Biosynthesis Regulation of Secondary Metabolite Production in Fusarium Fungi

Abstract

Fusarium fungi are prolific producers of a wide array of structurally and functionally diverse secondary metabolites (SMs), ranging from harmful mycotoxins to beneficial phytohormones and medicines. Many of these compounds show significant promise for use as agrochemicals, pharmaceuticals and food additives. The biosynthesis of these SMs in Fusarium fungi is strictly regulated by a complex network composed of various regulatory components. This review highlights recent advances in understanding how secondary metabolism in Fusarium fungi is regulated at various levels, particularly through the regulation of environmental factors (e.g., light, temperature, pH, carbon, and nitrogen sources), global and pathway-specific transcriptional factors (e.g., LaeA, LaeB, AreA, Tri6, and ZEB2), epigenetic modifications (e.g., histone acetylation and methylation, DNA and RNA modifications), and signal transduction pathways (e.g., cAMP, TOR, and MAPK pathways). Furthermore, the biological significances and potential applications of some metabolites (e.g., beauvericin, bikaverin, gibberellins, fumonisins, fusaric acid, and trichothecenes) produced by Fusarium fungi were discussed. Biosynthesis regulation on SM production offers a powerful approach to either unlock silent or cryptic biosynthetic gene clusters (BGCs) for the discovery of new SMs, to boost the yiled of low-abundance beneficial metabolites, or suppress specific BGCs to eliminate the production of toxic compounds in Fusarium fungi.

1. Introduction

Fusarium fungi are extremely widespread and ubiquitous in terrestrial and marine environments. They belong to the Nectriaceae, Hypocreales, Sordariomycetes of ascomycetous fungi [1,2]. Fusarium fungi include marine-derived, soil-derived, endophytic, and pathogenic species. Since 1809, when Link initially described and defined F. roseum from Malavaceous plants, more than 1000 Fusarium species have been described in this genus [3,4].

Many Fusarium species can produce structurally diverse secondary metabolites (SMs) to provide protection and survival for themselves in the environment. Furthermore, these metabolites have multiple biological activities including antimicrobial, cytotoxic, antioxidant, nematocidal, and plant growth regulatory activities. On the one hand, Fusarium fungi are the producers of mycotoxins, but on the other hand, they are an important source of bioactive compounds. This has attracted increasing attention for the study of bioactive metabolites of Fusarium fungi [5,6]. Fusarium-derived secondary metabolites mainly include polyketides, terpenoids, nitrogen-containing compounds, phenolics, and steroids [7,8,9]. Some of these metabolites (e.g., deoxynivalenol, zearalenone, fusaric acid, fusariotoxin T2 and fumonisin B1) are called mycotoxins or phytotoxins due to the toxicity on animals or plants, causing their diseases [10,11,12]. However, most Fusarium species can produce valuable bioactive SMs, demonstrating potential applications in the pharmaceutical, agricultural and food industries. This makes them a treasure trove of bioactive compounds [6,8,13,14].

In order to either reveal additional bioactive SMs, increase the yield of useful known SMs, or inhibit toxic metabolite production, the regulation of biosynthetic pathways in Fusarium fungi offered a promising strategy to enhance the production of low-yield valuable SMs, suppress toxic compounds, and uncover novel bioactive molecules. Biosynthesis regulation mainly includes environmental factor regulation [15,16,17,18], transcriptional factor regulation [19,20], epigenetic regulation [21,22], and signal transduction regulation [23]. They can effectively regulate Fusarium secondary metabolite production. These fungal species, mainly including F. avenaceum [24], F. fujikuroi (teleomorph: Gibberella fujikuroi) [25], F. graminearum (teleomorph: G. zeae) [26,27], F. oxysporum [28,29,30], F. proliferatum (teleomorph: G. intermedia) [31,32], F. pseudograminearum [33], F. sulphureum [34], F. verticillioides (teleomorph: G. moniliformis) [31,35], have been well studied for their secondary metabolite production via biosynthesis regulation.

In the past three decades, many advances have been achieved in the regulation on secondary metabolite production with the continuous revelation of biosynthetic gene clusters (BGCs) related to secondary metabolism in Fusarium fungi. To our knowledge, the specific reviews about the strategies for secondary metabolite production via biosynthesis regulation on Fusarium fungi have not been reported. This review summarizes diverse strategies, including regulations of environmental factors, global and pathway-specific transcriptional factors, epigenetic modification, signal transduction, the use of organic chemicals, and plant/microorganism-derived extracts to modulate secondary metabolite production in at least 50 Fusarium species. These approaches aim to discover novel bioactive secondary metabolites, inhibit harmful mycotoxins, and expedite the practical application of valuable metabolites derived from Fusarium fungi.

2. Regulation of Environmental Factors on Fusarium SM Production

The environmental factors can activate or suppress fungal BGCs to either increase or decrease production of SMs for fungal physiological or ecological adaptation to the environment. These environmental factors mainly include light, temperature, water availability (activity), ambient pH, carbon and nitrogen sources, and other media components [36,37]. Sometimes, they are known as the OSMAC (one strain many compounds) approach that the fungal cryptic metabolite BGCs are activated. OSMAC means the modulation of the optimal culture conditions (i.e., media composition, carbon and nitrogen sources, light, temperature, pH, and osmolarity) for SM production in fungi [38]. In addition, the transcriptional, signal transduction and epigenetic regulations of the genes involved in the biosynthetic pathways of SMs in fungi are responsive to environmental stimuli. Therefore, the environmental factors can affect the production of fungal SMs including their composition and relative contents. Some fungal species such as F. fujikuroi [25,39,40], F. graminearum [41], F. proliferatum [31], F. temperatum [42] and F. verticillioides [31,43] have been well studied for the environmental factors that affect their SM production. In fact, various environmental factors synergistically regulate the biosynthesis of SMs in fungi. Here, we summarize the effects of environmental factors on fungal secondary metabolism mainly based on the similar regulation mechanisms that a certain type of environmental factor may have.

2.1. Regulation by Light

By sensing and regulating gene expression modulated through light, fungi can produce various bioactive metabolites [16,17,18,44,45]. The impacts of light regulation on the production of some Fusarium SMs, such as carotenoids and gibberellins, have been reviewed previously [46].

Light repressed fusarin production via white collar protein WcoA in F. fujikuroi [47]. Further study showed that WcoA and WcoB regulated the mRNA levels of various genes in F. fujikuroi, including the genes for the photoreceptors OpsA and CryD, the regulatory proteins Csp1 and Hog1, and some key enzymes in the biosynthesis of different SMs, such as beauvericin (BEA), carotenoids, and fusarins. However, the deletion of WcoA and WcoB genes had little effect on the key biosynthetic genes of bikaverin and gibberellins in F. fujikuroi [48].

F. fujikuroi contained a gene encoding a speculated cry-DASH, namely CryD. The expression of the gene cryD in the wild-type (WT) strain was induced by light. The WT strain exhibited moderate photo-induction of gibberellin production, while the ΔcryD mutant did not [49]. VvdA was involved in the light regulation of fungal development and affected the accumulation of carotenoids in F. fujikuroi. The absence of vvdA in F. fujikuroi resulted in a shallow pigmentation under constant light exposure. The targeted ΔvvdA mutants accumulated fewer carotenoids than the WT strain [50]. In addition, F. fujikuroi could produce carotenoids which were terpenoid pigments acting as antioxidants and photoprotectants. The biosynthesis of carotenoids was stimulated by light via the regulation of the gene carS, which demonstrated that the transcription of the gene carS was positively regulated by light [51].

Both Fgwc-1 and Fgwc-2 genes were essential for light-dependent processes of F. graminearum. If these two genes were deleted, the biosynthesis of aurofusarin and trichothecene was derepressed [52].

The photolyase gene phr1 from the phytopathogen F. oxysporum f. sp. lycopersici was induced by visible light. Both the expression of the gene phr1 and the presence of α-tomatine were detected in the fungus. α-Tomatine was a glycoalkaloid from tomato damaged cell membranes, indicating that phr1 was induced by this cellular stress [53].

Red and green lights favored the production of antimicrobial compounds 8-deoxyjavanicin and fusolanone A in F. solani, an endolichenic fungus. It indicated that the adaptability of fungi to light offered an alternative in using light as an effective and low-cost approach to regulate and induce biosynthesis of beneficial bioactive compounds in fungi [54].

2.2. Regulation by Temperature

Temperature significantly affected the growth and metabolism of Fusarium fungi. The optimal temperature for the production of fumonisin was between 15 °C and 25 °C for F. proliferatum, and between 20 °C and 30 °C for F. verticillioides [55,56]. The expression of the fumonisin biosynthetic FUM1 gene was markedly induced at 20 °C in both F. proliferatum and F. verticillioides. However, the optimal temperature for fungal growth was 25 °C for these two Fusarium species [57].

F. subglutinans, which was isolated from maize ear rot materials in Poland, was cultured at different temperatures on a few cereal substrates (barley, maize, wheat, rye, oat, and rice kernels) as media. Among these substrates, rye substrate favored fusaproliferin production, wheat and rice substrates favored BEA production, and rice substrate favored moniliformin production. When F. subglutinans was cultured on rice substrate, the fungus produced the highest levels of BEA and fusaproliferin at 20–25 °C, while moniliformin production was the most suitable at 30 °C [58].

Fusarium species had the maximal production of fumonisins B1 (FB1) and B2 (FB2) at 20–25 °C on Czapek yeast agar plus 5% salt or potato dextrose agar (PDA) [59]. Similar results were observed for F. verticillioides to produce FB1 and FB2 at 20–35 °C [60]. Another study showed that the suitable conditions for F. verticillioides growth were 25 °C with water availability as 0.98 a_w, whereas the highest FB1 yield was observed at 15 °C with water availability as 0.98 a_w [61].

2.3. Regulation by Water Availability

Regulation of water availability/activity (a_w) on the expression of biosynthesis genes of Fusarium SMs has also been studied. Generally, temperature and water availability (relative humidity) synergistically affected fungal growth and SM accumulation. The optimal water availability for fumonisin production was 0.97–0.98 a_w for F. proliferatum and F. verticillioides [55,56].

The influences of different water availability and temperature on the contents of free and conjugated zearalenone (ZEA or ZEN) and deoxynivalenol (DON) for the stored wheat inoculated with F. graminearum were studied. As an important conjugate of DON, there was a significant difference in the content of DON-3-glucoside and its precursor DON in naturally contaminated wheat at 0.93 a_w and 25 °C, with a ratio of 56:44, respectively. The high contents of DON-3-glucoside could be influenced by the wheat varieties, harvesting seasons, fungal strain types, and locations. Unexpectedly, the content of ZEN-14-sulfate was three times higher than that of ZEN in naturally contaminated wheat at 0.98 a_w. The contents of emerging mycotoxins such as moniliformin were increased with increasing temperature and reached their highest levels at 0.95 a_w and 25 °C. In general, water availability had a significant impact on the content of each mycotoxin, while temperature changes had no significant effect on the content of each mycotoxin [62].

The effects of water availability (a_w), temperature, incubation time and their interactions on the accumulation of mycotoxins as well as the expression levels of biosynthetic genes in F. graminearum species complex strains from maize samples were studied. At 0.98 a_w/30 °C or 0.99 a_w/25 °C, the highest contents of DON, 3-AcDON and 15-AcDON of the F. boothii and F. graminearum strains were observed. The maximum contents of nivalenol (NIV) and 4-AcNIV were achieved at 0.99 a_w and 30 °C in F. asiaticum and F. meridionale [63]. Another example was the effect of temperature and water availability on mycotoxin production by F. oxysporum and F. sambucinum responsible for dry rot in potato tubers. The mycotoxins, including T-2, HT-2, diacetoxyscirpenol (DAS), 15-acetoxyscirpenol (15-AS), neosolaniol, and BEA were easily examined when potato tubers were stored at 10 °C and 0.99 a_w for 21 days. The relative contents of mycotoxins of the potato tubers at 0.99 a_w/10 °C were much higher than those of the potato tubers at 0.97 a_w/5 °C [64].

The highest level of fusaric acid (FA) was detected at 0.995 a_w/25 °C in grain contaminated with F. temperatum. Drying grain was the best strategy to reduce FA and fusarinolic acid contamination of grains, as the fungal growth and mycotoxin accumulation were typically at low levels [42].

The effects of water availability (0.955 a_w and 0.990 a_w) on the expression of five genes (i.e., FUM3, FUM8, FUM13, FUM14 and BIK1) in F. verticillioides were investigated after 14 and 21 days of cultivation, respectively. The FB production and biosynthetic gene expression reached their maximum values at 0.990 a_w, and the bikaverin production and BIK1 expression also showed the same trend. FUM3 and FUM14 were the most highly expressed genes, positively correlated with the production of FB1, FB2, and FB3 [65].

2.4. Regulation by Ambient pH

Ambient pH is an important environmental factor affecting the growth and development of Fusarium fungi, and their SM production. In F. fujikuroi, acidic environments (pH 4–5) promoted production of fusaric acid (FA), a virulence factor that disrupted plant membrane integrity [66,67]. Conversely, alkaline conditions suppressed melanin synthesis, reducing fungal survival under UV stress in F. fujikuroi [68].

In the liquid culture of F. proliferatum, the optimal pH range for fumonisin production was from 3.0 to 3.5. However, when pH was higher than 3.5, the growth of fungi would be enhanced [69].

Both Tri (TRI) gene expression and trichothecene B biosynthesis were induced by acidic pH (i.e., pH 3, 4 and 5) in F. graminearum, the pathogen of wheat Fusarium head blight and maize Fusarium ear rot. When the expression of the FgPAC1 gene, a zinc finger transcription factor, was high, the expression of Tri genes was repressed [70,71].

The ambient pH 6 was beneficial for the growth, pathogenicity, and diacetoxyscirpenol (DAS) production of F. sulphureum, which was the pathogen to cause Fusarium rot of muskmelon. Ambient pH 6 was also more conducive to the secretion of cell wall-degrading enzymes of the pathogen to degrade the cell wall of the host plant and upregulated the expression of DAS biosynthesis genes [34].

2.5. Regulation by Carbon Sources

Seven monosaccharides (i.e., fructose, arabinose, galactose, xylose, sorbose, mannose, and glucose), five disaccharides (i.e., sucrose, cellobiose, trehalose, maltose, and lactose), and three polysaccharides (i.e., dextrin, xylan, and inulin) were used as the carbon sources in the media to test their influences on secondary metabolism in three F. avenaceum strains. The fungal strains could grow and produce aurofusarin on the tested carbon sources. Enniatins (ENs) and moniliformin were produced on all carbon sources except on lactose, which indicated a common conserved regulatory mechanism. Differences in the production of fusarin C, chlamydosporol, 2-AOD-3-ol, and antibiotic Y were observed among Fusarium strains, indicating that the carbon source played a regulatory role in their biosynthesis [72].

Glucose promoted the growth of F. fujikuroi by shutting down SMs. Thus, the production of gibberellins was affected by the addition of glucose as the sole carbon source. However, the mixture of carbon sources could promote the slow assimilation of gibberellic acid (GA3) but increase its yield, demonstrating the influence exerted by the carbon metabolism [73].

