Involvement of LaeA and Velvet Proteins in Regulating the Production of Mycotoxins and Other Fungal Secondary Metabolites

Abstract

Fungi are rich sources of secondary metabolites of agrochemical, pharmaceutical, and food importance, such as mycotoxins, antibiotics, and antitumor agents. Secondary metabolites play vital roles in fungal pathogenesis, growth and development, oxidative status modulation, and adaptation/resistance to various environmental stresses. LaeA contains an S-adenosylmethionine binding site and displays methyltransferase activity. The members of velvet proteins include VeA, VelB, VelC, VelD and VosA for each member with a velvet domain. LaeA and velvet proteins can form multimeric complexes such as VosA-VelB and VelB-VeA-LaeA. They belong to global regulators and are mainly impacted by light. One of their most important functions is to regulate gene expressions that are responsible for secondary metabolite biosynthesis. The aim of this mini-review is to represent the newest cognition of the biosynthetic regulation of mycotoxins and other fungal secondary metabolites by LaeA and velvet proteins. In most cases, LaeA and velvet proteins positively regulate production of fungal secondary metabolites. The regulated fungal species mainly belong to the toxigenic fungi from the genera of Alternaria, Aspergillus, Botrytis, Fusarium, Magnaporthe, Monascus, and Penicillium for the production of mycotoxins. We can control secondary metabolite production to inhibit the production of harmful mycotoxins while promoting the production of useful metabolites by global regulation of LaeA and velvet proteins in fungi. Furthermore, the regulation by LaeA and velvet proteins should be a practical strategy in activating silent biosynthetic gene clusters (BGCs) in fungi to obtain previously undiscovered metabolites.

1. Introduction

Fungal secondary metabolites are highly complex and have a rich diversity that makes fungi a treasure of bioactive secondary metabolites [1,2,3,4]. Some fungal metabolites are harmful to plants, humans, and animals; these metabolites are usually called mycotoxins [5,6,7,8]. Many bioactive metabolites derived from fungi display their broad potential as agrochemicals, pharmaceuticals, cosmetics, and food additives [9,10].

In recent years, it has become easier, through genome mining, to identify and functionally predict fungal metabolites [11]. Many strategies can regulate (i.e., promote or inhibit) the biosynthesis of secondary metabolites in fungi, such as one strain-many compounds (OSMAC), global regulation, epigenetic regulation, heterologous expression, and combinatorial biosynthesis [12,13,14]. Among them, global regulation for secondary metabolite production has been thought to be the most practical strategy. Global regulation is a complex upper-level regulatory network in which microorganisms respond comprehensively to external environmental stimuli such as light intensity, temperature, ambient pH, redox status, and carbon and nitrogen sources. LaeA and velvet proteins belong to global regulators, and are mainly regulated by light [15], which focus on the secondary metabolism of several fungal species. The velvet proteins, which included VeA, VelB, VelC, VelD and VosA, are widely distributed in the fungal kingdom. At least 21 major velvet clades, with their highly conserved domains, were found in fungi to show functional and type diversity in their velvet proteins [16]. Both LaeA and velvet proteins can form multimeric complexes. They are involved in fungal development and secondary metabolism [15,16,17,18,19,20].

In the past 20 years, many advances have been achieved in regard to the regulation of LaeA and velvet proteins in fungal secondary metabolism and development. Though some reviews have been published [15,16,21], many other recent achievements have not been included. In this review, we focused on the regulation of LaeA and velvet proteins in secondary metabolite production in fungi by either inhibiting the production of mycotoxins, promoting the production of useful metabolites, or revealing previously undiscovered metabolites in order to accelerate their applications.

2. Regulation of LaeA on Secondary Metabolite Production in Fungi

LaeA (loss of aflR expression) is also called Lae1 or LAE1. It was first identified as a nuclear protein in Aspergillus. Compared to the velvet proteins, LaeA has no velvet domain [17]. LaeA functions as a global regulator of secondary metabolism and morphogenetic development in various filamentous fungi. The LaeA protein sequence contains an S-adenosylmethionine binding site, so it has been proposed to have methyltransferase activity and might be linked to the remolding of chromatin structure to regulate gene transcription by lysine or arginine methylation of histone [22,23]. In most cases, the laeA genes in fungi positively regulated production of secondary metabolites. Only a few laeA genes were found to negatively regulate secondary metabolite production (

Table 1. Some examples of LaeA regulating secondary metabolite production in fungi.

