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IDT production
IDT production in various fungi
To date, more than 50 fungal species have been demonstrated to produce paspaline-derived IDTs (Supplementary Table S1). The great majority of IDT producer fungi belong to only two ascomycetous classes in the Pezizomycotina subphylum: the Eurotiomycetes (Aspergillus, Penicillium, Emericella and Eupenicillium species in the Aspergillaceae and Trichocomaceae families within the Eurotiales order) and the Sordariomycetes (Claviceps, Epichloë, Escovopsis, Neotyphodium, Periglandula and Tolypocladium species in the Clav ic ipi t a cea e , Hypoc r eace ae and Ophiocordycipitaceae families in the Hypocreales order).
In the order Eurotiales (class Eurotiomycetes), at least 24 Penicillium and Eupenicillium species have been shown to produce various paspaline-derived IDTs (Supplementary Table S1). The most common IDT in these fungi is penitrem A, a metabolite that is of paramount importance for the con- tamination of foodstuffs with mycotoxins. One of the most important model organisms used in IDT biosynthetic studies is the saprophytic species P. paxilli, which produces paxilline. A set of bioactive paxilline analogues were also isolated from Penicillium camemberti, a white mold that is widely used in cheese ripening.
Among the more than 10 Aspergillus and Emericella spe- cies that produce paspaline-derived IDTs, the notorious afla- toxin producer A. flavus is also capable of synthetizing aflatrem A and at least two additional aflatrem congeners. Aflatrem and paspaline were also detected in cultures of two other Aspergillus species, A. minisclerotigenes and
A. parvisclerotigenus. Importantly, the koji mold Aspergillus oryzae was shown to produce 13-desoxypaxilline, a common intermediate for aflatrem biosynthesis. Some other Aspergilli, including A. desertorum, A. foveolatus, and A. striatus are verified paxilline producers while A. alliaceus synthesizes two paxilline-like IDTs in axenic cultures.
In the order Hypocreales (class Sordariomycetes), at least 13 species produce paspaline-derived IDTs (Supplementary Table S1). In the family Clavicipitaceae, C. paspali and
C. cynodontis yield paspalitrems while the well-known ergot alkaloid producer C. purpurea synthesizes only less complex IDTs such as paspaline. Epichloë gansuensis produces paxilline, while the IDT spectra of two additional clavicipitaceous fungi, N. lolii and E. festucae are rather dif- ferent. These two fungi form IDTs with the C11(12) epoxide, giving rise to terpendoles and the more elaborate lolitrems. Meanwhile, Periglandula ipomoeae strains, symbionts of morning glory, also produce terpendole analogues such as terpendoles C, K, and E (Schardl et al. 2013; Lee et al. 2017; Gardner et al. 2018). Fungi in two other hypocrealean families also produce paspaline-derived IDTs. Thus, T. album (basionym Chaunopycnis alba; family Ophiocordycipitaceae) is another important terpendole producer, while Escovopsis weberi (family Hypocreaceae) produces shearinines.
Interestingly, the zygomycetous fungus Mucor irregularis has recently been shown to yield penitrems and the related rhizovarins. M. irregularis (family Mucoraceae, order Mucorales, phylum Mucoromycota) is the only currently known fungus outside the Ascomycota phylum that produces paspaline-derived IDTs (Gao et al. 2016).
Gene clusters for IDT biosynthesis
To date, ten gene clusters for the biosynthesis of the paspaline- derived IDTs have been identified (Fig. 3), including those for penitrems (P. crustosum and P. simplicissimum) (Liu et al. 2015; Nicholson et al. 2015), paxilline (P. paxilli) (Scott et al. 2013), shearinines (P. janthinellum) (Nicholson et al. 2015), aflatrems (A. flavus and A. oryzae) (Zhang et al. 2004; Nicholson et al. 2009), paspalitrems (C. paspali) (Kozák et al. 2018), terpendoles (T. album) (Motoyama et al. 2012), and lolitrems (N. lolli and its anamorph E. festucae) (Young et al. 2006; Saikia et al. 2012). All these gene clusters contain orthologues of the paxG, paxM, paxC and paxB genes whose enzyme products are collectively responsible for the biosynthesis of paspaline as described in section 3.1 (Fig. 1, Supplementary Table S2). The only exception is the terpendole K gene cluster of T. album that is missing a GGPP synthase gene dedicated to IDT biosynthesis, indicat- ing that this precursor is supplied by the primary metabolic GGPP synthase of the strain (Motoyama et al. 2012).
