The Secondary Metabolites and Biosynthetic Diversity From Aspergillus ochraceus

Aspergillus ochraceus, generally known as a food spoilage fungus, is the representative species in Aspergillus section Circumdati. A. ochraceus strains are widely distributed in nature, and usually isolated from cereal, coffee, fruit, and beverage. Increasing cases suggest A. ochraceus acts as human and animal pathogens due to producing the mycotoxins. However, in terms of benefits to mankind, A. ochraceus is the potential source of industrial enzymes, and has excellent capability to produce diverse structural products, including polyketides, nonribosomal peptides, diketopiperazine alkaloids, benzodiazepine alkaloids, pyrazines, bis-indolyl benzenoids, nitrobenzoyl sesquiterpenoids, and steroids. This review outlines recent discovery, chemical structure, biosynthetic pathway, and bio-activity of the natural compounds from A. ochraceus.


INTRODUCTION
Filamentous fungi in the genus Aspergillus are well known for their important roles in lifesaving drugs, devastating toxins, or mass-produced industrial enzymes. Aspergillus is currently subdivided into 27 sections by the physiologic, phenotypic, and DNA sequence data (Houbraken et al., 2020). A. ochraceus, the representative species in Aspergillus section Circumdati (Visagie et al., 2014), is generally known as a food spoilage fungus and is widely isolated from cereal, coffee, fruit, beverage, soil, and marine environments due to their environmental tolerance and fast growth. Ochratoxin A (OTA) makes A. ochraceus notorious for their role as contaminants in mycotoxigenic food and feed. Moreover, increasing cases have suggested A. ochraceus acts as human and animal pathogens causing onychomycosis (Xu et al., 2021), allergic bronchopulmonary aspergillosis (Hassanzad et al., 2019), and otomycosis (Ghibaudo and Peano, 2010). Some novel IgE-binding proteins have been identified from A. ochraceus, indicating the allergenic potency of mycelial proteins (Roy et al., 2021). Recently, A. ochraceus has been found in a SARS-CoV-2 positive immunocompetent patient in Iran (Koehler et al., 2020), and also can cause COVID-19 associated pulmonary aspergillosis (Hakamifard et al., 2021). On the other hand, the adaption of Aspergillus to shifting environments lead to the formation of a particular set of proteins (Day and Quinn, 2019;Wang et al., 2022b). A. ochraceus has been an important source of industrial enzymes like protease (El-Khonezy et al., 2021;Komarevtsev et al., 2021;Zhu et al., 2021), esterase (Romero-Borbón et al., 2018) and tannase (Aracri et al., 2019).
Several medicinal metabolites, such as an intermediate for the synthesis of desogestrel and eplerenone, are characterized from A. ochraceus Li et al., 2021). Some A. ochraceus strains have shown the mycoremediation potential to remove petroleum hydrocarbons (Bilen Ozyurek et al., 2021), and the remarkable capability for converting biodegradable waste to value-added end products for commercial applications (Jathanna and Rao, 2022).
Secondary metabolites (SMs) play important roles both as a food spoilage fungus and as an industrial strain for bioproduction ( Figure 1). A. ochraceus as a food spoilage fungus exhibits a remarkably versatile secondary metabolism. Most SMs are derived from polyketides synthases, non-ribosomal peptides synthases, and terpene synthases, and used to defend their habitat or inhibit the growth of competitors (Macheleidt et al., 2016). And these compounds are likely to remain in the food chain after the occurrence of A. ochraceus in food substrate. From the perspective of drug discovery, many compounds have been isolated from A. ochraceus and screened for bio-activities. In fungi, the genes required for the biosynthesis of SMs are generally clustered on the chromosome. A growing number of Aspergillus genomes impressively shows numbers of unknown biosynthetic gene clusters (BGCs) of SMs, which considerably exceed the number of identified SMs, indicating their potential production of novel structural compounds (Keller, 2019). It has been well summarized that the linkage between SMs with their BGCs in the different Aspergillus spp. (Frisvad and Larsen, 2015;Romsdahl and Wang, 2019;Yu et al., 2021). However, although lots of compounds have been isolated from A. ochraceus, no other BGCs have been identified except for the OTA BGC. Reviewing the SMs and biosynthetic diversity from A. ochraceus would give insights into the understanding and utilization of this fungus.
In this review, we have critically scrutinized the existing reports to provide an overview of the various SMs produced by A. ochraceus. Considering the structural characteristics and biogenetics, these compounds could be classified as polyketides, nonribosomal peptides, diketopiperazine alkaloids, benzodiazepine alkaloids, pyrazines, bis-indolyl benzenoids, nitrobenzoyl sesquiterpenoids, steroids, et al. In addition, we present the bioactivities and the possible biosynthetic pathway of some compounds, which are ignored due to the shading of mycotoxin OTA.