Fungus	Overexpression/ Deletion of laeA	Positive/Negative Regulation	Production of Secondary Metabolites	Ref.
Alternaria alstroemeria	Overexpression	Positive	Increased production of myricetin (1), geraniol (2), ergosterol (3) and other compounds determined by metobolomic analysis.	[24]
Alternaria alstroemeria	Overexpression	Negative	Decreased production of the antitumor compounds via controlling the transcription of AaFla1.	[25]
Alternaria alternata	Deletion	Positive	Decreased production of alternariol (4) and alternariol monomethyl ether (5).	[26]
Alternaria alternata	Overexpression	Positive	Increased production of the anti-inflammatory meroterpenoid tricycloalternarene O (6).	[27]
Arthrobotrys flagrans	Overexpression and deletion	Positive	Increased production of the secondary metabolites by overexpression of AflaeA, and decreased production of the secondary metabolites by deletion of AflaeA.	[28]
Aspergillus sp. Z5	Overexpression	Positive	Increased production of diorcinol (7).	[29]
Aspergillus sp. FKI-5362	Overexpression	Positive	Increased production of MS-347a (8).	[30]
Aspergillus carbonarius	Deletion	Positive	Decreased production of citric acid (9).	[31]
Aspergillus carbonarius	Deletion	Positive	Decreased production of ochratoxin A (10).	[32]
Aspergillus carbonarius	Deletion	Positive	Decreased production of ochratoxin A (10) in ΔlaeA strain colonized in nectarines and grapes.	[33]
Aspergillus carbonarius	Inhibtion of LaeA	Positive	Decreased production of ochratoxin A (10) by treatment with eugenol through inhibiting LaeA expression.	[34]
Aspergillus cristatus	Overexpression	Positive	Increased production of multiple secondary metabolites including terpenoids and flavonoids.	[35]
Aspergillus flavipes	Deletion	Negative	Increased production of flavipamides A (11) and B (12), asperphenamate (13), 4′-OMe-asperphenamate (14), and cyclic Pro-Gly-Val-Gly-Try(8-OH, 3-prenyl)-Gly-Trp (15).	[36]
Aspergillus flavus	Deletion	Positive	Decreased production of sterigmatocystin (16).	[37]
Aspergillus flavus	Deletion	Positive	Decrease production of aflatoxins.	[38]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxin B1 (17).	[39]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxins.	[40]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxins, cyclopiazonic acid (18) and ustiloxin B (19).	[41]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxins.	[42]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxins and kojic acid (20).	[43]
Aspergillus fumigatus	Deletion	Positive	Decreased production of gliotoxin (21) and endocrocin (22).	[37]
Aspergillus fumigatus	Deletion	Positive	Decreased production of several mycotoxins including gliotoxin (21).	[44]
Aspergillus fumigatus	Deletion	Positive	Decreased production of gliotoxin (21), fumagillin (23), fumagatin (24) and helvolic acid (25).	[45]
Aspergillus fumigatus	Deletion	Positive	Decreased production of gliotoxin (21).	[46]
Aspergillus fumigatus	Deletion	Positive	Decreased production of endocrocin (22).	[47]
Aspergillus fumisynnematus	Overexpression	Positive	Increased production of cyclopiazonic acid (18).	[48]
Aspergillus luchuensis mut. kawachii	Deletion	Positive	Decreased production of citric acid (9).	[49]
Aspergillus nidulans	Overexpression	Positive	Increased production of terrequinone A (26).	[50]
Aspergillus nidulans	Deletion	Positive	Decreased production of sterigmatocystin (16) and norsolorinic aid (27).	[51]
Aspergillus nidulans	Deletion	Positive	Decreased production of streigmatocystin (16) and penicillin G (28).	[37]
Aspergillus nidulans	Deletion	Positive	Depressed expression of genes involved in biosynthesis of sterigmatocystin (16), terrequinone A (26) and penicillin G (28).	[52]
Aspergillus nidulans	Deletion	Negative	Increased production of sterigmatocystin (16).	[53]
Aspergillus nidulans	Overexpression	Positive	Increased production of sterigmatocystin (16).	[54]
Aspergillus niger	Deletion	Positive	Decreased production of asperrubrol (29), atromentin (30) and JBIR 86 (31).	[55]
Aspergillus niger	Deletion	Negative	Increased production of aspernigrin A (32) and BMS-192548 (33).	[55]
Aspergillus niger	Overexpression	Positive	Increased production of flaviolin (34), orlandin (35) and kotanin (36).	[56]
Aspergillus niger	Overexpression and deletion	Positive	Decreased production of OTA (10) in the deleted mutant, but increased production of OTA (10) in the overexpressed mutant.	[57]
Aspergillus ochraceus	Deletion	Positive	Decreased production of OTA (10).	[58]
Aspergillus oryzae	Deletion	Negative	Increased production of kojic acid (20).	[59]
Aspergillus oryzae	Overexpression	Positive	Increased production of monacolin K (37) and terrequinone A (38).	[60]
Aspergillus pachycristatus	Deletion	Positive	Decreased production of sterigmatocystin (16) and echinocandin B (39).	[61]
Aspergillus pseudoterreus	Overexpression	Positive	Increased production of itaconic acid (40).	[62]
Aspergillus terreus	Overexpression	Positive	Increased production of lovastatin (41).	[63]
Aspergillus terreus	Overexpression	Positive	Increased production of dihydroisoflavipucines 1 (42) and 2 (43).	[64]
Beauveria bassiana	Overexpression and deletion	Positive	Decreased production of beauvericin (44) and bassiatin (45) in the BbLaeA disruption strain, but increased production in the overexpressed strain.	[65]
Botrytis cinerea	Deletion	Positive	Decreased production of oxalic acid (46).	[66]
Botrytis cinerea	Deletion	Positive	Decreased production of abscisic acid (47).	[67]
Chaetomium globosum	Overexpression	Positive	Increased production of seven cytochalasans including chaetoglobosins A (48), B (49), D (50), E (51), O (52), V (53) and Z (54).	[68]
Chaetomium globosum	Overexpression and deletion	Positive	Decreased production of chaetoglobusin A (48) in ΔCglaeA mutant, restored production of chaetoglobusin A (48) in CglaeA-C strain, and increased production of chaetoglobusin A (48) in CglaeA-OE strain.	[69]
Cladosporium fulvum	Deletion	Negative	Increased production of cladofulvin (55).	[70]
Cochliobolus heterostrophus	Deletion	Positive	Decreased production of T-toxin (56).	[71]
Coprinopsis cinerea	Deletion	Negative	Increased production of coprinoferrin (57).	[72]
Daldinia eschscholzii	Replacement of a strong pgdA promoter	Positive	Induced production of dalestones A (58) and B (59).	[73]
Dothistroma septosporum	Deletion	Negative	Increased production of dothistromin (60).	[74]
Fusarium fujikuroi	Deletion	Negative	Increased production of bikaverin (61).	[75]
Fusarium fujikuroi	Deletion	Positive	Decreased production of gibberellins A3 (63) and A4 (64), fusarin C (65), fumonisins B1 (66), B2 (67), B3 (68) and B4 (69), deoxynivalenol (70), and 15-acetyl deoxynivalenol (71).	[75]
Fusarium fujikuroi	Deletion	Positive	Decreased production of fusaric acid (62), fusarinolic acid (72), and dehydrofusaric acid (73).	[76]
Fusarium fujikuroi	Deletion and overexpression	Positive	Deletion of laeA led to decreased production of gibberellins, fumonisins, and fusarin C (65). Overexpression of laeA led to increased production of gibberelins.	[77]
Fusarium fujikuroi	Deletion	Negative	Increased production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79).	[78]
Fusarium graminearum	Deletion and overexpression	Positive	Deletion of FglaeA led to a dramatic reduced production of trichothecenes and zearalenone (80). Overexpression of FglaeA caused the increased production of trichothecenes and zearlenone (80).	[79]
Fusarium oxysporum	Deletion	Positive	Decreased production of beauvericin (44) and fusaric acid (62).	[80]
Fusarium oxysporum f.sp. niveum	Deletion	Positive	Decreased production of bikaverin (61) and fusaric acid (62).	[81]
Fusarium verticillioides	Deletion	Positive	Decreased production of bikaverin (61), fusaric acid (62), fusarin C (65), and fumonisins.	[82]
Ganoderma lingzhi	Deletion and overexpression	Positive	Decreased production of ganoderic acids in the deleted mutant and increased ganoderic acids in the overexpressed mutant.	[83]
Magnaporthe oryzae	Overexpression	Positive	Decreased production of melanin and increased production penicillin G (28).	[84]
Magnaporthe oryzae	Overexpression	Positive	Increased production of secondary metabolites.	[85]
Monascus pilosus	Overexpression	Positive	Increased production of monacolin K (37) and unidentified pigments.	[86]
Monascus purpureus	Overexpression	Positive	Increased production of monacolin K (37).	[87]
Monascus ruber	Deletion	Positive	Decreased production of citrinin (81) and six pigments rubropunctamine (82), monascorubramine (83), monascin (84), rubropunctatin (85), ankaflavin (86), and monascorubrin (87).	[88]
Penicillium sp. LC1-4	Overexpression	Positive	Increased production of quinolactacin A (88).	[29]
Penicillium sp. MB	Deletion	Positive	Inhibited production of the members with the 1-oxa-7-aza-spiro [4,4] non-2-ene-4,6-dione skeleton, including pseurotins A (89), B (90), C (91), D (92), and E (93).	[89]
Penicillium brocae HDN-12-143	Overexpression	Positive	Increased production of fumigatin chlorohydrin (94), iso-fumitatin chlorohydrin (95), spinulosin (96), and pyranonigrin F (97).	[90]
Penicillium chrysogenum	Overexpression and deletion	Positive	Overexpression of PclaeA gene led to increased production of penicillin G (28). Deletion of PclaeA led to decreased production of penicillin G (28).	[22]
Penicillium chrysogenum	Deletion	Positive	Decreased production of penicillin G (28).	[91]
Penicillium chrysogenum	Deletion	Positive	Decreased production of penicillin G (28).	[92]
Penicillium chrysogenum	Deletion	Positive	Small reduction in penicillin G (28).	[93]
Penicillium citrinum	Deletion	Positive	Decreased production of compactin (98).	[94]
Penicillium digitatum	Deletion	Positive	Reduced expression of several secondary metabolite BGCs.	[95]
Penicillium dipodomyis YJ-11	Overexpression	Positive	Increased production of sorbicillinoids including 10,11-dihydrobislongiquinolide (99), 10,11,16,17-tetrahydrobislongiquinolide (100), bislongiquinolide (101), 16,17-dihydrobislongiquinolide (102), sohirnone A (103), and 2′,3′-dihydrosorbicillin (104).	[96]
Penicillium expansum	Deletion	Positive	Decreased production of patulin (105).	[97,98]
Penicillium oxalicum	Deletion	Positive	Decreased production of secondary metabolites.	[99]
Penicillium oxalicum	Deletion	Positive	Four of the 28 secondary metabolite BGCs were significantly downregulated.	[100]
Penicillium roqueforti	Deletion	Positive	Decreased production of roquefortine C (106), mycophenolic acid (107), and andrastin A (108).	[101]
Pestalotiopsis microspore	Deletion	Positive	Decreased production of pestalotiollide B (109).	[102]
Pleurotus ostreatus	Deletion	Positive	Decreased production of the intracellular polysaccharide (IPS).	[103]
Pyricularia oryzae	Deletion and overexpression	Positive	Deletion of PoLAE1 reduced the production of tenuazonic acid (110). Overexpression of PoLAE1 led to increased production of tenuazonic acid (110).	[104]
Trichoderma afroharzianum	Overexpression	Positive	Induced production of (1R,3E,5E)-1-(3,5-dihydroxy- 2,4-dimethylphenyl)-1-hydroxyhepta- 3,5-dien-2-one (111) and (1R,3E,5E)-1-(3,5-dihydroxy- 2,4-dimethylphenyl)-1-methoxyhepta- 3,5-dien-2-one (112).	[105]
Trichoderma longibrachiatum	Deletion and overexpression	Positive	Deletion of Tllae1 reduced the production of peptaibols. Overexpression of Tllae1 led to two-fold increased production of petaibols.	[106]
Trichoderma reesei	Overexpression	Positive	Increased production of sorbicillinoids.	[107]
Trichoderma reesei	Deletion	Positive	Decreased production of sterigmatocystin (16).	[108]
Trichoderma reesei	Deletion	Negative	Increased production of ophiobolin F (113).	[109]
Valsa mali	Deletion	Positive	Decreased production of toxic metabolites.	[110]

) [21]. The examples of LaeA regulating secondary metabolite production in fungi are shown in Table 1. The structures of the metabolites are shown in Figure S1.

Overexpression of AaLaeA increased production of antitumor compounds, including myricetin (1), geraniol (2), ergosterol (3) and other compounds in the endophytic fungus, Alternaria alstroemeria by metabolomic analysis [24]. In contrast, overexpression of AaLaeA in another endophytic fungus A. alstroemeria derived from the medicinal plant Artemisia annua decreased production of antitumor compounds. Unfortunately, these antitumor compounds were not further identified [25]. Many species in the Alternaria genus usually belong to the plant pathogenic fungi to produce host-specific toxins (HSTs) and cause host plant diseases [2]. A. alternata was the pathogen of some plants, such as tomato, apple and strawberry, helping to produce AAL-, AM-, and AF-toxins, respectively. It was found that LaeA positively regulated production of the HSTs as well as the development and pathogenicity of A. alternata [111]. In other instances, deletion of laeA in A. alternata greatly decreased the production of alternariol (AOH, 4) and alternariol monomethyl ether (AME, 5) [26]. Furthermore, overexpression of an LaeA-like global transcriptional regulator in the marine-derived fungus A. alternata JJY–32 led to the discovery of an anti-inflammatory meroterpenoid, namely tricycloalternarene O (6) [27].