In addition to the paxGMCB orthologues, all biosynthetic gene clusters for the paspaline-derived IDTs elucidated so far also contain genes similar to paxP and paxQ (Supplementary Table S2). The encoded cytochrome P450 monooxygenases are responsible for the oxidations that channel paspaline to- wards the paxilline, paspalinine or terpendole E-type cores of the various paspaline-derived IDT families (Fig. 2). All bio- synthetic gene clusters for paspaline-derived IDTs also feature additional genes encoding enzymes for the subsequent modi- fication of the angled hexacyclic skeleton (Fig. 3, Supplementary Table S2).
The known biosynthetic gene clusters for the different paspaline-derived IDT families show little synteny. The only exception to date is the pair of clusters for paxilline and the shearinines (Fig. 3), where the order and orientation of the genes are the same, except for the absence of a janJ orthologue in the paxilline gene cluster (Liu et al. 2016). In contrast, clusters that are derived from closely related species that produce metabolites belonging to the same IDT family are highly syntenic. For example, A. flavus and A. oryzae RIB40 contain aflatrem-type gene clusters with identical organiza- tions and chromosomal locations, with the corresponding Atm enzymes of the two species showing at least 95% pairwise identities. Nevertheless, A. oryzae produces non- prenylated paspalenes due to a single nucleotide insertion into exon 7 that renders atmQ nonfunctional (Nicholson et al. 2009; Qiao et al. 2010; Rank et al. 2012).
Unusually for fungal secondary metabolite biosynthetic gene clusters, several of the IDT clusters seem to be distribut- ed into multiple loci, although sequence closure of draft ge- nomes may lead to the revision of this notion in some cases. Thus, the penitrem biosynthetic genes of P. simplicissimum, but not those of P. crustosum, are separated into two subclus- ters. The ptm2 and ptm1 subclusters of P. simplicissimum are syntenic with genes PC-23 - penO and penG - PC-05, respec- tively, within the penitrem cluster of P. crustosum. The aflatrem biosynthetic gene clusters of A. flavus and
A. oryzae RIB40 are divided into two loci that reside on dif- ferent chromosomes (the atm1 subcluster is located on chro- mosome 5, while atm2 is on chromosome 7) (Nicholson et al. 2009). The paspalitrem gene cluster also spans two separate contigs on the genome sequence assembly of C. paspali RRC- 4128 (Schardl et al. 2013). These two contigs contain all the genes necessary for the biosynthesis of paspalitrems, except for the one that catalyzes C32 hydroxylation of paspalitrem A to yield the end product paspalitrem B (Kozák et al. 2018). Finally, the 11 known genes for lolitrem biosynthesis are dis- persed into three ltm subclusters in N. lolli and E. festucae. Subcluster 1 is separated from subcluster 2 by a 35-kb geno- mic region, while subcluster 3 is located at 16 kb from subcluster 2. The interspersed genomic regions are rich in AT repeats and retrotransposon elements, indicating that the com- posite ltm1-3 cluster resides in a rapidly evolving region of the genome, such as a chromosomal sub-telomeric region. This capacity for rapid evolution is underlined by the observation that the ltm1-3 composite cluster may have been duplicated in its entirety in N. lolii strain Lp19, while subcluster 3 could not be detected in the very closely related strain Lp1. In contrast,
E. festuceae F11 contains a single copy of the ltm1-3 compos- ite cluster (Young et al. 2006).
Regulation of IDT production
The regulation of IDT production has received surprisingly little attention up till now. Thus, no cluster-specific regulator has been validated in any IDT gene cluster so far. Although the penitrem gene clusters of P. simplicissimum and P. crustosum feature putative regulatory genes (ptmS and PC-06), the func- tion of these genes has not been determined experimentally (Liu et al. 2015). Similarly, the integration of IDT biosynthesis with other metabolic and morphogenetic processes also re- mains opaque. In A flavus, abundant aflatrem production is associated with sclerotia formation (Ehrlich and Mack 2014) while reduced aflatrem production results from the deletion of the ndsC gene encoding a global regulator of secondary metab- olism and asexual development (Gilbert et al. 2016).
While N. lolii and E. festucae produce lolitrems in planta only (Young et al. 2006), other IDT producers also biosynthesize these compounds in axenic cultures (Motoyama et al. 2012; Nicholson et al. 2015; Kozák et al. 2018). A. oryzae produces the paxilline precursor 13-desoxypaxilline under specific growth conditions only (Rank et al. 2012; Fountain et al. 2016). In contrast,
T. album produces large amounts of terpendoles in various me- dia, suggesting that this biosynthetic pathway is not subject to a strict regulation in this strain (Motoyama et al. 2012). Similarly,
P. crustosum strains isolated from different environments and substrates consistently produce penitrems (Frisvad and Filtenborg 1983; Yamaguchi et al. 1993; Sonjak et al. 2005). However, a variety of abiotic factors were still found to influence the production of these IDTs (Kalinina et al. 2017). Thus, culti- vation in the dark at relatively low temperatures (22 °C); glucose or rhamnose as the carbon source; and supplementation of the medium with glutamate all increase penitrem production by
P. crustosum. Interestingly, supplementation with tryptophan has the opposite effect in this fungus (Kalinina et al. 2017), in agreement with the notion that indole-3-glycerol phosphate and not tryptophan is the real precursor for IDT biosynthesis (Liu et al. 2015). In contrast, tryptophan serves as both a precursor and an inducer for ergot alkaloid biosynthesis in C. purpurea (Řeháček et al. 1971). In P. nigricans, penitrem production and sporulation are both induced by calcium chloride (Mantle et al. 1984), while CuSO4 increases penitrem production by
