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  • Review Article
  • OPEN ACCESS

Ophiocordyceps sinensis: A Potential Caterpillar Fungus for the Production of Bioactive Compounds

  • Paulchamy Chellapandi*  and
  • Shanmugam Saranya
 Author information  Cite
Exploratory Research and Hypothesis in Medicine   2024;9(3):236-249

doi: 10.14218/ERHM.2023.00040

Abstract

Ophiocordyceps sinensis is widely used in traditional Asian medicine and grows at high altitudes (3,000–4,000 meters) on the Qinghai-Tibet Plateau. This fungus is an expensive and rare species that is difficult to cultivate. Increasing global demand, limited commercial trade, and precious resources drive an urgent need for the development of artificial cultivation techniques to produce bioactive compounds. This paper reviews the genome biology, culture systems, solid-state and submerged fermentation processes used to produce bioactive compounds in O. sinensis. It also elucidates its biological properties at the genome level for the development of synthetic media. We performed a bibliometric analysis, retrieving information on various aspects of this fungus from NCBI PubMed. A total of 135 research articles on O. sinensis were collected, of which 104 focused on the production of bioactive compounds and 26 focused on ‘x-omics’ studies. Next-generation sequencing data provides a genetic basis for fungal biology and host specificity. Recent developmental transcriptomic studies described mechanisms underlying the transcriptional regulation of fruiting body development and cold adaptation. Metabolic data indicate that many bioactive compounds are produced by cultured mycelia or fruiting bodies. The biological properties of this fungus can be used to design and develop synthetic media for fruiting body development and enhance the production of bioactive compounds. Several bioactive compounds and their pharmacological properties have been studied in the mycelia and culture supernatants. Since cultured O. sinensis is an alternative to natural and cultured C. militaris strains, research on the design and formulation of solid media for the production of fruiting bodies and bioactive compounds is currently attracting attention.

Graphical Abstract

Keywords

Artificial cultivation, Genomics, Caterpillar fungus, Bioactive compounds, Systems biology, Medicinal mushroom

Introduction

The growth of the medicinal mushroom market is increasing globally owing to its use in therapeutic and cosmetic applications.1 The global medicinal mushroom market is expected to reach 7,246 kilotons by the end of the year 2023. The Asia-Pacific region will dominate the medicinal mushroom market and is estimated to reach 6,184 kilotons by 2023 at a compound annual growth rate of 13.5%. (https://www.grandviewresearch.com/industry-analysis/mushroom-market ). Advancements in cultivation and extraction techniques can create opportunities for the key players in the medicinal mushroom market.

Cordyceps is a genus of parasitic fungi that lives on certain caterpillars in mountainous regions of Indo-China. The global cordyceps market has experienced significant growth in the medicinal mushroom market. It offers industrial investment in R&D to generate massive revenue. The global cordyceps extract market was valued at US$ 473.4 million in 2018 at a compound annual growth rate of 10.4%. The Asia-Pacific region dominates the cordyceps market, with strong economic growth, significant investments, and commercialization of cordyceps-derived bioproducts. Cordyceps are confined to the Tibetan Plateau and India at 3,000 - 4,000 meters altitude.2,3 Consequently, limited natural resources, the price of wild cordyceps, and increased market demand have stimulated interest in the artificial cultivation of cordyceps and its fermented products.

Ophiocordyceps sinensis

Cordyceps (an ascomycete genus) is a caterpillar fungus that has been further separated into four genera: Cordyceps, Ophiocordyceps, Metacordyceps, and Elaphocordyceps. Ophiocordyceps sinensis is the best-known caterpillar fungus.4Cordyceps sinensis was transferred to O. sinensis based on a molecular phylogenetic study.5O. sinensis is a single, root-like structure consisting of dark brown fruiting spores and white tendrils known as mycelium. It infects a host larva (Lepidoptera: Hepialidae) and forms a worm-like sclerotium, from which the infected fungus forms one or more perithecial fruiting bodies or stromata. The stroma with the host larva-shaped sclerotium is called “Chinese Cordyceps”.6 20-hydroxyecdysone is an insect hormone that was recently found to be involved in hyphal formation in O. sinensis, and has provided insights into the regulation of dimorphism.7

O. sinensis is currently the world’s highest-priced biological commodity. This caterpillar fungus produces a variety of pharmacological properties, including anti-tumor, anti-aging, anti-fatigue, anti-inflammation, anti-atherosclerosis, and antioxidant activities. It is also used to treat male sexual disorders and to improve athletic performance. It is grown on grain-based substrates and can be cultured by submerged fermentation. Compared to its counterpart, C. militaris, efforts toward large-scale cultivation of O. sinensis for fruiting body production have met with less success.8,9 Therefore, the development of artificial culture techniques of O. sinensis has attracted worldwide attention in recent years.

