Metabolites from Clonostachys Fungi and Their Biological Activities

Clonostachys (teleomorph: Bionectria) fungi are well known to produce a variety of secondary metabolites with various biological activities to show their pharmaceutical and agrochemical applications. Up to now, at least 229 secondary metabolites, mainly including 84 nitrogen-containing metabolites, 85 polyketides, 40 terpenoids, and 20 other metabolites, have been reported. Many of these compounds exhibit biological activities, such as cytotoxic, antimicrobial, antileishmanial, antimalarial activities. This mini-review aims to summarize the diversity of the secondary metabolites as well as their occurrences in Clonostachys fungi and biological activities.

Clonostachys fungi are abundant in many classes of secondary metabolites, mainly including nitrogen-containing compounds, polyketides, and terpenoids. Many metabolites exhibit biological activities, such as antimicrobial, insecticidal, nematocidal, antiparasitic, phytotoxic and cytotoxic activities. Until now, secondary metabolites of Clonostachys fungi and their biological activities have not been reviewed. This mini-review describes the classification, occurrences, and biological activities of the secondary metabolites from Clonostachys fungi.

Nitrogen-Containing Metabolites and Their Biological Activities
The nitrogen-containing metabolites from Clonostachys fungi mainly include linear oligopeptides, cyclopeptides, and piperazines. The nitrogen-containing metabolites, their isolated Clonostachys fungi and biological activities are shown in Table 1. Table 1. Nitrogen-containing metabolites in Clonostachys fungi and their biological activities.

Metabolite Class
Metabolite Name Fungal Species Biological Activity Ref.

Cyclopeptides
Cyclopeptides are cyclic compounds formed mainly by the amide bonds between either proteinogenic or non-proteinogenic amino acids [13,39]. The structures of the cyclopeptides isolated from Clonostachys fungi are shown in Figure 2

Cyclopeptides
Cyclopeptides are cyclic compounds formed mainly by the amide bonds between either proteinogenic or non-proteinogenic amino acids [13,39]. The structures of the cyclopeptides isolated from Clonostachys fungi are shown in Figure 2.
Clonostachysins A (7) and B (8) were two cyclic nonapeptides isolated from Clonostachys rogersoniana. They exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at 30 μM but had no effect on other microalgae and bacteria, even at 100 μM [10].
Clonostachysins A (7) and B (8) were two cyclic nonapeptides isolated from Clonostachys rogersoniana. They exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at 30 µM but had no effect on other microalgae and bacteria, even at 100 µM [10].

Piperazines
The piperazines (also called 2,5-diketopiperazines) are formed by the condensation of two amino acids [41]. Piperazines are the common nitrogen-containing metabolites as monomers or dimers in Clonostachys fungi, and most of them contain disulfide bonds. The structures of piperazines isolated from Clonostachys fungi are shown in Figure 3.
Haematocin (46) was isolated from the culture broth of Nectria haematococca, the blight disease pathogen of ornamental plants. Haematocin (46) inhibited the germ-tube elongation and spore germination of rice blast pathogen Pyricularia oryzae at the IC 50 values of 30 and 160 µg/mL, respectively [26].
Haematocin (46) was isolated from the culture broth of Nectria haematococca, the blight disease pathogen of ornamental plants. Haematocin (46) inhibited the germ-tube elongation and spore germination of rice blast pathogen Pyricularia oryzae at the IC50 values of 30 and 160 μg/mL, respectively [26].
Gliocladiosins A (67) and B (68), the dipeptides conjugated with macrolides, were isolated from an O-methyltransferase gene, verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. These two compounds showed moderate antibacterial activity on Klebsiella pneumonia and Bacillus subtlilis [32]. Similarly, rogersonins A (69) and B (70) were two indole-polyketide hydrids isolated from verG disruption mutant of Clonostachys rogersoniana [33]. Blocking the biosynthesis of secondary metabolites through the disuption of the biosynthesis-related genes provide a method to activate cryptic or silent secondary metabolites in fungi.
Penicolinate A (83) was induced from the endophytic fungus Bionectria sp. through bacterial co-culture. Penicolinate A (83) exhibited potent cytotoxic activity against the human ovarian cancer cell line A2780 with an IC 50 value of 4.1 µM [28].

