Secondary Metabolites of Purpureocillium lilacinum

Fungi can synthesize a wealth of secondary metabolites, which are widely used in the exploration of lead compounds of pharmaceutical or agricultural importance. Beauveria, Metarhizium, and Cordyceps are the most extensively studied fungi in which a large number of biologically active metabolites have been identified. However, relatively little attention has been paid to Purpureocillium lilacinum. P. lilacinum are soil-habituated fungi that are widely distributed in nature and are very important biocontrol fungi in agriculture, providing good biological control of plant parasitic nematodes and having a significant effect on Aphidoidea, Tetranychus cinnbarinus, and Aleyrodidae. At the same time, it produces secondary metabolites with various biological activities such as anticancer, antimicrobial, and insecticidal. This review attempts to provide a comprehensive overview of the secondary metabolites of P. lilacinum, with emphasis on the chemical diversity and biological activity of these secondary metabolites and the biosynthetic pathways, and gives new insight into the secondary metabolites of medical and entomogenous fungi, which is expected to provide a reference for the development of medicine and agrochemicals in the future.


Introduction
The genus Purpureocillium in the Ophiocordycipitaceae family was structured by Luangsa-Ard et al. In 2011, based on the medical importance, the Purpureocillium lilacinum was designated as the type species of the Paecilomyces genus [1]. This species was nominated as Penicillium lilacinum by Thom in 1901, and then it was revised as Paecilomyces lilacinus by Samson in 1974 [2]. After comparing the 18S rRNA gene, internal transcribed spacer, and partial translation elongation factor 1-a sequences with P. lilacinus, Luangsa-Ard proposed a new genus name Purpureocillium and made the new combination P. lilacinum in 2011. The fungus was found in a wide range of land and marine environments [3][4][5]. They are often isolated from insects, nematodes, and the rhizosphere of many crops [6][7][8]. The species can grow in a wide range of temperatures from 8 to 38 • C with optimal temperatures of 26-30 • C [3]. It also has a wide pH tolerance and can grow on a variety of substrates [9]. This fungus has promising potential as a biocontrol agent to control crops' root-knot nematodes [10,11]. The parasitism of nematodes is that the hyphae directly invade the surface of nematodes' eggs and then produce appressoria on the surface, which infects the nematodes' eggs after adsorption. In the process of infection, P. lilacinum secretes a variety of enzymes, such as serine protease and chitinase, which can lead to the degradation of protein and chitin components of the nematode epidermis, which is conducive to the invasion of fungi and destruction of cell components. It has been shown that the fermentation filtrate of P. lilacinum can inhibit the mycelial growth of the pathogenic fungi Helminthosporium maydis and Fusarium graminearum, and has a significant inhibitory effect on the spore germination of Fusarium oxysporum [12,13]. Currently, there are eight registered pesticide products of P. lilacinum in China used to control root-knot nematodes (http://www.chinapesticide.org. cn/hysj/index.jhtml, accessed on 23 February 2021); similar pesticides are also registered in the USA (https://iaspub.epa.gov/apex/pesticides, accessed on 23 February 2021) and

