Introduction

The genus Aspergillus is rich in species and these species are able to produce a large number of extrolites, including secondary metabolites, bioactive peptides/proteins, lectins, enzymes, hydrophobins and aegerolysins. Extrolites are outward-directed chemical compounds from organisms that are secreted or anchored on the cell wall or in the membrane and accumulated. The word comes from extro (outwards directed and -ite: a chemical compound. The term is ecological rather than a metabolism term. The Aspergilli are also capable of biotransforming extrolites from other species. A xenoextrolite is an extrolite from another species than that in question. Because of the production of such diverse extrolites, many different Aspergillus species have been used in biotechnology, both for bulk and fine chemical production (Meyer et al. 2010), and also for exoenzyme production, and certain species stand out as working horses of biotechnology, especially Aspergillus niger, Aspergillus oryzae and Aspergillus terreus. Aspergillus species have also been used as heterologous producers of proteins and exometabolites and for bioremediation. Species of Aspergillus can also have negative effects and be pathogenic (Buzina 2013; Sugui et al. 2014a, b), give health problems in buildings (Miller and McMullin 2014) and deteriorate other materials.

Aspergillus species produces a wide array of small molecule extrolites (secondary metabolites or specialized metabolites, all abbreviated SM here), but also other bioactive molecules such as large peptide ribotoxins and lectins. The ribotoxins appear to be restricted to Aspergillus subgenus Fumigati sections Fumigati and Clavati (Ng and Wang 2006; Varga and Samson 2008; Abad et al. 2010), but bioactive peptides have also been reported from subgenus Aspergillus, for example eurocin production by Aspergillus montevidensis (Oeemig et al. 2012). Lectins have been found in phylogenetically distant subgenera of Aspergillus such as Circumdati, Nidulantes, Fumigati and Aspergillus (Singh et al. 2014a, b). Most known extrolites are small molecules, however, and these molecules will be emphasized here.

Specialized metabolites, as the name indicates, have evolved because of ecological challenges. Species with no competitors, such as the extromephile Xeromyces bisporus, do not produce any specialized metabolites and there are no gene clusters coding for such metabolites in the genome (Leong et al. 2015). Since Aspergillus species are usually very efficient specialized metabolite producers, we will also examine whether species in the different sections produce extrolites that have evolved with their species based on ecology or phylogeneny or both (Gibbons and Rokas 2013; Wisecaver and Rokas 2015).

Taxonomy and phylogeny of Aspergillus

The classification of Aspergillus has traditionally been based on morphology and colony colours including conidium colour, as was done in the latest full revision and identification-manual of Aspergillus by Raper and Fennell (1965) (Samson et al. 2006). A partial revision of some Aspergillus species by Kozakiewicz (1989) was heavily based on micromorphology, including conidium and ascospore characterization by scanning electron microscopy. Taxonomic characters based on ecophysiology, nutrition, secondary metabolites and extracellular enzymes were for many years used occasionally, but rarely incorporated into broad taxonomic schemes. However, all these ecologically relevant taxonomic features were promising, often giving clear-cut differences between closely related species. For example, the first use of secondary metabolites in Aspergillus taxonomy (Frisvad 1985; Frisvad and Samson 1990; Samson et al. 1990) was promising, as was the use of isoenzyme profiling (Cruickshank and Pitt 1990) and the use of simple ecophysiological and nutritional characters (Klich 2002; Pitt and Hocking 2009; Samson et al. 2010). It is now well established that profiles of small molecule extrolites are species-specific (Larsen et al. 2005; Frisvad 2015). In addition, large molecule extrolites appear also to be species specific (Varga and Samson 2008).

Cladistic analysis of the sequences of rDNA was used by Peterson (2000) to give an overview of potential phylogenetic relationships between species in Aspergillus and this has later been followed by a series of papers on sequence-based cladification of Aspergillus species, using nucleotide sequences of ITS1 and 2 from rDNA, β-tubulin, calmodulin and other genes (Geiser et al. 2007). Since analyses based on classification of functional characters were generally in agreement with sequence-based cladifications, a polyphasic approach using all these characters has been proposed for taxonomy, phylogeny, species descriptions and identifications (Frisvad et al. 2007a, b; Geiser et al. 2007; Samson et al. 2014).