In this study, using the NCBI PubMed database, we collected information from 135 research articles and manually refined the species-specific relevance. Only articles describing genomic, transcriptomic, metabolomic, and fermentation aspects of this fungus were included (Fig. 1). Ninety-one articles focused on the production of various bioactive compounds, particularly cordycepin, during submerged and solid-state fermentation processes. Twenty-six articles conveyed genome-related information. A bibliographic study indicated that most investigators have been interested in life cycle and host infection research. As such, the present review summarizes genome biology, artificial culture systems, and fermentation processes for bioactive compound production from O. sinensis. This review also highlights the biological characteristics of O. sinensis at the genomic scale for the development of artificial culture systems or synthetic media in the future.

Evolutionary trend in the number of publications covering <italic>O. sinensis</italic> in the NCBI-PubMed.
Fig. 1  Evolutionary trend in the number of publications covering O. sinensis in the NCBI-PubMed.

Artificial cultivation

Artificial cultures have become a favorable solution for the biotechnological production of O. sinensis for high cordycepin yield, but large-scale artificial cultivation is currently limited.3O. sinensis produces blastospores (budding yeast-like single cells) to facilitate dispersal in the fermentation broth.10 It grows as filamentous hyphae or even as fibers in a stroma structure in a solid medium. The gene-controlling mechanism for the formation of a single cell and filamentous hyphae is not yet known in artificial cultures.

Submerged cultivation

The cultivation of O. sinensis anamorph mycelia is a useful alternative for the large-scale production of fruiting bodies.11 Several artificial cultivation media and conditions have been optimized to enhance the yield of mycelial biomass, exopolysaccharides, and cordycepin in submerged cultures.12 It is reported to produce 20.9 g/L mycelial biomass, 4.1 g/L exopolysaccharides, and 18.2 mg/L cordycepin in submerged culture.13 Mycelial biomass increases (22 g/L) in submerged cultures under optimal culture conditions.14 Cha et al. optimized a culture medium containing 2% sucrose for increased production of mycelial biomass (54 g/L) and exopolysaccharides (28.4 g/L) in shaking-flask culture.15O. sinensis produced the maximum mycelial biomass (62.3 g/L) and exopolysaccharides (22 g/L) when the agitation speed was maintained at 350 rpm.15 Mycelial biomass and exopolysaccharide production increased with the addition of palmitic acid,16 Tween-80,17 and ammonium salts.18 Antioxidant and cholesterol esterase inhibitory responses of O. sinensis were improved by adding coconut water as a medium ingredient.19

Solid-state cultivation

The medicinal and health benefits of the fruiting bodies of O. sinensis have increased to meet the high demand for commercialization as a nutraceutical.20 However, its extreme specificity in the host range and confined geographic distribution hinder the natural formation of fruiting bodies. The fruiting bodies of C. militaris have been extensively cultivated in germinated cereal grains, soybean seeds, and silkworm pupae. The price of fruiting bodies cultured on pupae is almost 10 times higher than that cultured on cereal and soybean substrates. Comparatively, only a limited number of studies have reported the cultivation of fruiting bodies of O. sinensis under solid-state conditions. O. sinensis strains produce a large number of conidia on solid media by the freezing-shock method and peat soil medium.21,22 Silkworm pupae, rice grains, waste stale rice, wheat grains, and germinated soybeans have been intensively used as solid substrates for the artificial cultivation of mycelia and fruiting bodies.23,24 Germinated soybean medium has been developed to improve the production of polysaccharides with antioxidant and immunomodulatory effects in the fruiting bodies of O. sinensis.25 Low-value or waste stale rice grains are used as a substrate for the development of bioactive food materials from O. sinensis.23 Methanol extracts of O. sinensis mycelia cultured on rice contain linoleic acid, oleic acid, mannitol, tyrosine, alanine, and urea.26 A methanol-water (4:1 v/v) extract was obtained from the fruiting bodies of cultured O. sinensis on rice.27 This suggests the suitability of solid substrates for induced biosynthesis of bioactive compounds from fermented mycelia or fruiting bodies.

Production of bioactive compounds

A variety of bioactive compounds have been isolated from wild fungi, fermented mycelia, and culture supernatants and studied for different pharmacological activities in the last ten years (Table 1).28–81 The absorption, distribution, metabolism, and excretion properties of the bioactive compounds identified in O. sinensis mycelia and cultures are presented in Table 2. The chemical structures of some potential bioactive compounds identified from mycelia and artificial cultures are shown in Figure 2.