Arthrobotrys flagrans (synonym Duddingtonia flagrans) was a typical nematode trapping fungus which has been used for nematode biocontrol. Overexpression or deletion of AfLaeA positively regulated production of secondary metabolites and their antinematodal activity, whereas some metabolites were not produced due to the absence of AfLaeA. The antinematodal activity of these secondary metabolites needed further confirmation [28].

There are many examples of Aspergillus species regulated by LaeA to produce secondary metabolities (Table 1). Overexpression of Az5LaeA in Aspergillus sp. Z5 exhibited increased production of diorcinol (7). The laeA (AnLaeA) gene from A. nidulans was heteroexpressed in Aspergillus sp. Z5 and was also found to increase production of diorcinol (7) [29]. Introduction of laeA from A. nidulans to Aspergillus sp. FKI-5362 led to an increase in production of an antifungal compound MS-347a (8) which could inhibit the growth of broad plant pathogenic fungi, including Botrytis cinerea, Colletotrichum gloeosporoides, Leptosphaeria maculans, and Pyricularia oryzae [30]. MS-347a (8) was previously screened to inhibit myosin light chain kinase from Aspergillus sp. KY52178 [112]. When the laeA gene was deleted in A. carbonarius, the production of citric acid (9) [31] and ochratoxin A (OTA, 10) [32,33] was greatly decreased, respectively. If the deleted mutant ΔlaeA of A. carbonarius was colonized in nectarines and grapes, OTA (10) was significantly reduced [33]. When A. carbonarius was treated with eugenol at 0.2 μL/mL, OTA (10) production was decreased at 87.7%. The transcription of the clustered genes for OTA biosynthesis was significantly reduced under eugenol stress and was further confirmed by RT-qPCR analysis. The expression of LaeA was markedly downregulated by eugenol, which indicated that eugenol was probable through inhibiting LaeA expression to decrease OTA (10) production in A. carbonarius [33].

Overexpression of llm1 encoding LaeA-like methyltransferase in A. cristatus led to an increase in the contents of multiple secondary metabolites, including terpenoids and flavonoids. Unfortunately, these metabolites were not identified [35]. With a scaled-up fermentation of the ΔflLaeA strain of A. flavipes, five metabolites, including two previously undescribed piperazine derivatives flavipamides A (11) and B (12), along with three known nonribosomal peptides asperphenamate (N-benzoylphenylalaniny-N-benzoylphenyl-alaninate, 13), 4′-OMe-asperphenamate (14), and cyclic Pro-Gly-Val-Gly-Try(8-OH, 3-prenyl)-Gly-Trp (15), were obtained by LC-MS guided isolation [36].

Aflatoxins (AFs) have been thought of as the most potent carcinogens, and some fungal species from Aspergillus genus are their main producers, especially A. flavus. LaeA was revealed to positively regulate production of alfatoxins in A. flavus [38,39,40,42]. Deletion of laeA in A. flavus resulted in the significant upregulation of the NAD⁺-dependent histone deacetylase sirA involved in silencing secondary metabolism clusters via chromatin remodeling. Accompanying the chromatin modification, the enzymes participating in secondary metabolism, including biosynthesis of sterigmatocystin (ST, 16), aflatoxin B1 (17), cyclopiazonic acid (18), and ustiloxin B (19), were drastically decreased [37,39,41]. The enzymes in the ustiloxin B (19) biosynthesis pathway might be indirectedly regulated by LaeA [41]. An interaction partner of LaeA, namely kinetochore protein Spc105, was revealed to regulate development and secondary metabolism in A. flavus. Moreover, Spc105 positively regulated the production of secondary metabolites, such as aflatoxins and kojic acid (20), and negatively regulated the production of cyclopiazonic acid (18). Transcriptome analysis of the Δspc105 mutant revealed that 23 backbone genes for secondary metabolism were differentially expressed, corresponding to 19 of the predicted 56 secondary metabolite BGCs, suggesting broad regulatory roles for Spc105 in secondary metabolism in A. flavus [43].

The production of several mycotoxins was positively regulated by LaeA in A. fumigatus. For examples, the deletion of laeA led to decreased production of gliotoxin (21), endocrocin (22), fumagillin (23), fumagatin (24), and helvolic acid (25) in several strains of A. fumigatus [44,45,46,47]. Production of cyclopiazonic acid (18) was increased when laeA was overexpressed in A. fumisynnematus [48]. Deletion of laeA gene in A. luchuensis mut. kawachii led to the reduced production of citric acid (9). LaeA was considered as the methyltransferase through regulating the citrate exporter-encoding cexA gene [49].

Overexpression of laeA in A. nidulans led to an increased production of sterigmatocystin (ST, 16) [54] and terrequinone A (26) [50]. Accordingly, the deletion of the laeA gene in A. nidulans reduced the production of ST (16) and norsolorinic aid (NOR, 27) [51]. The deletion of laeA also led to a depressed expression of genes involved in the biosynthesis of ST (16), terrequinone A (26), and penicillin G (benzylpenicillin, 28) [52]. However, a contradictory example was that the deletion of laeA gene in A. nidulans also led to an increased production of ST (16) [53]. Generally, LaeA or LaeA-like methyltransferase F (LlmF) negatively regulated biosynthesis of ST (16) in A. nidulans.

Aspergillus niger is a biotechnologically important filamentous fungus and has been thought of as an industrial cell factory for the production of secondary metabolites with a broad spectrum of application fields, covering the agriculture, food, and pharmaceutical industries [113]. Deletion of laeA in A. niger decreased production of asperrubrol (29), atromentin (30), and JBIR 86 (31) but increased production of aspernigrin A (32) and BMS-192548 (33), which meant that LaeA positively regulated production of asperrubrol (29), atromentin (30), and JBIR 86 (31) and negatively regulated production of aspernigrin A (32) and BMS-192548 (33) in A. niger [55]. Overexpression of laeA gene in A. niger led to an activation of secondary metabolite BGCs in the mutant. Three compounds, including flaviolin (34), orlandin (35) and kotanin (36), were identified [56]. LaeA could influence the secondary metabolite profile in A. niger FGSC A1279 based on the genome sequencing and transcriptome analysis [114]. The production of ochratoxin A (OTA, 10) in A. niger was decreased in the deleted mutant and increased in the overexpressed mutant. Another similar example was that deletion of laeA gene in A. ochraceus led to the reduced production of OTA (10) [58]. This indicated that LaeA positively regulated the gene expression of the OTA (10) BGC in A. niger and A. ochraceus. In contrast, it was found that the upregulation of gene expression of OTA BGC did not necessarily increase OTA (10) production in A. niger [57].

Kojic acid (20) production was inhibited in the laeA disruption strain of A. oryzae, and restored in the laeA complement strain, which meant that LaeA positively regulated the biosynthesis of kojic acid (20) in A. oryzae [59]. In the expression system of A. oryzae, LaeA also showed its positive regulation on the heterologous BGCs. Overexpression of laeA resulted in the increased production of monocolin K (MK, 37) and terrequinone A (TQ, 38). The successful production of secondary metabolites belonging to different structural groups, namely MK (37) as a polyketide, and TQ (38) as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene clusters such as the BGC of MK (37) from Monascus pilosus and the BGC of TQ (38) from A. nidulans [60]. For A. pachycristatus and A. pseudoterreus, the production of secondary metabolites was also positively regulated by LaeA [61,62]. Production of sterigmatocystin (16) and echinocandin B (ECB, 39) was decreased in the laeA of the deleted mutant of A. pachycristatus [61]. Overexpression of laeA in A. pseudoterreus improved itaconic acid (40) yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that might be present in inexpensive sugar containing feedstocks, such as excess phosphate or complex nutrient sources [62].

A. terrreus is the main industrial producer of lovastatin (41), a drug that lowers cholesterol. Lovastatin (41) is also used as a precursor for simvastatin production. In A. terreus, the overexpression of the laeA gene triggered the increase in gene transcription related to penicillin G (28) and lovastatin (41) biosynthesis [37]. It has been observed that overexpression of the laeA gene in A. terreus increased the production of lovastatin (41) [63,115]. The chemical epigenetic modifiers 1,3-diaminopropane and spermidine also upregulated lovastatin (41) production and expression of lovastatin (41) biosynthetic genes in A. terreus via LaeA regulation [116]. Overexpression of laeA in A. terreus resulted in the activation of a silent secondary metabolite cluster without corresponding known metabolites. The yields of two antibacterial alkaloids dihydroisoflavipucines 1 (42) and 2 (43) reached 183 mg/L and 1.55 mg/mL, respectively. Both compounds showed obviously anti-Vibrio activities, with the MIC values ranging from 16 to 64 μg/mL against Vibrio anguillarum, V. campbellii, V. harveyi, and V. vulnificus [64].