Aspergillus species have widely different sexual states (Table 1), and it has been shown that Aspergillus fumigatus and allied species are nearly as molecularly divergent from A. niger and Aspergillus flavus as humans are from fish, based on average protein sequence identity (Fedorova et al. 2008). This is indeed reflected in the large differences between their sexual states: The small hard lightly-coloured sclerotioid ascomata of Aspergillus fischeri (Samson et al. 2007a, b) are very different from the black sclerotial stromatoid ascomata, in which many cleistothecial locules (2–8) are developing, in Aspergillus alliaceus (Raper and Fennell 1965), A. flavus (Horn et al. 2009a), Aspergillus parasiticus (Horn et al. 2009c) or Aspergillus nomius (Horn et al. 2009b). Furthermore, Aspergillus sensu lato as circumscribed by Raper and Fennell (1965) is paraphyletic, with a genus such as Polypaecilum (dichotomomyces-morph) placed between Aspergillus section Fumigati (neosartorya-morph) and Aspergillus section Clavati (neocarpenteles-morph) (Varga et al. 2007a, b, c; Houbraken and Samson 2011). With the accepted new nomenclatural system for fungi (one fungus one name) (Hawksworth 2011; Hawksworth et al. 2011), there have been discussions whether we should use the genus designation Aspergillus for all species in the monophyletic clade comprising Aspergillus sensu Raper and Fennell (1965), but including further species with different morphologies as dictated by DNA sequences (Samson et al. 2014) or to use the established names Eurotium, Neosartorya, Emericella etc. for distinct Aspergillus sections, as recommended by Pitt and Taylor (2014). If the latter solution to the nomenclatural problem in Aspergillus sensu Raper and Fennell (1965) was to be adopted, Aspergillus will have to be neo-typified, by for example A. niger (Pitt and Taylor 2014), because Aspergillus at present is typified by Aspergillus glaucus and keeping the name Eurotium will require such a neo-typification. In this review, we have decided to follow the decision of Samson et al. (2014) to include species of Aspergillus in the monophyletic clade including A. glaucus (Hubka et al. 2013) and nearly all species accepted by Raper and Fennell (1965). This has had the consequences that Penicillium inflatum had to be transferred to Aspergillus as Aspergillus inflatus, Aspergillus paradoxus, Aspergillus malodoratus and Aspergillus crystallinus had to be transferred to Penicillium as Penicillium paradoxum, Penicillium malodoratum and Penicillium crystallinum, Aspergillus zonatus and Aspergillus clavatoflavus had to be excluded from Aspergillus and finally that the species in the genera Dichotomomyces, Phialosimplex, Polypaecilum and Cristaspora had to be transferred to Aspergillus (Houbraken and Samson 2011; Houbraken et al. 2012; Samson et al. 2014). In this system, 354 species of Aspergillus have been accepted (Samson et al. 2014). As an example of proper naming of the well-known species in the former two-names for a species system A. fumigatus/Neosartorya fumigata and Aspergillus fischerianus/Neosartorya fischeri should now be named A. fumigatus and A. fischeri. If the sexual state has been observed for an isolate, the name can be more informative in calling them A. fumigatus (neosartorya-morph) and A. fischeri (neosartorya-morph). In two species in Aspergillus, Aspergillus monodii and Aspergillus arxii, only the sexual state has been found, making it difficult to recognize these species as Aspergillus, and in such cases sequencing of several house-hold genes is necessary for correct cladification, classification and identification (Samson et al. 2014). Several Aspergillus species have been genome sequenced (Andersen et al. 2011; Baker 2006; Pel et al. 2007; Gibbons and Rokas 2013), and many clusters coding for new Aspergillus secondary metabolites have been discovered (Chiang et al. 2010; Brakhage 2013).

Table 1 Sections of Aspergillus and their associated sexual states
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Being so different, the sections of Aspergillus could be hypothesized to produce widely different small molecule extrolites. Below, we will investigate whether this is the case.

Chemodiversity of Aspergillus

Chemotaxonomy based on secondary metabolites has been very valuable in Aspergillus taxonomy, and secondary metabolites are often included in species descriptions (Larsen et al. 2005; Frisvad et al. 2007a, b; 2008; references in Table 1). Independent analysis of Aspergillus species identified either using morphology plus physiology or using DNA sequences shows that the profile of secondary metabolites is species specific, while individual secondary metabolites may occur in closely related species, in less closely related species within a genus and even in completely unrelated species. Papers by Patron et al. (2007), Khaldi et al. (2008), Schmitt and Lumsch (2009), Ma et al. (2010), Slot and Rokas (2010), Khaldi and Wolfe (2011), Campbell et al. (2012), Wisecaver et al. (2014) and Wisecaver and Rokas (2015) indicate that at least in some cases horizontal gene cluster transfer is a possibility. Within a particular section of Aspergillus, often a large number of species share the ability to produce a given secondary metabolite. In Aspergillus section Flavi 14 out of 24 species can produce sterigmatocystins and 13/24 can produce aflatoxins (Fig. 1). In the same section all species except A. avenaceus can produce kojic acid (Varga et al. 2009; 2011b). Within a section the ability to produce a particular secondary metabolite seems to be laterally transferred (inherited from a common ancestor). Most secondary metabolites from Aspergillus are produced by species in only one or few sections. Some well know bioactive secondary metabolites, such as penicillin, viridivatin, mevinolin, pseurotin A and cyclopiazonic acid are present in phylogenetically different sections of Aspergillus (Fig. 1).

