Fungal Co-culture Research Articles

Exploring the Synergistic Secretome: Insights from Co-Cultivation of Aspergillus brasiliensis and Trichoderma reesei RUT-C30.

The spectrum of enzymes required for complete lignocellulosic waste hydrolysis is too diverse to be secreted by a single organism. An alternative is to employ fungal co-cultures to obtain more diverse and complete enzymatic cocktails without the need to mix enzymes during downstream processing. This study evaluated the co-cultivation of Aspergillus brasiliensis and Trichoderma reesei RUT-C30 in different conditions using sugarcane bagasse as the carbon source. The resulting enzymatic cocktails were characterized according to the impact of strain inoculation time on enzymatic activities and proteome composition. Data revealed that the profile of each enzymatic extract was highly dependent on the order in which the participating fungi were inoculated. Some of the co-cultures exhibited higher enzyme activities compared to their respective monocultures for enzymes such as CMCase, pectinase, β-glucosidase, and β-xylosidase. Analysis of the T. reesei RUT-C30 and A. brasiliensis co-culture secretome resulted in the identification of 167 proteins, with 78 from T. reesei and 89 from A. brasiliensis. In agreement with the enzymatic results, proteome analysis also revealed that the timing of inoculation greatly influences the overall secretome, with a predominance of T. reesei RUT-C30 proteins when first inoculated or in simultaneous inoculation.

Fungal Coculture of Herpotrichia sp. and Trametes versicolor Induces Production of Diverse Metabolites with Anti-Parkinson's Neuroprotective Activity.

Co-cultivation of isopod-associated fungi Herpotrichia sp. SF09 and Trametes versicolor SF09A led to the reciprocal induction of thirteen new compounds (1-7 and 9-13) with diverse architectures. Importantly, compounds 1 and 2 are rare fungal sesquiterpene-saccharide hybrids incorporating a xylopyranose moiety, compound (±)-3 represents the first example of a natural linear sesquiterpene racemate, and compound 7 is a rare α-pyrone derivative with a xylopyranose motif. Their structures were elucidated by analysis of NMR and mass spectrometry data, and their absolute configurations were determined by Mosher's method, microscale derivatization, and single-crystal X-ray diffraction, as well as ECD calculations. All the isolated compounds ameliorated MPP+-induced oxidative damage in PC12 cells in a dose-dependent fashion. Among them, compounds 5 and 15 showed significant protective action against neuronal injury by MPP+ at 5 μM. Meanwhile, transcriptome sequencing was performed to evaluate the molecular mechanism of the neuroprotective activity for compound 5. Results indicated that compound 5 might mitigate MPP+-induced neuronal injury through the regulation of multiple signaling pathways, including the PI3K-Akt and MAPK pathways. Our findings suggested that compound 5 could be a promising neuroprotective agent for the treatment of Parkinson's disease.

Evaluation of co-culture of cellulolytic fungi for enhanced cellulase and xylanase activity and saccharification of untreated lignocellulosic material.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

Enhancing quality of ruminant feed through fungal treatment: Usage of bamboo shoot residues.

In this investigation, we explore the harnessing of bamboo shoot residues (BSR) as a viable source for ruminant feed through fungal treatment, with the overarching objective of elevating feed quality and optimizing bamboo shoot utilization. The white-rot fungi (Wr.fungi), Aspergillus niger (A.niger), and its co-cultures (A.niger&Wr.fungi) were employed to ferment BSR. And the impact of different fermentation methods and culture time on the chemical composition (Crude protein Ash, neutral detergent fibre and acid detergent fibers), enzyme activity (Cellulase, Laccase, Filter paperase and Lignin peroxidase activities), and rumen digestibility in vitro were assessed. The findings reveal a nota ble 30.39% increase in crude protein in fermented BSR, accompanied by respective decreases of 13.02% and 17.31% in acid detergent fiber and neutral detergent fibre content. Enzyme activities experienced augmentation post-fermentation with A.niger&Wr.fungi. Specifically, the peak Cellulase, Laccase, and Lignin peroxidase activities for BSR with Wr.fungi treatment reached 748.4 U/g, 156.92 U/g, and 291.61 U/g, respectively, on the sixth day of fermentation. Concurrently, NH3-N concentration exhibited an upward trend with prolonged fermentation time. Total volatile fatty acids registered a decline, and the Acetate/Propionate ratio reached its nadir after 6 days of fermentation under the A.niger&Wr.fungi treatment. These outcomes furnish a theoretical foundation for the development of ruminant feeds treated via fungal co-culture.

