Pestalotiopsis Diversity: Species, Dispositions, Secondary Metabolites, and Bioactivities

Pestalotiopsis species have gained attention thanks to their structurally complex and biologically active secondary metabolites. In past decades, several new secondary metabolites were isolated and identified. Their bioactivities were tested, including anticancer, antifungal, antibacterial, and nematicidal activity. Since the previous review published in 2014, new secondary metabolites were isolated and identified from Pestalotiopsis species and unidentified strains. This review gathered published articles from 2014 to 2021 and focused on 239 new secondary metabolites and their bioactivities. To date, 384 Pestalotiopsis species have been discovered in diverse ecological habitats, with the majority of them unstudied. Some may contain secondary metabolites with unique bioactivities that might benefit pharmacology.


Introduction
As human society entered a new century, many new problems have to be faced, such as global warming, public health, and food crisis. Especially, novel coronavirus burst in 2020 spring caused a severe effect on global public health and the world economy. More effective novel medications will be investigated to respond to emerging public health challenges. Many bioactive components were already isolated and identified from plants, animals, bacteria, and fungi. Because of the great numbers of bacteria and fungi and their various habitats, they are important sources of bioactive components. Subramanian and Marudhamuthus (2020) [1] isolated and identified the endophytic bacteria, such as Bacillus flexus (DMTMMB08), Bacillus licheniforms (DMTMMB10), and Oceanobacillus picturae (DMTMMB24) from marine macroalgae Sargassum polycystum and Acanthaphora specifera in the benthic region of the Gulf of Mannar, and found that they are taxol-producing. The endophytic fungus Taxomyces andreanae was isolated from the outer bark of Taxus brevifolia and was first found to have the ability to produce taxol in a culture medium, at approximately 24-25 ng/L [2]. Since then, a great number of taxol-producing fungi, acting as endophytic fungi, have been isolated and identified, such as the endophytic fungus Chaetomella raphigera from a medicinal plant, Terminalia arjuna [3], the endophytic fungus Epicoccum nigrum TXB502 [4], and the endophytic fungus Penicillium polonicum from Ginko biloba [5]. Among them, Pestalotiopsis species have been widely studied. The fungal genus Pestalotiopsis was first established by Steyaert R. L. [6]. Since then, many Pestalotiopsis species have been isolated and identified. To date, 384 Pestalotiopsis species are listed in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, assessed on 1 August, 2022). All the described species in the Pestalotiopsis genus are differentiated primarily on morphological characteristics of conidia, conidiogenesis, teleomorph, and host associations. In addition, the presence or absence of basal and apical appendages can be used as additional taxonomic characters for identifying Pestalotiopsis species. They are widely distributed in tropical and temperate regions [7][8][9][10]. As early as 1996, taxol was first isolated and identified in Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana [11]. Besides Table 1. Identified metabolites from Pestalotiopsis species and their bioactivities.
P. diploclisia also produces the compound scylatone (5) [73]. Scylatone (5) is one of the melanin biosynthesis intermediates. P. microspora and Pestalotiopsis fici were reported to produce melanin pigment [13,84,90,114,116]. Melanin biosynthesis is complex in fungi, and some fungi have more than one biosynthesis pathway for melanins [185]. Melanization in mycelia and appressoria plays crucial roles in the protection of pathogens from antibiotic stressors and the pathogenicity or interaction with host plants [185,186], and melanin is essential not only for the protection of spores from biotic and abiotic stresses but also structural spore development [84]. Thus, scylatone and melanin are related to the infection of host plants.