Table 1

Pharmacological property of bioactive compounds produced by O. sinensis mycelia and cultures

ClassCompoundSourcePharmacological activitiesReferences
PolysaccharidesExtracellularCulture supernatantImmunomodulatory and antitumor activitiesZhang et al.29; Cheung et al.30; Song et al.31; Qi et al.32
MyceliumAntioxidant activityLeung et al.33
Fermentation brothPrebiotic candidateOhkuma et al.34; Song et al.35; Mao et al.36; Ying et al.37
Antitumor activitySheng et al.38
Reducing insulin metabolismLi et al.39
Cell proliferationWang et al.40
Inhibitory effects on sphingomyelinaseWang et al.41
Fermentation brothAntiinflammatory activityLi et al.42
IntracellularMyceliumImmunostimulatory and antitumor activitiesYan et al.43
Antioxidant activityChen et al.44
Immunomodulatory effectChen et al.45
Hypoglycemic activityLi et al.46
Protection of chronic renal failureWang et al.47
Cholesterol esterase inhibitory activityKim48
Lower plasma triglyceride and cholesterolKiho et al.49
Fruiting bodyAntioxidant activityWang et al.50
CordycepinCulture supernatantSteroidogenesisPao et al.51
Antimetastatic activityKubo et al.52
Antitumor activityXu et al.53
Immunomodulatory effectZhou et al.54
MyceliumAntioxidant and anti-inflammation effectsLiu et al.55
Cardiac hypertrophyWang et al.56
PeptidesCordyminFermentation brothAntioxidant and anti-inflammationWang et al.41
against cerebral ischemia-reperfusion injuryWang et al.41
Beneficial effect on diabetic osteopeniaQi et al.57
MyriocinFermentation brothImmune inhibitorXiao et al.58
Serine proteaseCulture supernatantFibrinolytic activityLi et al.59
Cordyceamide A, BCulture supernatantCytotoxic and antitumor activitiesJia et al.60,61
TryptophanCulture supernatantSedative-hypnotic effectLiu et al.55
Cordycepic acidCulture supernatantInhibition of liver fibrosisOuyang et al.62
Cordycedipeptide AFermentation brothCytotoxic effectJia et al. 200560
CordysinocanAnticancer and antioxidant activityCheung et al.30
NucleosidesAdenosineMyceliumImmunomodulatory effectYang et al.63
GuanosineMyceliumImmunomodulatory effectYu et al.64
Inhibition of renal fibrosisDong et al.65
Secondary metabolitesLovastatinMyceliumHypolipidemic effectLi et al.66
γ-Aminobutyric acidMyceliumNeurotransmitterLi et al.66
ErgosterolMyceliumCytotoxic and antitumor activitiesKobori et al.67; Matsuda et al.68
MelaninFermentation brothAntioxidant activityDong and Yao69
CordysininMyceliumAnti-inflammatory; inhibits superoxide anion generation and elastase releaseYang et al.70
SaponinsAntitumor activityZhu et al.71
UnknownPhotoprotective effectCheng et al.72
Alleviation of diabetic nephropathy and podocyte injuryWang et al.73
5α,8α-epidioxy-22-E-ergosa-6,9,22-trien-3β-olAnticancer activityMatsuda et al.68
Ergosteryl-3-O-β-D-glucopyranosideAnti-inflammatory and antioxidant activityYang et al.70
5α,8α-epidioxy-24®-methylcholesta-6,22-dien-3β-DglucopyranosideAnticancer activityBok et al.74
CerevisterolAnti-asthemia activityLin et al.75
Ergosta-4,6,8(14),22-tetraen-3-oneAnticancer activityZhao et al.76
ergosta-5-8(14),22-trien-7-one, 3β-ol (H1-A)HepatoprotectionLin et al.77
β-SitosterolAnticancer activityMatsuda et al.68
StigmasterolAnti-inflammatory activityGabay et al.78
CampesterolHypocholesterolemic activityOstlund79
3′,4′,7-TrihydroxyisoflavoneAntioxidant activityYang et al.70
VerticillinAnticancer activityChen et al.80
Butylated hydroxytoulineAntioxidant activityBabu and Wu81
Table 2

ADME properties of bioactive compounds identified from O. sinensis mycelia and cultures