Fungal co-culture enabled co-fermentation of food waste for production of endoglucanase enzyme

The primary scenarios of biochemical industries include diverse applications with high demand and the costly manufacturing of microbial cellulolytic enzymes. Using these enzymes to turn cellulosic waste into fuel is one of the prime applications of these enzymes to produce green fuel. Nevertheless, low production and weak functional abilities make these enzymes expensive and technically non-viable. This study presents multifactor bioprocess approaches to enhance cellulolytic enzyme production and efficiency using a low-cost strategy. Co-fermentation of pea pods (PPs) and potato peel (PP) wastes as food wastes has been conducted for solid-state fermentation (SSF) using fungal strain-based co-culture. To set the SSF, the nutrient medium and moisture balance of SSF have been maintained using an extract of PPs and PP waste, which are found to be rich in sugar and starch composition. Starch may function as a natural inducer in the SSF process to alleviate enzyme production in the presence of sugar composition. At 37 oC and pH 5.0, a maximum of 2.4 U/mL FPA (filter paper activity) has been produced at a substrate ratio of 4:04 (PPs:PP) using 50% of the moisture content on day 5 of the harvesting period. Additionally, 24.7 IU/mL of CMCase (carboxymethyl cellulase) in day 8 and 18.8 IU/mL of BGL (β-Glucosidase) in day 7 have been obtained. A temperature of 37 oC was found to be optimal, while a pH range of 5.0–9.0 was noticed to be optimal for maximum CMCase activity. Thus, the study may provide a sustainable approach to valorizing domestic solid food waste for the economic production of hydrolytic enzymes.

Metabolomics-based analysis of the interaction effects and stress response mechanisms in the co-culture system of fungi and microalgae under cadmium stress

Research on the co-culture of fungi and microalgae to harvest microalgae and enhance the adsorption capacity for heavy metals to reduce contamination is well demonstrated, but the study of the correlation mechanism among critical metabolites, system stability, and stress response in the system under cadmium stress based on metabolomics analyses has not been reported in detail. In this study, the co-culture system of Aspergillus allahabadii and Synechocystis sp. PCC6803 was taken as the object of investigation, and isotope labelling (13C–Na2CO3 and 15N-(NH4)2SO4) and nanoscale secondary ion mass spectrometry were used to confirm the existence of carbon and nitrogen nutrient exchange. The adsorption behavior of the system after Cd treatment was investigated by Fourier Transform Infrared Spectroscopy, X-ray Photoelectron Spectroscopy, and Scanning Electron Microscope/Transmission Electron Microscope. The content of extracellular polysaccharides in the system was increased from 40.8 mg/g to 80.12 mg/g, and the content of extracellular proteins was increased from 22.3 mg/g to 55.68 mg/g after 96 h of Cd treatment. Metabolomics results revealed 235 differential metabolites co-expressed after Cd treatment, with 95, 15, and 77 unique differential metabolites. In particular, the synthesis of extracellular polymeric substances was a factor affecting the stability of the symbiotic system, the up-regulation of the expression of the transporter system was one of the reasons for the improvement of the system's tolerance and resistance to heavy metals; and the nutrient metabolism between the fungus and the microalgae was the main defense mechanism to reduce the extent of the impairment of the system's carbon sequestration capacity by Cd. This provides a theoretical basis for the green and economically friendly application of the symbiotic system in microalgae harvesting, efficient carbon dioxide fixation and reduction of heavy metal pollution.

Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

The Impact of the Culture Regime on the Metabolome and Anti-Phytopathogenic Activity of Marine Fungal Co-Cultures.

Co-cultivation, coupled with the OSMAC approach, is considered an efficient method for expanding microbial chemical diversity through the activation of cryptic biosynthetic gene clusters (BGCs). As part of our project aiming to discover new fungal metabolites for crop protection, we previously reported five polyketides, the macrolides dendrodolides E (1) and N (2), the azaphilones spiciferinone (3) and 8α-hydroxy-spiciferinone (4), and the bis-naphtho-γ-pyrone cephalochromin (5) from the solid Potato Dextrose Agar (PDA) co-culture of two marine sediment-derived fungi, Plenodomus influorescens and Pyrenochaeta nobilis. However, some of the purified metabolites could not be tested due to their minute quantities. Here we cultivated these fungi (both axenic and co-cultures) in liquid regime using three different media, Potato Dextrose Broth (PDB), Sabouraud Dextrose Broth (SDB), and Czapek-Dox Broth (CDB), with or without shaking. The aim was to determine the most ideal co-cultivation conditions to enhance the titers of the previously isolated compounds and to produce extracts with stronger anti-phytopathogenic activity as a basis for future upscaled fermentation. Comparative metabolomics by UPLC-MS/MS-based molecular networking and manual dereplication was employed for chemical profiling and compound annotations. Liquid co-cultivation in PDB under shaking led to the strongest activity against the phytopathogen Phytophthora infestans. Except for compound 1, all target compounds were detected in the co-culture in PDB. Compounds 2 and 5 were produced in lower titers, whereas the azaphilones (3 and 4) were overexpressed in PDB compared to PDA. Notably, liquid PDB co-cultures contained meroterpenoids and depside clusters that were absent in the solid PDA co-cultures. This study demonstrates the importance of culture regime in BGC regulation and chemical diversity of fungal strains in co-culture studies.