In addition, nigrosporolide (85) and 4,7-dihydroxy-13-tetradeca-2,5,8-trienolide (86) were isolated in P. microspora and the mold Nigrospora sphaerica [118,193]. The two compounds (85 and 86) showed weak bioactivities against lymphoma cell line L5178Y with an IC 50 value of 21 µM, while they exhibited no biological activities against ovarian cancer cell line A2780 with an IC 50 value of more than 40 µM [118]. Furthermore, Harwooda et al. [193] reported that nigrosporolide (85) caused 100% inhibition in the growth of etiolated wheat coleoptile sections at 10 −3 M; however, it showed no effect at lower concentrations. Since auxins have a significant impact on plant development, nigrosporolide (85) could be analyzed as a new component in the auxin signaling pathway, such as the TOR kinase found via rapamycin produced by the soil bacterium Streptomyces hygroscopicus in the Easter Island [194,195]. cell line A2780 with an IC50 value of more than 40 μM [118]. Furthermore, Harwooda et al. [193] reported that nigrosporolide (85) caused 100% inhibition in the growth of etiolated wheat coleoptile sections at 10 −3 M; however, it showed no effect at lower concentrations. Since auxins have a significant impact on plant development, nigrosporolide (85) could be analyzed as a new component in the auxin signaling pathway, such as the TOR kinase found via rapamycin produced by the soil bacterium Streptomyces hygroscopicus in the Easter Island [194,195].  (88), and pitholide E (89) were isolated and identified in P. microspora and Pithomyces sp. [67,196]. However, Pitholide E (89) did not exhibit any significant antifungal activity against Cladosporium cladosporioides, while Pitholide B (87) and Pitholide D (88) were not analyzed for their bioactivity [67]. In addition, Pitholides A (90) and C (91) were isolated from Pithomyces sp. derived from the marine tunicate Oxycorynia fascicularis, and their bioactivities were also not evaluated [196].  (88), and pitholide E (89) were isolated and identified in P. microspora and Pithomyces sp. [67,196]. However, Pitholide E (89) did not exhibit any significant antifungal activity against Cladosporium cladosporioides, while Pitholide B (87) and Pitholide D (88) were not analyzed for their bioactivity [67]. In addition, Pitholides A (90) and C (91) were isolated from Pithomyces sp. derived from the marine tunicate Oxycorynia fascicularis, and their bioactivities were also not evaluated [196].  Yang XL et al. [21] introduced ambuic acid and several of its derivatives, and m compounds have been discovered since then. For example, three new ambuic acid der atives (98-100) were isolated and characterized in P. microspora found from the leaves Corylus chinensis [122]. Among them, microsporol A (98) and microsporol C (100) exh ited moderately inhibitory effects on human 5-lipoxygenase (5-LOX), by 48.32% a 58.72%, respectively. Yang XL et al. [21] introduced ambuic acid and several of its derivatives, and more compounds have been discovered since then. For example, three new ambuic acid derivatives (98-100) were isolated and characterized in P. microspora found from the leaves of Corylus chinensis [122]. Among them, microsporol A (98) and microsporol C (100) exhibited moderately inhibitory effects on human 5-lipoxygenase (5-LOX), by 48
The hypothetical biosynthetic pathways for chlorotheolides A (126) and B (127) are shown in Figure 3.
Molecules 2022, 27, x FOR PEER REVIEW 40 of 69 also found, which were not introduced by Yang XL et al. [21] and Xu J et al. [10]. Bioactivity assay showed that the four compounds (151-154) did not exhibit significant antifungal activities against the three fungal species, Fusarium solani, Ustilago maydis, and C. albicans [179]. were found in the fungus P. zonata (CGMCC 3.9222). However, their bioactivities were not analyzed [184].

Pestalotiopsis sp.
Many Pestalotiopsis strains were isolated; however, they were not identified carefully, and some bioactive compounds were found in these strains.