Mannose, used as the sole carbon source, significantly blocked the production of FB1 and FB2 by F. proliferatum as compared with the addition of sucrose. The RT-qPCR analysis indicated that expression of several key genes involved in the FB biosynthetic pathway and in transcription regulation was significantly downregulated in F. proliferatum with mannose as the carbon source, whereas the expression of histone deacetylation-related genes was significantly upregulated. These results indicated that the blockade of FB biosynthesis by mannose was related to the reduced conversion of acetyl-CoA to polyketide biosynthesis [74,75]. Further investigation showed that F. proliferatum cultivated in the Czapek’s broth (CB) without sucrose greatly induced production of fumonisins, while additional sucrose supplementation in the medium significantly decreased the production of fumonisin. In addition, cellulose, hemicellulose, and other polysaccharides extracted from banana peels replaced sucrose as a carbon source, reducing the production of fumonisins by F. proliferatum. Correspondingly, the genes related to fumonisin synthesis, such as FUM1 and FUM8, were significantly upregulated in the culture medium without sucrose [76]. In contrast, compared with sucrose, glucose had no significant effect on the fungal growth and fumonisin production of F. proliferatum, indicating that the glucose-responsive repressor CreA might not be a key regulatory factor in fumonisin biosynthesis [74].

It was reported that the starch content in maize affected the production of FB1, and the deletion of the α-amylase gene, AMY1, in F. verticillioides resulted in a decrease in FB1 production in starchy kernels [77]. Another investigation showed that among five carbon sources (i.e., glucose, amylopectin, amylose, starch, and amylose), the maximum production of FB1 was achieved with glucose, while the maximum production of sesquiterpenes was achieved with amylopectin in F. verticillioides [78].

F. verticillioides was a producer of useful SMs such as naphthoquinone pigments, monoterpenes, and sesquiterpenes. Their biosyntheses were stimulated in the cultures with the addition of fructose, lactose, and xylose at their optimal concentrations, respectively, with fumonisins being absent or present in trace amounts. However, the highest biosynthesis of fumonisins occurred in the medium with the addition of sucrose. The concentrations of FB1 and FB2 reached 7.85 mg/g dw and 0.38 mg/g dw, respectively [79].

It was concluded that carbon sources significantly affected SM production in Fusarium fungi. In general, fungi grew better with glucose than with other carbon sources. For the SM production in Fusarium fungi, the carbon catabolite repression (CCR) should be a major influence on the carbon-source-mediated regulation of metabolite biosynthesis [78,79].

2.6. Regulation by Nitrogen Sources

In fungi, different sources of nitrogen supplements resulted in physiological, morphological and metabolic alterations [15]. Some Fusarium fungi can produce naphthoquinone pigments such as fusarubin and bikaverin. The production of fusarubin was favoured in nitrogen -limited conditions by F. chlamydosporum [80].

The production of some PKS-NRPS-derived mycotoxins (i.e., fusarin C) in F. fujikuroi was usually regulated under high-nitrogen and acidic pH conditions [81]. The production of GA3 was repressed by the presence of nitrogen in high amounts of glutamine in F. fujikuroi [82]. Nitrogen starvation increased carotenoid accumulation in wild-type (WT) and carotenoid-overproducing strains [83].

With some amine compounds such as arginine, ornithine, putrescine and agmatine added in the media for cultivation of F. graminearum, the DON concentrations in the media were extremely high, and were equal to, or greater than 1000 mg/L [84]. Further mechanism study showed that two of the most strongly Tri6-dependent and agmatine-co-regulated genes appear to negatively regulate DON production [85]. When the medium pH was maintained at 4.0 in F. graminearum, and three amino acids including glycine, serine and threonine were added to the medium, respectively, they all suppressed the production of trichothecenes [86].

The production of phytohormone cytokinin in F. pseudograminearum was enhanced by PM3 as the nitrogen source. DON production was also increased in both F. graminearum and F. pseudograminearum by specific nitrogen sources [87].

The UHPLC-MS/MS analysis indicated that nitrogen sources (i.e., urea, NaNO₃, and (NH₄)₂SO₄) affected gibberellin biosynthesis and metabolic flux in F. sacchari. Additionally, the transcriptome analysis elucidated the potential impact of nitrogen availability on the expression of several genes involved in the synthesis of F. sacchari mycotoxins [88].

2.7. Regulation by Other Environmental Factors

Other environmental factors such as oxidative stresses [89], osmotic stresses [90], metal ions [91], medium components [43,92], and their complex factors, can regulate fungal SM production as well.

2.7.1. Regulation by Oxidative Stresses

When the liquid cultures of F. graminearum were treated with H₂O₂, the accumulation of trichothecenes including DON and 15-AcDON was rapidly and strongly enhanced. Due to H₂O₂ being the main factor causing oxidative bursts in pathogen-host interactions, this supported the theory that trichothecenes served as the virulence factors in the pathogenetic process [89].

2.7.2. Regulation by Osmotic Stresses

The production of trichothecenes in F. graminearum was markedly inhibited by NaCl which had no obvious effect on fungal growth. Both the osmosensor histidine kinase and osmotic stress-activated protein kinases were found to positively regulate aurofusarin production and negatively regulate trichothecene production [90].

2.7.3. Regulation by Metal Ions

Some metal ions have been revealed to affect SM production in Fusarium fungi. Mg²⁺ and Mn²⁺ suppressed trichothecene production at relatively low concentrations, while Fe²⁺, Zn²⁺ and Co²⁺ enhanced trichothecene production at relatively high concentrations in F. graminearum [91,93,94]. Mechanism study showed that Co²⁺ stimulated production of trichothecene by activating Tri6 transcription in F. graminearum [91].

Iron starvation induced biosynthesis of ferrichrome in F. oxysporum f. sp. cubense (Foc) TR4. It indicated that Foc TR4 produced hydroxamate, siderophore, and ferrichrome in response to iron starvation [95].

2.7.4. Regulation by Other Additives

Four PDA media from different manufacturers such as VWR, Fluka, Sigma, and Oxoid were used to examine their effects on the metabolite profiles of four Fusarium species (i.e., F. pseudograminearum, F. graminearum, F. fujikuroi, and F. avenaceum) using HPLC-HRMS analysis, from which the significant differences in intensity of nine out of ten metabolites were observed [92].

The poor nitrogen sources, alkaline pH, low iron availability, and CWI MAPK signaling were proven to be associated with increased production of fusaric acid in F. oxysporum [96].

Fusarielin-type polyketides are a therapeutically promising class of Fusarium metabolites. The combination of disaccharides, dextrin and arginine significantly increased the yield of fusarielin in F. graminearum and F. tricinctum [97].

The endophytic fungus F. tricinctum was cultured on the solid rice medium supplemented with fruit and vegetable juices, which resulted in an 80-fold increase in the accumulation of fusarielins A, B, J, K and L. However, when the fungus was grown in rice media lacking vegetable juice or fruit juice, fusarielin J was screened. In the presence of apple juice and carrot juice, the accumulation of fusarielin J was observed to be the most, while the stimulating effect of banana juice was weaker [98]. A similar study found that fermentation of F. tricinctum in solid beans and liquid Wickerham medium versus cultivation on solid rice medium resulted in an increase in the production of enniatins in the cultures of F. tricinctum with solid beans [99].

The influences of the substrates on mycotoxin production by F. verticillioides were studied. Maize meal agar medium was beneficial to the production of fumonisins A1 and B1, while malt extract agar was conducive to the production of fumonisins A2 and B2. Other mycotoxins such as fusarins, bikaverin derivatives and fumonisin analogs with different growth conditions were also identified [43].

3. Global and Pathway-Specific Transcriptional Factor Regulation on Fusarium SM Production

In the biosynthesis of fungal SMs, transcriptional factors (TFs) are generally divided into two groups. The first group is global TFs, which are basically located outside SM BGCs, controlling a variety of secondary metabolic pathways. These TFs mediate fungal responses to environmental signals. Another group are pathway-specific TFs, which are basically located in the specific SM BGCs and affect gene expression in the clusters. In Fusarium fungi, the global TFs are in response to carbon and nitrogen sources, ambient light, and pH [100], the pathway-specific TFs mainly regulate the expression of secondary metabolism-related genes in the cluster where they are located [101].

3.1. Global Transcriptional Factor Regulation

The global TFs are able to regulate secondary metabolism in addition to regulating mycelial differentiation, sporulation, and other developments. The global TFs can not only sense the changes of nutrient components in the media, but also the changes of environmental signals. They are capable of associating with other TFs for transmission, finally regulating multiple secondary metabolic BGCs in fungi to adapt to environmental changes [100]. Some global TFs have been reported to regulate secondary metabolism including LaeA, LaeB, velvet proteins, AreA, AreB, and PacC in Fusarium fungi.

3.1.1. Regulation of LaeA, LaeB and Velvet Proteins

Global transcriptional regulators in response to ambient light in Fusarium fungi included LaeA, LaeB and velvet proteins (i.e., VeA, VelB and VelC). Among these transcriptional factors, VeA was considered the central player of the light regulatory network in Fusarium species [18].

1. Regulation of LaeA and LaeB

F. fujikuroi was the pathogen of rice bakanae to produce many SMs such as bikaverin, gibberellins, fusaric acid, fusarubins, and fusarins. Among them, fusaric acid and fusarins belonged to the mycotoxins, and gibberellins belonged to phytohormones [66]. LaeA had a positive regulatory effect on the production of certain metabolites in F. fujikuroi. For example, deletion of the FflaeA gene in F. fujikuroi resulted in a decrease in the production of gibberellins A3 and A4, fusarin C, fumonisins B1, B2, B3 and B4, DON, and 15-AcDON [102]. Subsequently, similar results have been confirmed. Deletion of the Fflae1 (FflaeA) gene led to a reduction in the production of fusaric acid, fusarinolic acid, and dehydrofusaric acid in F. fujikuroi [103]. In addition, the deletion of Fflae1 led to a decrease in the production of gibberellins, fumonisins and fusarin C. Overexpression of Fflae1 resulted in an increase in the production of gibberellins in another F. fujikuroi strain [104]. Sometimes, LaeA negatively regulated some metabolite production in F. fujikuroi. The deletion mutant ΔFflaeA of F. fujikuroi showed an increase in the yield of bikaverin [102]. Another example was that deletion of the lae1 gene in F. fujikuroi led to upregulation of gibepyrone BGC expression as well as increased production of gibepyrones A, B, C, D, E, and F [105].

LaeA positively regulated the production of metabolites in the following plant pathogenic Fusarium species. Deletion of FglaeA in F. graminearum led to a dramatic decrease in the production of trichothecenes and zearalenone. Overexpression of FglaeA caused an increase in the production of trichothecenes and zearalenone, which indicated that FgLaeA positively regulated production of phytotoxins by F. graminearum [28]. For F. oxysporum f. sp. niveum, the deletion of the FoLae1 gene led to a decrease in conidia yield, as well as a reduction in the production of bikaverin and fusaric acid. In addition, all these changes in the deleted mutants were restored in the corresponding complementation strains [29]. For F. oxysporum, the deletion of the gene laeA led to a decrease in the production of BEA and fusaric acid (FA), which contributed to the virulence to plant hosts such as tomato plants [106]. For F. verticillioides, the deletion of the laeA gene reduced production of fusarin C, bikaverin, fusaric acid and fumonisins [107].

LaeB, an orthologue similar to LaeA, was first identified using a forward genetic screening in Aspergillus nidulans [108]. LaeB was involved in regulating the production of sterigmatocystin and other polyketides [109]. FpLaeB, an orthologue of LaeB protein, was required to regulate the secondary metabolism in F. pseudograminearum. The generation of DON was impaired in the FpLaeB deletion mutant via UHPLC-MS/MS assay. FpLaeB was also important for the formation of conidia as the FpLaeB deletion mutant formed fewer conidia in the induced medium. In addition, the FpLaeB deletion mutant showed reduced sensitivity to the cell wall integrity inhibitors, and its growth was more severely inhibited by the cell membrane inhibitor sodium dodecyl sulfate (SDS) than that of the wild-type strain. More importantly, when the ΔFpLaeB mutant was inoculated on the stem base or head of wheat, its virulence was decreased. These results indicated that FpLaeB played an important role in the growth, development, and maintenance of the cell walls, as well as membrane integrity. More importantly, FpLaeB was necessary for SM production and complete virulence of F. pseudograminearum [110]. Some examples of LaeA and LaeB modulating SM production in Fusarium fungi are shown in Table 1.

Table 1.

Some examples of LaeA and LaeB regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of laeA or laeB	Positive/Negative Regulation	Production of SM	Ref.
F. fujikuroi	Deletion of laeA	Negative	Increased production of bikaverin.	[102]
F. fujikuroi	Deletion of laeA	Positive	Decreased production of gibberellins A3 and A4, fusarin C, fumonisins B1, B2, B3 and B4, DON, and 15-AcDON.	[102]
F. fujikuroi	Deletion of laeA	Positive	Decreased production of fusaric acid, fusarinolic acid, and dehydrofusaric acid.	[103]
F. fujikuroi	Deletion and overexpression of laeA	Positive	Deletion of laeA led to decreased production of gibberellins, fumonisins and fusarin C. Overexpression of laeA led to increased production of gibberellins.	[104]
F. fujikuroi	Deletion of laeA	Negative	Increased production of gibepyrones A, B, C, D, E, and F	[105]
F. graminearum	Deletion and overexpression of laeA	Positive	Deletion of FglaeA led to a dramatic reduction in production of trichothecenes and zearalenone. Overexpression of FglaeA caused the increased production of trichothecenes and zearalenone.	[28]
F. oxysporum	Deletion of laeA	Positive	Decreased production of BEA and FA	[106]
F. oxysporum f. sp. niveum	Deletion of laeA	Positive	Decreased production of bikaverin and FA	[29]
F. verticillioides	Deletion of laeA	Positive	Decreased production of bikaverin, FA, fusarin C and fumonisins.	[107]
F. pseudograminearum	Deletion of laeB	Positive	Decreased production of DON in the ΔFpLaeB mutant	[110]

• 2.Regulation of Velvet Proteins VeA, VelB and VelC

The deletion of the veA gene in F. fujikuroi resulted in a decrease in the production of fusarin C, fumonisins B1, B2, B3 and B4, DON, 15-AcDON, and gibberellins A3 and A4. However, in the veA deletion strain, there was an increase in the production of bikaverin [102]. The subsequent results showed that the deletion of Ffvel1 (FfveA) resulted in a decrease in the production of fusarinolic acid, fusaric acid, and dehydrofusaric acid in F. fujikuroi strain [103]. Deletion of vel1 resulted in a decrease in the production of gibberellins, fumonisins and fusarin C. Overexpression of lae1 increased in the production of gibberellins in another F. fujikuroi strain [104]. The deletion of vel1 gene in F. fujikuroi led to the upregulation of gibepyrone BGC expression, and an increase in the production of gibepyrones A, B, C, D, E, and F as well [105].

Deletion of veA in F. graminearium resulted in a decrease in the production of DON (also called vomitoxin) [111], and also led to a decrease in the production of trichothecenes [112].

