Microbial Steroids: Novel Frameworks and Bioactivity Profiles

Abstract

Microorganisms have emerged as prolific and versatile producers of steroidal natural products, displaying a remarkable capacity for structural diversification that extends far beyond classical sterol frameworks. This review critically examines steroidal metabolites isolated from microbial sources, with a particular emphasis on marine-derived and endophytic fungi belonging to the genera Aspergillus and Penicillium, alongside selected bacterial and lesser-studied fungal taxa. Comparative analysis reveals that these organisms repeatedly generate distinctive steroid scaffolds, including highly oxygenated ergostanes, secosteroids, rearranged polycyclic systems, and hybrid architectures arising from oxidative cleavage, cyclization, and Diels–Alder-type transformations. While many reported compounds exhibit cytotoxic, anti-inflammatory, antimicrobial, or enzyme-inhibitory activities, the biological relevance of these metabolites varies considerably, highlighting the need to distinguish broadly recurring bioactivities from isolated or strain-specific observations. By integrating structural classification with biosynthetic considerations and bioactivity trends, this review identifies key steroidal frameworks that recur across taxa and appear particularly promising for further pharmacological investigation. In addition, current gaps in mechanistic understanding and compound prioritization are discussed. Finally, emerging strategies such as genome mining, biosynthetic gene cluster analysis, co-culture approaches, and synthetic biology are highlighted as powerful tools to unlock the largely untapped potential of microbial genomes for the discovery of novel steroidal scaffolds. Together, this synthesis underscores the importance of microorganisms as a dynamic and expandable source of structurally unique and biologically relevant steroids, and provides a framework to guide future discovery-driven and mechanism-oriented research in the field.

1. Introduction

Endophytic fungi are microorganisms that inhabit plant tissues for all or part of their life cycle without causing apparent disease. This symbiotic association is often mutualistic, conferring a range of physiological and ecological benefits to the host plant. Endophytes are highly diverse and have been documented in nearly all plant species, colonizing roots, stems, leaves, flowers, and seeds [1,2,3].

The interaction between endophytic fungi and their hosts is particularly compelling due to the multifaceted advantages these microorganisms provide. In exchange for shelter and nutrients, endophytic fungi promote plant growth by enhancing biomass accumulation, height, root development, and the uptake of water and nutrients. These effects are mediated through mechanisms such as the production of phytohormones and the solubilization of essential nutrients, including phosphorus and nitrogen. Endophytes also improve plant tolerance to abiotic stresses—such as drought, salinity, and extreme temperatures—by regulating osmotic balance, modulating hormone levels, and synthesizing antioxidant compounds. Furthermore, several endophytic fungi function as biological control agents, protecting plants against pathogens, insects, and herbivores through the production of antimicrobial and insecticidal secondary metabolites [4,5,6,7,8,9].

Among endophytic fungi, species belonging to the genera Aspergillus [10,11,12] and Penicillium [13,14,15] are particularly noteworthy. These genera are ubiquitous in nature and are recognized for their remarkable capacity to biosynthesize structurally diverse and pharmacologically active secondary metabolites, including steroids, terpenoids, alkaloids, and polyketides. Many of these compounds have demonstrated significant biological activities, such as anti-inflammatory, antibacterial, cytotoxic, and antitumor effects [16,17,18,19,20,21,22].

This review summarizes recent advances in the discovery, structural diversity, and biological activity of bioactive and structurally unusual steroids produced by microbial sources.

2. The Genus Aspergillus

Aspergillus (Ascomycota) is a genus of filamentous fungi characterized predominantly by asexual reproduction. Species within this genus are cosmopolitan and ecologically versatile, occupying terrestrial, freshwater, and marine habitats. They play important roles in natural ecosystems as decomposers and symbionts and are also widely exploited in the food, pharmaceutical, and biotechnology industries. Owing to their capacity to produce a broad spectrum of extracellular enzymes and organic acids, Aspergillus species have long attracted scientific interest. In parallel, they are prolific producers of structurally diverse secondary metabolites, including polyketides, alkaloids, terpenoids, and steroids, many of which exhibit notable biological activities [23,24,25,26].

Despite their beneficial applications, several Aspergillus species are opportunistic or obligate pathogens of plants, animals, and humans. In humans, aspergillosis encompasses a range of diseases caused by members of this genus, with invasive aspergillosis posing a significant threat to immunocompromised patients. The increasing incidence of such infections has intensified both clinical and biochemical investigations of Aspergillus-derived metabolites, including steroids that may contribute to virulence, chemical defense, or host–pathogen interactions [26,27,28].

From a natural products perspective, Aspergillus represents one of the most productive microbial genera for the discovery of novel steroidal frameworks. Compared with other fungal genera, Aspergillus species recurrently yield ergostane-derived steroids, but frequently with unusual modifications, such as extensive oxidation, rearrangement of the core tetracyclic scaffold, ring cleavage (secosteroids), or fusion with heterocyclic or polyketide-derived moieties. These structural variations suggest a high degree of enzymatic plasticity in Aspergillus steroid biosynthesis, likely driven by tailoring enzymes encoded within strain-specific or conditionally expressed biosynthetic gene clusters.

Across the literature surveyed, certain structural motifs recur among Aspergillus-derived steroids, including highly oxygenated ergostanes, C-ring–rearranged skeletons, and hybrid steroid–polyketide architectures. These recurring frameworks contrast with more isolated or singular structures found in other microbial taxa and indicate that Aspergillus species may rely on a limited number of core biosynthetic pathways that are diversified through post-synthetic modifications. Notably, many of these steroids are isolated from endophytic or marine-derived strains, suggesting that ecological niche and host association play a role in shaping steroidal chemical diversity.