Endophytic Pestalotiopsis sp. BC55 produces exopolysaccharide (EPS), with a maximum EPS value of 4.320 g/L in a 250 ml Erlenmeyer flask containing 75 mL potato dextrose broth supplemented with 7.66 g%/L glucose, 0.29 g%/L urea, and 0.05 g%/L CaCl 2 with medium pH 6.93, after 3.76 days of incubation at 24 • C [152]. The EPS is a homopolysaccharide of (1 → 3)-linked-d-glucose. EPSs are also produced by other fungi and bacteria, such as F. solani [212], F. oxysporum [213], Stemphylium sp. [214], the mangrove endophytic fungus Aspergillus sp. Y16 [215], lactic acid bacteria [216,217], Bacillus mycoides [218], and Bacillus licheniformis [219]. EPS bioactivity greatly varied due to chain length, molecular weight, branching, etc. A bioactive EPS with Mw~1.87 × 105 Da was isolated from endophytic fungus F. solani SD5 [212]. The isolated EPS showed in vitro anti-inflammatory and anti-allergic activity, and the EPS (1000 µg/mL) protects 55% of erythrocytes from hypotonic solution-induced membrane lysis. EPS produced by B. mycoides exhibited an anti-tumor effect [218]. EPS produced by B. mycoides showed low cytotoxicity against normal cells of baby hamster kidney (BHK) with an IC 50 value of 254 µg/mL, while it exhibited an inhibitory effect against cancer cells of human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma cells (Caco-2) with IC50 of 138 µg/mL and 159 µg/mL, respectively. Ren Q et al. [220] purified EPSs with a molecular weight of 2.7 × 10 6 Da to 1.7 × 10 7 Da from Lactobacillus casei and found that EPSs promote the differentiation of CD4 T lymphocytes into T-helper 17 cells in BALB/c mouse Peyer's patches in vivo and in vitro. Thus, it is reasonable to speculate that EPSs produced by Pestalotiopsis species might exhibit various similar bioactivities.

Accurate Biosynthesis Pathways and Enhanced Accumulation of Secondary Metabolites in Pestalotiopsis
Nutritional and environmental factors greatly promote secondary metabolite biosynthesis in Pestalotiopsis species [80,83,85]. Under the best nutritional and environmental conditions, how to maximize the yield of secondary metabolites in Pestalotiopsis species is a key problem. Genetic modification in biosynthesis pathways of important secondary metabolites is the best choice with certainty. The aim of genetic modification is to increase or inhibit the activities of key enzymes in biosynthesis pathways of wanted secondary metabolites in order to increase their yield. Some key enzymes in the biosynthesis of secondary metabolites in Pestalotiopsis species have been identified to date, improving our knowledge about accurate biosynthesis pathways and enhancing the accumulation of novel secondary metabolites.

Transcription Factors Involved in Secondary Metabolite Biosynthesis in Pestalotiopsis
Given the roles of transcription factors in gene expression, transcription factors involved in the biosynthesis of secondary metabolites in Pestalotiopsis species have been widely studied. Two transcription factors, PfmaF and PfmaH, cooperatively regulate 1,8dihydroxy naphthalene (DHN) melanin biosynthesis in P. fici. PfmaH, as a pathway-specific regulator, mainly regulates melanin biosynthesis, and PfmaF functions as a broad regulator to stimulate PfmaH expression in melanin production [90]. In addition, PfmaH directly regulates the expression of scytalone dehydratase, which catalyzes the transition of scytalone to 1,3,8-trihydroxynaphthalene (T3HN), which is reduced to vermelone, and vermelone is converted into DHN. Zhang P et al. [90] disrupted the gene PfmaF using the CRISPR/Cas9 system. They found that the disruption affected neither DHN melanin distribution nor conidia cell wall integrity in P. fici. Yet, the overexpression of PfmaF leads to heavy pigment accumulation in P. fici hyphae. Recently, two new transcription factors, Pmr1 and Pmr2, were identified in P. micropspora [221]. Pmr1 and Pmr2 were located in the gene cluster for melanin biosynthesis and both of them regulated the expression of genes in the melanin biosynthesis cluster. In ∆pmr1 and ∆pmr2 mutant strains, most genes in the gene cluster (including 21 genes, i.e., GEM11355_g-GEM11375_g) were significantly upregulated. Their upregulation is related to increased yield of secondary metabolites in the mutant strains ∆pmr1, compared with the wild type (WT). Meanwhile, HPLC analysis showed that the pestalotiollide B peak at 3.3 min was much greater in the ∆pmr1 and ∆pmr2 strains than that in WT; moreover, this increment in ∆pmr1 was significantly greater than that in ∆pmr2. In addition, Pmr1 played a larger regulatory role in secondary metabolism than Pmr2.