The overexpression of FnveA in F. nematophilum led to an increase of the antitumor activity of the crude extract against A549 cancer cells. Unfortunately, the antitumor compounds needed further identification [113].

The deletion of veA in F. oxysporum led to a decrease in the production of fusaric acid and beauvericin, which resulted in virulence to plant hosts such as tomato plants [106]. The deletion of the Fovel1 gene in F. oxysporum f. sp. niveum resulted in a decrease in the number of conidia, and a decrease in the production of fusaric acid and bikaverin. Furthermore, all these alterations in the deleted strains were restored in the corresponding complementary strains [29].

The global regulator VeA in F. solani, an endophytic fungus isolated from the medicinal plant Nothapodytes pittosporoides (Icacinaceae), was overexpressed. The antitumor activity of the crude extract was greatly increased. Metabolomics analysis showed that there were 48 key genes related to antitumor activity. Unfortunately, the antitumor compounds were not identified in the extract [114]. Another global regulator VeA was found to negatively regulate the transcription factor MtfA, which in turn targeted negatively regulating transcriptional levels of PRPS2 to mediate acadesine (AICAR) biosynthesis in F. solani [115].

When the Fvvel1 (FvveA) gene was deleted in F. verticillioides, the production of gibberellins, fusarin C, fumonisins B1, B2, B3 and B4, DON, and 15-AcDON was decreased. However, in the ΔFvvel1 mutant, the production of bikaverin was increased. The mechanisms of the gene Fvvel1 on the aforementioned metabolite production should be similar to those of FvlaeA in F. verticillioides [102]. The deletion of veA in maize pathogen F. verticillioides led to a decrease in the production of fusarin C, fumonisins B1, B2 and B3 [116]. Further investigation indicated that VeA was necessary for causing symptoms and mycotoxin synthesis in maize seedlings by F. verticillioides [117].

When the vel2 (velB) gene was deleted in F. fujikuroi, gibepyrone BGC expression was upregulated, and the production of gibepyrones A, B, C, D, E, and F was also increased in F. fujikuroi [105].

When the gene FgvelB was deleted in F. graminearum, the production of DON was decreased [118]. Production of trichothecenes and ZEN in the ΔFgvelB mutant of F. graminearum was also significantly reduced compared with the WT strain [119]. A similar example was that the deletion of FpvelB led to singificant differences in growth, conidiation, virulence and production of DON in F. pseudograminearum. In addition, FpVelB positively regulated another SM BGC associated with pathogenesis by modulating the expression of the gene PKS11. FpVelB regulated pathogen virulence by influencing DON biosynthesis in F. pseudograminearum [33].

F. proliferatum was the causative agent of rice spikelet rot disease. The disruption of FpvelC enhanced the production of fumonisin B1 and fusaric acid concomitantly. The transcripts of the BGC genes responsible for the biosynthesis of two mycotoxins were also significantly increased [120]. Some examples of VeA, VelB and VelC regulating SM production in Fusarium fungi are shown in Table 2.

Table 2.

Some examples of VeA, VelB and VelC regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of veA or velB	Positive/Negative Regulation	Production of SM	Ref.
F. fujikuroi	Deletion of veA	Negative	Increased production of bikaverin.	[102]
F. fujikuroi	Deletion of veA	Positive	Decreased production of gibberellins A3 and A4, fusarin C, fumonisins B1, B2, B3 and B4, DON, and 15-AcDON.	[102]
F. fujikuroi	Deletion of veA	Positive	Decreased production of fusaric acid, fusarinolic acid, and dehydrofusaric acid.	[103]
F. fujikuroi	Deletion of veA	Positive	Decreased production of gibberellins, fumonisins and fusarin C.	[104]
F. fujikuroi	Deletion of veA	Negative	Increased production of gibepyrones A, B, C, D, E, and F.	[105]
F. graminearium	Deletion of veA	Positive	Reduced production of DON.	[111]
F. graminearium	Deletion of veA	Positive	Decreased production of trichothecenes.	[112]
F. nematophilum	Overexpression of veA	Positive	Increased production of antitumor compounds.	[113]
F. oxysporum	Deletion of veA	Positive	Decreased production of BEA and FA.	[106]
F. oxysporum f. sp. niveum	Deletion of veA	Positive	Decreased production of bikaverin and FA.	[29]
F. solani	Overexpression of veA	Positive	Increased production of antitumor substances	[114]
F. solani	Deletion of veA	Negative	Increased production of acadesine	[115]
F. verticillioides	Deletion of veA	Positive	Decreased production of fusarin C and fumonisins B1, B2 and B3.	[116]
F. verticillioides	Deletion of veA	Positive	Decreased production of fumonisins.	[117]
F. fujikuroi	Deletion of velB	Positive	Decreased production of gibberellins, fumonisins and fusarin C.	[104]
F. fujikuroi	Deletion of velB	Negative	Increased production of gibepyrones A, B, C, D, E, and F	[105]
F. graminearum	Deletion of velB	Positive	Decreased production of DON.	[118]
F. graminearum	Deletion of velB	Positive	Decreased production of trichothecenes and ZEN.	[119]
F. proliferatum	Deletion of velC	Negative	Enhanced production of FB1 and FA	[120]
F. pseudograminearum	Deletion of velB	Positive	Decreased production of DON.	[33]

3.1.2. Regulation of AreA and AreB

AreA and AreB are global regulators belonging to the GATA transcriptional factor family in response to nitrogen sources that can modulate the production of SMs in fungi [121].

AreA could activate GA3 biosynthetic genes under nitrogen limitation in F. fujikuroi [122]. Both AreA and AreB were GATA-type transcriptional factors, which were studied in detail in F. fujikuroi. FfAreA shared 98% homology with the Cys₂/Cys₂ zinc finger domain of homologous fungi and acted as a GATA TF, directly binding to GATA/TATC elements in the promoter regions of six biosynthetic genes in the GA biosynthetic gene cluster. Therefore, AreA was considered the main regulator of GA3 biosynthesis, explaining the relationship between dynamic nitrogen status and yield [123]. By isolating, characterizing, and destroying AreA from F. fujikuroi, the dominant role of AreA in primary and secondary metabolic regulation was further revealed [124]. The transcriptional level and subcellular localization of AreA were preliminarily determined by the extracellular/intracellular nitrogen status. The accumulation of AreA in nuclear localization could continuously stimulate nitrogen metabolism, serving as a transcriptional factor targeting nitrogen-availability related genes [123].

AreB was identified as a negative regulatory factor of AreA-dependent genes in F. fujikuroi [125]. AreB acted as both repressor and activator of AreA-dependent genes, and three transcripts, including FfareB-a, FfareB-b, and FfareB-c, were discovered through alternative splicing with differential expression levels [126]. Further exploration was conducted on the subcellular localization of three transcripts and their interaction patterns with AreA. In most cases, the localization patterns of AreB-b and AreB-c were similar to AreA, while the localization of AreB-a under nitrogen-inhibited growth conditions was different. Fluorescence microscopy of AreB subcellular localization showed that AreB-a was the only nuclear localization. On the contrary, only a few F. fujikuroi hyphae showed nuclear localization fluorescence signals of AreB-b and AreB-c [126]. The nuclear localization heterodimers of AreA and AreB bound to the GATA/TATC elements of the target gene promoter and upregulate the biosynthesis of GA3 under nitrogen deprivation conditions [126]. The deletion of areA in F. fujikuroi revealed that nearly 24.5% of annotated transcription factors (TFs) were affected by nitrogen starvation, and 30% of TFs were affected by areB- deficiency during nitrogen starvation or nitrogen sufficiency [127].

AreA exhibited critical roles in regulating the production of DON by ammonium and cyclic adenosine monophosphate (cAMP) signaling in F. graminearum. The vegetative growth and DON yield of the ΔareA mutant were significantly reduced in F. graminearum cultures. The interaction between AreA and Tri10 (TRI10) might be related to its role in regulating Tri gene expression. Further research suggested that AreA participated in regulating DON production through ammonium inhibition and the cAMP-PKA pathway [128].

Another study showed that the ΔFgareA mutation triggered loss of trichothecene biosynthesis but did not affect zearalenone biosynthesis in F. graminearum [129]. Furthermore, overexpression of area increased production of gibberellins and bikaverin in F. graminearum [130].

AreA contributed to chromatin accessibility and expression of two velvet-regulated BGCs, encoding the biosynthesis of BEA and ferricrocin in F. oxysporum [131].

F. proliferatum was the pathogen of rice spikelet disease. The ΔFpareA mutant of F. proliferatum did not utilize nitrate as the N source, but instead utilized ammonium (NH₄⁺) or glutamine as the N source. Except for using 120 mmol/L of ammonium chloride (NH₄Cl) as the N source, the ability of the ΔareA mutant to biosynthesize fumonisin was significantly reduced [132].

The ΔareA mutant could not produce FB1 under either low or high nitrogen levels in F. verticillioides, which indicated that AreA profoundly affected fumonisin biosynthesis [133]. The deletion of the gene FUG1 reduced the production of fumonisins (i.e., FB1, FB2, and FB3) in F. verticillioides. Further RNA-seq analysis showed that AreA was downregulated in the FUG1 deletion strain of F. verticillioides, indicating that FUG1 might affect fumonisin biosynthesis by directly or indirectly regulating AreA. These results collectively provided important evidence that AreA and/or nitrogen sources regulated fumonisin biosynthesis [134]. Some examples of AreA and AreB regulating SM production in Fusarium fungi are shown in Table 3.

Table 3.

Some examples of AreA and AreB regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of areA and areB	Positive/Negative Regulation	Production of SM	Ref.
F. graminearum	Deletion of areA	Positive	Decreased production of DON	[128]
F. graminearum	Deletion of areA	Positive	Decreased production of trichothecene biosynthesis	[129]
F. graminearum	Overexpression of areA	Positive	Increased production of gibberellins and bikaverin	[130]
F. oxysporum	Deletion of areA	Positive	Decreased production of ferricrocin and BEA	[131]
F. proliferatum	Deletion of areA	Positive	Significantly decreased production of fumonisin	[132]
F. verticillioides	Deletion of areA	Positive	Lack of FB1 production	[133]

3.1.3. Regulation of PacC

PacC belonged to the Cys₂His₂ zinc finger family and recognized the DNA sequence 5-GCCARG-3. It was a key TF for fungal pH regulators [135]. PacC could regulate the production of various SMs in Fusarium fungi. Deletion of the pH regulatory gene pacC in F. fujikuroi resulted in partial derepression of the bik gene at acidic ambient pH, and led to a significant reduction in bikaverin synthesis [136].

The production of trichothecene was induced only under acidic pH conditions in F. graminearum. The ΔFgPac1 mutant was constructed, which showed decreased development at neutral and alkaline pH, increased sensitivity to H₂O₂ and an earlier induction of Tri gene and toxin accumulation at acidic pH. The strain expressing the FgPac1c constitutively active form of Pac1 exhibited strongly repressed Tri gene expression and reduced mycotoxin accumulation at acidic pH. The results demonstrated that Pac1 negatively regulated Tri gene expression and mycotoxin production in F. graminearum [137].

The pH of the environment surrounding F. proliferatum cells could affect the production of FB1 and FB2 as well as the expression of FUM. Further investigation on the molecular mechanism of fumonisin synthesis in F. proliferatum indicated that different pH conditions led to the production of different fumonisins. It was also noted that some changes in protein accumulation were paralleled by the production pattern of fumonisins. Further analysis of the potential functions of these proteins suggested that they might be related to SM biosynthesis and the structural modifications of fumonisins. Therefore, these differential responses indicated that the biosynthesis of fumonisin played a mediating role under different pH conditions [138].

Deletion of PAC1 in F. verticillioides induced an increase in the production of FB1 and in the expression of FUM1 when the fungus was cultured on maize kernels under acidic pH conditions, indicating the regulatory role of PAC1 in FB1 biosynthesis [139].

3.2. Pathway-Specific Transcriptional Factor Regulation

According to the genome information obtained, about 60% of fungal secondary metabolism BGCs contain pathway-specific transcriptional factors (TFs) that regulate the expression of secondary metabolism-related genes in the cluster. 90% of pathway-specific TFs belong to zinc finger proteins [140].

Overexpression of the pathway-specific TF gene FvFum21 in F. fujikuroi strongly activated the FUM cluster genes, leading to a 1000-fold increase in FBx levels [141].

Two pathway-specific Zn(II)₂Cys₆-type TFs, namely Fub10 and Fub12, were involved in fusaric acid BGC in F. fujikuroi. Fub10 positively regulated the expression of all FUB genes, while Fub12 participated in the bioconversion of the two fusaric acid derivatives, i.e., dehydrofusaric acid and fusarinolic acid, as a biotransformation detoxification [142].

GIP2 was a pathway-specific TF that regulated the aurofusarin BGC in F. graminearum. The analysis of targeted gene deletion and complementation confirmed that GIP2 was needed for the biosynthesis of aurofusarin. Overexpression of GIP2 in the wild-type strains increased aurofusarin production and reduced mycelial growth [143].

Activation of the local transcription factor FSL of the polyketide synthase 9 (PKS9) cluster led to production of fusarielins F, G and H in F. graminearum. The cytotoxicity of the three fusarielins was studied against colorectal cancer cell lines. Among them, fusarielin H showed more cytotoxic activity than fusarielins F and G [144].

Trichothecenes are isoprenoid mycotoxins isolated from wheat materials infected with the F. graminearum. Deletion of two pathway-specific TFs Tri6 and Tri10 led to greatly reduced production of toxins [71,145]. The expression of both Tri6 and Tri10 genes was later found to be stimulated by cyclic adenosine monophosphate (cAMP) treatment, which indicated that Tri6 and Tri10 genes were crucial for the regulation of DON biosynthesis by cAMP signaling in F. graminearum [146]. Tri6 (TRI6) was previously considered a global TF in F. graminearum [147,148], and later was recognized as a pathway-specific TF [100]. Tri6, a pathway-specific Zn(II)₂Cys₆-type TF in F. graminearum, directly bound to trichothecene BGC promoters under host-mimicking conditions, coordinating DON synthesis with infection stages [41,149].

ZEB2 was a pathway-specific TF belonging to the bZIP family. ZEB2 expression positively regulated zearalenone production in F. graminearum [150].

The FUM cluster gene fum21 encoded a Zn(II)₂Cys₆-type TF. The production of fumonisins was decreased in the knockout mutant ΔFvfum21 of F. verticillioides [151]. Some examples of the pathway-specific transcriptional regulation of Fusarium secondary metabolism are shown in Table 4.

Table 4.