In terms of biological activity, steroids from Aspergillus display a broad but non-random distribution of bioactivities. Cytotoxic, anti-inflammatory, antimicrobial, and enzyme-inhibitory activities are most frequently reported, whereas activities such as antiviral or neuroprotective effects are comparatively rare. Importantly, several studies indicate that subtle structural changes—such as hydroxylation pattern, side-chain truncation, or ring rearrangement—can markedly influence bioactivity, implying emerging structure–activity relationships (SARs) within specific steroid subclasses. However, these relationships are often discussed only at the level of individual compounds, and broader cross-species or cross-class comparisons remain underdeveloped.

In the following subsections, steroids produced by Aspergillus species are discussed with an emphasis not only on structural novelty but also on recurring scaffolds, biosynthetic logic, and biological relevance. Where possible, comparisons are drawn between compounds isolated from different Aspergillus species and ecological sources to distinguish generalizable trends from strain-specific discoveries, thereby providing a more integrative view of steroid biosynthesis and function within this prolific fungal genus.

2.1. Steroid Production in Aspergillus aculeatus

Recent work by Yue and colleagues [29] reported the isolation of a series of steroidal metabolites from a single endophytic fungal strain, Aspergillus aculeatus, obtained from the internal tissues of a surface-sterilized leaf of Pseudostellaria heterophylla collected in Liaoning Province, China. P. heterophylla (Caryophyllaceae), commonly known as “hai er shen” or “tai zi shen,” is widely used in traditional Chinese medicine as a qi tonic and yin-nourishing herb [30,31]. The endophytic origin of this strain is noteworthy, as plant-associated Aspergillus species are increasingly recognized as prolific producers of structurally diversified steroids, potentially reflecting host-driven ecological or metabolic pressures.

Chemical investigation of an ethyl acetate extract obtained after solid-state fermentation of the fungus on rice medium for 15 days led to the isolation of six new steroids (compounds 1–5 and 7; Figure 1). These metabolites, designated camphoratins L–P, were structurally elucidated using comprehensive spectroscopic methods and comparison with reported data. Structurally, the camphoratins belong to highly oxygenated ergostane-derived frameworks, featuring unusual oxidation patterns and substitutions that distinguish them from canonical fungal sterols. Such modifications are consistent with extensive post-cyclization tailoring, including regioselective hydroxylation and peroxide or ether formation, which are hallmarks of Aspergillus steroid biosynthesis.

The biological evaluation of selected compounds focused on anti-inflammatory activity, assessed via inhibition of nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages. Compounds 1, 2, and 6 exhibited moderate inhibitory effects on NO release, whereas other congeners were inactive or weakly active [29,32]. Although the activity profile is modest, comparison among structurally related metabolites suggests that specific oxygenation patterns and functional group placement may influence anti-inflammatory effects, pointing to an emerging but still preliminary structure–activity relationship within this small compound set.

In addition to the new metabolites, several known steroids were identified from the same strain, including (22E,24S)-5α,8α-epidioxy-24-ethylcholesta-6,22-dien-3β-ol (6), previously reported from Lactarius volemus [33]; ergosta-7,22-dien-6β- methanoxy-3β,5α-diol (8), earlier isolated from Aspergillus awamori [34]; and cerevisterol (9), known from Penicillium brasilianum [35]. The co-occurrence of both novel and widely distributed fungal steroids highlights A. aculeatus as a biosynthetically versatile producer, capable of generating conserved sterol scaffolds alongside rare, strain-specific derivatives.

Further analysis of additional chromatographic fractions from this same A. aculeatus culture expanded the inferred steroidal repertoire. HSQC datasets were exported as CSV files and processed using the SMART system to predict likely structural types based on spectral matching. This computational approach suggested the presence of additional steroid-like metabolites (putatively compounds 10–24) corresponding to entries in existing spectral databases [29,32]. While these compounds were not isolated in pure form, the results underscore the latent chemical diversity of this strain and suggest that conventional isolation captured only a subset of its steroidal metabolome.

Taken together, this study illustrates several broader points relevant to Aspergillus-derived steroids. First, even a single endophytic strain can produce a cluster of structurally related yet chemically diversified steroids, supporting the concept of pathway branching and enzymatic promiscuity. Second, although the observed bioactivities are moderate, the recurrence of anti-inflammatory effects among oxygen-rich ergostane derivatives suggests that this scaffold class may represent a generalizable bioactivity trend within Aspergillus. Finally, the combination of classical isolation with computational spectral prediction highlights an effective strategy for revealing hidden metabolite space, foreshadowing the value of integrating metabolomics and genome-guided approaches in future studies.

2.2. Steroids from Other Aspergillus Species

Beyond A. aculeatus, a wide range of Aspergillus species have been shown to produce structurally diverse steroids, reflecting both conserved sterol biosynthetic pathways and extensive post-cyclization diversification. These metabolites can be broadly grouped into (i) polyoxygenated ergostane derivatives, (ii) highly rearranged or cyclized sterols, and (iii) hybrid or adduct-type steroids, often associated with ecological or experimental triggers such as co-culture or marine-derived habitats.