Fig. 1
figure 1

Well-known secondary metabolites produced by Aspergillus species in different sections of the genus

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Chemical uniqueness and differences between subgenera and sections of Aspergillus

There are six major subgenera in Aspergillus: Circumdati, Nidulantes, Fumigati, Polypaecilum/Phialosimplex (not officially named yet), Cremei (only named as a section at present) and Aspergillus. As mentioned by Fedorova et al. (2008), these are distantly related, but with the necessary transfers of misplaced Aspergilli and Penicillia (Samson et al. (2014); Visagie et al. 2014c), they form a monophyletic clade (Houbraken and Samson 2011; Houbraken et al. 2012; Samson et al. 2014). The last three subgenera have the common feature in that they grow well at very low water activities and often tolerate high concentrations of sodium chloride (most pronounced in subgenus Aspergillus section Aspergillus and Restricti, most of the species formerly in the genus Eurotium) (Pitt and Hocking 2009). Halotolerance or xerotolerance is also reflected in the halotolerant Polypaecilum pisci being transferred to Aspergillus pisci, and Basipetospora halophile = Oospora halophile = Scopulariopsis halophilica = Phialosimplex halophila (Pitt and Hocking 1985; Greiner et al. 2014) being transferred to Aspergillus baarnensis and Phialosimplex salinarum obviously also to be transferred to Aspergillus (Samson et al. 2014). Subgenus Circumdati and and its sister subgenus Nidulantes are closely related, for example hülle cells, aflatoxins, kojic acid, indole diterpenes, and bicyclo[2.2.2]diazaoctanes are found in both subgenera (Raper and Fennell 1965; Yaguchi et al. 1994; Varga et al. 2009; Finefield et al. 2012; Cai et al. 2013). Some known secondary metabolites, present in cladistically different sections of Aspergillus, are shown in Fig. 1.

Unique extrolites in subgenus Circumdati

The subgenus Circumdati contains most biotechnologically important Aspergilli, such as A. niger, A. oryzae, Aspergillus. tamarii and A. terreus. Apart from species in subgenus Fumigati, subgenus Circumdati also contains the most important pathogenic species and mycotoxin producers. Within subgenus Circumdati, the sections have quite few SMs in common, but they do have many analogous SMs in common. Section Nigri species can produce the unique compounds: calbistrins, fumonisins, malformins, naphtho-γ-pyrones, nigerloxins, nigragillins, okaramins, pyranonigrins, tensidols, and yanuthones (Nielsen et al. 2009 (Fig. 2)). Section Flavi species can produce the unique compounds asperfurans, asperlicins, cyclopiamins and griseofulvins (Varga et al. 2011a, b); section Circumdati species can produce the unique compounds aspochraceins/sclerotiotides, aspyrones, chlorocarolides, destruxins, melleins, ochrindols, penicillic acid, petromindols, preussins, sulpinins and xanthomegnins (Visagie et al. 2014a); section Candidi species can produce chloroflavonins and xanthoascins; and section Terrei and Flavipedes species can produce the unique compounds aspochalasins, asterriquinols, butyrolactones, citreoviridin, citrinins, geodins, mevinolins and terreic acids (Samson et al. 2011a) (Fig. 2). Species in these sections produce many more SMs, but some of these will be mentioned as similar or analogous SMs in different sections. An overview of SMs that are unique in the subgenus Circumdati sections Nigri, Flavi, Circumdati, Terrei and Flavipedes are presented in Fig. 2. A large number of these extrolites are very bioactive.

Fig. 2
figure 2

A selection of unique secondary metabolites produced by species in Circumdati, sections Nigri, Flavi, Circumdati, Flavipes and Terrrei

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Unique extrolites in subgenus Nidulantes

Among the unique SMs in subgenus Nidulantes are aspernidins, asperugins, asteltoxins, austins, austocystins, cordycepins, echinocandins/mulundocandins, emecorrugatins, ethericins, falconensins, falconensons, emericellins, ophiobolins, shamixanthones, stromemycin, sydowinins and ustic acids (Fig. 3.) (Turner 1971; Turner and Aldridge 1983; Cole and Scheweikert 2003, Cole et al. 2003). However, many other SMs are shared with species in other Aspergillus subgenera and sections.