CompoundCompound IDLog Po/wSolubility (mol/L)Cytochrome P450 inhibitorLog Kp (skin permeation)Synthetic accessibility
Cordycepin6303−0.80.398no−8.27 cm/s3.67
Myriocin64383941.640.00119no−8.85 cm/s4.84
Cordyceamide A251792673.377.78e-09CYP2C19, CYP2C9, CYP2D6, CYP3A4−6.21 cm/s3.69
Cordyceamide B251792663.193.03e-08CYP2C19, CYP2C9, CYP2D6−6.55 cm/s3.73
Tryptophan63050.170.00175no−8.30 cm/s2.09
Cordycepic acid6251−2.21368no−9.61 cm/s3.3
Adenosine60961−1.612.56no−8.68 cm/s3.86
Guanosine135398635−2.023.21no−9.37 cm/s3.86
Lovastatin532323.890.00065CYP2C9, CYP3A4−5.74 cm/s5.76
γ-Aminobutyric acid119−0.720.923no−9.18 cm/s1
Ergosterol4446796.490.00000869CYP2C9−3.44 cm/s6.58
Melanin63256101.28.93e-08CYP1A2, CYP3A4−9.11 cm/s2
Ergosterol peroxide53515165.763.07e-05no−4.15 cm/s7.61
Ergosteryl-3-O-β-D-glucopyranoside441763974.80.00052no−5.56 cm/s7.94
Cerevisterol123027664.990.00013no−4.96 cm/s6.53
Ergosta-4,6,8(14),22-tetraen-3-one6441416−0.720.923no−9.18 cm/s1
β-Sitosterol2222847.190.000000649No−2.20 cm/s6.3
Stigmasterol52807946.960.00000339CYP2C9−2.74 cm/s6.21
Campesterol1731836.90.0000016No−2.50 cm/s6.17
Perlolyrine1601792.550.00000071CYP1A2, CYP2D6, CYP3A4−6.33 cm/s2.98
3′,4′,7-Trihydroxyisoflavone52846481.960.0000394CYP1A2, CYP3A4, CYP2D6−6.45 cm/s2.92

ADME, absorption, distribution, metabolism, and excretion; CYP, Cytochrome P450.

Chemical structures of some potential bioactive compounds identified from <italic>O. sinensis</italic> mycelia and cultures.
Fig. 2  Chemical structures of some potential bioactive compounds identified from O. sinensis mycelia and cultures.

Cordycepin

Cordycepin is an adenosine analog with potential antineoplastic, antioxidant, and anti-inflammatory activities.55 Cordycepin, however, is not the main bioactive component of O. sinensis and its content in the fruiting body is low. The mycelium of O. sinensis produces 0.075 mg/g cordycepin in potato dextrose agar medium and 0.021 mg/g in finger millet medium.82 Fruiting bodies of the naturally occurring O. sinensis produce 1.64 mg/g of cordycepin,83 which is similar to the amounts of cordycepin in cultured mycelia of C. militaris.84 Some studies have reported that cordycepin content is abundant in natural O. sinensis and low in cultured O. sinensis.85–87 Nevertheless, the type of extraction method used can determine the cordycepin content in the mycelia or fruiting bodies of O. sinensis.88 The challenge is to understand the key factors influencing the growth and cultivation of O. sinensis on solid substrates. It is imperative to develop a bioprocessing system to produce fruiting bodies or mycelia to obtain sufficient amounts of bioactive compounds on a large scale.89,90

Nucleotides

The nucleotide content of O. sinensis may vary depending on cultivation method and environmental factors. O. sinensis contains notable amounts of nucleotides, with adenosine being a particularly prominent nucleotide. It is known to contain relatively high levels of adenosine, a bioactive compound. Adenosine has been studied for its potential health-promoting properties, including its anti-inflammatory and immunomodulatory effects. The adenosine content in the natural environment is relatively low compared to that in cultured O. sinensis.55,91 Nucleotides in both wild and cultured O. sinensis enhance human immunity.63