Penicysteroid C (25), a polyoxygenated steroid, was isolated from a co-culture of Aspergillus niger and Streptomyces pyomogenus AS63D under solid-state fermentation on rice medium. The production of this metabolite exclusively in coculture highlights the effectiveness of interspecies interactions in activating silent or weakly expressed biosynthetic pathways. Compound 25 exhibited antimicrobial and cytotoxic activities, reinforcing the relevance of cocultivation strategies for steroid discovery [36].

In contrast, structurally rearranged steroids frequently exhibit enhanced or more selective bioactivities. Aspersteroid A (26, Figure 2), together with related 18,22-cyclosterols (27 and 28), was isolated from A. ustus NRRL 275. Aspersteroid A features a highly unusual 6/6/6/5/5 fused-ring system resulting from A-ring cleavage, multiple 1,2-alkyl shifts, and intramolecular C-18/C-22 cyclization. This compound showed potent immunosuppressive and antimicrobial activities, underscoring the functional relevance of extensive skeletal rearrangement [37].

Hybrid steroidal frameworks, although less common, represent some of the most chemically striking metabolites within this genus. Tennessenoid A (29), produced by an endophytic Aspergillus strain inhabiting a red marine alga, is an unprecedented steroid–sorbicillinoid adduct linked via a C–C bond. Its broad-spectrum antifungal activity suggests that hybridization between polyketide and steroid pathways can generate compounds with enhanced ecological function [38].

Spectasterols A–E (30–34), isolated from A. spectabilis, further exemplify the diversity of rearranged sterols. These aromatic ergosterols display either contracted or expanded ring systems arising from alkyl migrations and D-ring modifications. Among them, compound 33 stood out due to its combined cytotoxic and anti-inflammatory activities, including COX-2 suppression and inhibition of NF-κB signaling, highlighting a scaffold with multifunctional bioactivity [39].

Ergosterdiacids A and B (35 and 36), isolated from a mangrove-derived Aspergillus species, are rare Diels–Alder adduct steroids featuring a pentacyclic 6/6/6/6/5 system. Both compounds inhibited Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) and showed strong anti-inflammatory activity, representing one of the more pharmacologically promising steroid classes identified within this section [40].

From A. nidulans, nidulanoids A (37) and B (38) represent the first natural examples of 30-norlanostane triterpenoids bearing a C-9 side chain at C-17 and a hemiacetal linkage between C-3 and C-19. These features suggest that they may serve as biosynthetic intermediates between lanostanes and conventional fungal steroids. Alongside these, compound 38, a rare pregnane aldehyde, exhibited moderate cytotoxicity (IC₅₀ = 7.34 μM), distinguishing it as a potential anticancer lead [41].

Several additional Aspergillus species yielded oxygenated ergostane-type sterols with varying degrees of biological activity. Ergosterimide B (39), isolated from A. tubingensis YP-2, showed weak cytotoxicity (IC₅₀ = 11.05 μM) [42].

Marine- and sponge-derived Aspergillus strains yielded structurally distinct sterols, including aspersterol A (40) from deep-sea A. unguis, aspersubrin A (41) from A. subramanianii, and ochrasterone (42) from A. ochraceus. These findings emphasize the influence of marine and symbiotic habitats on steroid diversification, although bioactivity data remain limited for several of these metabolites [43,44,45].

Aspersterol E (43), obtained from an endophytic Aspergillus associated with Hibiscus tiliaceus, displayed only modest activity against MFC mouse gastric cancer cells [42,46]. These findings suggest that simple oxygenation of the ergostane scaffold alone is often insufficient for potent bioactivity, unless combined with more extensive structural rearrangements. In addition, a newly isolated Aspergillus species from soil in Qalubiya Governorate, Egypt, displayed strong antimicrobial activity. Chemical analysis revealed the presence of a polyhydroxysterol (44), which demonstrated potent antimicrobial effects [47].

An endophytic fungal strain was isolated from the fruit of the mangrove tree Avicennia marina (see Figure 3), collected at Kilo 17, Safaga, Red Sea, Egypt. From this isolate, identified as Aspergillus versicolor, a new epoxy-ergostane sterol named versicolor (45) was obtained as a minor metabolite from fermented rice cultures. Versicolor exhibited inhibitory activity against the SARS-CoV-2 3CL protease (3CLpro), with an IC₅₀ value of 2.168 ± 0.09 μM, indicating its potential as a candidate inhibitor of SARS-CoV-2 3CLpro [48].

A second marine-derived fungus, Aspergillus sp. ZJUT223, was isolated from seawater collected in the Grant Trough near the Marceau Trough and identified through ITS sequencing. Extraction was performed using ethanol followed by purification with ethyl acetate. From this strain, a new steroid, ganodermanic acid (46), was isolated [49].

Similarly, aspersteroline A (47) and its analogue (48), obtained from A. versicolor QC812, represent additional examples of 18,22-cyclosterols. Both compounds exhibited moderate cytotoxicity toward HL-60 leukemia cells, suggesting that C-18/C-22 cyclization may represent a recurring bioactive motif within Aspergillus-derived steroids [50].

Emerging Bioactivities and General Trends

Among the metabolites described in this section, rearranged cyclosterols, Diels–Alder adducts, and hybrid steroids consistently show stronger or more diverse bioactivities than simple ergostane derivatives. Anti-inflammatory, cytotoxic, immunosuppressive, and enzyme-inhibitory activities recur across multiple species, suggesting that Aspergillus steroids may occupy a privileged chemical space for modulation of inflammation and cancer-related pathways. However, many activities remain isolated observations, underscoring the need for systematic comparative bioevaluation and biosynthetic studies to distinguish broadly applicable scaffolds from strain-specific anomalies.