Fig. 3
figure 3

A selection of unique secondary metabolites produced by species in subgenus Nidulantes, sections Nidulantes, Aenei, Usti, and Versicolores

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Unique extrolites in subgenus Fumigati

There are several unique SMs in subgenus Fumigati (Fig. 4) In section Fumigati, some important ones are fiscalins, fischerins, fumagillins, fumigaclavins, fumigatonins, fumiquinazolins, glabramycins, helvolic acids, pyripyropens, ruakuric acids, tryptoquivalins, viridicatumtoxins and viriditoxins and in section Clavati expansolides, cytochalasin E and patulin (Frisvad 1985; Varga et al. 2007a, b, c, Samson et al. 2007a, b; Hong et al. 2008; Frisvad et al. 2009).

Fig. 4
figure 4

A selection of unique secondary metabolites produced in subgenus Fumigati, section Fumigati and Clavati

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Unique extrolites in subgenus Aspergillus, section Cremei and subgenus ‘Polypaecilum/Phialosimplex’

In section Aspergillus and Restricti, unique SMs include asperglaucide, asperentins, auroglaucins, echinulins, epiheveadride, flavoglaucins and neoechinulins (Fig. 5) (Slack et al. 2009; Turner 1971; Turner and Aldridge 1983; Cole and Scheweikert 2003, Cole et al. 2003), while section Cremei species can produce asperolides, anthraquinone-derived bianthrons, leuconic acid, citraconic anhydrides and wentilactones uniquely (Fig. 5) (Verchère et al. 1969; Turner 1971; Assante et al. 1979; Dorner et al. 1980; Selva et al. 1980; Turner and Aldridge 1983; Cole and Scheweikert 2003, Cole et al. 2003; Sun et al. 2012). Asperglaucide from Aspergillus restrictus and Aspergillus penicillioides (Itabashi et al. 2006) has a clear resemblance to asperphenamate found in Aspergillus flavipes in subgenus Circumdati section Flavipedes (Clark et al. 1977).

Fig. 5
figure 5

A selection of unique secondary metabolites produced by species in subgenus Aspergillus sections Aspergillus and Restricti, and in section Cremei

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The same secondary metabolite produced in phylogenetically different subgenera and sections of Aspergillus

Despite the chemical differences between sections, there are several examples of the same SM being produced by species in different sections in Aspergillus, even phylogenetically more distantly related Aspergilli. This can be explained by lateral or horizontal SM gene cluster transfer or by reinvention of a gene cluster coding for the same secondary metabolite biosynthetic family. The results obtained so far indicate that lateral gene transfer is common within a series or section of a genus, while horizontal gene transfer (HGT) is more likely in phylogenetically more distant species in a genus or even very distantly related genera across the whole fungal kingdom (Rank et al. 2011; Campbell et al. 2012; Wisecaver and Rokas 2015). HGT of either a gene cluster or a whole mini-chromosome can then be a result of species occurring in the same habitat with a large degree of competition/collaboration and the same ecological challenge (Ma et al. 2010).

The polyketide sterigmatocystin (Fig. 1) has been found in widely different genera, including Aschersonia, Aspergillus, Bipolaris, Botryotrichum, Chaetomium, Humicola, Moelleriella, Monicillium and Podospora but also in widely different sections of Aspergillus including sections Flavi, Ochraceorosei, Aenei, Nidulantes, Versicolores and Cremei. Sterigmatocystin is most common in the two sister subgenera Circumdati and Nidulantes (Rank et al. 2011), while only A. inflatus in section Cremei produce it, and those Aspergillus sections are distantly related (Houbraken and Samson 2011). These genera span the Pezizomycotina, i.e. nearly all known filamentous ascomycetes. One further species Staphylotrichum boninense producer sterigmatocystin precursors, 5′-oxyaverantin, averantin and versicolorin B that are galactofuranosylated (Tatsuda et al. 2015), indicating that even sterigmatocystins and aflatoxins may be present as glycosides in foods (masked mycotoxins). Since it appears improbable that a common ancestor of all ascomycetes could produce sterigmatocystin or its precursors, the ability to produce this secondary metabolite must have evolved independently a large number of times, or the gene cluster or a chromosome carrying, it must have been horizontally transferred as suggested by Slot and Rokas (2011) for Aspergillus and Podospora. Secondary metabolites derived from sterigmatocystin, aflatoxins, are present in only two genera: Aspergillus (Varga et al. 2009) and Aschersonia (Kornsakulkarn et al. 2012, 2013). Within Aspergillus, there are some interesting differences between sections: Aflatoxins G1 and G2 has only been found in section Flavi, while species in other sections never produce aflatoxins G1 and G2, but accumulate both sterigmatocystin and aflatoxin B1 (Frisvad et al. 2005). Concomitant accumulation of aflatoxin B1 and sterigmatocystins is also seen in Aschersonia coffeae and Aschersonia marginata (Kornsakulkarn et al. 2012, 2013). Production of sterigmatocystin is restricted to the subgebera Circumdati section Flavi and Nidulantes sections Aenei, Ochraceorosei, Versicolores and Nidulantes, but has also been detected in the more distantly related A. inflatus in section Cremei (Rank et al. 2011; Samson et al. 2014). Interestingly sterigmatocystin and aflatoxins have never been found in Penicillium.

