactive compounds in C. militaris, was significantly enriched in stage 4 of aging period and is associated with glucose accumulation. The accumulation of cordycepin in stage 4 of aging period also seems to be related to the glutamine and glutamic acid pathway. Our
results
also showed enrichment of other bioactive compounds such as mannitol and xylitol in stage 4 of aging period. Our metabolomic profiling based on the developmental stages of C. militaris is useful for exploring bioactive compounds (e.g., cordycepin, mannitol, GABA, and xylitol) that are enriched in stage 4 of aging period and understanding the biosynthetic mechanisms associated with cordycepin production. Through optimization of fruit body cultivation by selecting stage 4 of aging period as a harvesting time, our findings can be utilized in food and medical applications of C. militaris in future.

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