Leucinostatins
Leucinostatins is a class of peptide mycotoxins that have some potent effects on liver cells after oral administration, and they were used as both antimicrobial and antitumor agents through interactions with the membrane phospholipids [26].
In the early 1970s, Tadashi and his team isolated a new antibiotic from the culture medium of P. lilacinum, named leucinostatin ( Figure 1, Table 1). It was found that leucinostatin A, B, C, and D were active against yeasts including pathogenic and nonpathogenic strains and filamentous fungi, and were moderately active against Gram-positive bacteria, but more important is its anticancer activity, which is cytotoxic to the HeLa cell culture and inhibits Ehrlich subcutaneous solid tumors and prostate cancer [27,28]. A new report indicated that leucinostatin has a significant antihypertensive effect on rabbit blood pressure, but does not affect the reaction of adrenaline and acetylcholine [29]. It is worth mentioning that leucinostatin is one of the active substances to kill nematodes. Manabu suggested that Leucinostatin A inhibited prostate cancer cell growth through the reduction in insulin-like growth factor I expression in prostate stromal cells [30]. In addition, the use of leucinostatin A-loaded nanospheres could represent a new promising therapeutic system against Candida infection [31]. Leucinostatin Y exhibited the promising importance of the C-terminal of leucinostatins on the preferential cytotoxicity and inhibition of mitochondrial function of cancer cells in the absence of glucose [32]. It is not difficult to see that leucinostatins are potential anticancer compounds, but it should also be noted that  50 values for leucinostatins A and B hydrochlorides were found to be 1.8 mg/kg in mice body weight. Thus, it should be evaluated more carefully when it comes to drug use [33,34]. In addition, Yuzuru separated to obtain leucinostatin A, B, C, D, H, and K in P. lilacinum and found that the different isolates all produced leucinostatins, which means that they may play an important role not only in the infection process of mycoses in the human case, but also with insects and nematodes [26].
Molecules 2022, 26, x 3 of 16 mitochondrial function of cancer cells in the absence of glucose [32]. It is not difficult to see that leucinostatins are potential anticancer compounds, but it should also be noted that the acute toxicities determined by intraperitoneal LD50 values for leucinostatins A and B hydrochlorides were found to be 1.8 mg/kg in mice body weight. Thus, it should be evaluated more carefully when it comes to drug use [33,34]. In addition, Yuzuru separated to obtain leucinostatin A, B, C, D, H, and K in P. lilacinum and found that the different isolates all produced leucinostatins, which means that they may play an important role not only in the infection process of mycoses in the human case, but also with insects and nematodes [26].

Acremonidins and Acremoxanthones
Acremonidins ( Figure 2, Table 1) and acremoxanthones ( Figure 3, Table 1) are kinds of polyketides (PKs), which contain alternating carbonyl and methylene groups, usually involved in cell defense or intercellular communication. mitochondrial function of cancer cells in the absence of glucose [32]. It is not difficult to see that leucinostatins are potential anticancer compounds, but it should also be noted that the acute toxicities determined by intraperitoneal LD50 values for leucinostatins A and B hydrochlorides were found to be 1.8 mg/kg in mice body weight. Thus, it should be evaluated more carefully when it comes to drug use [33,34]. In addition, Yuzuru separated to obtain leucinostatin A, B, C, D, H, and K in P. lilacinum and found that the different isolates all produced leucinostatins, which means that they may play an important role not only in the infection process of mycoses in the human case, but also with insects and nematodes [26].

Acremonidins and Acremoxanthones
Acremonidins ( Figure 2, Table 1) and acremoxanthones ( Figure 3, Table 1) are kinds of polyketides (PKs), which contain alternating carbonyl and methylene groups, usually involved in cell defense or intercellular communication.   Acremoxanthone and acremonidin are xanthone-anthraquinone heterodimers, both calmodulin inhibitors produced by P. lilacinum [35]. Both of them exhibited anti-Bacillus cereus, antibacterial, antifungal, antiplasmodial, and cytotoxic activity [36]. In addition, acremonidin A and C, and acremoxanthone C and D were found to be moderate for 20 s proteasome inhibitory activity. Acremonidin A and Acremoxanthone C also have high affinity with human calmodulin biosensors. This means that they are also calmodulin inhibitors [37]. In addition, researchers found that acremoxanthone C has anti-oomycete activities [38]. Two new compounds acremoxanthones F and G represented antimalarial activity against the Plasmodium falciparum K1 strain, a multidrug-resistant strain [39]. These studies indicate that acremonidins and acremoxanthones all have high medicinal and economic value, but their toxicological effects need to be evaluated in more detail.

Paecilomide
Teles found a new α-pyridone Paecilomide ( Figure 4, Table 1) by adding salmonella typhimurium to a flask containing P. lilacinum in two different concentrations and three different forms at two different stages of the development of P. lilacinum. He observed that adding a small amount of inoculum produced a living extract, while adding a large amount of inoculum led to the extraction of active extract, paecilomide [40]. It is an acetylcholinesterase inhibitor [41,42].