The bioactive bicyclo[2.2.2]diazaoctanes, such as aspergamides, stephacidins, aspergillimides and notoamides are produced by several species in closely related sections Circumdati, Nigri and Candidi (Finefield et al. 2012; Cai et al. 2013), but also by species in subgenus Nidulantes section Versicolores (Finefield et al. 2012; Kato et al. 2015). Some Aspergillus species produce both enantiomers of these bicyclo[2.2.2]diazaoctanes, and in some cases, the final biosynthetic product is only of one configuration (Kato et al. 2015).

The aspergillic acids are also produced by species in several sections in subgenus Circumdati, but has not been found outside this subgenus yet. Many species in section Flavi produce aspergillic acids (White and Hill 1943; Varga et al. 2011a, b), species in section Circumdati can produce neoaspergillic acids (Maebayashi et al. 1978) and A. flavipes (section Flavipedes) produces flavipucin (Findlay and Radics 1972).

The nephrotoxin ochratoxin A is produced by species in the closely related sections Circumdati, Flavi and Nigri in subgenus Circumdati only (Frisvad et al. 2004a, Samson et al. 2004; Varga et al. 2011a, b; Visagie et al. 2014a). This mycotoxin has also been found in Penicillium verrucosum and Penicillium nordium, however (Frisvad et al. 2004b), but not in species in any other fungal genus.

In several cases, certain SMs are produced by quite unrelated species of Aspergillus, for example pseurotin A (Fig. 1) has been found in A. fumigatus (Wenke et al. 1993) in section Fumigati, while the distanly related A. nomius in section Flavi also produce it (Varga et al. 2011a, b). Similarly, several species in section Flavi produce cyclopiazonic acid (Varga et al. 2011a, b), while Aspergillus lentulus and Aspergillus fumisynnematus in section Fumigati also produce this mycotoxin (Larsen et al. 2007).

Viridicatin (Fig. 1) and related compounds are produced by species in cladistically different sections of Aspergillus. It is produced by Aspergillus sclerotiorum in section Circumdati (Visagie et al. a, b, c), Aspergillus jensenii in section Versicolores (reported as Aspergillus nidulans by Ishikawa et al. 2014) and by A. fumigatus in section Fumigati (Frisvad and Dyer, unpublished).

Penicillins (Fig. 1) are also produce by phylogenetically different species in different sections: A. nidulans and other Aspergilli produce penicillins (Dulaney 1947a, b), while A. parasiticus and A. flavus in section Flavi and Aspergillus clavatus in section Clavati also produces penicillins (Arnstein and Cook 1947).

Analogous secondary metabolites are produced in different sections of Aspergillus (heteroisoextrolites)

The many secondary metabolites produced from one biosynthetic origin, a biosynthetic family of compounds, could be called small molecule isoextrolites. However, there are functionally and biosynthetically quite similar SMs that may be analogous. We call these metabolites for small molecule heteroisoextrolites. Given the large phylogenetic distance between the main subgenera of Aspergillus (Fedorova et al. 2008), it is to be expected that the species in those subgenera produce different versions of the functionally the same kind of secondary metabolite. An example is 6-methylsalicylic acid-derived antibiotically active secondary metabolites of similar, but not identical structures (Fig. 6). Species in section Flavi produce parasitenone (Son et al. 2002), in section Nigri some species produce the terpene-decorated yanuthones (Bugni et al. 2014; Holm et al. 2014), in section Terrei some species produce terreic acid (Guo and Wang 2014; Guo et al. 2014), in section Fumigati some species produce fumigatin oxide (Yamamoto et al. 1965), in section Clavati most species produce (+)-epoxydon and the end-product patulin is also produced (Varga et al. 2007c), while another species in the section, Aspergillus acanthosporus, produces (+)-isoepoxydon (Kontani et al. 1990). These epoxyquinones and epoxyquinols thus seem to be spanning the whole genus, except that species in sections Aspergillus and Restricti have not been reported to produce these compounds.