The production of beauvericin (44) and bassiatin (45) was reduced in the BbLaeA disruption strain of Beauveria bassiana but was increased in the overexpressed strain [65].

The production ability of oxalic acid (OA, 46) was lost in laeA disruption strain of Botryitis cinerea [66]. Another example was that the production yield of abscisic acid (ABA, 47) was decreased 90% in the laeA disruption strain of B. cinerea. It was considered that BcLAE1 was involved in epigenetic regulation as a methyltransferase, with enhanced H3K9me3 modification and attenuated H3K4me2 modification in the ΔBclae1 mutant of B. cinerea [67].

Overexpression of laeA in Chaetomium globosum CBS148.51 upregulated expression of the chaetoglobosin BGC and resulted in the isolation of seven cytochalasans, including chaetoglobosins A (48), B (49), D (50), E (51), O (52), V (53), and Z (54). Of them, chaetoglobosin Z (54) was a new cytochalasan. These cytochalasans displayed strong cytotoxic activity against the HepG 2 cell line [68]. Similarly, the production of chaetoglobusin A (48) in the ΔCglaeA mutant of another C. globosum strain was inhibited, its CglaeA-C strain restored the production of chaetoglobusin A (48), and the strain of CglaeA overexpression led to an increase in chaetoglobusin A (48). This indicated that LaeA positively regulated the production of chaetoglobusin A (48) in C. globosum [69].

Cladosporium fulvum was the non-obligate biotrophic fungal tomato pathogen. Deletion of laeA in C. fulvum led to the increased production of the mycotoxin cladofulvin (55), which meant that LaeA negatively regulated biosynthesis of cladofulvin (55) in this fungus [70].

T-toxin (56) was a host selective phytotoxin produced by the maize pathogen Cochliobolus heterostrophus. Deletion of Chlae1 decreased production of T-toxin (56) in C. heterostrophus [71].

Coprinoferrin (57) was an acylated tripeptide hydroxamate consisting of tandem aligned N⁵-hexanoyl-N⁵-hydroxy-L-ornithine with modifications of N-acetyl and C-carboxamide. Knockout of laeA in the mushroom fungus Coprinopsis cinerea upregulated the biosynthesis of a novel siderophore, namely coprinoferrin (57), which indicated that LaeA negatively regulated the production of coprinoferrin (57) [72]. The unique chemical properties made coprinoferrin (57) an iron (III) binder (siderophore), which helped iron acquisition from the environment and promoted hyphal growth as well as fruiting body formation in C. cinerea. In addition, coprinoferrin (57) could be chemically synthesized from the N-Boc-L-glutamic acid 5-benzyl ester [117].

Daldinia eschscholzii was an endophytic fungus from the guts of mantis (Tenodora aridifolia). Replacement of the native promoter of the global regulator LaeA-like gene of D. eschscholzii by a strong gpdA promoter led to the generation of two novel cyclopentenone metabolites named dalestones A (58) and B (59). Both dalestones inhibited the gene expression of TNF-α and IL-6 in LPS-induced RAW264.7 macrophages [73].

Deletion of DsLaeA resulted in enhanced production of dothistromin (60) in the pine needle pathogen Dothistroma septosporum and increased expression of the regulatory gene DsAflR in the dothistromin (60) biosynthetic pathway [74].

Fusarium fujikuroi (teleomorph: Gibberella fujikuroi) is the pathogen of rice bakanae disease that produces a series of secondary metabolites, such as bikaverin (61), fusaric acid (62), gibberellins, fusarins, and fusarubins. Among them, fusaric acid (62) and fusarins belong to the harmful mycotoxins [118]. LaeA positively regulated production of some metabolites in F. fujikuroi. For example,, deletion of laeA in F. fujikuroi led to decreased production of gibberellins A3 (63) and A4 (64), fusarin C (65), fumonisins B1 (66), B2 (67), B3 (68) and B4 (69), deoxynivalenol (70), and 15-acetyl deoxynivalenol (71) [75]. Similar results were subsequently confirmed. Deletion of the lae1 gene led to reduced production of fusaric acid (62), fusarinolic acid (72), and dehydrofusaric acid (73) in the F. fujikuroi strain [76]. Furthermore, deletion of the lae1 led to decreased production of gibberellins, fumonisins, and fusarin C (65). Overexpression of lae1 led to increased production of gibberelins in another F. fujikuroi strain [77]. However, LaeA also negatively regulated production of some metabolites in F. fujikuroi. The production of bikaverin (61) was increased in the deletion mutant of F. fujikuroi [75]. Another example was that deletion of the lae1 gene in F. fujikuroi upregulated the expression of gibepyrone BGC as well as increased the production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79) [78].

LaeA positively regulated mycotoxin production of the following phytopathogenic Fusarium species. Deletion of FglaeA in F. graminearum led to a dramatic reduced production of trichothecenes and zearalenone (80). Overexpression of FglaeA caused the increased production of trichothecenes and zearalenone (80). This indicated that FgLaeA positively regulated production of phytotoxins of F. graminearum [79]. For the fungus F. oxysporum, deletion of laeA caused the decreased production of beauvericin (44) and fusaric acid (62), which contributed to virulence in plant hosts, such as tomato plants [80]. For the fungus F. oxysporum f.sp. niveum, the deletion of the FoLae1 gene led to depressed conidiation and reduced production of fusaric acid (62) and bikaverin (70). In addition, all of these alterations in the deleted mutants were restored in the corresponding complementation strains. [81]. For the fungus F. verticillioides, the deletion of laeA reduced the production of fusaric acid (62), fusarin C (65), bikaverin (70), and fumonisins [82].

Ganoderic acids (GAs) are lanosterol-type triterpenoids produced by the Ganoderma species that possess multiple bioactivities, including anti-cancer, anti-inflammatory, antioxidant, and anti-HIV activities [119]. When a methyltransferase-like laeA gene was deleted in G. lingzhi, the production of ganoderic acids was reduced. RT-qPCR analysis further revealed that the transcription levels of genes involved in the biosynthesis of garnoderic acids were drastically lower in the ΔlaeA strain. In contrast, constitutive overexpression of laeA resulted in an increased concentration of GAs [83].

Magnaporthe oryzae causes blast disease, the most serious disease of cultivated rice affecting global rice production. MolaeA negatively regulated sporulation and melanin biosynthesis and positively regulated production of penicillin G (or called benzylpenicillin, 28) [84]. Metabolomic profiling analysis showed that overexpression of MolaeA led to the increased biosynthesis of secondary metabolites in M. oryzae. Unfortunately, these metabolites have not been identified [85].

Some Monascus species can produce edible pigments, with their structures bearing a highly oxygenated pyranoquinone bicyclic core and a quaternary carbon center. However, the mycotoxin citrinin (81) produced by some Monascus strains restricts application of the pigments [120]. Monacolin K (37), a cholesterol-lowering agent, was increased three times when laeA was overexpressed in Monascus pilosus. In addition, the pigment production was also remarkably increased [86]. The production of monacolin K (37) was also increased when laeA was overexpressed in M. purpureus [87]. For another Monascus species, the deletion of MrlaeA in M. ruber exhibited a drastically reduced production of toxin citrinin (CIT, 81) and six pigments, namely rubropunctamine (82), monascorubramine (83), monascin (84), rubropunctatin (85), ankaflavin (86), and monascorubrin (87) [88].

The laeA gene from Aspergillus nidulans was heteroexpressed in the fungus Penicillium sp. LC1-4. Overexpression of AnLaeA caused an increased production of a bioactive compound quinolactacin A (88). It indicated that heteroexpressed of AnLaeA in fungi was a simple and effective method to explore metabolic potential [29]. LaeA could also positively regulate production of antibacterial pseurotins in Penicillium sp. Deletion of the laeA gene in Penicillium sp. strain MB inhibited production of the members with a 1-oxa-7-aza-spiro [4,4] non-2-ene-4,6-dione skeleton. Among these deduced compounds, pseurotins A (89), B (90), C (91), D (92), and E (93) displayed obvious antibacterial activity. This was why cheese rind bacterial communities assembled with the laeA deletion mutant of Penicillium sp. strain MB had significantly higher bacterial abundances than the wild-type strain [89].