Some examples of pathway-specific transcriptional factors regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of the Pathway-Specific TF Gene	Positive/Negative Regulation	Production of SM	Ref.
F. fujikuroi	Overexpression of Fum21	Positive	Strongly activated the FUM cluster genes leading to 1000-fold elevated FBx levels.	[141]
F. fujikuroi	Deletion of Fub10	Positive	Decreased production of FA	[142]
F. graminearum	Deletion of GIP2	Positive	Decreased production of aurofusarin	[143]
F. graminearum	Overexpression of GIP2	Positive	Increased production of aurofusarin	[143]
F. graminearum	Activation of FSL	Positive	Induced production of fusarielins F, G and H	[144]
F. graminearum	Deletion of Tri6	Positive	Reduced production of trichothecenes	[145]
F. graminearum	Deletion of Tri10	Positive	Reduced production of trichothecenes	[145]
F. graminearum	Activation of ZEB2	Positive	Increased production of ZEN	[150]
F. verticillioides	Deletion of fum21	Positive	Decreased production of fumonisins	[151]

3.3. Miscellaneous Transcriptional Factor Regulation

Most eukaryotic transcriptional factors (TFs) could be classified into different groups based on the types of DNA-binding domains. They included basic region/leucine zipper (bZIP), MADS box, myb, homeobox helix–loop–helix, and zinc fingers. Among them, bZIP TFs were involved in fungal stress response, asexual development and other cell processes [152]. Most of the transcriptional factor regulations belong to the global regulations in response to environmental stresses. Other reported miscellaneous global regulations in Fusarium species include regulations of Sge1, HXK1, and AtfA.

The esyn1 gene was responsible for the modulation of enniatins (ENs) biosynthesis in F. avenaceum. Activation of esyn1 transcription led to increased production of ENs [24].

MeaB belonging to the bZIP TF demonstrated a completely different regulatory capability from Cpc1 [153]. Two distinct MeaB TFs, MeaBL and MeaBS from F. fujikuroi, were expressed in an AreA-dependent manner, which depended on the availability of nitrogen. During the nitrogen-sufficient period, especially with the addition of glutamine, MeaBL appeared more frequently in the nucleus, while MeaBS appeared to be dysfunctional as it remained isolated in the cytoplasm [154]. Under nitrogen starvation, blocking MeaB could slightly upregulate GA cluster genes and some intracellular nitrogen transport channels [153].

In F. fujikuroi, the global regulator FfSge1 was required for expression of SM gene clusters, but not for conidiogenesis and pathogenicity. Its overexpression in the wild-type background led to increased production of fumonisin (FUM), fusaric acid (FU) and apicidin F (APF) under the optimal conditions. It was noteworthy that FU, APF, and FUS were produced even under non-favorable conditions, which indicated that overexpression of FfSGE1 could override nitrogen regulation [155]. Deletion of sge1 in F. verticillioides also led to decreased production of fumonisins including FB₁, FB₂ and FB₃ [156].

Transcription factor ART1, a predicted Zn(II)₂Cys₆ zinc finger TF, mediated starch hydrolysis and mycotoxin production in F. graminearum and F. verticillioides. ART1 played an important role in the production of both trichothecene and fumonisin by the regulation of genes involved in starch hydrolysis [157].

FgStuA in F. graminearum was a TF gene that shared homology with key developmental regulators in fungi. The deletion mutant ΔFgStuA significantly reduced the pathogenicity on wheat heads and the production of SMs. The production of red pigment aurofusarin was decreased in the ΔFgStuA mutant. The ability of the ΔFgStuA mutant to synthesize 15-AcDON and DON was also decreased [158]. Further investigation showed that the TF FgStuA regulated virulence and mycotoxin biosynthesis via recruiting the SAGA complex in F. graminearum [159].

The transcription factor FoAce2 (encoding F. oxysporum angiotensin converting enzyme 2) was found to regulate vegetative growth, virulence, conidiation, and cell wall homeostasis of F. oxysporum f. sp. cubense. In the ΔFoAce2 mutant, three biosynthesis genes of BEA were down-regulated, resulting in a decrease in BEA production [160].

C₂H₂ was the most common TF in the zinc finger TF family, widely conserved from single-celled organisms to higher mammals [161,162]. F. oxysporum f. sp. lycopersici C₂H₂ TF FolCzf1 was needed for the production of conidia and fusaric acid, and early host infection. Compared with those of WT and ΔFolCZF1-C strains, the ΔFolCZF1 strain showed a significant decrease in FA production, indicating that FolCZF1 was involved in the biosynthesis of FA. In addition, under favorable conditions for FA production, the expression level of FA biosynthesis genes in F. oxysporum f. sp. lycopersici was significantly reduced, which further supported the role of FolCzf1 in regulating FA production [163].

Fp487 was a Zn₂Cys₆ transcription factor in F. pseudograminearum. Compared with the wild-type strain CF14047, the conidiation, pathogenicity, and production of 3-AcDON of the ΔFp487 mutant significantly decreased [164].

ZRF1 was a zinc binuclear cluster-type gene in F. verticillioides. The gene ZFR1 deletion mutant exhibited normal growth and development on maize kernels, but the production of fumonisin was decreased to less than 10% of that of the wild-type strain. Overexpression of ZFR1 in ΔFvzfr1 mutant restored FB1 production to wild-type levels, which indicated that ZFR1 was a positive regulator of FB1 biosynthesis in F. verticillioides [165].

The HAP complex was a conserved, heterotrimeric transcriptional regulator that bound the consensus sequence CCAAT to modulate gene expression in F. verticillioides. The Hap3 subunit linked the HAP complex to regulate fumonisin biosynthesis. Deletion of HAP3 suppressed fumonisin biosynthesis [166].

The gene hxk1 was a putative hexokinase-encoding gene to modulate carbon catabolism, sporulation, FB1 production and pathogenesis in F. verticillioides. The Δhxk1 mutant produced about 50% and 80% less trehalose than the WT strain, respectively [167].

The gene FvatfA from the maize pathogen F. verticillioides putatively encoded bZIP-type transcription factor FvAtfA, which was homologous to the Aspergillus nidulans AtfA and Schizosaccharomyces pombe Atf1. Deletion of FvatfA led to the overproduction of bikaverin and abolishment of fumonisin production in the ΔFvatfA strain [168].

MADS-box TFs played a role in virulence, and vegetative and sexual development of F. verticillioides. Two MADS-box TFs, Mads1 and Mads2, in terms of their roles in secondary metabolism and sexual mating. The MADS1 and MADS2 knockout mutants exhibited decreased vegetative growth and FB1 production when compared to the wild-type strain. Mads1 was a broad regulator of secondary metabolism in F. verticillioides, and might target regulons upstream of Mads2 to affect FB1 production [169].

FvOshC was identified as the specific protein that bound to ergosterol in F. verticillioides. Gene knockout complementation techniques confirmed that FvOshC acted as a global regulatory protein to play a positive role in the pathogenicity and FB1 biosynthesis in F. verticillioides [170].

Fusarium sp. was an endophytic fungus isolated from the ambrosia beetle Xylosandrus morigerus. FspTF was a member of the fungal-specific family of transcription factor KilA-N/APSES. The deletion mutant ΔFsptf could not synthesize the pigments javanicin and fusarubin which indicated that FspTF positively regulated pigment production [171]. Some examples of the miscellaneous TFs regulating SM production in Fusarium fungi are displayed in Table 5.

Table 5.

Some examples of the miscellaneous transcriptional factors regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of the TF Gene	Positive/Negative Regulation	Production of SM	Ref.
F. avenaceum	Overexpression of esyn1	Positive	Increased production of ENs.	[24]
F. fujikuroi	Deletion of meaBL and meaBS	Negative	Increased production of GAs	[153]
F. fujikuroi	Overexpression of FfSge1	Positive	Increased production of FUM, FU, and APF in the FfSge1-OE strain	[155]
F. verticillioides	Deletion of sge1	Positive	Decreased production of fumonisins.	[156]
F. verticillioides	Deletion of art1	Positive	Decreased production of fumonisins including FB1	[157]
F. graminearum	Deletion of Fgart1	Positive	Decreased production of trichothecenes	[157]
F. graminearum	Deletion of FgStuA	Positive	Reduced production of DON and 15-AcDON	[158]
F. oxysprum f. sp. cubense	Deletion of FoAce2	Positive	Decreased production of BEA.	[160]
F. oxysporum f. sp. lycopersici	Deletion of FolCzf1	Positive	Reduced production of FA	[163]
F. pseudograminearum	Deletion of Fp487	Positive	Reduced production of 3-AcDON	[164]
F. verticillioides	Deletion and overexpression of zfr1	Positive	Positive regulation of fumonisin production	[165]
F. verticillioides	Deletion of HAP3	Positive	Suppressed production of fumonisins.	[166]
F. verticillioides	Deletion of hxk1	Positive	Decreased production of trehalose and FB1	[167]
F. verticillioides	Deletion of FvatfA	Negative	Overproduction of bikaverin in the ΔFvatfA strain	[168]
F. verticillioides	Deletion of FvatfA	Positive	Abolishment of fumonisin production in the ΔFvatfA strain	[168]
F. verticillioides	Deletion of MADS1 and MADS2	Positive	Decreased production of FB1	[169]
F. verticillioides	Deletion of FvOshC	Positive	Decreased production of FB1	[170]
Fusarium sp.	Deletion of Fsptf	Positive	Decreased production of javanicin and fusarubin	[171]

4. Epigenetic Regulation on Fusarium SM Production

Epigenetic regulation on SM production in fungi mainly includes modifications of histone, DNA and RNA. DNA in chromatin is organized in an array of nucleosomes. Two copies of each histone protein subunit, including H2A, H2B, H3, and H4, are assembled into an octamer surrounding by DNA of 145 to 147 base pairs, forming the core of the nucleosome [172].

Chromatin structure is the basis for regulating gene expression. The loose structure of euchromatin is associated with transcriptional activity, while the tight structure of heterochromatin is related to transcriptional repression. The epigenetic regulatory mechanisms mainly include histone acetylation, histone methylation, recognition by reader modules, sumoylation, phosphorylation, ubiquitylation, and DNA methylation, which are involved in the control of DNA expression [45].

Histone acetylation modification is controlled by two classes of enzymes known as histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs catalyze the transfer of acetyl groups from acetyl-CoA to the ε-amino group on the side chain of lysine residues of the core histones, typically forming a part of the complex [173]. On the contrary, HDACs remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins, and thus significantly affect chromatin remodeling and transcriptional regulation in eukaryotes. Histone methylation modification is regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs), which are used to add and remove methyl groups on lysine and arginine residues, respectively. So the gene expression is regulated through the synergistic effect of HATs and HDACs, or HMTs and HDMs [22].

Chromatin modifications and heterochromatic labeling are associated with the regulation of fungal secondary metabolism BGCs [174]. In Fusarium fungi, some epigenetic regulation strategies have been reported to regulate secondary metabolism including the regulations of HATs, HDACs, HMTs, HDMs, chromatin readers, DNA methylation, and RNA modifications. Epigenetic regulation on Fusarium SM production should be an efficient strategy by activating useful metabolite production or inhibiting toxic metabolite production [21,22,175,176].

4.1. Regulation of HATs

Histone acetylation by HATs leads to activation of euchromatin to positively regulate gene expression of fungal secondary metabolism [22].

In the Δelp3 mutant of F. graminearum, the amount of perithecia formed was reduced and maturation of perithecia was delayed.

The main trichothecenes such as DON and 15-AcDON were not detected in the ΔFgelp3 mutant, while both DON and 15-AcDON were produced at detectable levels in the WT strain. The RT-qPCR analysis showed that transcription of the trichothecene biosynthesis genes Fgtri5 and Fgtri6 was significantly reduced in the ΔFgelp3 mutant compared with the WT strain. In a virulence test, 21 days after wheat head inoculation, the disease symptoms caused by the Δelp3 mutant were significantly reduced, while the wild-type and complementary strains caused typical wheat blight symptoms. This indicated that the HAT gene Fgelp3 was involved in various biological processes, including sexual and asexual reproduction, SM production, and virulence in F. graminearum [177].

DON was a mycotoxin produced by Fusarium species. This mycotoxin was a virulence factor that assisted fungi in colonizing and spreading within spikes. DON, originally known as vomitoxin, has severe vomiting effects on some animals such as humans, pigs, dogs and minks [178]. In F. fujikuroi, the ΔFfGcnE mutant showed an obvious deficiency of H3K9/K14/K27ac and the reduced production of 18 metabolites [179]. The deletion of FgGcnE in F. graminearum led to decreased production of DON [180,181,182]. In addition, the deletion of the bromodomain of FgGcn5 led to a significant reduction in DON production and virulence of F. graminearum [183].

The deletion of FgSas3 reduced production of DON, and reduced sporulation and perithecium formation, which demonstrated that Sas3 positively regulated DON biosynthesis and sporulation in F. graminearum [180].

Deletion of hat1 resulted in downregulation of GA gene expression and decreased GA production in F. fujikuroi. Instead, overexpression of hat1 resulted in an upregulation of GA gene expression and an increase in the production of GAs [104].

Although the Fghat1 deletion mutant (ΔFghat1) of F. graminearum was normal in fungal growth, asexual and sexual development, and pathogenicity, it had severe defects in DON production. Exogenous cAMP treatment rescued the defects of the ΔFghat1 mutant in DON production, indicating a relationship between FgHat1 and cAMP signaling in F. graminearum [184].

The ΔFghat2 mutant of F. graminearum produced significantly decreased levels of DON compared to the wild-type strain. Compared with the wild-type strain, the expression levels of Tri6 and Tri12 in the ΔFghat2 mutant were significantly reduced, indicating that FgHAT2 was essential for DON biosynthesis in F. graminearum [185]. Some examples of HATs regulating SM production in Fusarium fungi are displayed in Table 6.

Table 6.

Some examples of HATs regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of the HAT Gene	Positive/Negative Regulation	Production of SM	Ref.
F. graminearum	Deletion of elp3	Negative	Decreased production of trichothecenes such as DON and 15-AcDON	[177]
F. fujikuroi	Deletion of GcnE	Positive	Decreased production of 18 metabolites	[179]
F. graminearum	Deletion of gcn5	Positive	Decreased production of DON.	[180]
F. graminearum	Deletion of gcn5	Positive	Inhibited production of DON.	[181]
F. graminearum	Deletion of gcn5	Positive	Inhibited production of DON.	[182]
F. graminearum	Deletion of the bromodomain of FgGCN5	Positive	Significant reduction in DON production	[183]
F. fujikuroi	Deletion of hat1	Positive	Decreased GA production.	[104]
F. fujikuroi	Overexpression of hat1	Positive	Increased GA production.	[104]
F. graminearum	Deletion of sas3	Positive	Decreased production of DON.	[178]
F. graminearum	Deletion of hat1	Positive	Severe defects in DON production	[184]
F. graminearum	Deletion of hat2	Positive	Decreased production of DON	[185]

4.2. Regulation of HDACs

Histone deacetylation by HDACs leads to the formation of heterochromatin and suppresses gene expression. So HDACs negatively regulate gene expression of fungal secondary metabolism [22,45]. HDACs regulating SM production in Fusarium fungi included class I HDACs (i.e., Hda2, Hos2, and Rpd3), class II HDACs (i.e., HdaA, Hdf1, and Hdf2) and class III HDACs (i.e., Hst2, Sir2, and SirD).