Fig. 6
figure 6

Some 6-methylsalicylic acid (6-MSA)-derived secondary metabolites in different sections of Aspergillus (see text for the producers of each compound)

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Small organic acids (Fig. 7) should be classified as secondary metabolites when they are secreted and accumulated (Frisvad 2015). The gene cluster for itaconic acid has been characterized (Van den Straat et al. 2014), and in Aspergillus, this acid has been found in Aspergillus itaconicus (Kinoshita 1931) and Aspergillus gorakhpurensis (Busi et al. 2009) in section Cremei and in A. terreus in section Terrei (Van den Straat et al. 2014). It appears that most sections of Aspergillus have a unique profile of organic acid production. In Aspergillus section Flavi, most species produce kojic acid (Varga et al. 2011b), which is glucose derived (Terebayashi et al. 2010) and malic acid as the main acids (Peleg et al. 1988; Knuf et al. 2014). In the phylogenetically closely related Aspergillus section Nigri, A. niger, Aspergillus carbonarius and Aspergillus tubingensis predominantly produce citric acid, oxalic acid and gluconic acid, depending on pH (Goldberg et al. 2006). A. niger was originally reported to produce citric acid consistently (Moyer 1953a, b), but some of the acid-producing strains were later re-identified to A. carbonarius and A. tubingensis (Frisvad et al. 2011). Furthermore a citric acid producing Aspergillus wentii (Moyer 1953a, b), was later shown to be A. niger (Frisvad et al. 2011). Deletion of the glucose oxidase gene in A. carbonarius resulted in the production of citric acid, oxalic acid and malic acid (Yang et al. 2014), but apparently malic acid is not naturally overproduced in A. carbonarius. Although seemingly a major small acid produced by A. niger, citric acid has also been reported from Aspergillus lanosus in section Flavi, Aspergillus ochraceus and Aspergillus melleus in section Circumdati and in A. gorakhpurensis in section Cremei (Srivastava and Kamal 1980). However, citric acid production is much stronger and more consistent in A. niger. In section Circumdati, the dominant small acid seems to be malic acid (Srivastava and Kamal 1980; West 2011), but most species in that section produce the small polyketide acid penicillic acid (Frisvad et al. 2004a, b; Visagie et al. 2014a, b, c), not produced by species in any other Aspergillus section. The main acid produced by A. fumigatus appear to be epoxysuccinic acid (Martin and Foster 1955), but in general species in the unrelated sections Nigri, Terrei and Cremei are the most efficient producers of small organic acids.

Fig. 7
figure 7

Examples of small organic acids produced by species in different sections of Aspergillus

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A systematic study of all species in section Nigri has not been performed yet, but preliminary studies indicate that while the biseriate species in section Nigri produce large amounts of citric acid/oxalic acid/gluconic acid, the uniseriate species are much less productive.

Fumonisins were discovered in A. niger in 2007 (Frisvad et al. 2007b, 2011) and in a recent paper a motif-independent method for prediction of secondary metabolite gene clusters, A. fumigatus was predicted to produce fumonisins (top hit) based on the gene cluster in Fusarium graminearum (Takeda et al. 2014). However, fumonisins have never been detected in A. fumigatus. Interestingly, A. fumigatus and A. lentulus produce sphingofungins and fumifungin (Larsen et al. 2007), structurally related to fumonisins (Fig. 8), so this is probably reflecting some sequence similarities in the two gene clusters. Sphingofungins and fumonisins may also be heteroisoextrolites.

Fig. 8
figure 8

Analogous compounds in Aspergillus in the distantly related sections Nigri and Fumigati: Fumonisin B2 from A. niger and fumifungins and sphingofungins from A. fumigatus and A. lentulus

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Some unique chlorinated PKS-NRPS-derived molecules have been detected in sections Flavi, Circumdati, Nigri and Candidi. While ochratoxin A, a phenylalanine PKS hybrid, is present in species in Circumdati, Flavi and Nigri (Frisvad et al. 2011; Varga et al. 2011a, b; Visagie et al. 2014a, b, c); it has never been found in section Candidi. Interestingly the only flavonoid-type SM known in fungi, chlorflavonin, is produced by Aspergillus candidus and is also derived from a phenylalanine and a PKS hybrid that is chlorinated (Fig. 9) (Burns et al. 1979). This indicates that different section-specific analogous secondary metabolites may be produced in Aspergillus. A comparison of the gene clusters coding for production ochratoxins and chlorflavonins may throw light upon this interesting observation.