Two oxygenated ergostane-type steroids—one new compound, 3β-hydroxy-5α,6β-methoxyergosta-7,22-dien-15-one (49), and one known analogue, ergosta-6,22-dien-3β,5α,8α-triol (50)—were isolated from crude extracts of a marine sponge-derived Aspergillus sp. Notably, compound 49 represents a marine ergostane-type steroid featuring two methoxy groups at C-5 and C-6 and demonstrated antibacterial activity against Staphylococcus aureus [51].

3. The Genus Penicillium

Penicillium is a globally distributed genus of blue–green molds belonging to the kingdom Fungi. Species within this genus reproduce predominantly asexually (anamorphic or deuteromycetous forms) and play major ecological roles as decomposers of organic matter in terrestrial and marine environments. While many species are responsible for destructive food spoilage and the production of mycotoxins, others are of substantial industrial importance as sources of enzymes, antibiotics, and other value-added metabolites. In addition, several Penicillium species are recognized as common indoor allergens. Although DNA sequencing has become essential for accurate species identification, the absence of a fully comprehensive and universally curated reference database continues to complicate Penicillium taxonomy and strain-level assignment [52,53,54].

The implementation of the “one fungus, one name” principle under the International Code of Nomenclature for algae, fungi, and plants has substantially reshaped the taxonomic framework of Penicillium. Species formerly classified under genera such as Chromocleista, Eladia, Eupenicillium, Torulomyces, and Thysanophora have been reassigned to Penicillium, resulting in a broader monophyletic genus. As a consequence of ongoing taxonomic revision and intensified exploration of under-investigated habitats, particularly marine and endophytic niches, the number of accepted Penicillium species has expanded to 354. This revision also includes new combinations involving Aspergillus crystallinus, A. malodoratus, and A. paradoxus, which are now placed within Penicillium section Paradoxa [55,56,57].

To enhance taxonomic reliability and reproducibility, updated species lists now incorporate MycoBank numbers, live ex-type cultures, and GenBank accession numbers for ITS, β-tubulin, calmodulin, and RPB2 gene sequences. These resources provide a standardized reference framework that is particularly important for correlating metabolite profiles with correctly identified fungal strains [58,59,60,61,62,63].

From a natural products perspective, Penicillium species represent one of the most prolific microbial sources of structurally novel steroids (see Figure 4), rivaling Aspergillus in both chemical diversity and biosynthetic creativity. However, in contrast to Aspergillus, which frequently yields highly oxygenated ergostanes and rearranged core skeletons, Penicillium-derived steroids often exhibit distinctive structural motifs, including unusual side-chain modifications, ring-contracted or ring-expanded frameworks, and hybrid architectures incorporating polyketide- or aromatic-derived elements. These differences suggest divergent evolutionary trajectories in steroid biosynthesis between the two genera, despite their close phylogenetic relationship.

When considered collectively, steroids from Penicillium can be broadly grouped into several recurring structural classes, such as modified ergosterol derivatives, secosteroids, and highly rearranged polycyclic frameworks. While many individual compounds have been reported only once from single strains, certain scaffolds recur across phylogenetically distant Penicillium species, indicating conserved biosynthetic logic. At the same time, strain-specific metabolites with unprecedented architectures highlight the influence of ecological context, cultivation conditions, and silent gene cluster activation on steroidal output.

In terms of biological activity, Penicillium-derived steroids display a wide spectrum of reported effects, including cytotoxic, anti-inflammatory, antimicrobial, enzyme-inhibitory, and immunomodulatory activities. Notably, cytotoxicity against cancer cell lines and inhibition of inflammatory mediators are among the most frequently observed activities, whereas activities such as antiviral or neuroprotective effects remain comparatively rare. In several cases, subtle structural variations—particularly in oxidation patterns and side-chain functionality—correlate with pronounced differences in bioactivity, suggesting emerging structure–activity relationships (SARs). However, as with Aspergillus, these SARs are typically discussed only within individual studies, and systematic cross-comparisons across compound families remain limited.

Compared with Aspergillus, Penicillium appears to produce a higher proportion of structurally singular or “one-off” steroids, many of which lack close analogues in other fungal genera. While this underscores the genus as a source of exceptional chemical novelty, it also complicates efforts to generalize biosynthetic pathways or predict biological function. Future studies integrating metabolomics, genomics, and targeted biosynthetic investigations will be essential to determine which steroidal frameworks represent isolated curiosities and which constitute broader, exploitable biosynthetic themes.

In the following subsections, Penicillium-derived steroids are discussed with attention to structural classification, biological relevance, and comparison with related fungal genera, aiming to move beyond compound enumeration toward a more integrative understanding of their biosynthetic origin and pharmacological potential.

Steroidal Metabolites from Penicillium

A new steroid, persteroid (51, see Figure 4), was isolated from the marine-derived Penicillium sp. ZYX-Z-143. Persteroid exhibited inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), with an IC₅₀ value of 46 μM, and strongly suppressed nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages, suggesting potential anti-inflammatory properties [64].

Another new steroid, penivariod A (52), was isolated from Penicillium variabile EN-394, an endophytic strain obtained from the marine red alga Rhodomela confervoides. Penivariod A demonstrated potent antimicrobial activity, particularly against Escherichia coli and Pseudomonas aeruginosa, with MIC values of 1.0 and 2.0 μg/mL, respectively [65].