Novel Fungal Co-Culture Technique for Enhanced Bioconversion of Agro-Industrial Waste to Amylase

Global strategies for management of solid waste occasionally lead to the environmental pollution. Now a days environmental friendly methods to valorize this waste are more demand to reduce the global warming. Solid-state fermentation (SSF) which is a potential waste recycling method to convert these solid wastes into value-added products by microbial population. In the present study, SSF was carried out using four substrates namely castor husk, rice husk, groundnut fodder, sugarcane bagasse and saw dust for the selection of renewable and chief substrate for the industrial production of amylase enzyme. We used two indigenous strains i.e., Aspergillus protuberus and Aspergillus unguis. Maximum production of α-amylase 1.614 U/g of substrate and 0.958 U/g of substrate on 2nd day of incubation in rice husk respectively. Groundnut waste (0.847 U/g of substrate) and castor husk (0.692 U/g of substrate) were also showed highest production of glucoamylase on 1st day and 2nd day of incubation in SSF. Further, prominent increase in the production of α-amylase (12 U/ml) and glucoamylase (3 U/ml) and extra cellular secretion of protein (20 mg/ml) was noticed in co-culture system on 2nd day of incubation in submerged fermentation (SmF).

Degradation of lignocellulose by different bacterial and fungal co-cultures

Long seen as non-valorisable waste, agricultural co-products are increasingly used in biorefinery processes. Co-culture appears as new trend for to improve the degradation of lignocellulose and improve the production of bioproducts. The goal of the study was to setup inter-domain co-cultures with high capabilities of lignocellulose degradation using a pluridisciplinary approach combining bioinformatics, enzymology, transcriptomics. Different individual lignocellulolytic strains: Trichoderma reesei QM6a and three bacteria (Streptomyces coelicolor A3(2), Rhizobium sp.XylPr11 and Sphingobacterium prati AraPr2 affiliated from different phyla) were used in that study . Synergic activities have been observed and quantified in co-culture conditions, particularly for xylanases and peroxidases activities. The enzymatic activities for the co-cultures in the most interesting co-culture (T. reesei QM6a/S. coelicolor A3(2)) reached more up to 2 IU/mL and 430 IU/mL respectively for the xylanase and peroxidase. Furthermore, ATR-FTIR analysis showed a real impact of co-culture condition on the substrate compared to the monoculture specially for hemicellulose degradation. Transcriptomics of S. coelicolor A3(2) either in mono or co-culture showed a relative similar pattern profile whatever the condition analysed with a specific overexpression of certain CAZyme genes involved in glycolysis due to the hydrolytic role played by the fungal partner. This work provided the proof of concept for technological feasibility, pertinence and usefulness of interdomain co-culture.

Key metabolites and regulatory network mechanisms in co-culture of fungi and microalgae based on metabolomics analysis

Key metabolites and regulatory network mechanisms in co-culture of fungi and microalgae based on metabolomics analysis

Comparative bioactivity and metabolites produced by fungal co-culture system against myco-phytopathogens

Comparative bioactivity and metabolites produced by fungal co-culture system against myco-phytopathogens

Co-culture of Acinetobacter sp. and Scedosporium sp. immobilized beads for optimized biosurfactant production and degradation of crude oil

The widespread exploration and exploitation of crude oil has increased the prevalence of petroleum hydrocarbon pollution in the marine and coastal environment. Bioremediation of petroleum hydrocarbons using cell immobilization techniques is gaining increasing attention. In this study, the crude oil degradation performance of bacterial and fungal co-culture was optimized by entrapping both cells in sodium-alginate and polyvinyl alcohol composite beads. Results indicate that fungal cells remained active after entrapment and throughout the experiment, while bacterial cells were non-viable at the end of the experimental period in treatments with the bacterial-fungal ratio of 1:2. A remarkable decrease in surface tension from 72 mN/m to 36.51 mN/m was achieved in treatments with the bacterial-fungal ratio of 3:1. This resulted in a significant (P < 0.05) total petroleum hydrocarbon (TPH) removal rate of 89.4%, and the highest degradation of n-alkanes fractions (from 2129.01 mg/L to 118.53 mg/L), compared to the other treatments. Whereas PAHs removal was highest in treatments with the most fungal abundance (from 980.96 μg/L to 177.3 μg/L). Furthermore, enzymes analysis test revealed that catalase had the most effect on microbial degradation of the target substrate, while protease had no significant impact on the degradation process. High expression of almA and PAH-RHDa genes was achieved in the co-culture treatments, which correlated significantly (P < 0.05) with n-alkanes and PAHs removal, respectively. These results indicate that the application of immobilized bacterial and fungal cells in defined co-culture systems is an effective strategy for enhanced biodegradation of petroleum hydrocarbons in aqueous systems.