Fig. 9
figure 9

Ochratoxin A is a chlorinated PKS-NRPS-derived secondary metabolites produced from phenylalanine and a polyketide by species in section Nigri, Flavi and Circumdati. Chlorflavonin is also derived from phenylalanine and a polyketide and is chlorinated, so an example of analogous biosynthetic pathways

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Another group of antioxidative secondary metabolites abundant in species in section Candidi is terphenyllins and candidusins (Rahbaek et al. 2000; Yen et al. 2001), probably being overproduced to protect the white/yellow conidia of these fungi rather than via melanin, as opposed to species in section Nigri that produce very large amounts of melanins. However, the terphenyllins and candidusins have analogous SM molecules in section Nigri: cycloleucomelon and atromentin (Hiort et al. 2004) and aspulvinones in section Terrei (Gao et al. 2013). All these biosynthetic families are produced via the shikimic acid pathway (Turner 1971). Analogous alkaloidic shikimic acid derived SMs to the compounds in other sections of Aspergillus are emerin and epurpurins in section Nidulantes (Ishida et al. 1975; Takahashi et al. 1996), xanthoascin in section Candidi (Takahashi et al. 1976) and fumiformamide in Fumigati (Zuck et al. 2011) (Fig. 10). Thus, it seems that shikimic acid derived functionally quite similar SMs are produced by species in the different sections of Aspergillus.

Fig. 10
figure 10

Shikimic acid-derived analogous secondary metabolites from Aspergillus sections Candidi (terpenyillin, candidusin A, xanthoascin), Nigri (cycloleucomelon and atromentin), Nidulantes (emerin and epurpurin) and Fumigati (fumiformamide)

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Gliotoxin is an important secondary metabolite produced by A. fumigatus and related species in section Fumigati. Even though this epidithiodioxopiperazine has been reported in trace amounts from other potentially pathogenic Aspergilli, including A. niger, A. flavus and A. terreus (Lewis et al. 2005; Kupfahl et al. 2008). The results obtained by latter two groups suffered from unavailability of strains, so the results could not be verified, and there is some doubt whether this was just transient or non-production. Gliotoxin seems to be only produced in high amounts by species in section Fumigati in Aspergillus. However, the other species produce biosynthetically closely related epidithiodioxopiperazines: A. flavus, A. oryzae and A. tamarii can produce aspirochlorine = oryzachlorin (Berg et al. 1976; Sakata et al. 1982; 1983; Monti et al. 1999; Chankhamjon et al. 2014), A. terreus can produce acetylaranotin (Miller et al. 1968; Cosulich et al. 1968; Guo et al. 2013) and A. striatus and six other species in section Nidulantes can produce emestrin (Seya et al. 1985; Terao et al. 1990; Kawahara et al. 1994; Ooike et al. 1997) (Fig. 11). Interestingly, both aspirochlorine and acetylaranotin is biosynthesized via a phenylalanyl phenylalanine diketopiperazine, while gliotoxin is biosynthesized via phenylalanyl serine diketopiperazine (Amatov and Jahn 2014).

Fig. 11
figure 11

Analogous secondary metabolites in different Aspergillus sections. Gliotoxin, aspirochlorin, acetylaranotin and emestrin. Emestrin is fused with a polyketide and so differs from the others that are only derived from amino acids (phenylalaninyl phenylalanine diketopiperazine for aspirochlorin and acetylaranotin and phenylalanine and serine in gliotoxin

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Emodin has been found in many Aspergillus species across the whole genus, but is also common in Penicillium, Talaromyces and even in plants (Turner 1971; Turner and Aldridge 1983; Izhaki 2002; Yilmaz et al. 2014). It has multiple effects on other organisms; has an antibacterial, antifungal, antiparasitic and antiviral effects; is a feeding deterent on insects, birds and small mammals; and is also an antioxidant (Izhaki 2002). Regarding Aspergillus, it was early reported as a mycotoxin from A. wentii (section Cremei) (Wells et al. 1975), but usually emodin, biosynthesized via atrochrysone, is converted into more chemically elaborate end-products, depending on the Aspergillus section (Fig. 12). In subgenus Aspergillus, emodin is turned into anthrons (Turner 1971) and in section Cremei, several Aspergillus species turns emodin into emodin bianthrones and isosulochrin (Assante et al. 1980; Hamazaki and Kimura 1983; Rabie et al. 1986; Ji et al. 2014). In section Nigri and Circumdati, emodin is converted to secalonic acids (Yamazaki et al. 1971; Andersen et al. 1977; Turner and Aldridge 1983, Varga et al. 2011a). In A. fumigatus, emodin is converted to either trypacidin/3-O-methylsulochrin or into chloroanthraquinones (Yamamoto et al. 1968). In A. terreus, emodin is converted in to geodin (Nielsen et al. 2013). In subgenus Nidulantes, emodin is converted to emericellin and shamixanthones (Nielsen et al. 2011; Sanchez et al. 2011; Simpson 2012) and may also be involved in biosynthesis and the specific allocation of asperthecin in the ascomata (Brown and Salvo 1994; Szewczyk et al. 2008).