A series of unusual C₂₅ steroids (53–66), characterized by a distinctive bicyclo[4.4.1] A/B ring system, were isolated from an antitumor mutant strain of Penicillium purpurogenum G59 AD-1-2. The isolated metabolites included antineocyclocitrinols A (53) and B (54), and 23-O-methylantineocyclocitrinol (55), all featuring a bicyclo[4.4.1] A/B framework with a Z-configured Δ^20,22 double bond. Additional C₂₅ steroids—neocyclocitrinols A (57), B (56), C (59), and D (58), threo-23-O-methylneocyclocitrinol (60), erythro-23-O-methyl-neo-cyclocitrinol (61), 24-epi-cyclocitrinol (62), cyclocitrinol (63), 20-O-methyl-24-epi-cyclocitrinol (64), 20-O-methylcyclocitrinol (65), and isocyclocitrinol B (66)—were also identified. All compounds displayed varying degrees of cytotoxicity against multiple human cancer cell lines, highlighting the pharmacological potential of this structurally unique class of C₂₅ steroids [66].

Two new C₂₃-steroid derivatives, cyclocitrinic acid A (67) and cyclocitrinic acid B (68), were isolated from the mangrove-derived fungus Penicillium sp. SCSIO 41429. Cyclocitrinic acid B demonstrated moderate pancreatic lipase inhibition, with an IC₅₀ value of 32 μM, and exhibited both pancreatic lipase inhibitory and antioxidant properties [67].

From the plant-associated fungus Penicillium fellutanum, an unusual clathrate-type meroterpenoid, isoatlantinone A (69), along with two new steroids, acrocalysterols E (70) and F (71), was isolated. Isoatlantinone A is notable for its highly oxygenated meroterpenoid structure featuring a unique caged bioxatetracyclo-[6.3.2.0^1,6.0^1,12]-tridecane ring system. All isolates were screened for antifungal and cytotoxic activities, among which compound 71 exhibited potent cytotoxicity toward HCC-1806 human breast cancer cells (IC₅₀ = 18.15 ± 1.05 μM). These findings highlight P. fellutanum as a promising source of structurally novel and bioactive metabolites [68].

Chemical investigation of Penicillium oxalicum 2021CDF-3, an endophytic fungus associated with marine red algae, led to the discovery of a new polyoxygenated ergostane steroid, peniciloxatone A (72). This compound showed cytotoxic activity against FADU and HepG2 cell lines, with IC₅₀ values of 9.5 and 18.1 μM, respectively [69].

A new steroid with strong antibacterial activity, rubensteroid A (73), along with its decarboxylated analogue, solitumergosterol A (74), was isolated from the Magellan Seamount-derived fungus Penicillium rubens AS-130. Rubensteroid A features a rare 6/6/6/6/5 pentacyclic ring system, proposed to originate from a [4 + 2] Diels–Alder cycloaddition between 14,15-didehydroergosterol (14-DHE) and maleic acid or maleimide, followed by decarboxylation. Compound 73 demonstrated potent antibacterial activity against Escherichia coli and Vibrio parahaemolyticus, both with MIC values of 0.5 μg/mL [70].

From the lichen-associated fungus Penicillium aurantiacobrunneum, two new sterols—(20S)-hydroxy-24(28)-dehydrocampesterol (75) and 7α-methoxy-8β-hydroxy- paxisterol (76)—were obtained. Sterol 75 showed cytotoxicity against the HPAC pancreatic adenocarcinoma epithelial cell line (IC₅₀ = 17.76 ± 5.35 μM) [71].

A new cytotoxic steroid, 16α-methylpregna-17α,19-dihydroxy-(9,11)-epoxy- 4-ene-3,18-dione-20-acetoxy (77), was isolated from Penicillium citrinum SCSIO 41017, associated with the sponge Callyspongia sp. This compound displayed moderate cytotoxicity against MCF-7 human breast cancer cells, with IC₅₀ values of 13.5–18.0 μM [72].

From mangrove sediments collected in the Dongzhaigang Mangrove Reserve (Hainan, China), strain ABC190807 of Penicillium brefeldianum was isolated. Its EtOAc extract exhibited potent larvicidal activity against Aedes aegypti third-instar larvae (LC₅₀ = 0.089 mg/mL). A novel purinyl steroid, ergosta-4,6,8(14),22-tetraen-3-(6-amino-9H-purin-9-yl) (78), was isolated from this extract [73].

Sterolic acid (79, see Figure 5), an unusual sterol, was isolated from a deep-sea sediment-derived Penicillium sp. This metabolite features a diepoxy moiety within its A-ring and an oxabicyclo[2.2.2]octane system—structural motifs previously known only from plant-derived natural products. Sterolic acid additionally contains a carboxylic acid group at C-27, further distinguishing it from typical fungal sterols [74].

Penicillitone (80), a sterol with a rare tetracyclic skeleton, was obtained from Penicillium purpurogenum. Penicillitone exhibited notable cytotoxicity toward multiple cancer cell lines, including A549 (IC₅₀ = 5.57 μM), HepG2 (IC₅₀ = 4.44 μM), and MCF-7 (IC₅₀ = 5.98 μM), with adriamycin serving as a positive control [75].