To date, secondary metabolite production in various Penicillium species has been found to be positively regulated by LaeA. Overexpression of PbrLaeA led to the discovery of four compounds, namely fumigatin chlorohydrin (94), iso-fumitatin chlorohydrin (95), spinulosin (96), and pyranonigrin F (97), in the fungus P. brocae HDN-12-143. Among them, iso-fumitatin chlorohydrin (95) was a new compound. Both fumigatin chlorohydrin (94) and iso-fumitatin chlorohydrin (95) exhibited cytotoxic activity against HL-60, with IC₅₀ values of 18.63 µM and 24.83 µM, respectively [90]. Overexpression of PclaeA in P. chrysogenum gave rise to a 25% increase production of penicillin G (benzylpenicillin, 28). PclaeA knock-down mutants exhibited drastically reduced production and biosynthesis gene expression of penicillin G (28) [22]. Deletion of laeA in P. chrysogenum decreased production of penicillin G (28) [91]. However, epigenetic modifiers 1,3-diaminopropane (1,3-DAP) and spermidine completely restored the levels of penicillin G production in the laeA knock-down mutant. This indicated that LaeA in P. chrysogenum might act epigenetically on the expression of secondary metabolite genes by heterochromatin reorganization, which should be studied in detail [92]. Small reduction in penicillin G (28) was also reported in another ΔPclaeA mutant of P. chrysogenum [93]. The full-length laeA gene, namely Pci-laeA, with the sequence as 1340 bp, including an ORF of 1284 bp encoding 427 amino acids, was cloned from P. citrinum. The predicted molecular mass of Pci-LaeA was 48.72 kDa, with an estimated theoretical isoelectric point of 6.96. Pci-LaeA had a conserved S-adenosylmethionine binding site and a potential MlcR (a pathway specific regulator in mevastatin biosynthesis) binding site [121]. When an laeA gene was deleted in P. citrinum, production of compactin (also named ML-236B, mevastatin, 98) was suppressed [94]. Comparative transcriptome analysis revealed that the function loss of PdLaeA in P. digitatum resulted in the reduced expression of several secondary metabolite gene clusters [95].

Sorbicillinoids are important hexaketide metabolites derived from fungi. They have a variety of biological activities with unique structural features to make them attractive candidates for developing new pharmaceutical and agrochemical agents [122,123]. Overexpression of the laeA gene in the marine-derived fungus P. dipodomyis YJ-11 induced metabolic variations to afford a series of sorbicillinoids, including two new ones named 10,11-dihydrobislongiquinolide (99) and 10,11,16,17-tetrahydrobislongiquinolide (100), as well as four known analogues, bislongiquinolide (101), 16,17-dihydrobislongiquinolide (102), sohirnone A (103), and 2′,3′-dihydrosorbicillin (104). This indicated that regulation of LaeA is a useful strategy in activating silent gene clusters in fungal strains to obtain previously undiscovered compounds [96].

The mycotoxin patulin (105) was produced in the colonized tissue by P. expansum during the storage of apples. Deletion of laeA in P. expansum led to a decrease in patulin (105) production, which positively regulated patulin gene expression and patulin biosynthesis. Loss of LaeA affected the colonization of P. expansum in apple fruits. The ΔlaeA strains showed reduced virulence at all stages of apple maturity, and the disease severity was reduced by up to 22% in more mature fruits [97,98]. This demonstrated that patulin metabolism modulated by LaeA contributed in part to the pathogenicity of P. expansum [97].

The LaeA in P. oxalicum played an important role in asexual development, the expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis. Deletion of the laeA gene led to decreased production of secondary metabolites. Unfortunately, these differential metabolites have not been identified [99]. Four (i.e., cluster_1, cluster_5, cluster_14, and cluster_26) of the 28 secondary metabolic gene clusters were significantly downregulated in the ΔlaeA mutant compared with the wild-type strain (WT) of P. oxalicum. The LaeA was speculated to be the putative methyltransferase. Histone H2B lysine 122 and lysine 130 were considered as the putative targets of LaeA [100]. Another example was that the disruption of PrlaeA in P. roqueforti led to a substantial reduction in the production of the three metabolites roquefortine C (106), mycophenolic acid (107), and andrastin A (108). However, deletion of PrlaeA had little impact on asexual development [101].

Disruption of laeA in the Pestalotiopsis microspore led to the decreased production of pestalotiollide B (PB, 109) [102]. Similarly, deletion of PoLaeA1 in Pleurotus ostreatus decreased the intracellular polysaccharide (IPS) content by about 28–30% as well as the cellulose activity, which provided new insights into the regulation of polysaccharide biosynthesis and cellulose production in filamentous fungi [103]. PoLAE1 also positively regulated tenuazonic acid (TeA, 110) production of the rice blast pathogen Pyricularia oryzae (teleomorph: Magnaporthe oryzae) [104].

LaeA positively regulated the secondary metabolite production of the following Trichoderma species. Overexpression of TalaeA in T. afroharzianum led to the production of two new antifungal polyketides: (1R,3E,5E)-1-(3,5-dihydroxy-2,4-dimethylphenyl)-1-hydroxyhepta-3,5-dien-2-one (111) and (1R,3E,5E)-1-(3,5-dihydroxy-2,4-dimethylphenyl)-1-methoxyhepta- 3,5-dien-2-one (112). Both compounds showed strong antifungal activity on plant pathogenic fungi Botrytis cinerea, Colletotrichum lagenarium, and Fusarium oxysporum f.sp. nicotianae [105]. Deletion of Tllae1 in T. longibrachiatum reduced the production of peptaibols to a large degree. The peptaibols belonged to antimicrobial peptides and were named as trichokonins (TKs) which were mainly classified into 20-aa trichokonin A (TKA) and 12-aa trichokonin B (TKB). Overexpression of Tllae1 in T. longibrachiatum led to two-fold increased production of petaibols. Overexpression of laeA gene in T. reesei led to the increased production of sorbicillinoids, which were not identified. If the laeA gene in another T. reesei strain was deleted, the production of sterigmatocystin (16) was decreased [108]. T. reesei had a potential to produce terpenoids. If the lae1 gene along with major hemi-cellualse genes were deleted, the production of sesquiterpenoid ophiobolin F (113) in T. reesei was increased to 1187.06 mg/L by using the modified chassis [109].

Deletion of VmLaeA in apple canker pathogen Valsa mali led to greatly reduced virulence, with lesion length being reduced by 48% in apple twigs. The toxicity of secondary metabolites produced by VmLaeA deletion mutant (ΔVmlaeA) was markedly decreased in comparison with the wild-type strain. Unfortunately, these toxic metabolites have not been identified [110].

3. Regulation of Velvet Proteins on Secondary Metabolite Production in Fungi

The velvet proteins (or so-called velvet family proteins) included VeA (velvet A), VelB (velvet like B), VelC (velvet like C), VelD (velvet like D), and VosA (viability of spores A). These five proteins all contain the velvet and transactivation domains. They are highly conserved in dimorphic and filamentous fungi [16,39,124,125,126]. They mainly play important roles in fungal development, asexual sporulation, sexual development, secondary metabolism, and stress tolerance [127]. It has been revealed that LaeA and velvet proteins formed multimeric complexes, such as VelB-VeA-LaeA, VelB-VosA, and VelB-VelB, in fungi. The heterotrimeric VelB-VeA-LaeA complex controls sexual development and secondary metabolism in response to light [128,129,130,131,132]. The following is the research progress of velvet proteins on the regulation of secondary metabolite production in fungi.

3.1. Regulation of VeA on Secondary Metabolite Production in Fungi

The VeA (also called VelA, Ve1, and Vel1) proteins usually positively regulated production of secondary metabolites in fungi. Most of the regulated metabolites were polyketides. Some examples of VeA proteins regulating secondary metabolite production in fungi are shown in

Table 2. Some examples of VeA regulating secondary metabolite production in fungi.