PfZipA, on the other hand, is one of the bZIP transcription factors in P. fici. Without oxidative treatment, the ∆PfzipA mutant strain of P. fici produced less isosulochrin and ficipyrone A than wild type [78]. However, PfZipA mediates the sensitivity of P. fici to oxidative stress caused by the oxidative reagents that-butyl hydroperoxide (tBOOH), diamide, H 2 O 2 , and menadione sodium bisulfite (MSB). tBOOH treatment decreased the production of iso-A82775C and pestaloficiol M in ∆PfzipA strain; MSB treatment decreased the production of RES1214-1 and iso-A82775C; however, it increased pestaloficiol M production in the mutant; and H 2 O 2 treatment resulted in enhanced production of isosulochrin, RES1214-1, and pestheic acid (23), yet decreased ficipyrone A and pestaloficiol M in ∆PfzipA strain, compared to the wild type [78].

Histone Acetylation
Histone acetylation is an important modification of histone proteins, which plays an important role in condensing and relaxing DNA. Histone acetylation is also involved in the biosynthesis of secondary metabolites in Pestalotiopsis species. Zhang Q et al. [113] identified a B-type histone acetyltransferase, Hat1, in the P. microspora. Secondary metabolites dramatically decreased in a hat1 deletion mutant strain, suggesting HAT1 functions as a regulator of secondary metabolism. Therefore, it is reasonable to speculate that the overexpression of the gene hat1 improves the biosynthesis of secondary metabolites in the fungus, thus, its overexpression mutant strains might be used for specific metabolites. In P. microspora, an MYST histone acetyltransferase encoded by the gene MST2 modulates secondary metabolism and conidial development [222]. Deleting the gene (mst2) caused serious growth retardation and impaired conidial development, e.g., delayed and reduced conidiation and aberrant conidia capacity. At the same time, overexpression of mst2 triggered earlier conidiation and higher conidial production. Deletion of mst2 also reduced the production of secondary metabolites in P. microspora [222]. In P. microspora NK17, Niu X et al. [112] found that a putative histone deacetylase gene (HID1) played an important role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/L. In contrast, the deletion of gene hid1 resulted in a dramatic decrease in conidia production of P. microspora NK17. These results suggest that the histone deacetylase HID1 is a regulator, concerting secondary metabolism and development, such as conidiation, in P. microspora.

Polyketide Synthases
Polyketides possess diverse chemical structures and biological activities and are the most important sources of novel secondary metabolites in plants, bacteria, and fungi. Polyketide synthases (PKSs) catalyze the biosynthesis of polyketides. While type I and type II PKSs exist as large protein complexes, type III PKSs are relatively small homodimeric proteins (~45 kDa monomer). In Pestalotiopsis species, PKSs are involved in the biosynthesis of some secondary metabolites. For example, the biosynthesis of pestalotiollide B is controlled by polyketide synthase [111]. Chen L and co-workers successfully deleted 41 out of 48 putative PKSs in the genome of P. microspora NK17. Furthermore, they found that 9 of the 41 PKS deleted strains significantly increased the biosynthesis of pestalotiollide B, and the deletion of pks35 increased pestalotiollide B by 887% [111].
The fungal products dibenzodioxocinones promise a novel class of inhibitors against cholesterol ester transfer protein [112]. A gene cluster of 21 genes, including PKS8 encoding a polyketide synthase, was defined, and disruption of genes in the cluster led to the biosynthesis of loss of dibenzodioxocinones [120]. Of the 21 genes, 5 genes, i.e., GME11356, GME11357, GME11358, GME11365, and GME11367, were deduced to participate in the generation of the backbone structure, and three regulatory genes, i.e., GME11360, GME11369, and GME11370, were also identified.