P. crustosum (Kalinina et al. 2017).
Oxidative stress can also be an important factor for the regulation of tremorgenic IDT biosynthesis. Aflatrem biosyn- thesis is generally upregulated in A. flavus by hydrogen per- oxide, although different isolates react differently to varied concentrations of H2O2 (Fountain et al. 2016). In contrast, H2O2 has a strong inhibitory effect on penitrem biosynthesis by P. crustosum.
IDTs as threats to agriculture, public health, and the fermentation industries
Agricultural threats
Among the known IDTs, lolitrems and paspalitrems represent the most severe danger for livestock. These compounds cause a tremorgenic syndrome in grazing animals, referred to as Bryegrass stagger^ in the case of lolitrem B ingestion (Fletcher
and Harvey 1981; Gallagher et al. 1981), and BPaspalum
stagger^ or BBermuda grass stagger^ in the case of paspalitrem mycotoxicoses (Cole et al. 1977; Uhlig et al. 2009). Ryegrass
stagger is typically caused by grazing on Lolium perenne (peren- nial ryegrass) infected by N. lolii (Gallagher et al. 1981), since lolitrem B is abundant in the N. lolii - L. perenne association (Philippe 2016). Ryegrass stagger is most frequently reported in New Zealand and Australia, and the affected animals include sheep, cattle and horses (di Menna et al. 2012). To manage rye- grass stagger, different endophyte strains with altered mycotoxin production spectra were isolated and tested. Among these, the endophytic fungus AR37 does not produce lolitrem B, but biosynthesizes epoxy-janthitrem in high concentrations. While epoxy-janthitrem is just as toxic to insects as lolitrem B, it does not cause a tremorgenic syndrome on grazing animals (Thom et al. 2013).
Outbreaks of Paspalum stagger are frequent in South Africa but case reports from the Americas, Europe, and New Zealand were also published (Mantle et al. 1978; Moyano et al. 2010; Cawdell-Smith et al. 2010). C. paspali infecting Paspalum dilatatum (Dallis grass) and C. cynodontis infecting Cynodon dactylon (Bermuda grass) produce similar paspalitrem IDT pro- files (Uhlig et al. 2009). Ingestion of sclerotia containing these toxins causes a tremorgenic syndrome with very similar symp- toms to that of ryegrass stagger (Moyano et al. 2010). Just as with lolitrem B intoxication, the affected animals usually recover rap- idly after being removed from the infected pasture (Moyano et al. 2010; Cawdell-Smith et al. 2010).
Grazing on morning glories, most frequently on Ipomoea asarifolia and Ipomoea muelleri, may also cause a tremorgenic syndrome on livestock (Gardiner et al. 1965; Medeiros et al. 2003; Dorling et al. 2004; Carvalho de Lucena et al. 2014). This toxicosis is also associated with the presence of IDTs (Lee et al. 2017), produced by seed- transmitted endophytic fungi, most likely P. ipomoeae (Schardl et al. 2013; Lee et al. 2017). The main IDT congeners isolated from endophyte-infected I. asarifolia and I. muelleri are terpendole K, 11-hydroxy-12,13-epoxyterpendole K and 6,7-dehydroterpendole A (Lee et al. 2017).
A ryegrass stagger-like syndrome, Bhuecu’s disease^ has been observed on sheep, horses, cattle, and goats in Argentina.
This is caused by the ingestion of the grass Poa huecu con- taminated with penitrem A and B, produced by Penicillium species (Scuteri et al. 1992). Penitrem A may also contaminate the soil, and ingestion of such soil by grazing animals can also cause a tremorgenic syndrome (Patterson et al. 1979). However, most case reports of penitrem A intoxications in- volve the ingestion of moldy food by pets, most frequently dogs (Hayes et al. 1976; Richard et al. 1981; Hocking et al. 1988; Walter 2002). Symptoms on dogs include generalized convulsion, ataxia, vomiting, tremors, frequent urination and defecation, mydriasis (dilation of the pupil), polypnea (panting), and hyperthermia (Richard and Arp 1979; Richard et al. 1981). Typically, the affected animals recover within a few weeks or months, but ataxia may still remain observable even years later in severe cases of intoxication (Eriksen et al. 2010).