Fungus	Overexpression/ Deletion of veA	Positive/Negative Regulation	Production of Secondary Metabolites	Ref.
Acremonium chrysogenum	Deletion	Positive	Decreased production of cephalosporin C (114).	[133]
Alternaria alternata	Deletion	Positive	Decreased production of alternariol (4) and alternariol monomethyl ether (5).	[26]
Alternaria alternata	Deletion	Positive	Decreased production of alternariol (4) and alternariol monomethyl ether (5).	[134]
Aspergillus carbonarius	Deletion	Positive	Production of ochratoxin A (10) was decreased to almost zero.	[32]
Aspergillus carbonarius	Deletion	Positive	Decreased production of ochratoxin A (10).	[135]
Aspergillus flavus	Deletion	Positive	Decreased production of cyclopiazonic acid (18), aflatrem B (115), and aflatoxins.	[136]
Aspergillus flavus	Deletion	Positive	Decreased production of asparasone A (116).	[137]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxin B1 (17).	[138]
Aspergillus flavus	Deletion and overexpression	Positive	Decreased production of aflatoxins in the deletion mutant and increased production of aflatoxins in the overexpression mutant.	[139]
Aspergillus fumigatus	Deletion	Positive	Decreased production of gliotoxin (21).	[140]
Aspergillus fumigatus	Overexpression	Negative	Decreased production of gliotoxin (21).	[140]
Aspergillus fumigatus	Deletion	Positive	Decreased production of fumagillin (23), gumitremorgin G (117), fumigaclavine C (118), and glionitrin A (119).	[141]
Aspergillus fumigatus	Overexpression	Negative	Decreased production of fumagillin (23), gumitremorgin G (117), fumigaclavine C (118), and glionitrin A (119).	[141]
Aspergillus nidulans	Deletion	Positive	Decreased production of sterigmatocystin (16) and penicillin G (28).	[142]
Aspergillus nidulans	Deletion	Positive	Decreased production of penicillin G (28).	[143]
Aspergillus nidulans	Deletion	Positive	Decreased production of sterigmatocystin (16) and norsolorinic aid (27)	[51]
Aspergillus nidulans	Deletion	Negative	Increased production of orsellinic acid (120), F9775A (121) and F9775B (122).	[144]
Aspergillus nidulans	Deletion	Positive	Decreased production of aspernidines.	[145]
Aspergillus niger	Deletion	Positive	Decreased production of ochratoxins A (10), α (123), and β (124).	[146]
Aspergillus ochraceus	Deletion	Positive	Decreased production of ochratoxin A (10).	[58]
Aspergillus oryzae	Deletion	Positive	Decreased production of kojic acid (20).	[147]
Aspergillus pachycristatus	Deletion	Positive	Decreased production of sterigmatocystin (16) and echinocandin B (39).	[61]
Aspergillus parasiticus	Deletion	Positive	Decreased production of sterigmatocystin (16).	[148]
Aspergillus parasiticus	Deletion	Positive	Reduced aflatoxin BGC gene expression and aflatoxisome development.	[149]
Chaetomium globosum	Deletion and overexpression	Positive	Decreased production of chaetoglobusin A (48) in the veA deleted mutant and increased production of chaetoglobusin A (48) in the veA overexpressed mutant.	[150]
Cladosporium fulvum	Deletion	Negative	Increased production of cladofulvin (55).	[70]
Cochliobolus heterostrophus	Deletion	Positive	Decreased production of T-toxin (56).	[71]
Fusarium fujikuroi	Deletion	Negative	Increased production of bikaverin (61).	[75]
Fusarium fujikuroi	Deletion	Positive	Decreased production of gibberellins A3 (63) and A4 (64), fusarin C (65), fumonisins B1 (66), B2 (67), B3 (68) and B4 (69), deoxynivalenol (70), and 15-acetyl deoxynivalenol (71).	[75]
Fusarium fujikuroi	Deletion	Positive	Decreased production of fusaric acid (62), fusarinolic acid (72), and dehydrofusaric acid (73)	[76]
Fusarium fujikuroi	Deletion	Positive	Decreased production of gibberellins, fumonisins, and fusarin C (65).	[77]
Fusarium fujikuroi	Deletion	Negative	Increased production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79).	[78]
Fusarium graminearium	Deletion	Positive	Reduced production of deoxynivalenol (69).	[151]
Fusarium graminearium	Deletion	Positive	Decreased production of trichothecenes.	[152]
Fusarium nematophilum	Overexpression	Positive	Increased production of antitumor compounds.	[153]
Fusarium oxysporum	Deletion	Positive	Decreased production of beauvericin (44) and fusaric aicd (73)	[80]
Fusarium oxysporum f.sp. niveum	Deletion	Positive	Decreased production of bikaverin (61) and fusaric acid (62)	[81]
Fusarium verticillioides	Deletion	Positive	Decreased production of fusarin C (65) and fumonisins B1 (66), B2 (67) and B3 (68).	[154]
Mycosphaerella graminicola	Deletion	Positive	Decreased production of melanin.	[155]
Neurospora crassa	Deletion	Positive	Decreased production of carotenoids.	[156]
Neurospora crassa	Deletion	Positive	Decreased production of siderophore coprogen (125) and carotenoids.	[157]
Penicillium chrysogenum	Deletion	Positive	Decreased production of penicillin G (28).	[91]
Penicillium chrysogenum	Deletion	Positive	Decreased production of penicillin G (28).	[158]
Penicillium chrysogenum	Deletion	Positive	Small reduction in penicillin G (28).	[93]
Penicillium citrinum	Deletion and overexpression	Positive	Decreased production of compactin (98) in the deletion mutant, and increased production of compactin (98) in the overexpressed mutant.	[94]
Penicillium citrinum	Overexpression	Positive	Increased production of compactin (98).	[159]
Penicillium expansum	Deletion	Positive	Decreased production of citrinin (81) and patulin (105).	[127]
Penicillium expansum	Deletion	Positive	Blocked production of patulin (105).	[160]
Penicillium expansum	Deletion	Positive	Lost production of patulin (105).	[161]
Pestalotiopsis microspora	Deletion	Negative	Increased production of pestalotiollide B (103).	[102]

. The structures of the metabolites are shown in Figure S1.

Disruption of the AcveA gene in Acremonium chrysogenum resulted in a reduction in cephalosporin C (114) production, which meant that AcVeA positively regulated cephalosporin C (114) biosynthesis in A. chrysogenum [133].

Deletion of the veA gene in Alternaria alternata greatly reduced sporulation and the production of alternariol (AOH, 4) and alternariol monomethyl ether (AME, 5) [26]. The production of both AOH (4) and AME (5) in A. alternata was significantly stimulated by blue light. The disruption of AaVeA resulted in a marked decrease in AOH (4) and AME (5) production under blue light illumination [134].

Aspergillus carbonarius was the pathogen of grape Aspergillus rot [162]. The fungus could produce ochratoxin A (OTA, 10). Upon the deletion of veA in A. carbonarius, the production of OTA (10) almost disappeared [32]. Deletion of veA in A. carbonarius resulted in significant reduction in OTA (10) production. During both in vitro growth and the infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. The OTA (10)-defective ΔveA mutant was considered as the potential biocontrol agent [135].

VeA affected the biosynthesis of mycotoxins in Aspergillus flavus. Deletion of veA in A. flavus decreased the production of cyclopiazonic acid (18), aflatrem B (115), and aflatoxins [136]. Deletion of veA also decreased the production of asparasone A (116) [137] and aflatoxin B1 (17) in A. flavus [138]. VepN contained a septin-type guanine nucleotide-binding domain, representing a conserved protein family from yeast to humans belonging to the P-loop GTPase superfamily. It was found that the global regulation gene veA positively regulated vepN to influence aflatoxin production, morphological development, and pathogenicity in A. flavus [139].

Both deletion and overexpression of veA in Aspergillus fumigatus led to decreased production of gliotoxin (21). The RNA sequencing data provided evidence supporting this pattern. It was possible that both deletion and overexpression of veA downregulated fumR transcription, suggesting that veA influenced the activation of the fumagillin gene cluster through regulation of fumR [140]. The similar regulation pattern was also observed in A. fumigatus to produce other secondary metabolites. Both deletion and overexpression of veA in A. fumigatus decreased the production of fumagillin (23), gumitremorgin G (117), fumigaclavine C (118), and glionitrin A (119) [141].

The deletion of veA in A. nidulans suppressed the production of sterigmatocystin (16). The veA deletion mutant produced less penicillin G (28) than the regular strain. The veA gene was also required for sexual development [142]. The deletion of the veA gene in A. nidulans reduced the production of sterigmatocystin (16) and norsolorinic aid (NOR, 27) [51]. VeA was thought to be involved in the penicillin G (28) biosynthesis via repression of the expression of the acvA gene, which led to reduced penicillin production in A. nidulans [143]. Upon further investigation, it was found that VeA repressed the expression of the cryptic orsellinic acid (120) BGC in A. nidulans through histone 3 acetylation. The deletion of veA led to increased production of orsellinic acid (120), F9775A (121), and F9775B (122) in A. nidulans [144]. Further investigation revealed that A. nidulans VeA was a multi-phosphorylated protein, and it was hypothesized that at least four specific amino acids (T167, T170, S183 and Y254) underwent reversible phosphorylation to trigger development and sterigmatocystin (16) biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regard to sexual development and veA gene expression [163]. In the dark, VeA entered the nucleus of A. nidulans, formed a VelB-VeA-LaeA heterotrimeric complex, controlled sexual development, and enhanced sterigmatocystin (16) production [128]. The downstream transcription factor regulatory gene of veA was revealed as mtfA in A.nidulans. Deletion of mtfA could decrease the expression of the genes in the penicillin gene cluster, reducing penicillin production. In this case, overexpression of mtfA enhanced the transcription of the penicillin BGC, increasing penicillin production more than five-fold with respect to the control. However, it was detrimental for the expression of the terrequinone BGC in regard to either deletion or overexpression of mtfA. In addition to its effect on secondary metabolism, mtfA also affected asexual and sexual development in A. nidulans. Deletion of mtfA resulted in a reduction in the conidiation and sexual stages [164]. Another example of the downstream transcription factor regulatory gene of veA1 was revealed in sclB in A. nidulans. Deletion of sclB also decreased production of aspernidines [145].

The deletion of veA in Aspergillus niger suppressed the production of ochratoxins A (OTA, 10), α (OTα, 123), and β (OTβ, 124). The veA gene acted as the positive regulator of conidia production, OTA (10) biosynthesis, and oxidative stress tolerance in A. niger regardless of light conditions. Darkness promoted conidial production and OTA (10) biosynthesis in the wild-type strain of A. niger [146]. The deletion of veA in A. oryzae also decreased production of kojic acid (20) [147]. Disruption of veA significantly reduced the production of echinocandin B (39) and sterigmatocystin (16) in A. pachycristatus [61]. Deletion of the veA gene in A. parasiticus reduced aflatoxin BGC gene expression and aflatoxisome development [149].