4.2.1. Regulation of Class I HDACs

If the gene Ffhda2 in F. fujikuroi was deleted, the production of bikaverin, fusarubin, fusaric acid, and gibberellins (GAs) including GA3, GA4 and GA7 was decreased. It was estimated that FfHda2 was necessary for the virulence of F. fujikuroi by regulating the production of SMs in rice seedlings [186].

An increase in H4K16ac levels was observed in the Fvhos2 deletion mutant of F. verticillioides. The fumonisin B1 production was decreased in the ∆Fvhos2 strain, which meant FB1 biosynthesis was positively regulated by FvHos2. Accordingly, in the ∆Fvhos2 strain, the expression of FUM genes such as FUM1, FUM8, FUM19, and FUM21, was significantly reduced [35].

Overexpression of FvRpd3 in F. verticillioides increased FB1 production. Therefore, FUM genes such as FUM1, FUM8, FUM19, and FUM21, showed significantly high expression in the FvRpd3-OE strain [35].

4.2.2. Regulation of Class II HDACs

If the gene Hda1 was deleted in F. fujikuroi, the production of BEA would increase by 1000 times. However, the deletion of the gene Ffhda1 in F. fujikuroi led to a decrease in the production of plant hormones GAs including GA3, GA4 and GA7, as well as a decrease in the production of bikaverin, fusarubin and fusaric acid. Only the production of fusarin A was increased. It was estimated that FfHda1 was required for the virulence of rice seedlings [187].

Deletion of Fvhda1 in F. verticillioides increased in the production of FB1, indicating that FvHda1 negatively regulated the biosynthesis of fumonisins. In addition, the RT-qPCR revealed an increase in FUM1 expression in the ΔFvhda1 mutant [35].

The absence of Fghdf1 significantly reduced the virulence and DON production of F. graminearum. Furthermore, the ∆Fghdf1 mutant had stronger tolerance to H₂O₂ than the WT strain [188].

If the gene Fahdf2 was deleted in F. asiaticum, the production of 4-AcNIV and 4,15-diAcNIV would increase [189].

4.2.3. Regulation of Class III HDACs

Class III HDACs belong to sirtuin type NAD⁺-dependent deacetylases whose activities are sensitive to intracellular NAD⁺ availability [190]. Class III HDACs including Hst2 (HstB, SirT2), Hst4 (SirT4), SirT5, Hst4, SirA, Sir2, SirD (Sir4), and SirE have been reported to regulate fungal secondary metabolism [22].

Hst2 was also named HstB or SirT2. If the gene Fvhst2 was deleted in F. verticillioides, the causal agent of destructive diseases of maize, the level of H4K16ac was increased. Correspondingly, the production of FB1 was increased, which indicated that the FB1 biosynthesis was negatively regulated by Fvhst2. In addition, when sugarcane and maize were infected with the ∆Fvhst2 mutant, the vegetative growth, conidiation, and virulence of the mutant were increased [35].

When the gene Fvsirt4 was overexpressed in F. verticillioides, FB1 production was decreased. Accordingly, the key FUM genes (i.e., FUM1, FUM8, and FUM19) involved in FB1 toxin synthesis were significantly decreased in the FvSirt4-OE strain [35].

SirT5 and Sir2 were involved in histone acetylation at H3K9, H3K14, H3K27, and H4K16 residues in fungi. Fumonisin B production was significantly reduced in the ΔFvsirt5 mutant of F. verticillioides, and the expression of genes (FUMs and PKSs) involved in secondary metabolism was also significantly down-regulated [191].

Sir2 was known as SirB. The absence of Fvsir2 in F. verticillioides led to an increase in FB1 production, which indicated that FvSirB negatively regulated fumonisin biosynthesis [35]. However, fumonisin B production was significantly reduced in the Δsir2 mutant of F. verticillioides, the expression of genes related to the biosynthesis of fumonisin was also significantly downregulated [191]. Some examples of HDACs regulating SM production in Fusarium fungi are shown in Table 7.

Table 7.

Some examples of HDACs regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of the HDAC Gene	Positive/Negative Regulation	Production of SM	Ref.
Overexpression /Deletion of Class I HDAC Gene
F. verticillioides	Deletion of hos2	Positive	Decreased FB1 production.	[35]
F. verticillioides	Overexpression of rpd3	Positive	Increased FB1 production.	[35]
F. fujikuroi	Deletion of hda2	Positive	Decreased production of GA3, GA4, GA7, BIK, FSR, and FU	[186]
Overexpression/Deletion of Class II HDAC Gene
F. fujikuroi	Deletion of hda1	Positive	Decreased production of GA3, GA4, GA7, bikaverin, fusarubin, and FA	[186]
F. fujikuroi	Deletion of hda1	Negative	Increased production of fusarin A	[186]
F. fujikuroi	Deletion of hda1	Negative	Increased production of BEA	[187]
F. verticillioides	Deletion of hda1	Negative	Increased production of FB1	[35]
F. graminearum	Deletion of hdf1	Positive	Decreased production of DON	[188]
F. asiaticum	Deletion of hdf2	Negative	Increased production of 4-ANIV and 4,15-diAcNIV.	[189]
Overexpression/Deletion of Class III HDAC Gene
F. verticillioides	Deletion of hst2	Negative	Increased production of FB1	[35]
F. verticillioides	Overexpression of sirt4	Negative	Decreased production of FB1	[35]
F. verticillioides	Deletion of sir2	Negative	Increased production of FB1	[35]
F. verticillioides	Deletion of sirt5	Positive	Decreased production of FB1	[191]
F. verticillioides	Deletion of sir2	Positive	Decreased production of FB1	[191]

4.3. Regulation of HMTs

Histone methylation is the process of primarily adding methyl groups from S-adenosyl-1-methionine (SAM) to lysine or arginine residues through histone methyltransferases (HMTs). Histone methylation leads to chromatin tightening, making it difficult for related gene regions to be bound by TFs, RNA polymerases and other proteins, thereby inhibiting gene transcriptional activity. Therefore, HMTs negatively regulate gene expression of fungal secondary metabolism [192].

H3K27me3 was a major histone post-translational modification (PTM) in F. fujikuroi. Deletion of the methyltransferase Kmt6 reduced the burden of H3K27me3 and resulted in the induction of cryptic and silent SM BGCs in F. fujikuroi. One of the four putative SM BGCs, named STC5, was analyzed in more detail thereby revealing a new sesquiterpene (1R,4R,5S)-guaia-6,10(14)-diene [193]

The deletion of H3K27me3 had a more significant impact on the expression of SM BGCs than the intensely studied regulation by nitrogen. Deletion of kmt6 led to production of mycotoxins, pigments and other SMs in F. graminearum [194].

Lack of COMPASS component Ccl1 reduced H3K4 trimethylation levels and affected biosynthetic gene transcription and production of gibberellic acid in F. fujikuroi, and biosynthetic gene transcription and production of DON in F. graminearum [195].

Set2 and Ash1 were two HMTs targeting H3K36 in different chromatin regions in F. fujikuroi. In addition to HMT activity, Set2 also interacted directly with the elongation form of RNAPII through its SRI domain to activate gene transcription. H3K36me mediated by Ash1 in the sub-telomeric regions could inhibit the HMT activity of PRC2 on H3K27 and prevent the formation of heterochromatin. In addition, Set2 and Ash1 controlled the expression of multiple TFs and histone modifier-encoding genes, indirectly regulating the production of metabolites [196].

The methylation of lysine 20 of histone 4 (H4K20me) in F. fujikuroi and F. graminearum was functionally characterized. FfKMT5 in F. fujikuroi and FgKMT5 in F. graminearum were identified as solely responsible for H4K20 mono-, di- and trimethylation. The deficiency of Kmt5 had a significant impact on the secondary metabolism in two plant pathogens F. fujikuroi and F. graminearum with the most positive regulation on the biosynthesis of fusarin C in F. fujikuroi and zearalenone biosynthesis in F. graminearum [197].

FgSet1 was mainly responsible for monomethylation, dimethylation and trimethylation of H3K4 in F. graminearum [198]. The FgSet1 deletion mutant (ΔFgSet1) was impaired in hyphal growth and virulence. H3K4me was required for the active transcription of genes involved in DON and aurofusarin biosynthesis. Deletion of FgSet1 decreased production of DON and aurofusarin, which indicated that FgSet1 positively regulated biosynthesis of DON and aurofusarin [199].

Disruption of H3K27 methylation via Δkmt6 mutants of F. graminearum led to the production of a cyclic lipopeptide fusaristatin A. Overexpression of the gene kmt6 led to the production of three pyrone derivatives gibepyrone A, and fusapyrones A and B, which highlighted the role of chromatin remodeling in metabolic diversity [200].

The post-translational trimethylation of histone 3 lysine 9 (H3K9me3) was considered a marker of heterochromatin, and was established by the SET-domain protein Kmt1. FmKmt1 participated in H3K9me3 in F. mangiferae. The absence of FmKmt1 significantly affected fungal growth and stress response, which was essential for wild-type-like conidiation. Although FmKmt1 was essentially unnecessary for the biosynthesis of most known SMs, the deletion of FmKmt1 resulted in an almost complete loss of fusapyrone and deoxyfusapyrone [201].

F. proliferatum was a member of the F. fujikuroi species complex (FFSC). In the wild-type strain, the deletion of Fpkmt6 encoding the H3K27-specific histone methyltransferase resulted in the elevated expression of 49% of genes. However, the genes involved in the biosynthesis of the gibberellins (GAs) were among the most upregulated genes in the ΔFpkmt6 mutant. This indicated that H3K27me3 was involved in GA gene expression in F. proliferatum. The H3K27me3-specific methyltransferase FpKmt6 was the key regulator that inhibited the expression of secondary metabolism genes in F. proliferatum [32].

The deletion of FvSet1 in F. verticillioides led to various defects in fungal growth and pathogenicity. Furthermore, the ΔFvSet1 mutant exhibited a significant defect in FB1 biosynthesis with lower expression levels of FUM genes. FvSet1 played an important role in F. verticillioides in the responses to various environmental stresses by regulating the phosphorylation of FvMgv1 and FvHog1 [202].

FvSet2 in F. verticillioides was an ortholog of S. cerevisiae Set2. FvSet2 was responsible for the trimethylation of histone 3 lysine 36 (H3K36me3). The ΔFvSet2 mutant exhibited significant defects in vegetative growth, FB1 biosynthesis, pigmentation, and fungal virulence. In addition, trimethylation of H3K36 was important for the active transcription of genes related to the biosynthesis of FB1 and bikaverin [203]. Some examples of HMTs regulating SM production in Fusarium fungi are shown in Table 8.

Table 8.

Some examples of HMTs regulating SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of the HMT Gene	Positive/Negative Regulation	Production of SM	Ref.
F. graminearum	Deletion of kmt6	Negative	Increased production of SMs	[194]
F. fujikuroi	Deletion of Ccl1	Positive	Decreased production of GA3	[195]
F. fujikuroi	Deletion of set2 and ash1	Positive	Decreased production of SMs	[196]
F. fujikuroi	Deletion of kmt5	Positive	Decreased production of fusarin C	[197]
F. graminearum	Deletion of Ccl1	Positive	Decreased production of DON	[195]
F. graminearum	Deletion	Positive	Decreased production of ZEN	[197]
F. graminearum	Deletion of FgSet1	Positive	Decreased production of DON and aurofusarin	[199]
F. graminearum	Deletion of kmt6	Negative	Led to the production of fusaristain A	[200]
F. graminearum	Overexpression of kmt6	Positive	Led to the production of three pyrone derivatives gibepyrone A, and fusapyrones A and B	[200]
F. mangiferae	Deletion of Fmkmt1	Positive	An almost complete loss of fusapyrone and deoxyfusapyone	[201]
F. proliferatum	Deletion of Fpkmt1	Negative	Increased production of GAs	[32]
F. verticillioides	Deletion of FvSet1	Positive	Significant defect in FB1 biosynthesis.	[202]

4.4. Regulation of HDMs

Histone demethylation by HDMs leads to methyl groups being removed from lysine and arginine residues of histone. The chromatin is activated. So HDMs positively regulate gene expression of fungal secondary metabolism.

There are a few reports about the regulation of HDMs on fungal secondary metabolism. FgKdm5 in F. graminearum was a homolog of KDM5 proteins belonging to the diverse JmjC domain-containing superfamily. It had the function of the histone demethylase. Lack of FgKdm5 resulted in a significant decrease in the production of five SMs including DON, fusarin C, zearalenone, fusarielin H and chrysogine, which indicated that Fgkdm5 positively regulated SM production in F. graminearum [204].

4.5. Regulation of Chromatin Readers

The bromo-adjacent homology (BAH)-plant homeodomain (PHD) containing protein BAH–PHD protein 1 (BP1) was a reader for H3K27 methylation in F. graminearum, which was the cereal fungal pathogen. BP1 interacted with the core polycomb repressive complex 2 (PRC2) component Suz12 and directly bound methylated H3K27. BP1 was distributed in a subset of genomic regions marked by H3K27me3 and co-repressed gene transcription. The BP1 deletion mutant showed the same phenotypes in fungal growth and virulence. The expression profile of secondary metabolism genes was similar to that of strains lacking H3K27 methyltransferase Kmt6. Furthermore, BP1 could directly bind to DNA through its PHD finger, which might increase the residence of nucleosomes and enhance transcriptional inhibition of H3K27me3-labeled target regions. So BP1 negatively regulated secondary metabolism. BP1 was considered a novel methylated H3K27 reader that played important roles in fungal development and pathogenicity, as well as the production of SMs. Its orthologs were widely distributed in ascomycetes, indicating that the compounds which actively targeted BP1 could be used to manage plant fungal diseases, especially those caused by Fusarium species [27].

4.6. Regulation of DNA Methylation

DNA methylation plays important roles in eukaryotic gene expression and silencing, cell differentiation, and phylogeny. It may lead to changes in chromatin structures, DNA stability, and DNA-protein interactions, thereby affecting gene expression [205].

FgDIM-2 and FgRID were two DNA methyltransferases (DNMTs) in F. graminearum. FgDIM-2 was a homologue to DIM-2 (deficient in methylation) from N. crassa, and FgRID was a homologue to RID (repeat-induced point (RIP) deficient) from N. crassa. The production of 15-AcDON was increased by the dual-deletion strain ΔFgDim-2ΔFgRid, which indicated that the DNMTs negatively regulated 15-AcDON production [206].

4.7. Regulation of RNA Modifications

The regulation of RNA modifications mainly includes RNA methylation, RNA interference, and non-coding RNA. Two non-coding RNAs (ncRNAs) including long non-coding RNA (lncRNA) and small interfering RNA (siRNA), were found to regulate secondary metabolism in Fusarium fungi.

Both FgTri5 expression and DON biosynthesis were regulated by a long non-coding RNA (lncRNA), namely RNASP in F. graminearum. By replacing the promoter region of Tri5 with the promoter region of Tri12 to delete RNA5P, the expression of Tri5 and the biosynthesis of DON were increased [207].

Small interfering RNA (siRNA) mediated gene silencing led to the overproduction of bikaverin in Fusarium sp. HKF15 [208].