Enhanced biovalorization of palm biomass wastes as biodiesel feedstocks through integrated solid-state and submerged fermentations by fungal co-cultures

Palm empty fruit bunches (EFB) were valorized into fungal lipids by oleaginous fungus Aspergillus tubingensis TSIP9 under solid-state fermentation (SSF) and submerged fermentation (SmF). An integrated SSF-SmF process increased lipid production from 116.2 ± 0.1 mg/g-EFB under SSF and 60.1 ± 0.2 under SmF up to 124.9 ± 0.5 mg/g-EFB, possibly due to the combined benefits of dispersed mycelia forming during SSF and better mass transfer during SmF. As A. tubingensis lacks sufficient β-glucosidase, it was co-cultured with high β-glucosidase-producing Trichoderma reesei QM 9414. The co-cultures improved overall lipid yields likely due to synergistic interaction of the two fungi. After inoculum size was optimized and the co-cultures were performed in bioreactors, the lipid yield was increased up to 205.1 ± 1.1 mg/g-EFB. The fatty acid composition of fungal lipids indicated their potential use as biodiesel feedstocks. The fungal fermentation of EFB also provided cellulose pulp residues. These strategies could be practical options for low-cost biovalorization of biomass wastes.

The Potential Use of Fungal Co-Culture Strategy for Discovery of New Secondary Metabolites

Fungi are an important and prolific source of secondary metabolites (SMs) with diverse chemical structures and a wide array of biological properties. In the past two decades, however, the number of new fungal SMs by traditional monoculture method had been greatly decreasing. Fortunately, a growing number of studies have shown that co-culture strategy is an effective approach to awakening silent SM biosynthetic gene clusters (BGCs) in fungal strains to produce cryptic SMs. To enrich our knowledge of this approach and better exploit fungal biosynthetic potential for new drug discovery, this review comprehensively summarizes all fungal co-culture methods and their derived new SMs as well as bioactivities on the basis of an extensive literature search and data analysis. Future perspective on fungal co-culture study, as well as its interaction mechanism, is supplied.

Fungal co-culture improves the biodegradation of hydrophobic VOCs gas mixtures in conventional biofilters and biotrickling filters

The present study systematically evaluated the potential of Candida subhashii, Fusarium solani and their consortium for the abatement of n-hexane, trichloroethylene (TCE), toluene and α-pinene in biofilters (BFs) and biotrickling filters (BTFs). Three 3.2 L BFs packed with polyurethane foam and operated at a gas residence time of 77 s with an air mixture of hydrophobic volatile organic compounds (VOCs) were inoculated with C. subhashii, F. solani and a combination of thereof. The systems were also operated under a BTF configuration with a liquid recirculating rate of 2.5 L h-1. Steady state elimination capacities (ECs) of total VOCs of 17.4 ± 0.7 g m−3 h−1 for C. subhashii, 21.2 ± 0.8 g m−3 h−1 for F. solani and 24.4 ± 1.4 g m−3 h−1 for their consortium were recorded in BFs, which increased up to 27.2 ± 1.6 g m−3 h−1, 29.2 ± 1.9 g m−3 h−1, 37.7 ± 3.3 g m−3 h−1 in BTFs. BTFs supported a superior biodegradation performance compared to BF, regardless of the VOCs. Moreover, a more effective VOC biodegradation was observed when C. subhashii and F. solani were grown as a consortium. The microbial analysis conducted revealed that the fungi initially introduced in each BF represented the dominant species by the end of the experiment, with C. subhashii gradually overcoming F. solani in the system inoculated with the fungal consortium.