CgVeA in Chaetomium globosum was thought of as a light-signaling responsive regulator. It was involved in the regulation of chaetoglobusin A (48) biosynthesis. Deletion of CgveA caused an obvious decrease in chaetoglobusin A (48) production from 51.32 to 19.76 mg/L under dark conditions. In contrast, CgveA overexpression resulted in a dramatic increase in chaetoglobusin A (48) production, reaching 206.59 mg/L under illumination, which was higher than that noted in darkness. The RT-qPCR results confirmed that CgVeA, as a light-responsive regulator, positively regulated chaetoglobusin A (48) biosynthesis by controlling the expression of core genes of the chaetoglobusin A (48) biosynthetic gene cluster and other relevant regulators [150].

Deletion of veA in tomato fungal pathogen Cladosporium fulvum led to increased production of the pigment cladofulvin (55), which meant that VeA negatively regulated biosynthesis of cladofulvin (55) in this fungus [70].

Deletion of veA in Fusarium fujikuroi led to decreased production of gibberellins A3 (63) and A4 (64), fusarin C (65), fumonisins B1 (66), B2 (67), B3 (68) and B4 (69), deoxynivalenol (70), and 15-acetyl deoxynivalenol (71). However, the production of bikaverin (61) was increased in the deletion mutant [75]. The similar results were confirmed later. Deletion of vel1 led to reduced production of fusaric acid (62), fusarinolic acid (72), and dehydrofusaric acid (73) in the F. fujikuroi strain [76]. Deletion of vel1 led to decreased production of gibberellins, fumonisins, and fusarin C (65). Overexpression of lae1 led to increased production of gibberelins in another F. fujikuroi strain [77]. Deletion of the vel1 gene in F. fujikuroi led to upregulation of gibepyrone BGC expression as well as the increased production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79) [78].

Deletion of veA in Fusarium graminearium, the causal agent of Fusarium head blight, led to reduced production of deoxynivalenol (vomintoxin or DON, 68) [151] and decreased production of trichothecenes [152].

Upon the overexpression of FnveA in Fusarium nematophilum, the antitumor activity of the crude extract was increased on A549 cancer cells. Unfortunately, the antitumor compounds were not identified [153].

Deletion of veA in Fusairum oxysporum caused the decreased production of beauvericin (44) and fusaric acid (62), which contributed to virulence on plant hosts such as tomato plants [80]. Deletion of the FoVel1 gene in F. oxysporum f.sp. niveum led to depressed conidiation and reduced production of bikaverin (61) and fusaric acid (62). In addition, all of these alterations in the deleted mutants were restored in the corresponding complementation strains [81].

Deletion of Ffvel1 in Fusarium verticillinoides led to decreased production of gibberellins (63) and A4 (64), fusarin C (65), fumonisins B1 (66), B2 (67), B3 (68) and B4 (69), deoxynivalenol (70), and 15-acetyl deoxynivalenol (71). However, the production of bikaverin (61) was increased in the deletion mutant. The regulation mechanisms of vel1 in the above metabolite production should be similar to those of laeA in this fungus [75]. Deletion of veA in maize pathogen Fusarium verticillioides led to decreased production of fusarin C (65) and fumonisins B1 (66), B2 (67), and B3 (68) [154]. Further investigation showed that VeA was necessary for causing symptom and mycotoxin synthesis in maize seedlings by F. verticillioides [165].

Deletion of the mve1 gene in Mycosphaerella graminicola decreased production of melanin. The Δmve1 mutant displayed an albino phenotype with a significant reduction in melanin biosynthesis and less production of aerial mycelia on agar plates [155].

Deletion of the veA gene in Neurosopora crassa decreased both asexual conidiation and carotenoid production [156]. Further investigation showed that the production of siderophore coprogen (125) was also decreased in the ΔveA mutant of N. crassa [157].

Deletion of the veA gene in Penicillium chrysogenum decreased the production of penicillin G (28) [91,158]. A small reduction in penicillin G (28) was also reported in another ΔPcvelA mutant of P. chrysogenum [93]. P. citrinum is known to produce compactin (also called ML-236B or mevastatin, 98). This polyketide exhibited a potent inhibitory activity on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Compactin (98) was industrially converted into pravastatin by microbes. Currently, pravastatin has been widely used as a pharmaceutical drug for the treatment of hypercholesterolemia [158]. Deletion of veA in P. citrinum led to suppressed production of compactin (98), and overexpression of veA led to increased production of compactin (98). This indicated that veA is the key regulation factor of compactin (98) biosynthesis [94]. Overexpression of veA in P. citrinnum led to compactin (98) production being increased [159].

Penicillium expansum is the causal agent of apple blue mold disease [166]. It produces the mycotoxins citrinin (81) and patulin (105). The disruption of veA in P. expansum drastically reduced the production of citrinin (81) and patulin (105) in synthetic media, which is associated with a marked downregulation of all genes involved in the biosynthesis of the two mycotoxins. Moreover, the null mutant ΔPeveA strain was unable to produce patulin (105) in apples [127]. Deletion of veA in P. expansum led to the production of patulin (105) in the ΔveA mutant being completely blocked [160,161]. The ΔveA mutants also exhibited reduced growth and conidiation when exposed to stressors, including cell membrane stress, oxidative stress, osmotic stress, and different pH values, which indicated that patulin (105) contributed to the fungal anti-stress ability. The non-mycotoxigenic strain ΔveA of P. expansum showed its biocontrol capability against a postharvest pathogen of pome fruit during postharvest handling and storage [161].

Deletion of the veA gene in Pestalotiopsis microspora led to increased production of pestalotiollide B (103). The veA gene appeared to negatively regulate the biosynthesis of pestalotiollide B (103) [102].

3.2. Regulation of VelB on Secondary Metabolite Production in Fungi

VelB (also called Vel2) mainly coordinates with other members, such as LaeA, VeA and VosA, to regulate the production of fungal secondary metabolites in fungi, as VelB lacks a site for a nuclear localization signal (NLS) [128]. Some examples of VelB regulating secondary metabolite production in fungi are shown in

Table 3. Some examples of VelB regulating secondary metabolite production in fungi.

Fungus	Overexpression/ Deletion of velB	Positive/Negative Regulation	Production of Secondary Metabolites	Ref.
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxins.	[167]
Aspergillus flavus	Deletion	Positive	Decreased production of aflatoxin B1 (17).	[138]
Aspergillus nidulans	Deletion	Positive	Decreased production of sterigmatocystin (16).	[128]
Aspergillus nidulans	Deletion	Negative	Increased production of sterigmatocystin (16).	[168]
Aspergillus ochraceus	Deletion	Positive	Reduced production of ochratoxin A (10).	[58]
Aspergillus oryzae	Deletion	Negative	Increased production of kojic acid (20).	[147]
Botrytis cinerea	Deletion	Negative	Increased production of melanin.	[169]
Colletotrichum siamense	Deletion	Positive	Decreased production of melanin.	[170]
Curvularia lunata	Deletion	Positive	Decreased production of methyl 5-hydroxymethylfuran-2-carboxylate (126).	[171]
Fusarium fujikuroi	Deletion	Positive	Decreased production of gibberellins, fumonisins and fusarin C (65).	[77]
Fusarium fujikuroi	Deletion	Negative	Increased production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79)	[78]
Fusarium graminearum	Deletion	Positive	Decreased production of deoxynivalenol (68).	[171]
Fusarium graminearum	Deletion	Positive	Decreased production of trichothecenes and zearalenone (80).	[172]
Fusarium pseudograminearum	Deletion	Positive	Decreased production of deoxynivalenol (68).	[173]
Neurospora crassa	Deletion	Positive	Reduced biosynthesis of carotenoids.	[157]
Penicillium chrysogenum	Deletion	Negative	Increased production of penicillin G (28).	[174]
Penicillium Expansum	Deletion	Positive	Blocked production of patulin (105).	[160]
Penicillium expansum	Deletion	Positive	Decreased production of chaetoglobusin A (48), citrinin (81), and patulin (105).	[175]
Penicillium expansum	Deletion	Negative	Increased production of fumarylalanine (127).	[175]
Pestalotiopsis microspora	Deletion	Positive	Decreased production of pestalotiollide B (103).	[102]

. The structures of the metabolites are shown in Figure S1.

Deletion of velB in Aspergillus flavus abolished aflatoxin production and sclerotial formation either under illumination or in darkness. VelB may have a dual role and likely coordinates with FluG to modulate its functions [167]. A similar result was reported by Eom et al., indicating that inactivation of velB led to decreased production of aflatoxin B1 (17) in A. flavus [138].