Ochratoxins
The most common A. ochraceus product described is ochratoxin A (1) (Figure 2). It was first discovered from A. ochraceus isolated from sorghum grain in South Africa in 1965(van der Merwe et al., 1965b. Since then, more than 90 kinds of foodstuffs such as cereal, beer, coffee, cheese, and meat products have been found to contribute to OTA dietary exposure (Ostry et al., 2013). Recently, some A. ochraceus strains were re-classified as A. westerdijkiae based on the β-tubulin sequence and morphological identification Durand et al., 2019). Surprisingly, a genome mining study demonstrated the integral cluster of OTA was not found in the A. ochraceus genome, (Gil-Serna et al., 2020), while the strain A. ochraceus fc-1, re-classified as A. westerdijkiae, has been reported to contain an intact cluster Wang et al., 2022a).
Biological toxic studies performed on rats, trout, and mice demonstrated the carcinogenic potency of OTA (Kanisawa and Suzuki, 1978). The International Agency for Research on Cancer evaluated the experimental evidence for carcinogenicity as sufficient and classified OTA as a possible human carcinogen (group 2B) (IARC, 1993). Additionally, OTA was well documented in its nephrotoxicity, immunotoxicity, myelotoxicity, genotoxicity, embryotoxic, and teratogenicity in many species (Pfohl-Leszkowicz and Manderville, 2007;Malir et al., 2013a;Malir et al., 2013b).
The biosynthetic pathway of (1) was first investigated by exploring the related metabolites. Labeling study by the introduction of [1-14 C] L-phenylalanine into the culture of A. ochraceus lead to the detection of radioactivity in the phenylalanine moiety of (1), indicating L-Phenylalanine was the precursor of (1) (Steyn et al., 1970). [2-14 C]acetate and [2-14 C]malonic acid radiolabeling experiments indicated malonic acid was involved in the isocoumarin moiety biosynthesis but not in the phenylalanine moiety biosynthesis (Ruhland et al., 1996), and the isocoumarin moiety most derived via acetate condensation.
Advances in sequencing technology make scientists realize the dihydrocoumarin moiety of (1) is catalyzed by polyketide synthase (PKS) and the polyketide moiety of (1) is linked to Lphenylalanine catalyzed by non-ribosomal peptide synthetase (NRPS) (Huffman et al., 2010;Wang et al., 2015). In 2018, a consensus biosynthetic gene cluster was identified by comparative genomic analyses among OTA-producing fungi. And the biosynthetic pathway was clarified by discovering the intermediate metabolites in OTA gene disruption mutants. Briefly, OtaA (PKS) utilized acetyl-CoA and malonyl-CoA to synthesize 7-methylmellein by condensation, which was oxidized to OTβ by cytochrome P450 OtaC. Then, OtaB (NRPS) combined OTβ and L-phenylalanine to synthesize (2) by catalyzing the formation of an amide-bond. (2) was chlorinated by the halogenase OtaD to form (1). Recently, a cyclase gene otaY was proved to be involved in the biosynthesis of (1) ( Figure 2). OtaY was speculated to catalyze the cyclization process of 7-methylmellein (Ferrara et al., 2020;Ferrara et al., 2021). Figure 3, mellein (4) and 4-hydroxymellein (5), structurally similar to the dihydroisocoumarin moiety of (1), are also produced by A. ochraceus (Cole et al., 1971;Moore et al., 1972). The biosynthesis of (4) starts with the condensation of acetyl-CoA and malonyl-CoA, and acetyl units would be added until the pentaketide is formed (Huff and Hamilton, 1979), just like the biosynthesis 7-methylmellein in OTA. Compound (4) and its derivatives exhibit an array of bio-activities such as antitumor, antifungal, antibacterial, and anti-inflammatory (Hussain et al., 2015;Mdachi, 2016).
Diaporthin (8) and orthosporin (9) were also characterized from A. ochraceus (Harris and Mantle, 2001). (8) was reported to reproduce symptoms of canker in Chestnut trees and (9) could cause irregular brown spots on the leaves of oats (Hallock et al., 1988). The difference between the two compounds is the replacement of the methoxy by hydroxyl moiety on the benzene ring. Structurally, we hypothesize that one PKS could be responsible for the biosynthesis of these compounds.