Pyrones
Elbandy isolated 13 compounds from the sponge-derived fungus P. lilacinum in 2009, four of which were α-pyrones: paecilopyrone A, paecilopyrone B, phomapyrone B, and phomapyrone C; and one γ-pyrone: kojic acid ( Figure 5, Table 1). Paecilopyrone A and its Acremoxanthone and acremonidin are xanthone-anthraquinone heterodimers, both calmodulin inhibitors produced by P. lilacinum [35]. Both of them exhibited anti-Bacillus cereus, antibacterial, antifungal, antiplasmodial, and cytotoxic activity [36]. In addition, acremonidin A and C, and acremoxanthone C and D were found to be moderate for 20 s proteasome inhibitory activity. Acremonidin A and Acremoxanthone C also have high affinity with human calmodulin biosensors. This means that they are also calmodulin inhibitors [37]. In addition, researchers found that acremoxanthone C has anti-oomycete activities [38]. Two new compounds acremoxanthones F and G represented antimalarial activity against the Plasmodium falciparum K1 strain, a multidrug-resistant strain [39]. These studies indicate that acremonidins and acremoxanthones all have high medicinal and economic value, but their toxicological effects need to be evaluated in more detail.

Paecilomide
Teles found a new α-pyridone Paecilomide ( Figure 4, Table 1) by adding salmonella typhimurium to a flask containing P. lilacinum in two different concentrations and three different forms at two different stages of the development of P. lilacinum. He observed that adding a small amount of inoculum produced a living extract, while adding a large amount of inoculum led to the extraction of active extract, paecilomide [40]. It is an acetylcholinesterase inhibitor [41,42].  Acremoxanthone and acremonidin are xanthone-anthraquinone heterodimers, both calmodulin inhibitors produced by P. lilacinum [35]. Both of them exhibited anti-Bacillus cereus, antibacterial, antifungal, antiplasmodial, and cytotoxic activity [36]. In addition, acremonidin A and C, and acremoxanthone C and D were found to be moderate for 20 s proteasome inhibitory activity. Acremonidin A and Acremoxanthone C also have high affinity with human calmodulin biosensors. This means that they are also calmodulin inhibitors [37]. In addition, researchers found that acremoxanthone C has anti-oomycete activities [38]. Two new compounds acremoxanthones F and G represented antimalarial activity against the Plasmodium falciparum K1 strain, a multidrug-resistant strain [39]. These studies indicate that acremonidins and acremoxanthones all have high medicinal and economic value, but their toxicological effects need to be evaluated in more detail.

Paecilomide
Teles found a new α-pyridone Paecilomide ( Figure 4, Table 1) by adding salmonella typhimurium to a flask containing P. lilacinum in two different concentrations and three different forms at two different stages of the development of P. lilacinum. He observed that adding a small amount of inoculum produced a living extract, while adding a large amount of inoculum led to the extraction of active extract, paecilomide [40]. It is an acetylcholinesterase inhibitor [41,42].

Phomaligols
Phomaligol A, phomaligol A1, methylphomaligol A, acetylphomaligol A, phomaligol A hydroperoxide, phomaligol A1 hydroperoxide, phomaligol B, and phomaligol C were all isolated from the culture of P. lilacinum ( Figure 6, Table 1). All of them are yellow oil. Phomaligol A and Methylphomaligol A both showed cytotoxicity against a small panel of human solid tumor cell lines [46]. Phomaligol A, phomaligol A1, phomaligol A hydroperoxide, and phomaligol A1 hydroperoxide were previously isolated from Phoma lingam and Phoma wasabiae, but their biological activity has not been reported yet [47,48].

Phomaligols
Phomaligol A, phomaligol A1, methylphomaligol A, acetylphomaligol A, phomaligol A hydroperoxide, phomaligol A1 hydroperoxide, phomaligol B, and phomaligol C were all isolated from the culture of P. lilacinum ( Figure 6, Table 1). All of them are yellow oil. Phomaligol A and Methylphomaligol A both showed cytotoxicity against a small panel of human solid tumor cell lines [46]. Phomaligol A, phomaligol A1, phomaligol A hydroperoxide, and phomaligol A1 hydroperoxide were previously isolated from Phoma lingam and Phoma wasabiae, but their biological activity has not been reported yet [47,48]. linear analog paecilopyrone B may be derived from pentaketide; unfortunately, the detailed synthetic pathway is still unclear [43]. Kojic acid showed antibacterial activities and tyrosinase inhibitory activity [44,45].