A novel inhibitor of anchorage-independent tumor cell growth was isolated from the culture broth of Penicillium aurantiogriseum TP-F0213. The compound, identified as 16-acetoxy-3,7,11-trihydroxyergost-22-en-6-one (81), known as anicequol, possesses an ergostane-type carbon skeleton with substituent configurations 3β, 5α, 7β, 11β, 16β, and 24S. Anicequol inhibited anchorage-independent proliferation of DLD-1 human colon cancer cells with an IC₅₀ of 1.2 μM while showing substantially lower activity against anchorage-dependent growth (IC₅₀ = 40 μM) [76].

A new polyoxygenated steroid, penicisteroid A (82), was isolated from the culture extract of Penicillium chrysogenum QEN-24S, an endophytic strain from an unidentified Laurencia species (see Figure 6) of marine red algae [77].

The steroid 8(14),22E-dien-3β,5α,6β,7α-tetraol (83) was isolated from a Penicillium sp. associated with South Pole sea moss. This compound showed anticancer activity toward HepG2 liver cancer cells, with an IC₅₀ of 10.4 μg/mL [78].

Two novel naturally occurring [4+2] Diels–Alder cycloaddition ergosteroids (84 and 85) were isolated from Penicillium herquei. These compounds represent the first known steroidal cycloadducts formed with 1,4,6-trimethyl-1,6-dihydropyridine-2,5-dione or 4,6-dimethyl-1,6-dihydropyridine-2,5-dione [79].

Several novel steroids—citristerones A (86), B (87), D (88), E (89), and a new series of 23,24-diol-containing ergosterols (90)—along with three known analogues, were isolated from Penicillium citrinum TJ507, an endophytic strain from Hypericum wilsonii. Citristerone B exhibited exceptional anti-neuroinflammatory activity (IC₅₀ = 0.60 μM) in LPS-stimulated BV-2 microglial cells. Further mechanistic studies revealed that citristerone B markedly reduced NO and cytokine release, inhibited TNF-α, iNOS, and NF-κB expression, and suppressed ROS accumulation [80].

Three andrastin-type meroterpenoids, hemiacetalmeroterpenoids A–C (91–93), were isolated from the mangrove-soil-derived Penicillium sp. N-5. Hemiacetalmeroterpenoid A (91) possesses a unique, highly congested 6,6,6,6,5,5 hexacyclic skeleton and exhibited strong antimicrobial activity against Penicillium italicum and Colletotrichum gloeosporioides (MIC = 6.25 μg/mL) [81].

Two novel nortriterpenoids (94 and 95) were isolated from the endophytic fungal strain Penicillium ochrochloron SWUKD4.1850, collected from healthy Kalmia angustifolia in Yunnan Province, China. Compound 95 represents the first naturally occurring 27-nor-3,4-secocycloartane shinortriterpenoid and exhibited moderate cytotoxicity toward HL-60, SMMC-7721, and MCF-7 cell lines (IC₅₀ = 6.5–17.8 μM) [82].

From Penicillium expansum WTJP1, isolated from Aconitum carmichaelii, a previously undescribed compound named expansinin (96) was discovered. Expansinin represents the first naturally occurring conjugate of an indole alkaloid and an ergosteroid, and was evaluated for cytotoxicity against five human cancer cell lines [83].

Scabrosteroid A (97), a novel steroidal heterodimer, was isolated from Penicillium scabrosum FXI744. This compound represents the first example of a naturally occurring pyrrolidinone–ergosterol hybrid, linked via a C-3/C-3′ bond. Scabrosteroid A inhibited NO production (IC₅₀ = 9.5 μM) and showed moderate immunosuppressive activity [84].

Penicildiones A (98) and B (99), two new steroids, were isolated from the soft-coral-derived fungus Penicillium sp. SCSIO 41201 cultured in 1% NaCl potato dextrose broth [85].

Finally, isopenicins A (100) and C (101)—novel meroterpenoids with unprecedented terpenoid–polyketide hybrid skeletons—were isolated from Penicillium sp. sh18. Compound 102 was identified as a potent inhibitor of the Wnt/β-catenin signaling pathway [86].

4. Steroids Produced by Miscellaneous Microorganisms

In addition to the extensively studied genera Aspergillus and Penicillium, a wide range of other fungi and microorganisms have been reported to produce structurally distinctive steroidal metabolites (Figure 7). These organisms are grouped here as “miscellaneous” not due to marginal importance, but because each is currently represented by a limited number of reports, often involving single strains or unique ecological niches (e.g., jellyfish-, sponge-, insect-, or plant-associated fungi). Collectively, however, these studies significantly expand the chemical and biosynthetic landscape of microbial steroids, frequently revealing architectures not yet observed in the dominant genera.

4.1. Secosteroids and Ring-Modified Steroids

Ring cleavage represents a recurring diversification strategy among microbial steroids. A structurally unique C₂₅ steroid, phomarol (103), isolated from Phoma sp. associated with the giant jellyfish Nemopilema nomurai, exhibits an unusual seven-membered A ring (1(10→19)-abeo), an aromatic B ring, and a cyclized side chain forming a fused pentacyclic framework. This level of rearrangement parallels the highly modified cyclosterols observed in Aspergillus, but differs in ring topology and aromatic incorporation [87].

Several fungi yielded 9,11-secosteroids, reinforcing this motif as a common route to structural novelty. Cyclosecosteroid A (104), from the mangrove endophyte Talaromyces sp., displayed moderate acetylcholinesterase inhibition [88]. Similarly, altersteroids A–D (105–108) from Alternaria sp. feature a γ-lactone fused to the D ring, with compound 107 demonstrating cytotoxicity and apoptosis induction, including activity against cisplatin-resistant tumor cells. These findings highlight secosteroids as functionally relevant scaffolds, particularly in anticancer contexts [89].