4.8. Miscellaneous Epigenetic Regulation

Miscellaneous epigenetic regulations include chromatin remodeling, histone phosphorylation, ubiquitylation, and sumoylation. In F. graminearum, if the genes Msg5 and Yvh1 of phosphatases were deleted, the production of DON was decreased, indicating the complex regulatory networks were involved in the production of Fusarium SMs [209].

Transcription activator FgDDT interacted with the chromatin remodeling factor FgISW1 to regulate the development and pathogenicity of F. graminearum [210].

FvHP1 was a speculated member of the heterochromatin protein 1 (HP1) family in F. verticillioides. FvHP1 retained the essential residues required for H3K9me2/3 recognition. Phenotypic analysis of the ∆FvHP1 mutant showed impaired vegetative growth, reduced conidiation and virulence, and altered FB1 production. In addition, the accumulation of red pigments (i.e., aurofusarin) in the mutant was linked to the deregulation of secondary metabolism, specifically the overproduction of fusarubin-type naphthoquinones, such as 8-O-methyl nectriafurone in F. verticillioides [211].

5. Regulation of Signal Transduction on Fusarium SM Production

Some signal transduction pathways have been revealed to regulate Fusarium SM production including the cAMP signaling pathway, TOR signaling pathway, MAPK signaling pathway, and G protein signaling pathway [23].

5.1. Regulation of cAMP Signaling Pathway

The cAMP signaling pathway (or called the cAMP pathway) includes regulatory subunits of cAMP-dependent protein kinase (PKA) to investigate their roles in fungal sexual development, colony structure, and the regulation of secondary metabolism.

The cAMP pathway had an impact on GA and bikaverin biosynthesis. cAMP inhibited fusarubin biosynthesis in F. fujikuroi [212].

The absence of the catalytic domain of the AcyA protein in F. fujikuroi resulted in various phenotypic changes, such as slower mycelial growth, increased synthesis of red pigments (i.e., aurofusarin), decreased synthesis of gibberellins, and partial activation of carotenoid biosynthesis even under dark conditions [213].

The biosynthesis of fusarin was subject to a complex control including regulators from diverse signaling pathways [214]. The gene FfacyA encoded an adenylate cyclase. It linked light regulation to cAMP signaling. AcyA was a positive regulator of fusarin biosynthesis in F. fujikuroi [214]. The gene carS encoded a RING finger protein repressor in F. fujikuroi [215]. It is involved in carotenoid regulation. CarS was a positive regulator of fusarin biosynthesis in F. fujikuroi [214]. The gene FfwcoA encoded a white collar photoreceptor in F. fujikuroi. It linked light regulation to cAMP signaling. WcoA was a positive regulator of fusarin biosynthesis [214].

F. graminearum contained two genes, FgCPK1 and FgCPK2, which encoded the catalytic subunits of cAMP-dependent protein kinase A (PKA). The deletion of cpk1 led to a marked reduction in conidiation, vegetative growth and DON biosynthesis, while simultaneously enhancing the fungal tolerance to elevated temperatures [216].

5.2. Regulation of TOR Signaling Pathway

The target of rapamycin (TOR) protein is a key signal-transducing component that regulates cell growth and metabolism. In the F. fujikuroi genome, a single TOR-encoding gene has been identified, and it was essential for viability. Through pharmacological inhibition, TOR disruption led to widespread disturbances in cellular function. This included the dysregulation of AreA-mediated secondary metabolism and a strong downregulation of genes associated with signal transduction, ribosome formation, protein translation and autophagy. Interestingly, when TOR was inhibited by rapamycin under nitrogen-limited conditions, the nitrogen catabolite repression was partially lifted, suggesting a modulatory role of TOR in nitrogen sensing. Despite this, the exact involvement of TOR in nitrogen regulation remained poorly understood [217]. Genome-wide analyses revealed that not all TOR-responsive genes were controlled by the transcription factor AreA, indicating a sophisticated regulatory network where both the glutamine synthetase (GS) and TOR pathways competed and collaborated [124,218]. While rapamycin-induced activation of GS occurred independently of AreA, the downstream targets of GS were indirectly and inversely affected by TOR inhibition. Nevertheless, both signaling routes converged to support GA₃ production [47]. The precise mechanism in F. fujikuroi needs further investigation [15].

5.3. Regulation of the MAPK Signaling Pathway

Mitogen-activated protein kinase (MAPK) pathways are crucial regulators of secondary metabolism in fungi. In F. graminearum, the involvement of MAPK cascades in the biosynthesis of trichothecenes, a class of toxic SMs that have been widely investigated, highlighting their significant role in modulating the expression and activity of genes associated with toxin production [100].

In standard laboratory culture conditions, the absence of MK1 (MAP kinase 1) in F. verticillioides resulted in lower expression levels of key biosynthetic genes FUM1 and FUM8, along with a significant reduction in fumonisin accumulation. Restoration of the Fvmk1 mutant by reintroducing the wild-type FvMK1 gene reversed these effects, demonstrating that FvMK1 played a positive regulatory role in fumonisin biosynthesis [219].

The deletion of Fphog1, a HOG-type MAP kinase encoding gene in F. proliferatum, was found to enhance both FB1 production and the expression of FUM genes when grown under nitrogen-limited conditions [220]. This suggested that MAPK signaling pathways were involved in modulating FB1 biosynthesis in response to environmental factors, particularly nutrient availability.

The elevated pH levels have been shown to enhance FB production in F. proliferatum through activation of the MAPK pathway [138]. Furthermore, the disruption of FvBCK1, a gene encoding a MAP kinase that acted as the primary upstream element of the cell wall integrity (CWI), resulted in reduced FB1 biosynthesis compared to the WT strain, indicating that the CWI MAPK pathway played a critical role in regulating FB1 production in F. proliferatum [221].

5.4. Regulation of Other Signaling Pathways

GAC1, a GTPase-activating protein, enhanced bikaverin production in F. verticillioides. It demonstrated a critical role of the signal transduction pathway in regulating bikaverin biosynthesis [222].

In F. verticillioides, FvMK1 was a mitogen-activated protein kinase gene that played a pivotal role in fungal development and virulence. Deletion of FvMK1 resulted in a complete loss of pathogenicity, with the mutant unable to colonize host tissues through wound sites. Moreover, it failed to induce stalk rot symptoms beyond the point of inoculation on corn stalks, highlighting the essential role of genes during the process of infecting plants. The ΔFvmk1 mutant also exhibited a marked reduction in fumonisin production along with significantly lower expression of FUM1 and FUM8, two key genes in the fumonisin biosynthetic pathway. These findings underscored the importance of FvMK1 in regulating multiple biological processes, including vegetative growth, asexual sporulation, mycotoxin production, and pathogenicity of F. verticillioides [219].

G protein signaling in F. fujikuroi played a critical role in fungal growth, secondary metabolism and sexual development. Specifically, Gα subunits FfG1 and FfG3 acted as the negative regulators of fusarubin biosynthesis [212].

6. Regulation of Organic Chemicals and Plant/Microorganism-Derived Extracts on Fusarium SM Production

Organic chemicals, which are known as low molecular weight organic compounds, can regulate the secondary metabolism of fungi [21]. Some organic chemicals are synthesized, while others are natural and derived from plants and microorganisms. Most chemicals are considered epigenetic modifiers, and other chemicals function as signaling compounds, precursors, inhibitors of SM biosynthesis. Furthermore, the regulatory functions of some chemicals remained unclear [21,223]. Currently, these studies were only conducted in vitro. The regulations of the chemicals with known structures, along with the plant/microorganism-derived extracts with the compound structures unclear on Fusarium SM production, are introduced as follows.

6.1. Regulation of Organic Chemicals

The production of both 4-aminobutyrate (GABA) and DON was increased in F. asiaticum by adding 2 mM agmatine. GABA might be biosynthesized from agmatine through putrescine as the intermediate, and DON biosynthesis was also influenced [224].

Compactin (known as 6-demethylmevinolin or 6-DMM), an HMG-CoA reductase inhibitor produced by Penicillium citrinum, was involved in the regulation of cholesterol biosynthesis. This compound has been shown to effectively suppress the biosynthesis of the polyketide mycotoxin aflatoxin B1 (AFB1) in Aspergillus flavus. Compactin also inhibited melanin synthesis and blocked spore development [225]. Furthermore, compactin suppressed the production of DON and ZEN in F. culmorum at 25 μg/mL in medium [226].

6-Pentyl-α-pyrone (6PAP) was an antifungal compound produced by the biological control fungus Trichoderma sp. The presence of 6PAP in the culture medium led to a significant reduction in DON production by F. graminearum [227].

Two plant-derived lignans, pinoresinol and secoisolariciresinol, showed inhibitory activity on mycelia growth and trichothecene biosynthesis in F. graminearum. Both pinoresinol and secoisolariciresinol at concentrations of 1.25 mg/L and 5.0 mg/L in the medium all inhibited radial growth and decreased production of DON and nivalenol (NIV) in F. graminearum. RT-qPCR analysis revealed that ligan treatment reduced trichothecene production in F. graminearum linked with downregulation of mRNA expression of the genes tri4, tri5 and tri11 [228].

The production of DON in F. graminearum was inhibited with 0.5 mM of caffeic acid supplementation, although the fungal growth was not affected at this dosage (0.5 mM) applied to the fungus [229]. NPD12671 was a synthetic furanocoumarin derivative. It stimulated production of 15-AcDON at 2 μM in F. graminearum. However, dihydroartemisinin (DHA) was screened to inhibit the production of 15-AcDON at 5 μM in F. graminearum. It was found that NPD12671 stimulated trichothecene production through activation of Tri6 expression, and DHA inhibited trichothecene production through repression of Tri6 transcription [230]. Nicotinamide at 500 µg/mL markedly inhibited the synthesis of DON and ergosterol peroxide in F. graminearum, the fungal pathogen responsible for wheat head blight [231].

The mycelia of F. graminearum were grown on PDB supplemented with two concentrations (3 µg/mL and 10 µg/mL) of trichostatin A (TSA) for 48 h, 72 h, and 96 h, respectively. After which, the mRNA levels were approximated via RT-qPCR analysis. It was then found that the HADC levels and trichodiene synthase gene Tri5 were different over time and the amount in response to the use of TSA. Treatment with TSA induced upregulation of Tri5 gene expression in the toxigenic isolate, with the obvious expression observed after 48 h at 3 µg/mL [232].

The histone acetyltransferase Gcn5 played a crucial role in epigenetic regulation. Phenazine-1-carboxamide (PCN) decreased DON production by inhibiting FgGcn5 in F. graminearum. The molecular mechanism of FgGcn5 inhibition by PCN was also unraveled by combined in silico and in vitro investigations [233]. 2-Hydroxy-4-methoxybenzaldehyde (HMB) was a SM with antimicrobial activity in many plant species [234]. The treatment of HMB at the minimum inhibitory concentration (MIC, 100 μg/mL) notably decreased the production of ergosterol and DON biosynthesis in F. graminearum. RT-qPCR investigation showed that the HMB treatment importantly modulated the expression of the key genes, including those of ergosterol biosynthesis (Erg2, Erg5, Erg6, etc.), DON biosynthesis (up to 16 genes), global regulators (LaeA, VeA, and VelB), the redox system (i.e., MnSOD, Cu/ZnSOD, GSS, and CAT), and stress signaling pathways (i.e., Hog1, Ssk1, Ssk2, and Pbs2) [234]. Exposure of F. graminearum to citric acid at 5 or 10 mM led to a marked decrease in biosynthesis of type B trichothecene mycotoxins, including DON, 3-AcDON, 15-AcDON, and NIV. However, the mycelial growth and pigment biosynthesis were enhanced [235].

The endophytic fungus F. oxysporum was treated with epigenetic modifier prednisone (300 μM), and the production of active compound umbelliferone was increased [236].

Treatment of F. oxysporum f. sp. conglutinans with 500 µM suberoyl bis-hydroxamic acid (SBHA) led to the induction of secondary metabolic pathways, resulting in the production of two novel fusaric acid derivatives, 5-butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 5-(but-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid [237].

The effects of different chemical modifiers, including 5-azacytidine (5-Aza), nicotinamide (NIC), sodium butyrate (SB), and sodium valproate (SV) on the metabolic profiles of F. verticillioides were assessed. After treatment, the fungal metabolome was analyzed using UHPLC–HRMS/MS in both untargeted and targeted metabolomics modes. The most pronounced changes in secondary metabolism were observed with SV, which significantly altered the metabolic fingerprint of F. verticillioides, likely through the activation of silent or cryptic biosynthetic gene clusters. Multivariate analysis highlighted 50 metabolites that most distinctly separated the five treatment conditions. Of these, twelve were annotated as fusarins or structural analogs. In comparison, NIC and SB induced only minor changes in the production of these compounds, indicating a weaker modulatory effect on the fungal secondary metabolism under the tested conditions [238].

Linoleate diol synthase 1 (LDS1) primarily generated 8-hydroperoxyoctadecenoic acids, which were further converted into various di-hydroxyoctadecenoic acids. The Fvlds1-deleted mutant of F. verticillioides exhibited enhanced growth, increased conidiation, higher fumonisins production, and faster maize cobs infection compared to the WT strain, which indicated that oxylipins produced by LDS1 acted as negative promoters of growth, conidiation, and fumonisin biosynthesis in F. verticillioides, a maize fungal pathogen [239].

The exposure of Fusarium sp. RK97-94 to the protein synthesis inhibitor hygromycin B, was found to induce the biosynthesis of several SMs, including lucilactaene, NG-391, fusarubin, 1233A, and 1233B. 1233A was identified as an inhibitor of HMG-CoA synthase. Genomic analysis led to the identification of the biosynthetic gene cluster responsible for 1233A production, which comprised four key genes. Notably, one of these genes played a role in providing self-resistance to the producing organism, protecting it from the inhibitory effects of 1233A [240].

The treatment of Fusarium sp. RK97-94 cultures with 30 µM NPD938 triggered the biosynthesis of three derivatives of lucilactaene including dihydroNG391, dihydrolucilactaene, and 13α-hydroxylucilactaene. Evaluation of their antimalarial potential against Plasmodium falciparum revealed significant differences in activity. DihydroNG391 showed only modest efficacy with an IC₅₀ of 62 µM, whereas dihydrolucilactaene demonstrated strong potency with an IC₅₀ of 0.0015 µM, and 13α-hydroxylucilactaene exhibited intermediate activity with an IC₅₀ of 0.68 µM. The structure–activity relationship studies indicated that the absence of the epoxide moiety, specifically, its reduction in NG391 to yield dihydrolucilactaene which led to a dramatic 1200-fold enhancement in antimalarial activity, highlighting the negative impact of the epoxide group on potency. Furthermore, opening the tetrahydrofuran ring in 13α-hydroxylucilactaene to generate dihydrolucilactaene resulted in a 100-fold increase in activity, suggesting that the integrity of the pyrrolidone ring and the lack of an epoxide are more crucial for antimalarial efficacy than the presence of the tetrahydrofuran ring. In cytotoxicity assays, dihydrolucilactaene showed low toxicity toward human cancer cell lines, with IC₅₀ values of 21 µM against HeLa cells and 37 µM against HL-60 cells [241]. Some examples of organic chemicals to regulate SM production in Fusarium fungi are displayed in Table 9.