The knock out of the velB gene in Aspergillus nidulans led to reduced content in sterigmatocystin (16) under illumination. However, the production yield of sterigmatocystin (16) in the deletion mutant was almost the same as that of the wild-type strain, which indicated that the mycelial growth rate of the deletion mutant was bigger than that of the wild-type strain [128]. Further investigation showed that the deletion of velB in A. nidulans resulted in decreased mRNA levels of vadJ throughout the life cycle. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin (16). This indicated that velB was necessary for proper coordination via vadJ to regulate sterigmatocystin (16) production [168].

Deletion of the velB gene in Aspergillus ochraceus led to drastically reduced production of ochratoxin A (OTA, 10) [58].

The deletion of BcvelB led to increased conidiation and melanin biosynthesis in Botrytis cinerea. The expression of the melanin biosynthesis gene cluster was also upregulated [169].

CsVelB positively regulated the melanin production of Colletotrichum siamense [170].

Deletion of the ClvelB gene in Curvularia lunata led to a decrease in the production of conidia and the phytotoxin methyl 5-hydroxymethylfuran-2-carboxylate (126). The ΔClvelB mutant was impaired in colonizing the host tissue. However, deletion of the ClvelB gene led to an increase in aerial hyphae and melanin production [176].

Deletion of the vel2 gene in F. fujikuroi led to upregulation of the gibepyrone BGC expression as well as increased production of gibepyrones A (74), B (75), C (76), D (77), E (78), and F (79) [78].

Deletion of FgvelB in Fusarium graminearum led to decreased production of deoxynivalenol (DON, 68) [171]. Production of trichothecenes and zearalenone (80) in the FgvelB-deleted strain of F. graminearum was also dramatically reduced compared with the wild strain [172]. A similar example is that the deletion of FpvelB led to notable differences in growth, conidiation, virulence, and deoxynivalenol (68) production in F. pseudograminearum. Furthermore, FpVelB positively regulated another secondary metabolite BGC associated with pathogenesis by modulating the expression of the PKS11 gene. FpVelB regulated the pathogen virulence by influencing deoxynivalenol (68) production in F. pseudograminearum [173].

Deletion of velB in Neurospora crassa led to reduced biosynthesis of light-dependent carotenoids [157].

Penicillium expansum is the pathogen of apple blue mold disease and the main producer of patulin (105). The ΔPevelB mutant colonized apples, albeit at a lower rate than the wild-type and complemented strains. Conidiation was significantly reduced in the ΔPevelB strain. Under light conditions, the ΔPevelB strain showed a reduced level of spore viability. Deletion of the velB gene strongly inhibited the production of mycotoxins chaetoglobusin A (48), citrinin (81), and patulin (105) in synthetic media or in planta but increased the production of fumarylalanine (127). In addition, the genes involved in siderophore biosynthesis, ergosterol biosynthesis, and nitrate assimilation were also upregulated in the ΔPevelB strain. This indicated that VelB was involved in the development, pathogenicity, and secondary metabolism of P. expansum [175].

Deletion of veA in Pestalotiopsis microspora led to a decrease in production of pestalotiollide B (103). The velB gene appeared to stimulate the biosynthesis of pestalotiollide B (103) [102].

3.3. Regulation of VelC, VelD and VosA on Secondary Metabolite Production in Fungi

In velvet proteins, the functions of VelC, VelD, and VosA in fungi have been seldom studied for their regulation in secondary metabolism. Some examples of VelC, VelD, and VosA regulating secondary metabolite production in fungal species of the genera Aspergillus and Penicillium are shown in

Table 4. Some examples of VelC, VelD, and Vos A regulating secondary metabolite production in fungi.

Overexpression/ Deletion	Fungus	Positive/Negative Regulation	Production of Secondary Metabolites	Ref.
Deletion of velC	Aspergillus Oyzae	Positive	Decreased production of kojic acid (20).	[147]
Deletion of velC	Penicillium Expansum	Positive	Decreased production of patulin (105).	[160]
Deletion of velD	Aspergillus flavus	Positive	Decreased production of aflatoxin B1 (17).	[138]
Deletion of velD	Aspergillus oryzae	Positive	Decreased production of kojic acid (20).	[147]
Deletion of vosA	Aspergillus nidulans	Positive	Lost production of trehalose (128) in spores.	[177]
Deletion of vosA	Aspergillus nidulans	Negative	Slightly increased production of sterigmatocystin (16) in ascospores and upregulation of the BGCs associated with the biosynthesis of other secondary metabolites, including asperthecin (129), microperfuranone (130), and monodictyphenone (131).	[178]
Deletion of vosA	Aspergillus nidulans	Negative	Increased production of emericellamides A (132), C (133) and D (134), austinol (135) and dehydroaustinol (136).	[125]
Deletion of vosA	Aspergillus nidulans	Negative	Increased production of sterigmatocystin (16).	[179]
Deletion of vosA	Aspergillus oryzae	Negative	Increased production of kojic acid (20).	[147]

. The structures of the metabolites are shown in Figure S1.

3.3.1. Regulation of VelC

The VelC (also called Vel3 and VE-3) protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production in fungi [180]. Deletion of the velC gene in Aspergillus oryzae led to decreased production of kojic acid (20) [147]. Deletion of the velC gene in Penicillium expansum led to markedly decreased production of patulin (105) [160]. For the above-mentioned two fungal species, VelC positively regulated secondary metabolite production.

3.3.2. Regulation of VelD

The VelD protein is also known as Vel4. Deletion of the velD gene in Aspergillus flavus led to decreased production of aflatoxin B1 (17) [138]. Another example was that deletion of the velD gene in A. oryzae led to decreased production of kojic acid (20) [147]. It indicated that VelD positively regulated secondary metabolite production in A. flavus and A. oryzae.

3.3.3. Regulation of VosA

The regulation of VosA in secondary metabolite production was studied in detail via a sample of Aspergillus nidulans. The deletion of vosA in A. nidulans resulted in a lack of trehalose (128) in spores, a rapid loss of the cytoplasm, organelles, and viability of spores, and a dramatic reduction in the tolerance of conidia to heat and oxidative stress [177]. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA in A. nidulans led to elevated expression of sterigmatocystin (16) biosynthetic genes and a slight increase in sterigmatocystin (16) production in ascospores. Moreover, the deletion of vosA caused the upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin (129), microperfuranone (130), and monodictyphenone (131) [178]. VosA in A. nidulans could interact with the downstream target SclB to negatively regulate production of secondary metabolites, including emericellamides A (132), C (133), and D (134), as well as austinol (135) and dehydroaustinol (136) [125]. The second instance of such a scenario was that the VosA-repressed dnjA gene negatively regulated metabolism in the Aspergillus species. The deletion of dnjA caused increased production of sterigmatocystin (16) and aflatoxin B1 (17) in A. nidulans and A. flavus, respectively [181]. The third instance was that the VosA-VelB-repressed mcrA gene negatively regulated sterigmatocystin (16) production in A. nidulans. The conidia of the ΔmcrA mutant contained more amounts of sterigmatocystin (16) [182]. The fourth instance involved the VosA-VelB targeted gene vidD, which was required for proper fungal growth, development, and sterigmatocystin (16) production in Aspergillus nidulans [183]. Furthermore, transcriptomic, protein–DNA interaction, and metabolomics studies of VosA, VelB, and WetA in A. nidulans played interdependent, overlapping, and distinct roles in governing morphological development and metabolic remodeling in the conida, leading to the production of vital conidia suitable for fungal proliferation and dissemination. The related secondary metabolites regulated by VosA, VelB, and WetA in A. nidulans asexual spores included sterigmatocystin (16), austinol (135), dehydroaustinol (136), norsolorinic acid (137), nidurufin (138), versiconol (139), and emericellamids A (132), C (133), D (134), E (140), and F (141) [184]. In addition, Vos-VelB could activate putative C₆ transcription factor VadZ to regulate development and sterigmatocystin (16) production in A. nidulans [179].

4. Conclusions

LaeA and velvet proteins could obviously regulate the production of fungal secondary metabolites by responding to the light conditions under which fungi are grown. We can manipulate fungal secondary metabolite production to inhibit the production of harmful mycotoxins while promoting the production of useful metabolites [157]. However, we only know a little about the regulation mechanisms between LaeA/velvet proteins and secondary metabolite BGC expression, which should be studied in detail in the future [11,14,64,185].

In summary, the regulation studies of LaeA mainly focus on the toxin-producing fungal species for control of mycotoxin production, as well as plant endophytic fungi and marine-derived fungi for mining novel bioactive compounds. This should be an effective strategy for promoting or inhibiting production of secondary metabolites through global regulation of LaeA and velvet proteins in fungi. Some cryptic BGCs for secondary metabolite production are possibly activated by LaeA and velvet proteins through the regulatory networks [68,90,96,105]. This is beneficial for the excavation of bioactive compounds from fungi. Furthermore, some non-mycotoxigenic fungal strains obtained by deletion or overexpression of laeA or velvet protein encoding genes could be used as the biocontrol agents during the application to plants to reduce mycotoxin contamination [161].