4.2. Ergostane and Pregnane Derivatives with Functional Diversification

Conventional ergostane frameworks remain prevalent among miscellaneous fungi but often incorporate unusual oxidation patterns or side-chain modifications. Acrocalysterols A and B (109–110) from Acrocalymma sp. displayed notable cytotoxicity, with compound 110 showing IC₅₀ values below 20 μM across multiple cancer cell lines [90]. Likewise, compound 111 from Periconia pseudobyssoides inhibited heme polymerization, indicating antimalarial potential [91].

Spartopregnenolone (112), isolated from Phaeosphaeria spartinae associated with a marine red alga, represents a biosynthetic bridge between triterpenes and steroids, echoing transitional metabolites such as nidulanoids from A. nidulans. Such compounds provide rare experimental insight into steroid biosynthetic evolution [92].

4.3. Rearranged Steroids and Novel Ring Systems

Rearrangement-driven structural innovation is particularly prominent in this group. Phomopsterones A (113) and B (114) from Phomopsis sp. exemplify this trend, with phomopsterone A containing an unprecedented bicyclo[3.3.1]nonane motif generated via B-ring scission and A-ring rotation. Phomopsterone B showed anti-inflammatory activity, consistent with trends observed for rearranged Aspergillus steroids [93].

Steresterones A and B (115–116) from Stereum hirsutum, along with compound 117 (IC₅₀ as low as 2.3 μM), further underscore the cytotoxic potential of rearranged fungal steroids, although most reports remain limited to in vitro assays [94]. Two unusual steroid-like metabolites, asterogynin A (118) and asterogynin B (119), were obtained from an endophytic fungus isolated from the small palm Asterogyne martiana [95].

4.4. Hybrid Steroids and Biosynthetic Cross-Talk

Hybridization between steroidal cores and other biosynthetic pathways emerges as a major theme. Trichosterol A (120), from Trichoderma koningiopsis, is the first reported steroid–alkaloid hybrid incorporating a rare 1,2-oxazine moiety and exhibited herbicidal activity, suggesting potential agricultural applications [96].

Marine-associated fungi continue to yield particularly complex steroidal architectures. Microascusteroids A and B (121–122) from Microascus sp. are rare 5,6- and 9,10-seco ergostanes with anti-inflammatory and enzyme-inhibitory activity [97]. Chaeglobol A (123), isolated from Chaetomium globosum, possesses an octacyclic framework generated via a proposed [4 + 2] cycloaddition, emphasizing the role of pericyclic reactions in microbial steroid biosynthesis [98].

Similarly, striasteroids A–C (124–126) from Striaticonidium cinctum represent polyketide–steroid hybrids, with striasteroid C being the first known adenine–steroid conjugate and showing potent neuraminidase inhibition. These hybrids parallel tennessenoid A from Aspergillus but expand the range of biochemical cross-talk observed in microbial steroid biosynthesis [99].

The sponge-derived fungus Gymnacella dankaliensis produced dankasterones A and B (127–128) only under modified culture conditions, reinforcing the importance of medium engineering in eliciting cryptic metabolites. Both compounds showed potent cytotoxicity, while the structurally related gymnasterone A (129) was inactive, illustrating sharp structure–activity contrasts [100].

Two undescribed steroids, bipolarsterols A (130) and B (131), were isolated from pathogenic fungus Bipolaris oryzae, along with nine known congeners. Bipolarsterol A represents the first example of a 19(10→5)-abeo-7(8→9)-abeo-ergostane featuring a spiro[4.5]decan-6-one system, while bipolarsterol B is a new member of the rare steroid–phenylpropanoid hybrid class [101].

Matsutakone (132), a novel sterol featuring an unprecedented polycyclic ring system, along with a new norsteroid, matsutoic acid (133), was isolated from the fruiting bodies of Tricholoma matsutake. Bioassay results demonstrated that both compounds exhibited inhibitory activity against acetylcholinesterase, with compound 132 displaying an IC₅₀ value of 20.9 μM [102].

4.5. Emerging Trends and Comparative Perspective

Across this section, several generalizable patterns emerge: (i) Secosteroids and rearranged frameworks are disproportionately associated with cytotoxic and anti-inflammatory activity; (ii) Hybrid steroids frequently exhibit enzyme inhibition or ecological bioactivity, suggesting adaptive advantages; (iii) Many findings remain strain-specific, with limited comparative bioevaluation, underscoring the need for standardized screening.

Compared to Aspergillus and Penicillium, these miscellaneous microorganisms contribute fewer compounds numerically but disproportionately expand structural novelty, particularly through ring cleavage, fusion, and hybridization. As genomic and cultivation strategies advance, these taxa are likely to become increasingly important contributors to microbial steroid discovery.

Three unusual C₃₀ ergosterols—cordycepsterols A–C (134–136)—were isolated from the medicinal fungus Cordyceps militaris. These sterols possess an uncommon 6/6/6/5/6 pentacyclic skeleton that is hypothesized to arise from a canonical ergosterol framework through extension by two additional skeletal carbons. All three metabolites showed potent inhibition of nitric oxide production, with IC₅₀ values of 3.0, 0.9, and 2.3 μM, respectively. At 5 μM, they significantly suppressed the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), underscoring their promising anti-inflammatory potential and highlighting C. militaris as a valuable source of bioactive sterols [103].