Phomaligols
Phomaligol A, phomaligol A1, methylphomaligol A, acetylphomaligol A, phomaligol A hydroperoxide, phomaligol A1 hydroperoxide, phomaligol B, and phomaligol C were all isolated from the culture of P. lilacinum ( Figure 6, Table 1). All of them are yellow oil. Phomaligol A and Methylphomaligol A both showed cytotoxicity against a small panel of human solid tumor cell lines [46]. Phomaligol A, phomaligol A1, phomaligol A hydroperoxide, and phomaligol A1 hydroperoxide were previously isolated from Phoma lingam and Phoma wasabiae, but their biological activity has not been reported yet [47,48].

Pigment
Lenta investigated the ethyl acetate extract of the mycelium of P. lilacinum isolated from the pigment purpureone ( Figure 7, Table 1), which was found to possess potent antileishmanial activity and antibacterial activity [49].

Pigment
Lenta investigated the ethyl acetate extract of the mycelium of P. lilacinum isolated from the pigment purpureone ( Figure 7, Table 1), which was found to possess potent antileishmanial activity and antibacterial activity [49].

Cerebrosides
Cerebrosides A, B, C, and D ( Figure 9, Table 1) were isolated from the P. lilacinum ZBY-1 strain by professor Cui in 2013, and they are all linear analogs. Cerebrosides function as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions [53]; cerebrosides A and B have significant effects of analgesia and brain protection [54]. Cerebrosides C and D were known to potentiate the activity of cell wall-active antibiotics [55]. In addition, cerebrosides A and C play a role in cell growth, differentiation, and apoptosis in animals [56].

Pigment
Lenta investigated the ethyl acetate extract of the mycelium of P. lilacinum isolated from the pigment purpureone ( Figure 7, Table 1), which was found to possess potent antileishmanial activity and antibacterial activity [49].

Cerebrosides
Cerebrosides A, B, C, and D ( Figure 9, Table 1) were isolated from the P. lilacinum ZBY-1 strain by professor Cui in 2013, and they are all linear analogs. Cerebrosides function as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions [53]; cerebrosides A and B have significant effects of analgesia and brain protection [54]. Cerebrosides C and D were known to potentiate the activity of cell wall-active antibiotics [55]. In addition, cerebrosides A and C play a role in cell growth, differentiation, and apoptosis in animals [56].

Cerebrosides
Cerebrosides A, B, C, and D ( Figure 9, Table 1) were isolated from the P. lilacinum ZBY-1 strain by professor Cui in 2013, and they are all linear analogs. Cerebrosides function as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions [53]; cerebrosides A and B have significant effects of analgesia and brain protection [54]. Cerebrosides C and D were known to potentiate the activity of cell wall-active antibiotics [55]. In addition, cerebrosides A and C play a role in cell growth, differentiation, and apoptosis in animals [56].