Exopolysaccharides

Polysaccharides content plays an important role in medical applications and is recognized as a potential prebiotic candidate.92,93 Exopolysaccharides have numerous pharmacological properties, including immunomodulatory, antioxidant, and antitumor effects.94 Additionally, their content is a greater constituent of the culture supernatant than O. sinensis.45,95–98 High molecular weight exopolysaccharide fractions from the cultured C. sinensis strain HK1 had a significant protective effect on the viability of probiotic bacteria.92,99 These fractions may be beneficial for the formulation of synbiotic products containing probiotic bacteria.35C. sinensis polysaccharides (CPS-1, CPS-2, and CS-F10) are novel water-soluble polysaccharides purified from the mycelia of O. sinensis. CME-1, a novel, water-soluble, 27.6-kD polysaccharide, is also purified from O. sinensis mycelia. CPS-1 is a water-soluble polysaccharide that stimulates pancreatic insulin release and/or reduces insulin metabolism.100 CPS-2 activates platelet-derived growth factor/signal-regulated kinase and transforms the growth factor β1/Smad pathway to reduce platelet-derived growth factor BB-induced cell proliferation. Both CPS-1 and CPS-2 are abundant in the mycelia of O. sinensis.40 CME-1 is a water-soluble polysaccharide isolated from the mycelia. It inhibits sphingomyelinase activity and protects cells against oxidative stress.16,101 CS-F10 and Cordysinocan are typical polysaccharides extracted from cultured mycelia which can lower plasma glucose levels and decrease the protein content of the facilitative glucose transporter.102 Cordysinocan produced by O. sinensis induces cell proliferation, increases phagocytosis, and increases acid phosphatase activity.30

Peptides

Cytotoxic serine protease with fibrinolytic activity purified from O. sinensis its culture supernatant may be linked to its pharmacological use in cardiovascular diseases.59 Cordymin has a putative beneficial effect on diabetic osteopenia.57 Cordycedipeptide A and cordyceamides A and B, isolated from culture supernatants, have exhibited cytotoxic effects on several tumor cell lines.60,61 Tryptophan produced from cultured cells has a sedative-hypnotic effect, as it is the precursor of serotonin.103 Cordycepic acid ameliorates the lipopolysaccharide-induced inflammatory phenotype and transforming growth factor-β1-induced fibrogenic response to inhibit and resolve liver fibrosis.62 Cordycedipeptide A is a cyclodipeptide that exerts cytotoxic activity against L-929, A375, and HeLa cells isolated from the fermentation broth of O. sinensis.60 Cordymin peptides isolated from mycelia have been used to treat cerebral ischemia-reperfusion injury.41 Myriocin is an atypical amino acid extracted from the culture of O. sinensis that has immunosuppressant activity.58

Sterols

Ergosterol is a provitamin form of vitamin D2, and cultured mycelia of O. sinensis effectively alleviates liver fibrosis induced by carbon tetrachloride.104 Ergosterol may influence various signaling pathways involved in fibrosis, such as those related to transforming growth factor-beta, which is a central mediator of fibrotic processes. H1-A, a pure sterol compound isolated from cultured O. sinensis cells, is regularly used in traditional Chinese medicine. This compound also modulates the balance between cell proliferation and apoptosis.77,105 Additionally, the methanol extract of O. sinensis is known to inhibit different tumor cell lines.68,74

Secondary metabolites

Cordysinins (A-E) are ß-carboline compounds isolated from the mycelia of cultured O. sinensis. These compounds inhibit superoxide anion generation and elastase release.106 Lovastatin, γ-aminobutyric acid, and ergothioneine are secondary metabolites isolated from mycelia that have different hypolipidemia, hypotension, and antioxidant activities.28 The melanin pigment isolated from the fermentation broth of this culture has antioxidant activity.107 Saponin is a class of chemical compounds found in the mycelium that exhibits good antitumor activity.71 Biomass supplementation prepared from O. sinensis decreases blood lipid concentration, increases hepatoprotective activity, and normalizes testosterone levels.108 Mycelial extracts of strains Cs-HK1 and CS-4 exhibit cosmetic and skincare benefits due to their anti-collagenase activity and photoprotective effects.72 These extracts also alleviate diabetic nephropathy and podocyte injury.73 Some volatile components, including 2,5,6-trimethyldecane, 2,3-dimethylundecane, and 2,2,4,4-tetramethyloctane, have been identified in the artificial culture of O. sinensis.109 However, the pharmacological properties of several bioactive compounds extracted from the fermented broth, mycelium, or fruiting body of O. sinensis have yet to be studied.

Genome structure and organization

Current ‘x-omics’ data have provided essential knowledge of the genome biology and pathogenesis of O. sinensis (Table 3).6,94,115–117O. sinensis is a homothallic fungus, capable of selfing.110 The α-domain protein mating type1-1 and high-mobility group box protein mating type1-2 in a single genome and their expression in vegetative mycelia bestows its selfing process.111 Longer intergenic regions and numerous introns enlarge the mitochondrial genome size of O. sinensis.112,113 Methylation of its mitochondrial genome confers adaptation to the cold and low partial pressure of the oxygen environment at high altitudes.114 The genome of the O. sinensis strain Co18 (Accession: ANOV00000000) was sequenced with ∼240-fold coverage and 88.7% completeness using a Roche 454 GS FLX system.115