Fig. 12
figure 12

Some emodin-derived secondary metabolites in different sections of Aspergillus. Secalonic acid A is produced by A. sclerotiorum in section Circumdati, and the optical antipode secalonic acid D is produced by several uniseriate Nigri species, sulochrin is produce by A. wentii in section Cremei, while geodin is produced by Aspergillus terreus in section Terrei, anthrons and bianthrons are produced by species in sections Aspergillus and Cremei and shamixanthone is produced by species in section Nidulantes. The chloro anthrone is produced by A. fumigatus in section Fumigati in addition to 3-O-methylsulochrin and trypacidin

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Dimeric diketopiperazines are also produced by fungi in different sections of Aspergillus: asperazine and similar compounds were isolated from A. tubingensis, Aspergillus vadensis and Aspergillus luchuensis in section Nigri (Varoglou et al. 1997; Varga et al. 2011a; Li et al. 2015) ditryptophenaline by A. flavus in section Flavi (Springer et al. 1977), aspergilazine A is produced by Aspergillus taichungensis in section Candidi (Cai et al. 2012), WIN 64821, probably from A. flavipes in section Flavipedes (Barrow et al. 1993), and eurocristatine is produced by by Aspergillus cristatus in section Aspergillus (Gomes et al. 2012).

The indoloterpenes are often produced in sclerotia only and occur in section Flavi, Nigri, Circumdati, Candidi and Nidulantes: Aflavinins are produced in sclerotia of section Flavi (Gallagher et al. 1980; Cole et al. 1981), 10,23-dihydro-24,25-dehydroaflavinins are produced in sclerotia by species in section Nigri (Tepaske et al. 1989, Frisvad et al. 2014), radarins and secopenitrems are produced in the sclerotia of species in Circumdati (Laakso et al. 1992) and emindole SB and similar compounds are produced in ascomata by species in section Nidulantes (Nozawa et al. 1988) and in Aspergillus cejpii in subgenus Fumigati (Harms et al. 2014), in addition to fischerindoline in Aspergillus thermomutatus in section Fumigati (Masi et al. 2013).

The bicoumarins, kotanins, aflavarins, isokotanins and desertorins are similar polyketides produced in the sclerotia of species in several sections of Aspergillus. Species in section Flavi, A. alliaceus and A. flavus produce isokotanins and aflavarins (TePaske et al. 1992; Laakso et al. 1994), A. clavatus in section Clavati and A. niger in section Nigri produce kotanins (Cutler et al. 1979; Varga et al. 2007c; Nielsen et al. 2009) and Aspergillus desertorum in section Nidulantes produces desertorins (Nozawa et al. 1987). However, the kotanins are produced in isolates of A. niger without sclerotia being produced (Frisvad et al. 2014), so there is no strict correlation between ascoma or sclerotium in different Aspergillus sections and these bicoumarins.

Other analogous specialized metabolites including siderophores (Yin et al. 2013) have been found in several sections of Aspergillus, but the examples above show that these heteroisoextrolites are shared by sections covering the whole genus Aspergillus.

Conclusions

The genus Aspergillus contains a large number of species that are capable of producing a large number of specialized metabolites. Some of these metabolites are produced on most common substrates, while others need special chemical signals (xenoextrolites) in order to be produced. In the different sections of Aspergillus, the species produce many specialized metabolites in species-specific profiles. These profiles contain unique SMs, SMs shared with related and distantly related Aspergilli and analogous SMs (heteroisoextrolites) which are biosynthetically related and often functionally similar extrolites. The many shared similar and analogous secondary metabolites across the genus Aspergillus indicates that this genus is broad, yet has similarities indicating it should not be split into several smaller genera. The unique metabolites in many of these sections of Aspergillus are only unique within the genus Aspergillus, as several of those occur in Penicillium species also. However, we hypothesize that many more unique secondary metabolites will be discovered in each of the Aspergillus sections, based on genome sequencing evidence. The ability to accumulate and secrete small molecule extrolites, therefore, is a reaction to challenges in the environments and competition and collaboration in species consortia, rather than being determined only by phylogeny. The secondary metabolites have probably evolved based on gene duplications, horizontal gene transfer and new gene cluster formations as a reaction to the environment.