After forming polyketides, they can be converted into other secondary metabolites. The pestheic acid biosynthetic gene (pta) cluster was identified through genome scanning of the fungus P. fici. The biosynthetic pathway was elucidated through gene disruption intermediate detection and enzymatic analysis [82]. The results showed that the pestheic acid biosynthesis proceeded through the formation of the polyketide backbone, cyclization of a polyketo acid to a benzophenone, chlorination, and construction of the diphenyl ether skeleton through oxidation and hydrolyzation. The gene PTAA is important in pestheic acid biosynthesis in P. fici. Pestheic acid was abolished in the ptaA disruption mutants of P. fici [82]. In the pestheic acid biosynthesis pathway, the gene PTAM encodes a flavin-dependent halogenase, catalyzing chlorination. Inactivation of flavin-dependent halogenase from the Chaetomium chiversii radicicol locus yielded dechloro-radicicol (monocillin I) [223]. Thus, in P. fici, PTAM (ptaM) disruption might result in a change in pestheic acid biosynthesis.

Other Regulatory Proteins and Enzymes
The Snf1/AMPK is highly conserved in the eukaryotes and acts as a central regulator of carbon metabolism and energy production. In the filamentous fungus P. microspora, SNF1 concerts carbon metabolism and filamentous growth, conidiation, cell wall integrity, stress tolerance, and the biosynthesis of secondary metabolites [224]. The Snf1 deletion strain of P. microspora NK17 (∆snf1) displayed remarkable retardation in vegetative growth and pigmentation. Furthermore, it produced a diminished number of conidia, even in the presence of glucose, and Snf1 deletion caused damage to the cell wall of P. microspora [224]. In addition, Pestalotiollide B was considerably reduced in the mutant strain ∆snf1. These results demonstrate that SNF1 is a regulator of secondary metabolism and may be involved in either the activation or silencing of certain gene clusters in P. microspora NK17. Therefore, the more accurate function of SNF1 should be elucidated in secondary metabolite biosynthesis research.
Evidence shows biosynthesis of secondary metabolites and development are correlated processes in fungi, and pleiotropic proteins regulate the equilibrium between the biosynthesis of secondary metabolites and development. A global regulator, RsdA (regulation of secondary metabolism and development), was identified through genome-wide analysis and deletion of the regulator gene in the endophytic fungus P. fici [225]. Deleting rsdA significantly reduced asexual development, resulting in low sporulation, abnormal conidia, and major secondary metabolites (such as asperpentyn, fificiolide A, and chloroisosulochrin) while remarkably increasing melanin pigment production. In addition, pestheic acid, a basic building block for a group of structurally diverse compounds, was completely abolished in the ∆rsdA strain, implying that the biosynthesis of pestheic acid analogs was dramatically reduced.
Canonical Gcn2/Cpc1 kinase is an amino acid sensor and regulates the expression of target genes in response to amino acid starvation. When the mutant strain ∆gcn2 of P. microspora was cultured in the presence of 3AT (5 mM) to mimic amino acid starvation conditions, biosynthesis of pestalotiollide B was almost inhibited [114]. Meanwhile, the loss of gcn2 led to a less-pigmented phenotype of P. microspora [114]. All the results demonstrate that the protein encoded by gcn2 is a regulator of secondary metabolism and may be involved in either activation or silencing of gene clusters in P. microspora.
G-protein-mediated signaling pathways regulate fungal morphogenesis, development, pathogenesis, and secondary metabolism [226][227][228][229][230][231]. In Pestalotiopsis species, G proteinmediated signaling regulates secondary metabolites. The gene pgα1 putatively encodes the α-subunit of a group I G protein in P. microspora NK17. The pgα1 deletion mutants showed retarded vegetative growth, mycelium aging, premature conidiation, deformed conidia, significantly increased melanin production, and a sharp decrease in the production of pestalotiollide B [13]. Meanwhile, the expression of pks1, which encodes melanin polyketide synthase involved in 1,8-dihydroxy naphthalene (DHN) melanin biosynthesis, was upregulated 55-fold in pgα1 deletion mutants. All the results imply obvious changes in the biosynthesis of different secondary metabolites in pgα1 mutants. In addition, the deficiencies of pestalotiollide B production and conidiation in ∆pgα1 mutants could not be rescued by deletion or overexpression of the gene hid1 encoding histone deacetylase, suggesting that the protein PGα1 can override the effect of hid1 on pestalotiollide B production and conidiation.