Table 9.

Some examples of chemicals to regulate SM production in Fusarium fungi.

Fungus	Chemical and Its Concentration	Production of SM	Ref.
F. asiaticum	Agmatine (2 mM)	Increased production of GABA and DON.	[224]
F. culmorum	Compactin (25 μg/mL)	Suppressed production of DON and ZEN.	[226]
F. graminearum	6PAP (62.5 μg/mL)	Reduced production of DON	[227]
F. graminearum	Pinoresinol (1.25, 5.0 mg/L)	Decreased production of DON and NIV	[228]
F. graminearum	Secoisolariciresinol (1.25, 5.0 mg/L)	Decreased production of DON and NIV	[228]
F. graminearum	Caffeic acid (0.5 mM)	Inhibited production of DON.	[229]
F. graminearum	NPD12671 (2 μM)	Stimulated production of 15-AcDON	[230]
F. graminearum	DHA (5 μM)	Inhibited production of 15-AcDON	[230]
F. graminearum	Nicotinamide (500 μM)	Decreased production of ergosterol peroxide and DON.	[231]
F. graminearum	TSA (10 μg/mL)	An increase in trichodiene synthase gene (Tri5) expression	[232]
F. graminearum	Phenazine-1-carboxamide	Decreased DON production by inhibiting FgGcn5.	[233]
F. graminearum	2-Hydroxy-4-methoxybenzaldehyde (100 μg/mL)	Reduced production of ergosterol and DON.	[234]
F. graminearum	Citric acid (5 or 10 mM)	Decreased production of type B trichothecenes	[235]
F. oxysporum	Prednisone (300 μM)	Increased production of umbelliferone	[236]
F. oxysporum f. sp. conglutinans	SBHA (500 μM)	Induced production of two FA derivatives namely 5-butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 5-(but-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid.	[237]
F. verticillioides	5-Azacytidine (25 μM)	5-Azacytidine had some impacts on these metabolites.	[238]
F. verticillioides	Sodium valproate (100 μM)	Induced the alteration of the metabolic profile by promoting the expression of cryptic genes.	[238]
F. verticillioides	Di-hydroxyoctadecenoic acids.	Reduced production of fumonisins	[239]
Fusarium sp. RK97-94	Hygromycin B (100 μg/mL)	Induced the production of SMs, including lucilactaene, NG-391, fusarubin, 1233A, and 1233B, in Fusarium sp. RK97-94.	[240]
Fusarium sp. RK97-94	NPD938 (30 μM)	Induced production of three lucilactaene analogures, namely dihydroNG391, dihydrolucilactaene, and 13α-hydroxylucilactaene.	[241]

6.2. Regulation of the Extracts from Plants and Microorganisms

The crude extracts were prepared from plants, bacteria and fungi. There were multiple chemicals (components) with their structures unknown in each crude extract. The regulatory mechanisms of the extracts from plants and microorganisms should be complex.

6.2.1. Regulation of Plant Extracts

Putrescine was a defense compound produced by wheat could induce hypertranscription of F. graminearum trichothecene biosynthetic genes (FgTRIs), leading to increased DON accumulation during fungal infection. Further investigation into the regulatory mechanisms revealed that the transcription factor FgAreA mediated putrescine-induced FgTRIs expression by promoting histone modifications specifically, histone H2B monoubiquitination (H2Bub1) and histone 3 lysine 4 di-/trimethylation (H3K4me2/me3) at the FgTRIs loci. This indicated that wheat defense compound putrescine triggered mycotoxin DON synthesis by regulating H2B ub1 and H3K4me2/3 deposition in F. graminearum [242].

The essential oils from Eucalyptus camaldulensis (Myrtaceae) green branches and Origanum majorana (Labiatae) whole plants were screened to reduce Tri4 gene expression and mycelia growth of F. oxysporum strains in vitro. The production of trichothecenes in F. oxysporum was also decreased. GC-MS analysis showed that 1,8-cineole and spathulenol were the main compounds in the essential oil of E. camaldulensis green branches, and tricyclene, and p-cymene were the main compounds in the essential oil of O. majorana whole plants. Further investigation was needed to determine whether these compounds were active in affecting mycelia growth and mycotoxin production in F. oxysporum [243].

The regulatory mechanisms of host plant metabolites in the plant–Fusarium interaction have been investigated [244]. The effects of selected plant metabolites on F. proliferatum metabolism were analyzed. Quercetin-3-glucoside (Q-3-Glc) and kaempferol-3-rutinoside (K-3-Rut) were found to enhance fungal growth, whereas compounds such as DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one), isorhamnetin-3-O-rutinoside (Iso-3-Rut), ferulic acid (FA), protodioscin, and neochlorogenic acid (NClA) suppressed F. proliferatum. The influence of these metabolites on the expression of key FUM genes was evaluated using RT-qPCR in liquid cultures supplemented with the compounds. Twenty-four hours after treatment, chlorogenic acid (ClA) upregulated the expression of CPR6 and SSC1, while DIMBOA and protodioscin downregulated these genes. By the third day of exposure, FUM1 transcription was increased by all metabolites except Q-3-Glc, relative to the control. FUM6 expression was induced by protodioscin, K-3-Rut, and ClA, but repressed by FA and DIMBOA. In contrast, FUM19 was upregulated by all tested metabolites except ferulic acid, highlighting the compound-specific modulation of fumonisin biosynthesis pathways in response to plant-derived chemicals [245].

Fumonisin production by F. proliferatum was evaluated in liquid cultures supplemented with extracts from various host plants, including pineapple fruit, white asparagus spears, garlic bulbs, six-week-old pea plants, and young maize cobs. Analysis of both mycelia and culture media revealed that asparagus extract triggered the highest increase in fumonisin (FB) levels, with garlic extract showing the second strongest stimulatory effect. Pineapple extract was particularly effective in upregulating the gene fum1 as a key gene in the fumonisin biosynthetic pathway and significantly enhanced fumonisin synthesis across multiple fungal strains. In contrast, pea plant extract suppressed both fungal growth and mycotoxin production, indicating an inhibitory influence on F. proliferatum metabolism [246].

F. proliferatum strains PEA1 and PEA2, and F. oxysporum strains 34 OX and 1757 OX were two plant fungal pathogens originally identified from infected pea (Pisum sativum). The main metabolites were isolated from pea. It was found that coumarin, spermidine, p-coumaric acid, isoorientin, and quercetin (each at 100 ng/mL) reduced the growth of the fungal pathogens. All the metabolites highly inhibited the biosynthesis of FB1 and BEA [247].

6.2.2. Regulation of Fungal Extracts

The production of Fusarium SMs was significantly influenced by fungal extracts [248]. Yeast extracts, which were rich in nitrogen-containing compounds, played a significant role in modulating the secondary metabolism of Fusarium species. Selected yeast strains isolated from wheat grains and bread have demonstrated the ability to suppress the production of key mycotoxins, including DON, nivalenol (NIV), and zearalenone (ZEA or ZEN), isolated from F. culmorum, F. graminearum, and F. poae [248]. Fusaristatin A production in F. graminearum was found to be highest when the fungus was cultured in yeast extract-sucrose (YES) medium. Subsequent analysis revealed that cultivation conditions significantly influenced yield, with fusaristatin A levels in stationary liquid cultures exceeding those in agitated cultures by more than fourfold, highlighting the importance of growth conditions on metabolite [249].

The influence of yeast extract on SM production was investigated in four Fusarium species including F. avenaceum, F. fujikuroi, F. graminearum, and F. pseudograminearum. Yeast extract significantly enhanced the synthesis of DON and zearalenone in F. graminearum and F. pseudograminearum, with certain extracts leading to high toxin levels. In F. avenaceum, the production of chlamydosporol, 2-AOD-3-ol, and enniatins was altered by yeast supplementation, while in F. fujikuroi, yeast extract influenced the biosynthesis of bikaverin, gibberellic acid, fumonisin, and fusaric acid. In contrast, the production of fusarin C and aurofusarin remained unaffected by yeast extract across all Fusarium strains capable of producing these metabolites, indicating a selective regulatory effect of yeast-derived components on fungal secondary metabolism [250].

6.2.3. Regulation of Bacterial Extracts

The SMs (i.e., iturin A, fengycin, surfactin and bacitracin) from Bacillus velezensis WB induced oxidative equilibrium damage and reduced fusaric acid synthesis in F. oxysporum f. sp. niveum. The expression of fusaric acid biosynthesis-related genes was also down-regulated [251].

7. Other Regulations of Fusarium SM Production

Other regulations of Fusarium SM production mainly include metabolic shunting [252,253], transporters [254], and development-related proteins [255].

7.1. Regulation of Metabolic Shunting

Metabolic shunting was also called genetic dereplication, is a strategy that has been previously utilized to uncover novel SMs, particularly low-abundance compounds from fungi species [256,257]. The overexpression of two GA3 biosynthetic pathway genes ggs2 and cps/ks enhanced 150% of GA3 production in F. fujikuroi. However, the overexpression of hmgR and fppS resulted in lower metabolite production, likely due to negative feedback regulation of HmgR. To overcome this, the transmembrane domains of HmgR were deleted, and the catalytic domain was overexpressed, which significantly enhanced GA3 production by 250% [258].

In F. fujikuroi, the simultaneous deletion of the bikaverin and fusarubin BGCs resulted in enhanced production of GA3, with the ΔBIKΔFSR strain showing a 31.67% increase compared to the wild type. Furthermore, the absence of these SM pathways was associated with improved mycelial growth and more efficient carbon utilization, suggesting a reallocation of metabolic resources toward primary growth and GA3 [253].

F. sporotrichioides, the causative agent of cereal plant disease Fusarium head blight, could produce mycotoxins including trichothecenes and T-2 toxin. 7-Hydroxyisotrichodermin was a shunt pathway metabolite of F. graminearum. If 7-hydroxyisotrichodermin was utilized in a cross-species feeding experiment with a trichodiene synthase-deficient mutant of F. sporotrichioides, leading to the production of 7-hydroxy T-2 toxin as the end product. When evaluated for cytotoxicity in HL-60 cells, 7-hydroxy T-2 toxin exhibited a potency that was ten-fold lower than that of the parent compound, T-2 toxin [252].

7.2. Regulation of Fusarium Co-Cultivation with Other Microorganisms

F. oxysporum and F. fujikuroi were Fusarium species complexes isolated from the nails of patients suffering from onychomycosis. The pure cultures of two Fusarium strains only produced trace amounts of fusaric acid. When they were grown in a co-cultivation system, large amounts of fusaric acid were produced. It indicated that there was a regulation between two fungal species [259].

The yield of BEA by F. oxysporum AB2 under solid-state fermentation was 22.8 mg/L. When F. oxysporum AB2 was co-cultured with another fungus Epicoccum nigrum TORT, BEA yield was greatly improved and reached 84.6 mg/L [260].

Two depsipeptides, namely subenniatins A and B, were induced to be produced by co-culturing F. tricinctum and F. begonia. Neither depsipeptides were observed when either of the two fungal species was cultured alone [261].

The co-cultivation of F. tritinctum with the bacterium Bacillus subtilis on solid rice medium resulted in the production of nine SMs including macrocarpon C, (−)-citreoisocoumarin, 2-(carboxymethylamino)benzoic acid, (−)-citreoisocoumarinol, lateropyrone, enniatin type cyclic depsipeptides enniatins B, B1 and A1, and lipopeptide fusaristatin A. These metabolites displayed strong antibacterial activity on B. subtilis, Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, with MIC values ranging from 2 to 8 μg/mL [262]. Another example was the co-cultivation of F. tritinctum with Streptomyces lividans. Four new dimeric naphthoquinones, fusatricinones A–D, and a new lateropyrone derivative, dihydrolateropyrone were induced in production of the co-culture system. In addition, several known metabolites such as enniatin derivatives, showed an enhanced accumulation in the co-cultures [263].

7.3. Regulation of Transporters

ZRA1, a putative ABC transporter gene, was required for zearalenone (ZEA) production in F. graminearum. Deletion of ZRA1 resulted in reduced ZEA production, which indicated that the ABC transporter gene ZRA1 positively regulated ZEA production [254].

PTR2s were peptide transporters. Three deletion mutants including ΔFgPTR2A, ΔFgPTR2C, and ΔFgPTR2D led to the higher synthesis of DON and ZEA and a reduced synthesis of fusarielin H compared to the wild-type strain of F. graminearum. The development of perithecium was actually decreased in these mutants, but not affected by the deletion of FgPTR2B. This indicated that PTR2 peptide transporters in F. graminearum influenced both sexual development and SM production [264].

7.4. Regulation of Development-Related Proteins

Kex2 was a kexin-like protease located in the Golgi apparatus that plays a key role in processing and activating precursor proteins. FvKex2 was required for the fungal normal vegetative growth in F. verticillioides. The ∆Fvkex2 mutant showed a reduced production of FB1 as well as a pathogenicity reduction compared to the wild-type and genetically complemented strains. It indicated that FvKex2 was required for development, virulence, and FB1 production in F. verticillioides [265].

7.5. Others

F. fujikuroi was capable of synthesizing a range of SMs derived from polyketides and non-ribosomal peptides, including fusarins, fusarubins, and bikaverins. The biosynthesis of these compounds relied on key enzymes such as polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs), which typically required post-translational activation. This modification was mediated by an Sfp-type 4′-phosphopantetheinyl transferase. The F. fujikuroi Sfp-type PPTase FfPpt1 was essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins. The ΔFfppt1 mutants revealed an increased production of terpenoid-derived like GAs and volatile compounds such as α-acorenol, which indicated that FfPpt1 negatively regulated the biosynthesis of terpenoids in F. fujikuroi [266].

FfCOX17 is a copper chaperone protein in F. fujikuroi. The fumonisin production in the ∆FfCOX17 mutant was significantly increased compared to the WT strain, but the pathogenicity of the ∆FfCOX17 mutant was not affected, which might be caused by that there was no significant change in the content of gibberellin [267].

HmbC belonged to the high-mobility group (HMG) family to participate in the regulation of carotenoid biosynthesis in F. fujikuroi. Deletion of the gene hmbC resulted in enhanced carotenoid accumulation and upregulated mRNA expression of genes involved in carotenoid biosynthesis [268].

Non-histone proteins belonging to the high-mobility group (HMG) family are essential structural components of chromatin and contributed to a variety of cellular processes in eukaryotes. In the plant-pathogenic fungus F. graminearum, the HMG protein FgNhp6 has been identified as a key regulator influencing pathogenicity, the production of DON, and overall fungal development. The ΔFgNhp6 deletion mutant showed markedly decreased ability to infect wheat tissues, including coleoptiles and floral spikes, indicating impaired virulence. Surprisingly, despite reduced pathogenicity, these mutants produced higher levels of DON. Genome-wide transcriptome profiling using RNA-seq demonstrated that disruption of FgNhp6 altered the expression of genes across multiple metabolic networks. Notably, pathways linked to secondary metabolism, such as those governing the biosynthesis of sterols and the pigment aurofusarin, were significantly suppressed in the mutant strain [269].