Table 3

Genome sequencing data of O. sinensis and C. militaris

Genome featuresO. sinensis strain
C. militaris CM01
Co181229ZJB12195IOZ07
Size (Mb)∼120∼139∼116.42∼12032.2
Sequence reads (Gb)1122.3–4.7
Coverage (fold)241x160x24x100X147 x
G+C content (%)46.144.7–45.151.4
Assembly size (Mb)87.7114116.4232.2
Predicted genes6,9729,6107,9398,9169,684
Exons per gene2.6–2.8––
Total sequence length78,515,811112,137,038101,068,960110,880,99232,268,578
Total ungapped length73,986,648111,803,71895,235,56232,212,078
Scaffolds10,6033,6876182332
Scaffold N5011,98670,939354,0454,551,492
Scaffold L501,829463813
Contigs25,8738,65721,76423597
Contig N505,39430,57011,49518,163,664108,187
Contig L503,6021,1032,380390
WGS or clone25,8733,68761823597
Genome accessionANOV00000000LKHE00000000LWBQ00000000JAAVMX000000000AEVU00000000
Assembly accessionGCA_000448365GCA_002077885GCA_001648815GCA_012934285GCA_000225605
Assembly methodNewbler v. 2.3ABySS ver. 1.2.3SOAPdenovo v. 2.0Canu v. 1.7Newbler v. 2.3
ReferencesHu et al.115Li et al.116Xia et al.6Shu et al.117Zheng et al.94

The genome of O. sinensis is approximately three times larger (∼120 Mb) than that of other entomopathogenic ascomycetes, due to the repeat-driven expansion of its genome. The massive proliferation of retrotransposable elements in gene-poor or gene-free regions and fragmented pseudogenes in this genome is an important context for genome size inflation.58 The protein-coding genes in its genome (6,972) are fewer than those in C. militaris (9,684).94 A complete genome assembly has been obtained for C. militaris without the requirement for multiple sequencing technologies.116 The genome of O. sinensis was also sequenced using a combination of Illumina HiSeqTM 2000 and Roche 454 sequencing technologies.6 The assembled genome size was 116.42 Mb (156 scaffolds) and covered ∼97% of the predicted genome size (∼120 Mb) with 7,939 predicted protein-coding genes. However, high-quality draft genome assembly, contiguity, gap-free sequences, and annotation of transposable elements and protein-coding genes for O. sinensis can be achieved using a combination of PacBio and Illumina reads.117

Gene expression during growth development

Genome and RNA sequencing (RNA-seq) data analyses have revealed the molecular mechanisms of fungal pathogenicity, specialized host infection, and cold tolerance.6 RNA-seq is the foremost approach for transcriptome profiling of many medicinal mushrooms to understand the molecular mechanisms underlying infection and fruiting body formation (Table 4).118–122 Comparatively, the number of expressed genes in the transcriptome of O. sinensis is greater than C. militaris, many of which are involved in sexual and fruiting body development.118 Transcriptome analysis has revealed that O. sinensis has typical mechanisms for biphasic pathogenesis and extreme cold adaptation with putative antifreeze proteins.115

Table 4

Summary of developmental transcriptomes (RNA Seq. data) and mapping of O. sinensis

Growth stageClean readsBase No. (G)GC%Mapped reads (% mapped)AccessionReferences
Fruiting body1,743,676––84.4SRX220584Xiang et al.118
Before infection of Thitarodes jiachaensis61,405,3966.1458.5785.06SRP068250Zhong et al.119
After infection of Thitarodes jiachaensis132,370,31813.2346.2912.84SRP068250
Asexual mycelium31,664,3143.9661.0477.52SRP103894Zhong et al.120
Sclerotium40,090,2425.0160.9283.56SRP103894
Fruiting bodies40,037,7605.060.679.71SRP103894
Asexual mycelium26,345,7987.9061.2390.40PRJNA382001Li et al.121
Sclerotium36,736,40611.0260.1291.55PRJNA382001
Primordium31,512,6209.4560.9289.07PRJNA382001
Young fruiting body27,822,2948.3560.4190.40PRJNA382001
Developed fruiting body32,871,7289.8661.1391.69PRJNA382001
Mature fruiting body34,059,00710.2260.9886.80PRJNA382001
Asexual mycelium36,788,1924.6360.0083.98SRR5282570Tong et al.122
Developing fruiting body38,627,9304.8760.7082.61SRR5282574
Mature fruiting body32,751,2204.5760.3585.12SRR5282577

RNA-seq, RNA sequencing.