Paecilaminols and Others
Cui  Table 1). Compounds paecilaminol and paecilaminol hydrochloride have the ability to inhibit human cancer K562, MCF-7, HL-60, and BGC-823 cells. Compound paecilaminol was a major antitumor metabolite of the strain P. lilacinum ZBY-1 and it is also a NADH-fumarate reductase inhibitor [57]. Me myristate was promised to be exploited as a hydrophobic medical carrier [58]. Me linoleate exhibited cytotoxic antibacterial activities against Bacillus subtilis and Staphylococcus aureus [59,60]. Modern medicine often makes acute respiratory distress syndrome models by injecting oleic acid into rabbits or rats [61].
Indole-3-carboxaldehyde has useful antimicrobial properties, and it can inhibit the atopic dermatitis-like inflammation induced by MC903 [62,63]. Hydroxybenzoic acids such as 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid have various functional biological properties, including anticancer, antibacterial, antiaging, antidiabetic, anti-inflammatory, and antiviral activities. They are widely used in food, cosmetic, and pharmaceutical industries [64]. In addition, 4-hydroxybenzoic acid increased after an organic intervention diet, and it may help with health-promoting qualities in the near future [65,66]. Table 1. SMs isolated from Purpureocillium lilacinum and their biological activities.
Indole-3-carboxaldehyde has useful antimicrobial properties, and it can inhibit the atopic dermatitis-like inflammation induced by MC903 [62,63]. Hydroxybenzoic acids such as 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid have various functional biological properties, including anticancer, antibacterial, antiaging, antidiabetic, anti-inflammatory, and antiviral activities. They are widely used in food, cosmetic, and pharmaceutical industries [64]. In addition, 4-hydroxybenzoic acid increased after an organic intervention diet, and it may help with health-promoting qualities in the near future [65,66]. Table 1. SMs isolated from Purpureocillium lilacinum and their biological activities.
We know that the typical NRPS modules have adenylation (A), thiolation (T) or peptidyl carrier (PCP), condensation (C), and thioesterase (TE) domains [70,71], which are, respectively, responsible for the activation of amino acids, the extension of peptide chains, the formation of amide bonds, and the release of peptide chains [72,73]. The synthesis mechanism of NRPSs is shown in Figure 11. Generally speaking, the A domain combines with the amino acid substrate under the action of ATP to form the corresponding aminoacyl AMP, and the aminoacyl AMP combines with the sulfhydryl group of the T domain to form the aminoacyl-s-carrier complex. Finally, the carriers carrying the aminoacyl group and the peptide acyl group combine with the specific region of the C domain, and the amino group on the aminoacyl-s-carrier complex attacks the acyl group on the peptidyl-s-carrier complex, forming a new peptide bond, and finally forming a complete peptide chain, through the action of multiple modules, wherein the amino acids in the peptide chain correspond to the modules in the NRPS one by one. Some NRPS modules also contain epimerization (E), formylation (F), methylation (M), heterocyclization (CY) [74], reduction (R), and oxidation (OX) domains, which are involved in the structural modification of peptide chains. Finally, mature peptide chains are released from the NRP assembly line under the action of the TE domain [75].
tidyl-s-carrier complex, forming a new peptide bond, and finally forming a complete peptide chain, through the action of multiple modules, wherein the amino acids in the peptide chain correspond to the modules in the NRPS one by one. Some NRPS modules also contain epimerization (E), formylation (F), methylation (M), heterocyclization (CY) [74], reduction (R), and oxidation (OX) domains, which are involved in the structural modification of peptide chains. Finally, mature peptide chains are released from the NRP assembly line under the action of the TE domain [75]. Figure 11. The biosynthesis of NRPSs [76].
In 2016, Wang sequenced and analyzed the whole genome of P. lilacinum, and predicted the knock-out of the NRPS synthetic gene (LcsA), PK synthetase (LcsC), Acyl CoA ligase (LcsD), and thioesterase (LcsE), using high-performance liquid chromatography (HPLC) to compare the crude extracts of wild-type and mutant strains of P. lilacinum. It was found that the crude extracts of ΔLcsA, ΔLcsC, ΔLcsD, and ΔLcsE had a lack of Leucinostatin A and Leucinostatin B, and then these enzymes were found to play a key role in the synthesis of Leucinostatin, and the synthesis of leucinostatin of P. lilacinum was suggested. This hypothetical biosynthesis is initiated by the assembly of 4-methylhex-2enoic acid via reductive PKs. However, they were unable to estimate which PKs were responsible for 4-methylhex-2-enoic acid [18].
In microorganisms, PK comes from the independent hypothesis of a variety of compounds. Polyketide compounds are assembled by repeated Claisen condensations between the activated acyl initiation unit and the chain extender unit derived from malonyl-CoA. This process is catalyzed by the synergistic action of keto synthase (KS), acyltransferase (AT), and phosphopan ethylation acyl carrier protein (ACP) or CoA linked to the primary chain. After each extension step, the functionality of β-keto can be reduced by further involved enzymes [76]. This general PK catalytic mechanism is realized by Figure 11. The biosynthesis of NRPSs [76].
In 2016, Wang sequenced and analyzed the whole genome of P. lilacinum, and predicted the knock-out of the NRPS synthetic gene (LcsA), PK synthetase (LcsC), Acyl CoA ligase (LcsD), and thioesterase (LcsE), using high-performance liquid chromatography (HPLC) to compare the crude extracts of wild-type and mutant strains of P. lilacinum. It was found that the crude extracts of ∆LcsA, ∆LcsC, ∆LcsD, and ∆LcsE had a lack of Leucinostatin A and Leucinostatin B, and then these enzymes were found to play a key role in the synthesis of Leucinostatin, and the synthesis of leucinostatin of P. lilacinum was suggested. This hypothetical biosynthesis is initiated by the assembly of 4-methylhex-2-enoic acid via reductive PKs. However, they were unable to estimate which PKs were responsible for 4-methylhex-2-enoic acid [18].
In microorganisms, PK comes from the independent hypothesis of a variety of compounds. Polyketide compounds are assembled by repeated Claisen condensations between the activated acyl initiation unit and the chain extender unit derived from malonyl-CoA. This process is catalyzed by the synergistic action of keto synthase (KS), acyltransferase (AT), and phosphopan ethylation acyl carrier protein (ACP) or CoA linked to the primary chain. After each extension step, the functionality of β-keto can be reduced by further involved enzymes [76]. This general PK catalytic mechanism is realized by different enzyme mechanisms ( Figure 12). Three types of PKs are described below, which are responsible for the biosynthesis of polyketide chains.
PKs can be divided into three types: type I PKs are modular enzymes composed of several functional domains, which are arranged linearly and covalently. Any functional domain is not reused in the process of chain synthesis and extension. They mainly synthesize polyether, polyene, and macrolides. PKs of type II are aromatic, starting from acetyl CoA. Polyketones with an aromatic ring structure are synthesized with malonyl coenzyme A as an extension unit. Type III PKs are chalcone synthetases, a kind of homologous dimer enzyme that can be reused. It catalyzes the condensation of acetyl-CoA molecules to synthesize one ring or multi-ring aromatic polyketones [74]. different enzyme mechanisms ( Figure 12). Three types of PKs are described below, which are responsible for the biosynthesis of polyketide chains. Figure 12. The biosynthesis of PKs [77].
PKs can be divided into three types: type I PKs are modular enzymes composed of several functional domains, which are arranged linearly and covalently. Any functional domain is not reused in the process of chain synthesis and extension. They mainly synthesize polyether, polyene, and macrolides. PKs of type II are aromatic, starting from acetyl CoA. Polyketones with an aromatic ring structure are synthesized with malonyl coenzyme A as an extension unit. Type III PKs are chalcone synthetases, a kind of homologous dimer enzyme that can be reused. It catalyzes the condensation of acetyl-CoA molecules to synthesize one ring or multi-ring aromatic polyketones [74].
It is generally believed that most α-pyrones are synthesized through the polyketide pathway [78]. Terpenoids are a kind of chain or cyclic secondary metabolites, which are composed of isoprene as the basic unit. Terpenoids are synthesized by terpene synthase and can be divided into: monoterpenes, with geranyl diphosphate as the synthetic precursor; sesquiterpenes, with farnesyl diphosphate as the synthetic precursor; diterpenes, with geranyl pyrophosphate as the precursor. According to the degree of reduction, it can be divided into reduced terpenoids and nonreduced terpenoids [79].