Both FgWhi2 and FgPsr1 are the stress regulators and phosphatases of F. graminearum. They played crucial roles in the regulation of ergosterol and DON biosynthesis, and the response to fungicides in F. graminearum. The knockout mutants including ΔFgwhi2, ΔFgpsr1, and ΔFgwhi2ΔFgpsr1 all reduced the production of ergosterol and DON, and increased the fungicide sensitivity which positively regulated the sensitivity of F. graminearum to fungicides (i.e., chlorothalonil, fluazinam, azoxystrobin, phenamacril, and oligomycin) [270].

Malate synthase is a crucial enzyme in the glyoxylate cycle, which was a supplementary pathway that enabled certain organisms, including many fungi, to synthesize malate from glyoxylate and acetyl-CoA. The deletion of the malate synthase gene FgMS in F. graminearum reduced mycelial growth rate, decreased sporulation, weakened spore germination, diminished virulence, lowered DON production, increased sensitivity to cell wall stress, and reduced sensitivity to the fungicides including carbendazim, pydiflumetofen, difenoconazole, tebuconazole and phenamacril [271].

In F. graminearum, the MAP kinase MGV1 and the transcription factor Tri6 jointly modulated the biosynthesis of DON and fusaoctaxins. This cooperative regulation suggested that MGV1 acted as a central signaling node, enabling selective control over multiple BGCs. Meanwhile, Tri6 was located within a specific BGC, targeted regulatory precision, and controlled the expression of genes within its resident cluster [272].

FvFUG1 is an unknown gene that has shown a role in pathogenicity and fumonisin biosynthesis in F. verticillioides. The fumonisin production was decreased in the ΔFvFUG1 mutant. Furthermore, the biosynthesis of DIBOA and DIMBOA in maize kernels was also decreased in the ΔFvFUG1 mutant. FUG1 was considered a novel fungal transcription factor or involved in signal transduction, which needed further confirmation [134].

In F. verticillioides, the vacuole and mitochondria patch (vCLAMP) component FvVam6 has been linked to both fungal development and the biosynthesis of FB1 through its role in regulating vacuolar structure. Disruption of the FvVam6 gene resulted in altered vacuole morphology, indicating its importance in maintaining organelle integrity. Additionally, the ΔFvVam6 mutant exhibited a substantial decrease in FB1 production, underscoring the connection between proper vacuolar organization and mycotoxin synthesis [254]. Some examples of these regulatory factors to regulate SM production in Fusarium fungi are shown in Table 10.

Table 10.

Some examples of other regulatory factors to regulate SM production in Fusarium fungi.

Fungus	Overexpression/ Deletion of Gene	Positive/Negative Regulation	Production of SM	Ref.
F. fujikuroi	Deletion of FgCOX17	Negative	Significant increase in fumonisin production	[267]
F. fujikuroi	Deletion of hmbC	Negative	Increased production of carotenoids	[268]
F. fujikuroi	Deletion of the bikaverin and fusarubin biosynthesis gene clusters	Negative	Significant increase in GA3 production	[253]
F. graminearum	Deletion of FgNhp6	Negative	Significant increase in DON production	[269]
F. graminearum	Deletion of Fgwhi2	Positive	Significant decrease in DON and ergosterol production	[270]
F. graminearum	Deletion of Fgpsr1	Positive	Significant decrease in DON and ergosterol production	[270]
F. graminearum	Deletion of FgMS	Positive	Decreased production of DON	[271]
F. graminearum	Deletion of FgPTR2A, FgPTR2C, and FgPTR2D	Positive	Increased production of DON and zearalenone, and decreased production of fusarielin H	[264]
F. verticillioides	Deletion of FUG1	Positive	Decreased production of fumonisin, DIMBOA and DIBOA	[134]
F. verticillioides	Deletion of FvVam6	Positive	Significantly reduced FB1 production	[255]
F. verticillioides	Deletion of Fvkex2	Positive	Reduced production of FB1	[265]

8. Discussion

In the early 2000s, the studies on secondary metabolite production through biosynthesis regulation on Fusarium fungi were focused on the characterization of single biosynthesis genes and the effects of environmental factors. Currently, the researches were focused more on the regulation of SM BGCs related to a systemic and multifactorial perspective in Fusarium fungi. In the past, Fusarium secondary metabolites were mainly obtained from plant pathogenic Fusarium species. The secondary metabolites usually belonged to mycotoxins or phytotoxins. However, in recent years, more and more SMs were isolated from plant endophytic, soil-derived, and marine-derived Fusarium species. In addition to phytotoxic and mycotoxic activities, the biological activities of Fusarum SMs are also manifested in many other aspects such as anti-virus, antimalarial, anti-inflammatory, and neuroprotective activities [9].

Many Fusarium fungi have been known to produce structurally diverse SMs with a wide range of biological activities that make them a treasure trove of bioactive compounds [273,274,275,276,277,278,279,280,281,282]. To harness the potential of these beneficial SMs, the strategies of biosynthesis regulation essential to activate secondary metabolic pathways, thereby boosting the production of valuable metabolites [283].

The typical example was GA3, a phytohormone synthesized by F. fujikuroi, which plays a key role in enhancing crop yield. Despite its agricultural importance, large scale GA3 production has struggled to meet market demand, largely due to insufficient optimization of fermentation conditions and intricate regulatory network governing its biosynthesis. Consequently, improving GA3 yield through targeted regulatory strategies has emerged as an innovative and high-priority approach [40]. Another notable metabolite is bikaverin, a red polyketide pigment produced F. fujikuroi and related species which exhibits antiprotozoal and antifungal activities to display its potential as an antibiotic [284]. It is worth noting that an increasing number of Fusarium-derived SMs have demonstrated phytotoxic activities [285,286]. These phytotoxins serve as promising lead compounds for the development of novel herbicides, which are the structural analogs synthesized based on these natural scaffolds to show strong herbicidal activity against various weed species [287,288,289,290]. Additionally, acadesine (AICAR) is a compound currently in phase III clinical trials as an anti-tumor agent produced by the endophytic fungus F. solani. Its biosynthesis was mediated by the global transcriptional factor VeA, which regulated adenylosuccinate lyase to facilitate acadesine production in F. solani [291].

The cryptic metabolite BGCs in fungi can be unlocked through biosynthesis regulation strategies [292]. This approach has successfully led to the discovery of new bioactive metabolites (e.g., dihydrolucilactaene, sansalvamide, apicidins, and fusarielins) from some Fusarium species [87,144,237,241,281,293].

For example, treatment of Fusarium sp. RK97-94 with the epigenetic modifier NPD938, induced the production of dihydrolucilactaene, a newly identified metabolite exhibiting potent antimalarial activity [241]. Another example was that three new cyclic tetrapeptides apicidins F, J and K (APF, APJ and APK) were produced in the deletion mutants and WT strain of rice pathogen F. fujikuroi under conditions of higher nitrogen and acidic pH in a manner dependent on two global regulators, which were nitrogen regulator AreB, and the pH regulator PacC. The BGC of apicidins was named as APF. In addition, over-expression of the atypical pathway-specific TF-encoding gene APF2 led to the elevated expression of cluster genes under inducing and even repressing conditions and to significantly increase yields of apicidins F, J and K. Among the three cyclic tetrapeptides, apicidin F showed the strongest cytotoxicity and anti-tumor potential [293].

Some examples of the metabolite discovery from Fusarium fungi by unlocking the cryptic BGCs through biosynthesis regulation are shown in Table 11.

Table 11.

Some examples of the metabolite discovery from Fusarium fungi by unlocking the cryptic BGCs through biosynthesis regulation.

Fusarium Species	New Metabolite	Biological Activity	Biosynthesis Regulation Strategy	Ref.
F. pseudograminearum	A novel cytokinin	Plant growth regulation	OSMAC strategy	[87]
F. graminearum	Fusarielins F, G and H	Cytotoxic activity	Pathway-specific transcriptional factor regulation	[144]
F. oxysporum f. sp. conglutinans	5-butyl-6-oxo-1,6-dihydropyridine-2-carboxylic acid and 5-(but-9-enyl)-6-oxo-1,6-dihydropyridine-2-carboxylic acid	Not mentioned	Treatment of epigenetic modifier SBHA	[237]
Fusarium sp. RK97-94	Dihydrolucilactaene	Antimalarial activity	Treatment of epigenetic modifier NPD938	[241]
Fusarium sp. CNL 292	Sansalvamide	Cytotoxic activity	OSMAC strategy	[281]
F. fujikuroi	Apicidins F, J and K	Cytotoxic activity	OSMAC strategy; regulation of global transcriptional factors AreB and PacC, and pathway-specific transcriptional factor APF2	[293]

For the toxic and harmful Fusarium SMs, the strategies of biosynthesis regulation should be applied to minimize secondary metabolism to decrease metabolite production [175,294]. These toxic Fusarium SMs are usually called mycotoxins, mainly include enniatins, fumonisins, fusaric acid, fusariumic acids, trichothecenes, and zearalenone.

Multiple strategies of biosynthetic regulation could be used to enhance Fusarium SM production. These regulation strategies include the OSMAC (one-strain-many compounds) approach [38], co-cultivation of microorganisms [259,262], epigenetic regulation [21,22,295], transcriptional regulation [20], signaling pathway regulation [216], and metabolic shunting [256,273,296,297,298], which have been proven to be effective in promoting the production of fungal SMs.

To reveal more quantities of SMs in Fusarium fungi, beyond biosynthesis regulation, other strategies may also be applied including heterologous expression of BGCs [299,300,301], promoter engineering [101,302,303], and combinatorial metabolic engineering [304,305] for either activating Fusarium silent BGCs or utilizing some key genes involved in biosynthesis of SMs to mine more metabolites from Fusarium fungi. Some examples of the toxic SMs from Fusarium fungi are shown in Table 12.

Table 12.

Some examples of the toxic SMs from Fusarium fungi.

Metabolite	Toxicity	Metabolite-Producing Fusrium Species	Ref.
Fumonisins	Causing oesophageal cancer and neural tube defects; Phytotoxic activity by inhibiting root and shoot growth and causing chlorosis, necrosis, and wilting.	F. fujikuroi, F. oxysporum, F. proliferatum, and F. verticillioides	[12]
Enniatins	Causing mitochondrial dysfunction, lysosomal alteration, cell cycle disruption, and lipid peroxidation; Disrupting cell membranes by increasing permeability by forming pore structures.	F. avenaceum	[306]
FA, 9,10-dehydrofusaric acid	Phytotoxic activity causes plant wilting.	F. oxysporum, F. moniliforme, F. heterosporum	[307]
MON	Inhibiting protein synthesis, causes chromosome damage.	F. proliferatum, F. fujikuroi, and F. nygamai	[307]
Fusariumic acids	Phytotoxic activity causes plant wilting.	F. oxysporum f. sp. radicis-lycopersici	[308]
Trichothecenes including HT-2 toxin, T-2 toxin, DON, NIV, 3-AcDON, and 15-AcDON	Causing hepatotoxicity, enterotoxicity, neurotoxicity, and reproductive toxicity in animals and humans.	F. graminearum, F. sporothichioides, F. langsethiae and F. culmorum	[309]
Zearalenone (ZEN), zearalanone (ZAN)	Estrogenic effects on animals and humans; cause genetic toxicity, reproductive toxicity, hepatotoxicity, immunotoxicity, carcinogenicity, and so on.	F. graminearum, F. oxysporum, F. equisetum, F. nivalis and F. sambucinum	[310]

9. Conclusions and Perspectives

In summary, Fusarium fungi, including marine-derived, soil-derived, endophytic and pathogenic species, can produce large amounts of SMs with a wide range of biological activities to make them a treasure trove of bioactive natural compounds with potential applications. Numerous valuable SMs have been successfully identified from Fusarium fungi by using strategies of biosynthesis regulation. The biosynthesis regulation has been considered an effective approach to reveal Fusarium metabolites. The strategies to regulate Fusarium secondary metabolism mainly include environmental factor regulation, transcriptional factor regulation, epigenetic regulation, and signal transduction regulation (Figure 1). However, Fusarium SM production through biosynthesis regulation at multiple levels is a complex process governed by environmental factors together with the complex signaling and regulatory networks involving primary and secondary metabolism in fungi. Most of these networks and their regulatory mechanisms remain unclear.

Figure 1.

Strategies for SM production via biosynthesis regulation on Fusarium fungi.

Overall, the biosynthesis regulation for the production of SMs in Fusarium fungi is an efficient strategy for either activating cryptic BGCs and discovering new bioactive metabolites, or increasing the production of low-yield SMs. Furthermore, it can inhibit specific BGCs and decrease toxic metabolite production. The significant efforts will be needed to address the complex regulatory mechanisms of SM biosynthesis in Fusarium fungi in future investigations, which may help us better manage the production of Fusarium SMs.

Abbreviations

The following abbreviations are used in this manuscript:

3-AcDON	3-acetyl deoxynivalenol
15-AcDON	15-acetyl deoxynivalenol
4-AcNIV	4-acetyl nivalenol
AFB1	aflatoxin B1
APF	apicidin F
15-AS	15-acetoxyscipenol
aw	water availability
BEA	Beauvericin
BGC	biosynthetic gene cluster
cAMP	cyclic adenosine monophosphate
COMPASS	Complex Proteins Associated with Set1
DAS	Diacetoxyscirpenol
4,15-diAcNIV	4,15-diacetyl nivalenol
DNMT	DNA methyltransferase
DON	Deoxynivalenol
EN	Enniatin
FA	fusaric acid
FB1	fumonisin B1
FB2	fumonisin B2
FB3	fumonisin B3
FU	fusaric acid
FUM	Fumonisin
GA3	gibberellic acid
GAs	Gibberellins
HAT	histone acetyltransferase
H4K20me	monomethylation of lysine 20 of histone 4
H3K9me3	trimethylation of lysine 9 of histone 3 (hitone 3 lysine 9)
HDAC	histone deacetylase
HDM	Histone demethylase
HMT	histone methyltransferase
MAPK	mitogen-activated protein kinase
MON	Moniliformin
MIC	Minimal inhibitory concentration
NIV	Nivalenol
NRPS	non-ribosomal peptide synthetase
OE	Overexpression
OSMAC	one strain many compounds
6-PAP	6-pentyl-α-pyrone
PDA	potato dextrose agar
PKS	polyketide synthase
PRC2	polycomb repressive complex 2
PTM	post-translational modification
SAM	S-adenocyl-1-methionine
SBHA	suberoyl bishydroxamic acid
SM	secondary metabolite
T-2 toxin	fusariotoxin T2
TF	transcriptional (regulatory) factor
TOR	target of rapamycin
WT	wild-type
ZEN, ZEA	Zearalenone

Author Contributions

L.Z. conceptualized this manuscript. P.A., X.P., X.H. and L.Z. collected literature and wrote this paper. P.A., X.P., X.H., Y.L., J.S., Y.H., P.W. and L.L. organized literature and built the tables. X.H. and D.L. added critical revisions and suggestions to the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by the National Key Research and Development Program of China (2023YFD1401400, 2023YFD1700700 and 2022YFD1700200).