Biosynthetic genes involved in the production of mycotoxins and their regulation

G Adithi and M Y Sreenivasa*

Applied Mycology Lab, Department of Studies in Microbiology, University of Mysore, Mysuru, India

*Corresponding author: sreenivasamy@gmail.com; mys@microbiology.uni-mysore.ac.in

Mycotoxigenic fungi and their mycotoxins are a main cause of contamination in cereals, food, animal, and bird feed.

The major fungal species include Aspergillus, Fusarium and Penicillium

The secondary metabolites secreted by these toxigenic fungi are mycotoxins, which have a serious impact on human and animal health, causing carcinogenicity, genotoxicity, nephrotoxicity, hepatotoxicity, and reproductive disorders.

The main mycotoxins include aflatoxins, fumonisins, trichothecenes, zearalenone, and ochratoxins, which are chemically stable and not easily degraded through normal processing or autoclaving, and, thereby, end up in the food chain (Adithi et al., 2022).

The genetic components of the fungi always play a vital role in the biosynthesis of secondary metabolites, including mycotoxins.

Understanding the mycotoxin biosynthetic genes and their regulation has become fundamental for elucidating their expression and control.

This information can be useful for developing effective approaches for the decontamination of mycotoxins in food and feed.

The identification and manipulation of biosynthetic genes and key regulatory pathways help to develop innovative and targeted control methods to inhibit the biosynthesis of these toxins in plants and their products, ensuring food safety (Deepa et al., 2016).

General features of mycotoxin biosynthetic gene clusters

In fungi, biosynthetic gene clusters (BGCs) are groups of genes that control mycotoxin production, confirming that the pathway functions in a coordinated manner, while polyketide synthase (PKS) acts as a backbone gene for most mycotoxins (Proctor et al., 2008; Caceres et al., 2020; Liew et al., 2023)

These clusters also include pathwayspecific regulators and multiple transcription factors.

They contain transporters responsible for exporting toxins out of the cell to reduce self-toxicity.

BGCs are also highly flexible, displaying gene losses or rearrangements that account for variations in toxin production among species and strains (Sultana et al., 2019).

Biosynthetic genes by mycotoxin types

In Aspergillus species, the production of aflatoxins is controlled by two key genes, aflR and aflS, which encode a zinc-finger transcription factor that activates signaling pathway genes and a co-activator that enhances aflR function, respectively (Meyers et al., 1998).

Recent findings show that aflS, apart from its co-activator role, also helps to stabilize AflR–DNA interactions, and aflatoxin production can be affected by its loss (Caceres et al., 2020).

The ratio between aflS and aflR varies in response to environmental factors such as water activity and temperature, contributing to changes in toxin levels and the types of aflatoxins produced (Medina et al., 2017) (Figure 1).

Figure 1. Representation of the aflatoxin biosynthetic cluster in Aspergillus flavus. The arrows indicate the direction of gene transcription. Grey arrows represent regulatory genes, while blue arrows indicate structural genes (Serna et al., 2020).

KEY GENES RESPONSIBLE FOR FUMONISINS (FUM GENES 1,6,8,21)

Fumonisins are among the major mycotoxins secreted by Fusarium species, representing common contaminants of most cereals and grains (Dass et al., 2007; Sreenivasa et al., 2011).

The FUM gene cluster regulates the biosynthesis of these toxins, with FUM1, FUM6, FUM8, and FUM21 as key genes (Glenn et al., 2008) (Figure 2).

FUM1 encodes a polyketide synthase responsible for backbone assembly.

FUM6 and FUM8 mediate hydroxylation and methylation, both essential for toxin activity.

FUM21 acts as a regulatory transcription factor.

Disruption of these genes significantly reduces or eliminates fumonisin production.

Figure 2. Representation of the fumonisin biosynthetic gene cluster in Fusarium verticillioides. Arrows indicate the position and transcriptional orientation of genes. The grey arrow represents the regulatory gene. In Fusarium proliferatum and Fusarium oxysporum, the order and orientation of genes are similar, although the distances between them can vary (Alexander et al., 2009).

KEY GENES RESPONSIBLE FOR TRICHOTHECENES (TRI CLUSTERS, TRI5, TRI4, TRI6, TRI7, TRI8, TRI11, AND TRI13)

Trichothecenes are toxic sesquiterpenoid mycotoxins produced by Fusarium species that pose a serious food contamination risk.

Trichothecene biosynthesis is controlled by the TRI gene cluster.

TRI5 encodes trichodiene synthase, initiating trichothecene formation.

TRI4 catalyzes multiple oxygenation steps.

TRI6 is a regulatory gene that governs cluster expression.

TRI7, TRI8, TRI11 and TRI13 encode enzymes that modify the trichothecene backbone through reactions such as hydroxylation, acetylation, and epoxidation, producing diverse analogs (Proctor et al., 2008; Caceres et al., 2020)

Collectively, these genes coordinate the biosynthetic process and result in the secretion of type A and type B trichothecenes (Figure 3).

Figure 3. Representation of the trichothecene biosynthetic cluster and the TRI1–TRI16 cluster in Fusarium, which leads to the formation of T-2 toxin. Grey arrows represent regulatory genes, while the other arrows indicate the position and transcriptional orientation of genes (Alexander et al., 2009).