Problems and Perspectives
Among the more than 40 metabolites reviewed in this paper, we can see that most SMs of P. lilacinus that have been reported so far have the functions of anticancer activity, antimicrobial activity, insecticidal activity, cytotoxicity, drug carriers, and so on. Most importantly, some of the compounds showed potent activities compared to those of the positive controls, which indicates that they could be used to develop new medicines. These include the anticancer lead compound leucinostatins, ergosterol peroxide, (22E,24R)-5α, 6α-epoxy-3β-hydroxyergosta-22-ene-7-one, and paecilaminol. Leucinostatins is cytotoxic to HeLa cells, Ehrlich subcutaneous solid tumors, and prostate cancer. However, it was found to be toxic to rats by intraperitoneal injection, so more attention should be paid to its safety assessment when developing the drug. The other three compounds have the ability to inhibit human cancer K562, MCF-7, HL-60, and BGC-823 cells, but their safety for other species is still unknown. Acremoxanthone and acremonidin were both calmodulin inhibitors; paecilomide is an acetylcholinesterase inhibitor and kojic acid showed tyrosinase inhibitory activity, indicating their potential as insecticides. These remarkable activities make many of these compounds suitable candidates for new drugs and insecticides discovery and may lead to future synthesis studies. However, some of the SMs of P. It is generally believed that most α-pyrones are synthesized through the polyketide pathway [78]. Terpenoids are a kind of chain or cyclic secondary metabolites, which are composed of isoprene as the basic unit. Terpenoids are synthesized by terpene synthase and can be divided into: monoterpenes, with geranyl diphosphate as the synthetic precursor; sesquiterpenes, with farnesyl diphosphate as the synthetic precursor; diterpenes, with geranyl pyrophosphate as the precursor. According to the degree of reduction, it can be divided into reduced terpenoids and nonreduced terpenoids [79].