Nonribosomal Peptides
Non-ribosomal peptides are synthesized by multi-modular NRPSs from building blocks of 20 kinds of proteinogenic amino acids and non-proteinogenic amino acids such as ornithine and β-alanine (Wang et al., 2016;Stevenson et al., 2019). They have been optimized for a certain function in the native producer during years of evolution, as well as represent a promising basis for the development of substances with excellent activities. Generally, most of the non-ribosomal peptides described from A. ochraceus are cyclic peptides.
As shown in Figure 4, aspochracin (36) is a cyclotripeptide composed of N-methy-L-alanine, N-methy-L-valine and Lornithine (Chang et al., 1969). (36) demonstrated contact toxicity to the first instar larvae and eggs of silkworm. However, the insecticidal activity completely diminished in hexahydroaspochracin (37), indicating the triene in the side chain has been involved in molecular bioactivity. JBIR-15 (38), of which the N-methyl alanine is replaced by alanine compared to (36), was isolated from A. sclerotiorum (Motohashi et al., 2009). Violaceotide A (39) was extracted from a solid rice medium of A. ochraceus (Frank et al., 2019) and its structure was elucidated as cyclic tetrapeptide with L-threonine, L-O-methy-tyrosine, N-methy-L-alanine and L-lsoleucine. (39) showed anti-inflammatory activity with a high inhibitory rate .
Most fungi can produce siderophores under iron deficiency or other iron-related conditions. Hydroxamate-type siderophores, classified into fusarinines, coprogens, or ferrichromes, were mostly found and characterized in fungi (Garnerin et al., 2017). A large number of ferrichromes were isolated from iron-deficient cultures of A. ochraceus (Jalal et al., 1984). Ferrichromes are cyclic hexapeptides composed of three N δ -acyl-N δ -hydroxy-L-ornithine, one glycine, and two variable amino acids (alanine, serine, or glycine) linked by peptide bonds. The structures of ferrichrysin (40), ferrirubin (41), and asperchromes (42-53) were shown in Figure 4. Serine and alanine participate in the formation of these Frontiers in Chemistry | www.frontiersin.org August 2022 | Volume 10 | Article 938626 5 molecules as the variable amino acids; the acetyl and anhydromevalonyl are acyl groups linked to ornithine. Des (diserylglycyl)ferrirhodin (54) does not follow the typical ferrichrome structure. It is a linear siderophore consisting of three N δ -cis-anhydromevalonic acid-N δ -hydroxy-Lornithine moieties linked by peptide bonds. The absence of a cyclic hexapeptide ring in des (diserylglycyl)ferrirhodin leads to a bathochromic shift withpH value decreasing from 2.0 to 1.7, indicating the change of iron-binding property (Jalal et al., 1984). It is rare among microorganisms that A. ochraceus produces various siderophores derived from a common cyclic hexapeptide ring with the N-acyl side chain surrounding the iron atom. Their diversity of function and structure is worthy to be further explored.

Diketopiperazine Alkaloids
Diketopiperazine alkaloids are commonly isolated from fungi with excellent biological activities such as anticancer, antimicrobial, antiviral, antioxidant, and immunomodulatory Wang M.-H. et al., 2020). Diketopiperazine alkaloids, with a stable six-membered ring backbone, are cyclic dipeptides formed by the condensation of two amino acids through peptide bonds by NRPS (Jia et al., 2019). A. ochraceus is capable of producing abundant diketopiperazine alkaloids with structural diversity and biological activity ( Figure 5).
Asperochramides A-D (68-71) is another group of indole diketopiperazine alkaloids isolated from A. ochraceus . (68) and (69) are a pair of epimers assigned with the same planar structure. The significant difference between (68) and (70) is the cyclization of 2-isopentenyl and the replacement of 1-isopentenyl by hydroxyl. Removing the two isopentenyl forms (71). Bio-activities studies demonstrated that (68) has anti-inflammatory potential .
Circumdatins demonstrated inhibitory activity similar to other inhibitors of the mammalian mitochondrial respiratory chain (Fontana et al., 2001). For example, the IC 50 value of (80) against NADH oxidase is around 1.5 μM, indicating its potential to develop new tools for insect control (Lopez-Gresa et al., 2005). (83), (85), and (86) exhibited an ultraviolet-A protecting activity, which was better than the sunscreen agent oxybenzone (Zhang et al., 2008). (90) showed great DPPH radical-scavenging activity, which was more potent than the well-known butylated hydroxytoluene with an IC 50 value of 9.9 mm (Cui et al., 2009). (78) demonstrated potential as an agent for neuroprotective effects by attenuating LPS-induced pro-Inflammatory responses . Several compounds have been evaluated for their cytotoxicity, while no evidence provides to prove their cytotoxicity.
The biosynthesis of circumdatin remains to be explored in A. ochraceus and other fungi due to its structural complexity. However, the biosynthetic gene cluster of anthramycin and sibiromycin, which belong to the benzodiazepine family, have been identified clearly (Hu et al., 2007;Li et al., 2009), indicating NRPS might be involved in the biosynthesis of circumdatins.
It is reported the pyrazine compounds are biosynthesized from NRPS. The identification of a dihydropyrazine N,N′dioxide metabolite proposes a noncanonical NRPS pathway for pyrazine derivatives through genome mining of Pseudomonas (Kretsch et al., 2018). A gene cluster containing an NRPS-like encoding gene in A. flavus is responsible for the synthesis of aspergillic acid (107) by NRPS-like gene inactivation experiment (Lebar et al., 2018). Structurally, (107) and (93) are closely related isomers. Many fungi from Aspergillus spp. can produce (93) and its hydroxylated analogs, and the genome mining shows they all harbor the homologs BGC of (107), indicating the same biosynthesis pathway between (107) and (93) (Lebar et al., 2019).