KEY GENES RESPONSIBLE FOR ZEARALENONE

(PKS 4, 13 AND ZEB1, ZEB2)

Zearalenone (ZEN) is an estrogenic mycotoxin produced by Fusarium species that contaminates cereals and their derived products (Yu et al., 2022)

Zearalenone biosynthesis depends on polyketide synthase (PKS) genes and regulatory transcription factors.

PKS4 and PKS13 catalyze the assembly of the polyketide backbone from acetyl-CoA units, forming the precursor of ZEN (Perincherry et al., 2019).

The expression of these genes is regulated by the key transcription factors ZEB1 and ZEB2, which coordinate secondary metabolism for efficient ZEN production (Villani et al., 2019).

Controlled expression of PKS4, PKS13, ZEB1, and ZEB2 plays a vital role in minimizing ZEN contamination in food, feed, and other agricultural products (Figure 4).

Figure 4. Representation of the ZEN gene cluster showing its genomic organization and flanking regions in Fusarium graminearum. The direction of the arrows indicates the predicted position and orientation of transcription for each gene/ORF. The boxed area comprises the genes required for ZEN biosynthesis in F. graminearum (Nahle et al., 2021).

KEY GENES RESPONSIBLE FOR OCHRATOXIN A (AcOTApks, PKS GENES)

Aspergillus and Penicillium species secrete ochratoxin A (OTA), which is responsible for the contamination of cereals and other agricultural products.

Ochratoxin biosynthesis involves a complex, coordinated series of enzymatic processes in which the polyketide synthase, AcOTApks, plays a fundamental role (Wang et al., 2016).

This gene encodes a PKS enzyme responsible for initiating the condensation of acetate and malonate units, resulting in the dihydroisocoumarin core of OTA (Gallo et al., 2012). The process is further anchored by the incorporation of L-phenylalanine and subsequent chlorination to complete the OTA molecule (El Khoury & Atoui, 2010)

OTA biosynthesis is also regulated by non-ribosomal peptide synthases and chlorinating enzymes, although the complete mechanism has not yet been fully elucidated.

Current findings provide insight into the complex genetic and enzymatic network required for OTA production (Figure 5)

Regulation of biosynthetic gene expression

Genetic and environmental factors greatly influence the biosynthesis of major mycotoxins such as aflatoxins, fumonisins, trichothecenes, zearalenones, and ochratoxins.

The transcription factors Tri6 and Tri10 in Fusarium species regulate trichothecene production, while zearalenone biosynthesis is governed by ZEB1 and ZEB2 (Seong et al., 2009; Nahle et al., 2021)

In Aspergillus flavus, the transcription factor Apa-2 controls key biosynthetic genes involved in aflatoxin production, and similar regulatory roles have been identified for AflR and other pathway-specific regulators that control ochratoxin biosynthesis in Aspergillus and Penicillium species (Chang et al., 1993).

FUM genes respond to environmental cues such as carbon and nitrogen availability, thereby regulating fumonisin production in Fusarium species.

Environmental factors such as temperature, pH, and nutrient limitation can trigger the activation of signaling pathways, including MAPK cascades, leading to the overexpression of mycotoxin biosynthetic genes across fungal families.

A better understanding of the genetic and environmental regulatory mechanisms is crucial for developing innovative strategies to mitigate mycotoxin contamination in food and feed.

Target specific strategies in key biosynthetic genes inhibition

Recombinant DNA (rDNA) technology has emerged as an effective strategy to reduce mycotoxin contamination through target-specific action on key biosynthetic genes.

Host-induced gene silencing (HIGS) and RNA interference (RNAi) technologies in crops have shown promising results in suppressing the expression of polyketide synthase and trichothecene (TRI) cluster genes, thereby reducing aflatoxin and trichothecene accumulation under natural conditions (Majumdar et al., 2017; Stakheev et al., 2024).

Gene-specific CRISPR/Cas9-mediated editing of fungal genomes has successfully disrupted toxin-related genes in Fusarium and Aspergillus species, leading to the loss of mycotoxin production and providing valuable insights into cluster regulation (Ferrara et al., 2019).

A cumulative approach integrating resistant transgenic crops, CRISPR, and HIGS technologies, together with enzymatic or microbial detoxification, represents a sustainable strategy for managing mycotoxin production across pre- and post-harvest phases (Gomes et al., 2024)

Applications and future prospects

Food safety is a major global concern and largely depends on developing countermeasures against toxin production by fungi.

In this regard, biosynthetic gene clusters (BGCs), responsible for producing toxins such as aflatoxins, fumonisins, trichothecenes, and zearalenones, have become a key target for developing strategies to improve food safety management.

The adoption of cutting-edge technologies such as CRISPR-based genome editing and RNA interference (RNAi) can effectively silence toxin gene expression. At the same time, host- or spray-delivered RNA molecules are emerging as promising and practical crop protection strategies (Stakheev et al., 2024; Chen et al., 2025).

Global regulators such as LaeA and the velvet complex, along with chromatin modifications, represent additional prospects for suppressing entire gene clusters (Zhang et al., 2024)

In the near future, integrating gene-based diagnostics, RNA technologies, and biocontrol agents with multi-omics surveillance may pave the way for long-lasting and safe mycotoxin mitigation (Liew et al., 2023).

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