Problems and Perspectives
Among the more than 40 metabolites reviewed in this paper, we can see that most SMs of P. lilacinus that have been reported so far have the functions of anticancer activity, antimicrobial activity, insecticidal activity, cytotoxicity, drug carriers, and so on. Most importantly, some of the compounds showed potent activities compared to those of the positive controls, which indicates that they could be used to develop new medicines. These include the anticancer lead compound leucinostatins, ergosterol peroxide, (22E,24R)-5α, 6α-epoxy-3β-hydroxyergosta-22-ene-7-one, and paecilaminol. Leucinostatins is cytotoxic to HeLa cells, Ehrlich subcutaneous solid tumors, and prostate cancer. However, it was found to be toxic to rats by intraperitoneal injection, so more attention should be paid to its safety assessment when developing the drug. The other three compounds have the ability to inhibit human cancer K562, MCF-7, HL-60, and BGC-823 cells, but their safety for other species is still unknown. Acremoxanthone and acremonidin were both calmodulin inhibitors; paecilomide is an acetylcholinesterase inhibitor and kojic acid showed tyrosinase inhibitory activity, indicating their potential as insecticides. These remarkable activities make many of these compounds suitable candidates for new drugs and insecticides discovery and may lead to future synthesis studies. However, some of the SMs of P. lilacinus are toxic to animals and humanity. Hocquette, Dr. Qian, Pastor, and others have reported infections caused by P. lilacinus in immunocompromised patients [80,81].
With the development of society, more and more attention has been paid to biological control, more and more fungal products will come out, and the safety of related products has also received great attention. Therefore, how to ensure the safety of fungal products has become particularly important.
Generally, in production and in life, there are six destinations (i.e., target organisms, nontarget organisms, soil, water, atmosphere, and humans) involved in the production and application of P. lilacinum pesticide formulations. The most important destination is target organisms, including pests and crops when preparations are released in fields. Soil is another important destination, especially when it is released through soil treatments for nematodes. Water and the atmosphere are the destinations of the drifting formulations. Humans contact P. lilacinum through direct and indirect pathways. There is no doubt that the biosafety risks of P. lilacinum are closely related to the sources and fates of the SMs produced by entomopathogenic fungi [24].
Surveying the SMs will be beneficial to improving the safety of P. lilacinum fungal products. Thus, developing the discovery, structure, function, and synthesis pathway of secondary metabolites of P. lilacinum are of great significance to biomedicine, human health, and agricultural disease control. For a long time, due to the gene silencing or low expression of most gene clusters in common culture medium, the research of fungal secondary metabolites has been hindered to some extent. There are only a few kinds of research on SMs of P. lilacinum, which are leucinostatins, acremoxanthones, and paecilomides, and their synthetic pathway and regulatory mechanism are still unclear. Therefore, it is necessary to use a super-expressing transcription factor, to replace the promoter in the synthetic gene cluster with an inducible strong promoter, to modify the histone, to heterologously express the gene cluster to activate the silent gene cluster, and to further discover that the structure is novel and biologically active. The SMs production yield of P. lilacinum needs to be improved by changing the culture conditions. First, gene knockout methods need to be used to further clarify the synthesis mechanism of secondary metabolites. In addition, it is necessary to continuously improve the efficiency and precision of chemical separation detection, in order to be more conducive to the separation of secondary metabolites and the identification of structural functions.