Bis-Indolyl Benzenoids
Ochrindoles A-D (108-111) were isolated from the sclerotia of A. ochraceus (de Guzman et al., 1994) (Figure 8). Structurally, (108-110) are bis-indolyl benzenoids, and (111) is bis-indolyl quinone. Members of these bis-indolyl structures have been reported from fungal metabolites, such as terriquinones from A. terreus (Balibar et al., 2007), kumbicins from A. kumbius (Lacey et al., 2016), asterriquinol from A. sclerotiorum (Whyte et al., 2000). These compounds typically contain prenyl groups at various positions on the indole moieties or the central benzenoid ring. The biosynthesis of bis-indolyl benzenoids and quinone has been extensively investigated. Briefly, an aminotransferase converts Ltryptophan to the indole pyruvic acid, and two molecules of indole pyruvic acid are dimerized by a single-module NRPS to form the bis-indolyl benzenoid skeleton. Then, the oxidoreductase, prenyltransferase, methytransferase successively play the catalytic function to form the corresponding products. Ochrindoles showed moderate activity against the corn earworm Helicoverpa zea, fungivorous beetle Carpophilus bemipterus, as well as bacterial Bacillus subtilis (de Guzman et al., 1994).

DISCUSSION
Common fungi, especially A. ochraceus, are regularly underestimated for their biosynthetic potential, which deserves our recognition. Here, we comprehensively review the known SMs produced by A. ochraceus, and discuss their bioactivities and biosynthetic pathway. A. ochraceus produces a range of polyketides, nonribosomal peptides, diketopiperazine, terpenes, and other alkaloids. Except for the mycotoxins OTA, (6) and (7), these compounds possess antimicrobial, antiviral, anti-insect, antitumor, antioxidant, and anti-inflammatory activities. Thus, A. ochraceus strains could be valuable sources of compounds in the areas of medicine and agriculture.
In terms of fungi, a large number of natural products have been isolated from A. ochraceus until now. Nonetheless, several strategies have been used for enhancing the chemical diversity of microorganisms. Different media used in the cultivation of A. ochraceus leads to the production of different compounds. For example, when several inorganic salts or organic supplements are added to the solid rice medium culture, A. ochraceus is found to produce different metabolites, resulting in discovering the novel compounds; and this strategy verifies the OSMAC (One Strain MAny Compounds) theory (Frank et al., 2019). Grimm-Allen ironlimited medium allows A. ochraceus to secrete the extracellular siderophores (Jalal et al., 1984). Furthermore, the cultivation of two different microbial strains (A. ochraceus and B. subtilis) together leads to the induction of (126) and (127), which are not previously observed in the independent culture of each strain (Frank et al., 2019). Genetically, the changes in the cultural environment alter the gene expression profiles, hence activating silent SM gene clusters (Keller, 2019). For example, Frontiers in Chemistry | www.frontiersin.org August 2022 | Volume 10 | Article 938626 a total of 64 backbone SM genes, which are responsible for the biosynthesis of the chemical skeleton, are differentially expressed when A. nidulans undergoes a fungal-fungal cocultivation, leading to the activation of 14 aspernidine derivatives (Wang et al., 2022b). Although a large number of compounds have been discovered in A. ochraceus, some of them are derived from the same biosynthetic pathway, e.g., (8) and (9) (Harris and Mantle, 2001). In fact, the number of genes encoding biosynthetic enzymes clearly outnumbers the identified compounds in A. ochraceus (Gonçalves et al., 2021). Finally, we remind the scientific community not to discredit the ability of A. ochraceus to produce natural products and encourage them to explore unexpected natural products through manipulating nutritional or environmental factors.

AUTHOR CONTRIBUTIONS
GW, EL, WW, JZ and XG wrote the manuscript. LC and FX revised the manuscript.