In the fungus P. microspora, two genes, choA and choC, encode two phospholipid methyltransferases. choC deletion mutants (choC ∆ ) resulted in defects in phosphatidylcholine production, vegetative growth, and development of asexual structure [49], suggesting that genetic modification might regulate secondary metabolite biosynthesis in Pestalotiopsis species. However, choA, but not choC, was required to produce pestalotiollide B [49], suggesting distinct roles of the two genes.
The earlier examples demonstrate changes in the biosynthesis of secondary metabolites in Pestalotiopsis species by molecular tools, especially gene editing. Therefore, key genes encoding important enzymes in secondary metabolite biosynthesis in Pestalotiopsis species should be cloned, and the overexpression or deletion of these key genes is useful for enhanced biosynthesis of important secondary metabolites. More importantly, accurate biosynthesis pathways of secondary metabolites are the premise. Based on these basic studies on the effects of secondary metabolites in Pestalotiopsis species on human health, animals, and plants and the identification of their accurate biosynthesis pathways, it is possible to enhance biosynthesis and the accumulation of key secondary metabolites in the future. The industrial production of important secondary metabolites in this way will become possible.

Concluding Remarks and Future Perspectives
Given the important effects of secondary metabolites from Pestalotiopsis species on human health, animals, and plants, two aspects, i.e., the effects of these secondary metabolites and their accurate biosynthesis pathways, are vital. Therefore, more studies should focus on their accurate biosynthesis pathways to enhance biosynthesis and accumulation, further establishing the foundation for the industrial production of secondary metabolites from Pestalotiopsis species. Gene editing is a valuable method for fully comprehending secondary metabolite biosynthesis processes; however, it is very difficult to establish geneediting systems for some Pestalotiopsis species, despite genome editing systems having been established for few Pestalotiopsis species, such as P. fici and P. microspora [90,[232][233][234]. Furthermore, more effective gene-editing tools are to be developed and, therefore, long-term efforts are in the pipeline.
In addition, improvements for the best growth conditions are useful for enhanced biosynthesis and accumulation of secondary metabolites. For example, the addition of some chemicals in the culture medium promotes the biosynthesis of secondary metabolites, such as salicylic acid [235]. Meanwhile, the co-cultivation of fungi and bacteria can also trigger the biosynthesis of secondary metabolites. For example, the co-cultivation of Aspergillus flavipes and B. subtilis triggers the biosynthesis machinery of taxol [236]. At present, are isa no reports on the co-cultivation of Pestalotiopsis species with other microbes. Many gene clusters for the biosynthesis of secondary metabolites in filamentous fungi often stay silent under some culture conditions because of the absence of interaction with bacteria. For instance, Brakhage and colleagues have discovered that the silent secondary metabolite gene cluster for orsellinic acid (ors) in the filamentous fungus Aspergillus nidulans is activated upon physical interaction with the bacterium Streptomyces rapamycinicus, and the interaction of the fungus with this distinct bacterium led to increased acetylation of histone H3 lysines 9 and 14 at the ors gene cluster, thus to its activation [237][238][239]. Then, they identified the Myblike transcription factor BasR, a master regulator of bacteria-triggered production of fungal secondary metabolites, by chromatin mapping [240]. However, the interaction between Pestalotiopsis species and bacteria and key regulator nodes for transduction of the bacterial signals in the fungi is unclear. Certainly, activating silent gene clusters in Pestalotiopsis species is a good strategy for enhanced biosynthesis and accumulation of fungal secondary metabolites, just as in the Brakhage and Schroeckh advocated strategies [241]. Furthermore, as mentioned above, gene editing is a good and useful approach to increase the yield of secondary metabolites. We should try our utmost to establish whole feasible systems of gene editing for important Pestalotiopsis species. At present, the Pestalotiopsis species investigated are only a small part of this genus, and more species are yet to be studied and developed for human health.