Lignin degradation by co-cultured fungi: current status and future perspectives

The lignocellulosic biomass is a highly abundant and renewable resource. However, its exploitation is limited by the recalcitrance of the lignin present in the plant cellwall. In the last three decades, fungal co-cultures have increasingly been applied to overcome lignin recalcitrance by enhancing the production of ligninolytic enzymes through microbial interactions. In this paper, we systematically compile studies on fungal co-cultures used in the degradation of lignin-containing substrates to clarify the advantages and limitations of this type of culture. Based on their different delignification rate potentials, co-cultures can be classified into synergistic, antagonistic, and neutral. Co-cultivation results are generally related to the balance or imbalance of antagonistic and synergistic effects arising from the specific compatibility between the species during the interaction. It is well known that the paired species and the microenvironmental system conditions are responsible for the reported degradations,however, the mechanisms underlying these interactions remain poorly understood. In conclusion, literature results demonstrate the promising application of fungalco-cultures in biotechnological sectors to improve the degradation of lignin and its derivatives, through their better understanding of the efficient exploitation ofbiological resources on ecological and industrial scales.

Co-biomass degradation of fluoranthene by marine-derived fungi; Aspergillus aculeatus and Mucor irregularis: Comprehensive process optimization, enzyme induction and metabolic analyses

The application and relevance of marine-derived fungi in the mycoremediation of environment polluted with polycyclic aromatic hydrocarbons (PAHs) is promising whilst limiting environmental hazards. The present study investigated the fluoranthene degradation efficiency of marine-derived fungal co-culture, Aspergillus aculeatus (AA) and Mucor irregularis (MI) in batch processes (Plackett-Burman experiments) enhanced with the addition of surfactants and solid-state substrates. Further optimization studies done through fractional factorial design revealed that the co-culture exhibited 98.4% fluoranthene degradation capacity after 7 days of incubation. The role played by enzymes was revealed with 93, 85 and 71% induction of laccase, lignin peroxidase and manganese peroxidase respectively during fluoranthene degradation. The Gas Chromatography-Mass Spectrometry analysis revealed the formation of five metabolites; 1,2- dihydroxyfluoranthene, 9H-fluorene-1,9-dicarboxylic acid, benzene-1,2,4-tricarboxylic acid, benzene-1,3-dicarboxylic acid and benzoic acid after fluoranthene degradation by AA + MI co-culture which was used in predicting a metabolic pathway. The findings of this study elucidated the promising potentials of marine-derived fungal co-biomass in the eco-friendly remediation of polycyclic aromatic hydrocarbons thus promoting green technology.

Fusaric acid detoxification: a strategy of Gliocladium roseum involved in its antagonism against Fusarium verticillioides.

Fungal co-culture has several biotechnological applications including the discovery or the enhanced production of secondary metabolites. It is also a powerful tool aiding to elucidate the involvement of secondary metabolism in fungus-fungus interactions. The aim of this work was to investigate secondary metabolites produced when Fusarium verticillioides is co-cultured with Gliocladium roseum. Secreted metabolites were analyzed by HPLC-MS, and fusaric acid (FA) was quantified by HPLC-DAD. Four FA derivatives were identified only in the F. verticillioides-G. roseum co-culture. Mass spectrometry and one- and two-dimensional NMR spectra indicated that they were 5-butylpyridine-2-carboxylic acid methyl ester (5B2CAM), 4-(5-butylpicolinamido) butanoic acid (45BBA), methyl 4-(5-butylpicolinamido) butanoate (M45BBA), and bis(5-butyl-2-pyridinecarboxylate-N1,O2)-copper (B52P). 45BBA and M45BBA are reported for the first time and were FA biotransformation products generated by G. roseum. The antifungal activity of 5B2CAM, 45BBA, and M45BBA was evaluated in vitro against Botrytis cinerea and Aspergillus niger. They were less fungitoxic than FA, with 45BBA as the least toxic. Our results suggest that the effective antagonism exerted by G. roseum against F. verticillioides is due, at least in part, to its detoxifying ability against FA.

Fungal-fungal co-culture: a primer for generating chemical diversity.

Covering: 2002 to 2020In their natural environment, fungi must compete for resources. It has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. In a quest to discover new chemical diversity from fungal cultures, a growing trend has been to recapitulate this competitive environment in the laboratory, essentially growing fungi in co-culture. This review covers fungal–fungal co-culture studies beginning with the first literature report in 2002. Since then, there has been a growing number of new secondary metabolites reported as a result of fungal co-culture studies. Specifically, this review discusses and provides insights into (1) rationale for pairing fungal strains, (2) ways to grow fungi for co-culture, (3) different approaches to screening fungal co-cultures for chemical diversity, (4) determining the secondary metabolite-producing strain, and (5) final thoughts regarding the fungal–fungal co-culture approach. Our goal is to provide a set of practical strategies for fungal co-culture studies to generate unique chemical diversity that the natural products research community can utilize.