RNA-seq data of O. sinensis before (anamorphic hyphae) and after infection (hyphal body) of Thitarodes jiachaensis larvae were obtained using the Illumina HiSeqTM 2000 technology.119 The transcriptome contained 1,640 differentially expressed genes, of which 818 were upregulated (49.9%) and 822 were downregulated (50.1%). Genes encoding transporter/permease, glycoside hydrolases, heat shock proteins, and dehydrogenases were upregulated in O. sinensis after infection. S-antigen protein, allergen, glucose-repressible protein, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase, and D-aminopeptidase were the most significantly downregulated genes during infection. The proteins responsible for binding with iron, heme, and tetrapyrrole were downregulated 2-fold compared to upregulated genes.119

RNA-seq analysis also revealed three stages (asexual mycelia and hyphae in deceased caterpillars and perithecial stroma) in the life cycle of O. sinensis.120 The O. sinensis transcriptome contained 3,049 differentially expressed genes in the teleomorph stage compared to the anamorph stage. Differentially expressed photosynthesis-related genes were enriched in the stroma groups, which may participate in light-regulated fruiting body development via the reactive oxygen species mediated pathway. O. sinensis has 18 differentially expressed genes, similar to the constituent proteins of photosystem I, which could respond to light stimuli. Therefore, light-dependent reactions in the stromal hyphae depend on the altered RNA levels in the transcriptome. Developmental transcriptome analysis described the induction of primordium, the sexual development of O. sinensis, and the molecular basis of its lifestyle.121

Comparative transcriptome analysis illustrated the different growth stages (asexual mycelium, developing fruiting bodies, and mature fruiting bodies) of O. sinensis cultured in an artificial medium.122 It describes the stage-specific splicing of genes, protein synthesis, and baseline metabolism that may have important functions during fruiting body development. The nucleoside diphosphate kinase, β-subunit of the fatty acid synthase, and superoxide dismutase are fruiting body development-associated genes that respond to ecological factors. Cytoskeleton genes play crucial roles in vegetative growth and fruiting body development. This study provides novel insights into the genetic basis of fruiting body development and host infection. Important proteins involved in the metabolic pathways of active ingredients were identified in different culture periods using comparative proteomics of O. sinensis.123–127 Recent investigations into gene expression-associated metabolic changes, particularly bioactive compound synthesis, developmental stages, and environmental adaptation in O. sinensis will be helpful for the exploration, application, and improvement of O. sinensis using metabolic engineering.

Design and development of artificial media

A comparative proteomic study identified 2,541 protein groups out of 22,829 peptides from the fruiting bodies of wild and cultured O. sinensis.128 Proteins involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation differ, but their nutritional value is virtually the same between natural and artificial cultivation. Therefore, the proteomic profile of O. sinensis provides useful virtual nutritional information for artificial cultivation. Natural and artificial cultivation of O. sinensis depends on 165 proteins involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. Lysine, threonine, serine, and arginine were significantly altered with changes in protein abundance. The levels of nucleosides, nucleotides, adenosine, and the composition of proteins and metabolites were the same in both natural and artificial cultivation. However, the mode of cultivation can affect amino acid synthesis and metabolic pathways. It also influences the synthesis of isopropylmalate dehydrogenase, pyruvate kinase, and nicotinamide adenine dinucleotide phosphate-binding proteins, which are involved in amino acid synthesis and metabolism.128 Peptide mass spectrometry has been employed to identify and authenticate wild O. sinensis and its related cultured Ophiocordyceps mycelia powder, as well as mixed commercial products.29 The higher content of bioactive compounds accumulated in O. sinensis facilitates its artificial cultivation.27 Metabolic profiling has shown that water-boiled extraction is a much faster method than ethanol-soaking.129

The complete genome of O. sinensis was sequenced using Illumina HiSeqTM 2000 technology and assembled into a genome with a size of 120 Mb.39,115,130 In addition, transcriptomic analysis of O. sinensis has provided novel insights into the genetic basis of fruiting body development and facilitated artificial cultivation.119–122 The growth of C. militaris and its cordycepin production is strongly dependent on the preferred carbon source and the upregulation of genes associated with cordycepin biosynthesis.131 It has a cooperative mechanism in transcriptional control of the precursor pool. This transcriptional control system regulates cordycepin biosynthesis via main and putative alternative metabolic routes.132 Hence, the design of the cultivation medium is crucial for both fungal growth and cordycepin production from suitable carbon sources. A high-quality genome-scale metabolic model of C. militaris, iNR1329, has been constructed to design an optimal cultivation medium.133 This metabolic model consists of 1,329 genes, 1,821 biochemical reactions, and 1,171 metabolites. This model was used as a platform for the rapid growth and overproduction of bioactive compounds using the rational design of synthetic media.133 The growth rate of C. militaris and cordycepin production was significantly increased by the optimized synthetic medium. Genome-scale metabolic models of O. sinensis strains have not yet been developed for rapid growth and overproduction of bioactive compounds. Hence, system-level modeling and simulation are promising approaches for the development of an efficient fungal cell factory based on the metabolic network of the O. sinensis genome.

