Fungal Hormone Hypothesis Gains Ground as Heptelidic Acid Loss Silences Metabolite Genes in Trichoderma virens

When the filamentous fungus Trichoderma virens loses its ability to make a single sesquiterpene lactone called heptelidic acid, the rest of its chemical arsenal falls silent. In a study published in Current Genetics, researchers report that deleting the gene for heptelidic acid synthase, has1, caused a sweeping collapse of secondary metabolism in this plant-beneficial biocontrol fungus: hundreds of genes were downregulated, including dozens belonging to the gene clusters that build viridin, gliovirin, and volatile sesquiterpenes, and the metabolites themselves vanished from the culture filtrates. The finding, the authors argue, is consistent with the provocative idea that heptelidic acid may act as a microbial hormone, a small molecule that switches on the fungus’s secondary metabolism machinery, a regulatory mode that is well established in bacteria but rarely documented in fungi.

Trichoderma virens is widely used in agriculture as a biological control agent against plant diseases, and its usefulness rests largely on the volatile and non-volatile secondary metabolites it secretes. These compounds have antimicrobial properties and contribute to promoting plant growth and development. Among the major non-volatile metabolites are gliovirin, viridin, viridiol, and heptelidic acid, the last also known by its older name koningic acid. Understanding how production of these compounds is regulated, the researchers note, could enable precise and context-specific modulation of the beneficial metabolites that make the fungus an effective biocontrol agent.

Heptelidic acid has a storied history beyond plant pathology. Originally isolated in 1980 from the culture broth of T. virens, the purified compound, with the formula C15H20O5, was identified as an antibacterial metabolite showing high toxicity against the anaerobic bacterium Bacteroides fragilis, and its structure was later elucidated by spectrometry and X-ray crystallography. Surveys of many T. virens isolates divided the species into two groups: P-type strains synthesize gliovirin and heptelidic acid, while Q-type strains produce gliotoxin and dimethylgliotoxin, and both types make viridin and viridiol. Heptelidic acid was also found to be active against oomycetes, broadening the antimicrobial spectrum of the metabolite.

The compound later attracted attention far from soil microbiology. Heptelidic acid turned out to be a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase, or GAPDH, the enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate to 3-phosphoglycerate in the glycolytic pathway. It binds irreversibly to two cysteine residues at the active site of GAPDH, inactivating the enzyme. As a self-resistance mechanism, the genomes of producing organisms carry an additional GAPDH gene encoding an isoform relatively insensitive to the inhibitor. Because the compound selectively kills highly glycolytic cells through glucose-dependent ATP depletion, it has been explored as a potential anti-cancer agent aimed at the Warburg effect, the preference of many tumors for glycolysis over oxidative metabolism, and it also exhibits apoptotic activity, antimalarial properties, and inhibitory effects on certain DNA polymerases.

The genetic groundwork for the new study was laid in earlier work. Using suppression subtractive hybridization in a T. virens mutant downregulated in secondary metabolism, the team had previously identified a gene cluster designated the vir cluster, with a GAPDH gene located adjacent to the terpene cyclase vir4. Genome analysis later confirmed two GAPDH-encoding genes in the species, one house-keeping and one associated with the vir cluster. Deleting vir4 eliminated the production of several volatile sesquiterpenes without affecting viridin, viridiol, or gliotoxin, and the cluster was subsequently shown, first in the heptelidic acid-producing mold Aspergillus oryzae and then in T. virens itself, to be involved in heptelidic acid biosynthesis. Gene knockout experiments then proposed that another terpene cyclase, Has1, performs the first committed step, the cyclization of farnesyl pyrophosphate, while vir cluster enzymes carry out downstream modifications. Notably, deleting the vir-cluster GAPDH abolished the major non-volatile metabolites and volatile sesquiterpenes, deleting has1 abrogated non-volatile metabolite production, and deleting the non-ribosomal peptide synthetase gene Tex7 in a Q strain previously thought to be a heptelidic acid non-producer resulted in abundant heptelidic acid production along with elevated levels of gliotoxin, viridin, and viridiol. Those patterns suggested that the cluster-associated, heptelidic acid-insensitive GAPDH acts as a positive regulator of heptelidic acid biosynthesis, Tex7 acts as a negative regulator, and the secondary metabolism machinery depends on heptelidic acid production itself.

To test that hypothesis directly, the researchers compared the wild-type strain IMI 304061 with a Δhas1 mutant lacking heptelidic acid biosynthesis, using both transcriptomics and metabolomics. The two strains were grown in triplicate in potato dextrose broth for three days, RNA was isolated and sequenced on an Illumina NovaSeq 6000 platform, and expression levels were estimated with the DESeq2 package. Sequencing generated 765.36 Mbp of data for the wild type at an average Q30 above 94 percent and 651.53 Mbp for the mutant, and after de novo assembly 84.15 percent and 82.02 percent of the reads aligned, respectively. The metabolome was profiled by extracting three-day culture filtrates with ethyl acetate and analyzing them by liquid chromatography coupled to a Q-TOF mass spectrometer, with untargeted processing in XCMS Online and targeted peak quantification in Agilent MassHunter.