Quinones
The quinones isolated from Clonostachys fungi were mainly naphthoquinones except for three p-benzoquinones. Their structures are shown in Figure 6.

Quinones
The quinones isolated from Clonostachys fungi were mainly naphthoquinones except for three p-benzoquinones. Their structures are shown in Figure 6.

Sorbicillinoids
Sorbicillinoids are important hexaketide metabolites produced by fungi [70]. Six dimeric and one monomeric sorbicillinoids were extracted from culture broth of Clonostachys rosea YRS-06 [44]. Their structures are shown in Figure 7

Other Polyketides
The structures of the other polyketides isolated from Clonostachys fungi are shown in Figure 8. These metabolites mainly belong to aliphatic polyketides. Some of them contain a glycosyl group and exist as glycosides.
Usnic acid (169) is a unique polyketide from Bionectria ochroleuca Bo-1 which was isolated as an endophytic fungus from rice. It showed antibacterial activity against Xanthomonas oryzae with MIC value of 200 µg/mL [68].
Usnic acid (169) is a unique polyketide from Bionectria ochroleuca Bo-1 which was isolated as an endophytic fungus from rice. It showed antibacterial activity against Xanthomonas oryzae with MIC value of 200 μg/mL [68].

Meroterpenoids
Meroterpenoids are metabolites that are partially derived from terpenoid biosynthetic pathways. The structures of meroterpenoids isolated from Clonostachys fungi are shown in Figure  14.

Meroterpenoids
Meroterpenoids are metabolites that are partially derived from terpenoid biosynthetic pathways. The structures of meroterpenoids isolated from Clonostachys fungi are shown in Figure 14.
Taxol (generic name paclitaxel, 209), the well-known anticancer agent, was isolated from the endophytic fungus Gliocladium sp. from Taxus baccata [83]. The backbone of taxol (209) is a diterpenoid, and the side chain is phenylalanine-derived. Both diterpenoid and phenylpropanoid pathways are required for taxol biosynthesis. Taxol (209) was found in both plants [85] and fungi [86]. It is a result of the co-evolution of plants and fungi in secondary metabolism [75].
Taxol (generic name paclitaxel, 209), the well-known anticancer agent, was isolated from the endophytic fungus Gliocladium sp. from Taxus baccata [83]. The backbone of taxol (209) is a diterpenoid, and the side chain is phenylalanine-derived. Both diterpenoid and phenylpropanoid pathways are required for taxol biosynthesis. Taxol (209) was found in both plants [85] and fungi [86]. It is a result of the co-evolution of plants and fungi in secondary metabolism [75].

Miscellaneous Metabolites and Their Biological Activities
The miscellaneous metabolites mainly including phenolics and fatty acids isolated from Clonostachys fungi are listed in Table 4, and their structures are shown in Figure 15.

Miscellaneous Metabolites and Their Biological Activities
The miscellaneous metabolites mainly including phenolics and fatty acids isolated from Clonostachys fungi are listed in Table 4, and their structures are shown in Figure 15.

Conclusions and Future Perspectives
In this mini-review, we summarized chemical structures, occurrences and biological activities of the secondary metabolites from Clonostachys fungi. The main metabolites belong to nitrogen-containing compounds, polyketides, and terpenoids. Some piperazines (i.e., bionetins,
Though major fungal species of Clonostachys fungi have been studied for their metabolites [1], the remaining fungi need to be revealed in detail. Moreover, the biological activities, structure-activity relationships, mechanisms of action, as well as biosynthesis of the metabolites from Clonostachys fungi need to be further investigated. Clarification of the metabolites of Clonostachys fungi could not only be in favor of discovering more compounds with novel structures and excellent biological activities, but also better understand the chemotaxonomy of the genus Clonostachys.