Mangrove endophytic fungi, as extremophilic microorganisms, are known to produce diverse and biologically active secondary metabolites. A strain of Dothiorella sp. ZJQQYZ-1, isolated from the mangrove plant Kandelia candel, yielded six metabolites, including three new benzofuran derivatives and three new steroids: phomosterol C (137), phomosterol B (138), and phomosterol A (139). Among these, phomosterol A exhibited significant anti-inflammatory activity with an IC₅₀ value of 4.6 μM. Mechanistic studies further revealed that compound 139 effectively suppressed the protein expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW264.7 macrophages [104].

5. Future Directions in Microbial Steroid Discovery

Despite the remarkable structural diversity and biological activities of microbial steroids reported to date, current knowledge likely represents only a small fraction of the biosynthetic potential encoded within microbial genomes. Rapid advances in genome sequencing technologies and bioinformatic tools have revealed that many fungi and bacteria harbor a large number of cryptic or silent biosynthetic gene clusters (BGCs) whose products remain unknown. Genome mining approaches, combined with comparative genomics and machine learning–assisted prediction of secondary metabolite pathways, are expected to play an increasingly central role in the discovery of novel steroidal frameworks.

In particular, the identification of genes encoding oxidosqualene cyclases, cytochrome P450 monooxygenases, and tailoring enzymes involved in steroid biosynthesis provides valuable entry points for predicting and prioritizing new steroid-producing strains. Systematic exploration of publicly available genomic databases, followed by targeted experimental validation, has already proven successful for the discovery of structurally unusual natural products and holds great promise for expanding the chemical space of microbial steroids.

Another important strategy involves activation of silent gene clusters through environmental and biological perturbations. Approaches such as the OSMAC (One Strain–Many Compounds) strategy, modulation of culture conditions, and co-culture experiments with competing or symbiotic microorganisms have been shown to induce the production of previously undetected metabolites, including highly modified steroids. These methods are particularly relevant for endophytic and marine-derived fungi, whose metabolic pathways are often regulated by complex ecological interactions.

Advances in synthetic biology and metabolic engineering further expand opportunities for microbial steroid research. Heterologous expression of complete or refactored biosynthetic gene clusters in genetically tractable hosts enables access to metabolites that are difficult to obtain from native producers. Moreover, pathway engineering and combinatorial biosynthesis allow for the generation of non-natural steroid analogues with improved or novel biological properties, offering new leads for drug discovery.

Finally, the integration of multi-omics approaches—including genomics, transcriptomics, metabolomics, and proteomics—will facilitate a deeper understanding of steroid biosynthesis, regulation, and ecological function. Coupled with advanced analytical techniques and high-throughput bioactivity screening, these strategies are expected to accelerate the discovery of new microbial steroids and enhance their translation into pharmaceutical, agricultural, and biotechnological applications.

6. Conclusions

This review demonstrates that fungal microorganisms—particularly endophytic, marine-derived, symbiotic, and extremophilic species—constitute an exceptionally prolific and still underexplored source of structurally diverse steroidal natural products. Across the literature surveyed (spanning approximately the last two decades), fungi belonging predominantly to the genera Aspergillus and Penicillium, as well as a wide array of taxonomically diverse microorganisms, have yielded an impressive range of steroidal frameworks. These include classical ergostane and lanostane derivatives, highly rearranged abeo- and nor-steroids, secosteroids, polycyclic and Diels–Alder-derived architectures, and rare hybrid structures incorporating polyketide, alkaloid, nucleobase, or phenylpropanoid motifs.

Importantly, this structural diversity is not merely decorative but frequently correlates with significant biological activity. Among the compounds reviewed, cytotoxic and antiproliferative effects against cancer cell lines are the most commonly reported bioactivities, followed by anti-inflammatory, antimicrobial, enzyme-inhibitory, and neuroactive properties. Certain scaffold classes—such as rearranged cyclosterols, secosteroids, and steroid–polyketide hybrids—recur across multiple fungal taxa and appear particularly enriched in bioactivity, suggesting that these frameworks may represent privileged structures for future pharmacological exploration. Conversely, many reported activities remain limited to single assays or isolated studies, underscoring the need for deeper mechanistic investigation and broader biological validation.

From a biosynthetic perspective, fungal steroids provide compelling evidence of extraordinary enzymatic plasticity. Unusual oxidative cleavages, ring expansions and contractions, skeletal rearrangements, and intermolecular cycloaddition reactions highlight the sophistication of fungal steroid biosynthetic pathways. The frequent discovery of structurally related metabolites within single strains further suggests the operation of modular or branching biosynthetic routes, which may be amenable to manipulation through genetic or environmental perturbation.

Looking forward, the future discovery of novel microbial steroids will increasingly rely on integrative strategies that combine genome mining, biosynthetic gene cluster analysis, epigenetic modulation, coculture approaches, and synthetic biology. The rapid expansion of publicly available fungal genomic data, coupled with improved bioinformatic tools, offers unprecedented opportunities to identify cryptic steroid biosynthetic pathways and to link gene clusters with specific structural motifs. In parallel, advances in metabolomics, NMR data mining, and MS-based molecular networking will further enhance the efficiency of dereplication and structural discovery.

In summary, fungal-derived steroids represent a chemically rich and biologically versatile class of natural products with substantial potential for pharmaceutical, agricultural, and biotechnological applications. By moving beyond compound cataloguing toward comparative analysis, biosynthetic understanding, and functional prioritization, future research can more effectively translate this remarkable chemical diversity into tangible therapeutic and industrial outcomes.