The transcriptomic comparison was strikingly lopsided. In the Δhas1 mutant, 255 genes were downregulated while only 96 were upregulated, using a cut-off of log2 fold change of at least 2 and a P-value of 0.05 or less. Among the downregulated genes were many belonging to secondary metabolism gene clusters: nine genes of the gliovirin cluster, 14 genes of the viridin cluster, and five genes from the cluster responsible for volatile sesquiterpene and heptelidic acid biosynthesis were significantly downregulated. A cytochrome P450 gene from the has cluster and a terpene cyclase were downregulated as well, along with a polyketide synthase cluster, an ABC transporter associated with another polyketide synthase cluster, and a transcription factor from the Tex10 cluster involved in ferricrocin siderophore biosynthesis. Only three genes associated with secondary metabolism clusters were upregulated: a polyketide synthase, a terpene cyclase, and a major facilitator superfamily transporter from the has cluster. None of the genes from the viridin, gliovirin, or volatile sesquiterpene biosynthesis clusters were upregulated. The expression patterns were validated by RT-qPCR for six genes, with all five downregulated genes and the one upregulated terpene cyclase showing consistent results.

The metabolomic data mirrored the transcriptomic collapse. Untargeted profiling detected 5442 metabolites in positive ionization mode and 4978 in negative mode, of which 1362 were downregulated in the positive mode and 1878 in the negative mode. Critically, gliovirin, viridin, viridiol, and heptelidic acid were not detected in the Δhas1 mutant, and another sesquiterpene metabolite, dihydroartemisin, previously unreported in T. virens, was also absent. The strong correlation between gene expression and metabolite abundance, the authors write, indicates that heptelidic acid, a sesquiterpene lactone, might function as a positive regulator of key secondary metabolism pathways in the fungus.

The hormone hypothesis has a clear precedent in bacteria. In Streptomyces griseus, the secondary metabolite A-factor, a gamma-butyrolactone, regulates secondary metabolism, and related butyrolactones act as microbial hormones across Streptomyces species. In the canonical mechanism, A-factor binds the receptor protein ArpA, a transcriptional repressor that sits on the promoter of the regulatory gene adpA; binding of A-factor releases ArpA, activating the AdpA regulon and triggering secondary metabolism and morphological differentiation. In fungi, regulation of secondary metabolism by an endogenous metabolite is rarely reported, although the metabolite aureonitol acts as a transcriptional regulator of secondary metabolite biosynthesis in Chaetomium globosum, and global regulators such as components of the Velvet complex, the methyltransferase LaeA, histone acetylases, and deacetylases are known to control secondary metabolism in filamentous fungi. Whether heptelidic acid operates through a comparable receptor-based mechanism in T. virens remains unknown.

The authors are careful about what the data do and do not prove. Their results are indicative of a possibility that heptelidic acid might function as a microbial hormone regulating secondary metabolism in this biotechnologically important fungus, but correlation between loss of the metabolite and silencing of the clusters does not by itself establish a direct regulatory role, and further studies, including chemical complementation and identification of the regulatory pathways and any receptor, are required to confirm it. The work also points to a complex regulatory network in which heptelidic acid biosynthesis is itself positively controlled by a heptelidic acid-insensitive GAPDH encoded within the vir cluster and negatively controlled by Tex7, which encodes an unknown non-ribosomal peptide. Even before the mechanism is resolved, the researchers suggest the observation might open avenues for genetic enhancement of secondary metabolite biosynthesis in T. virens, a fungus whose value to agriculture depends on the very compounds that went silent when heptelidic acid was removed. The RNA sequencing data have been deposited in NCBI under BioProject ID PRJNA1291810.

Subject of Research: Transcriptomic and metabolomic evidence that heptelidic acid may regulate secondary metabolism in the biocontrol fungus Trichoderma virens

Article Title: Is heptelidic (koningic) acid a microbial hormone that regulates secondary metabolism in the biocontrol fungus Trichoderma virens?

Article References: Bansal, R., Sethy, S. K., Barvkar, V. T., Mukherjee, P. K., & Mondal, S. (2025). Is heptelidic (koningic) acid a microbial hormone that regulates secondary metabolism in the biocontrol fungus Trichoderma virens?. Current Genetics, 71(1), Article 29. https://doi.org/10.1007/s00294-025-01334-9

Image Credits: AI Generated

DOI: 10.1007/s00294-025-01334-9

Keywords: Trichoderma virens, heptelidic acid, koningic acid, secondary metabolism, microbial hormone, has1, transcriptomics, metabolomics, biocontrol, viridin, gliovirin, GAPDH