Future direction

The growth and development of next-generation sequencing data provide a genetic basis for the life cycle and host specificity of O. sinensis. Currently, transcriptomic data provides a better understanding of fruiting body development and cold adaptation. Bioactive compounds are produced by cultured mycelia or fruiting bodies and can be illustrated using their metabolic data. The biological characteristics of such systems have been used to design synthetic media for fruiting body development, and to enhance the production of bioactive molecules. O. sinensis mycelia and its culture supernatants are sources for several bioactive compounds having important pharmacological properties. Hence, this review emphasizes the importance of ‘x-omics’ data in the design and formulation of artificial culture media for the production of fruiting bodies and bioactive molecules. Moreover, multi-omics approaches aid in identifying the key genes, proteins, and metabolic pathways involved in bioactive compound production, optimizing culture conditions, and enhancing fungal adaptability. Integration of multi-omics data provides a comprehensive understanding of fungal biology. Additionally, x-omics data guide strain improvement, quality control, and the study of host-pathogen interactions, offering valuable insights into the sustainable and efficient production of medicinal fungi and their bioactive compounds. Hence, multi-omics knowledge of O. sinensis can lead to improved cultivation techniques, enhanced yields of bioactive compounds, and the development of more sustainable and cost-effective production processes.

High amounts of arsenic are usually found in the natural fruiting bodies of O. sinensis. It is an environmental pollutant that decreases neuronal migration and maturation. Consequently, its manufacturing and sales were strictly regulated by the China Food and Drug Administration in 2016. The U.S. Food and Drug Administration and the European Union have similar requirements for strict limits of arsenic content. Because of the clear toxicity of arsenic, which is also related to its valence, dosage, and duration of administration, studies listing the results of long-term toxicity in rats do not necessarily indicate a lack of toxicological concern. Especially with regard to oral administration (1,000 mg/kg) of the fruiting body.134 Previous studies have indicated that conidial forms of artificially cultured O. sinensis fermentation mycelia can be used as substitutes for natural mycelia. Fermented O. sinensis mycelium powder and its standardized mycelial fermentation products have been produced and are widely employed in traditional Asian medicine. It facilitates human demand and the global economic growth of this fungus.

Conclusions

O. sinensis and its host insects are fascinating. There are still many unanswered questions concerning the molecular mechanisms of insect-fungus interactions and fruiting body development to resolve to improve yields during artificial cultivation. Current genomic and transcriptomic data pave the way for understanding the genetic basis underlying the fungal biology, host specificity, and genetic improvement of O. sinensis. Metabolomic studies facilitate the identification of key metabolites or bioactive compounds produced by cultured mycelia or fruiting bodies of O. sinensis in submerged or solid-state cultures. However, O. sinensis fruiting bodies can neither be cultured nor developed on a large scale. This can be resolved by designing and developing synthetic media based on the biological characteristics of O. sinensis. Several studies have optimized submerged cultivation media for the enhancement of bioactive compounds from mycelia or fermented broth. However, only a few studies have focused on the formulation of solid-state media to increase the fruiting bodies of O. sinensis compared to C. militaris strains. Current research has focused on the design and formulation of artificial cultivation media for the production of bioactive compounds from mycelia and fruiting bodies of O. sinensis cultured on solid-based substrates. Artificial cultivation therefore still presents as the best option for industrial production to alleviate the pressure of increasing global demand and to protect limited natural resources for sustainable utilization.

Declarations

Acknowledgement

There is nothing to declare.

Funding

The work was supported in part by a grant from the Science and Engineering Research Board, Department of Science and Technology (MSC/2020/000438), New Delhi, India.

Conflict of interest

The authors have no conflict of interests related to this publication.

Authors’ contributions

Study concept and design, critical revision of the manuscript (CP), data analysis, and interpretation of data (SS). All authors have made a significant contribution to this study and have approved the final manuscript.

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  • Exploratory Research and Hypothesis in Medicine
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  • eISSN 2472-0712

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Ophiocordyceps sinensis: A Potential Caterpillar Fungus for the Production of Bioactive Compounds

Paulchamy Chellapandi, Shanmugam Saranya
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