Contamination of animal feedstuffs by spoilage fungi that produce mycotoxins including IDTs such as penitrem A (Stoev et al. 2010) represents another worldwide threat to the livestock industry. Such problems are independent of the geographical location or the origin of the feed from industrial or family-owned farms (EFSA Panel on Contaminants in the Food Chain (CONTAM) 2012).
Public health threats
Among IDTs, the toxic effects of only penitrem A and its analogues have been studied in depth, mainly in mice (Eriksen et al. 2013). Valuable information on the adverse physiological effects of these compounds on mammals also came from case reports on poisoned dogs that consumed var- ious moldy foods infected by P. crustosum (Eriksen et al. 2010; Eriksen et al. 2013). Tremorgenic mycotoxicoses likely caused by penitrem A and/or other mycotoxins produced
P. crustosum have only been reported very rarely in humans (Eriksen et al. 2013), and were connected to either the con- sumption of mold-contaminated food (Lewis et al. 2005) or drink (Cole et al. 1983), or resulted from exposure to moldy silage (Gordon 1993). It is important to note that food wastes from private households may contain high concentrations of tremorgenic mycotoxins, e.g., as high as 35–7500 μg/kg of penitrem A (Rundberget et al. 2004).
The neurotoxic effects of penitrem A include tremors, con- vulsions, ataxia and nystagmus (involuntary eye movement) (Eriksen et al. 2013). In humans, symptoms affecting the gas- trointestinal tract, such as nausea, vomiting, and bloody diar- rhea have also been recorded (Cole et al. 1983). The toxicokinetic characteristics of this lipophilic molecule are relatively well-known and include (i) rapid absorption through biological membranes, (ii) rapid distribution within the body through the blood vessels to the liver, the kidneys and the central nervous system (penitrem A can penetrate the blood- brain barrier), (iii) metabolism in the liver to yield more hy- drophilic compounds, and (iv) excretion through the bile into the feces (Moldes-Anaya et al. 2009; Eriksen et al. 2010; Moldes-Anaya et al. 2012; Eriksen et al. 2013).
Penitrem A interferes with GABAergic neurotransmission in the central nervous system by inhibiting the GABA(A) re- ceptors in the forebrain and in the cerebellum in a region- specific manner (Moldes-Anaya et al. 2011; Eriksen et al. 2013). In addition, this IDT is a potent antagonist of the high-conductance Ca2+-activated potassium channels (BK channels) in smooth muscles and peripheral tissues (Knaus et al. 1994; Eriksen et al. 2013). Importantly, lolitrem B and paxilline produced by N. lolii and E. festuce also inhibit these potassium channels, leading to ryegrass staggers in livestock (Dalziel et al. 2005; Imlach et al. 2008). Penitrem A-induced neurotoxicity may also be linked to the oxidative stress- inducing effect of this compound, as shown in cerebellar gran- ule neurons in rats (Berntsen et al. 2017).
Additional harmful physiological effects of IDTs may in- clude genotoxicity, as observed for paxillin in human lympho- cytes (Sabater-Vilar et al. 2003). It is noteworthy that both
P. crustosum extracts and penitrem A showed considerable cytotoxicity in vitro against human lung cancer, human hepa- toma carcinoma, murine fibroblast and murine neuroblastoma cell lines (Bunger 2004). The physiological effects of IDTs might be even more varied and severe because toxigenic fungi typically produce a wide spectrum of harmful secondary me- tabolites (Bunger 2004; Andersen and Frisvad 2004; Santini et al. 2014; Prencipe et al. 2018). Unfortunately, the synergis- tic behavior of these compounds is a notoriously understudied area of mycotoxicology.
Paspalitrem IDTs are relatively rarely implicated in out- breaks of toxic syndromes in humans. One such case involved an outbreak of tremors in India in 1946. At that time, Paspalum scrobiculatum, a type of millet, was consumed in certain parts of India because of a shortage of rice. Tellingly, the paspalitrem producer C. paspali was isolated from the unwholesome grain (Aaronson 1988). Even today, another IDT producer, C. purpurea is often isolated from rye and barley, and paxilline can be detected in such specimen at the maximum concentration of 0.6 mg/kg (Bauer et al. 2017).
C. purpurea sclerotia contain at least seven paspalenes, in- cluding paspaline (Uhlig et al. 2014). This raises the interest- ing possibility that these toxins may have played a role in the development of the feared symptoms of ergotism in the Middle Ages. Some authors, including Bauer and coworkers hypothesized that IDTs might have contributed to outbreaks of convulsive ergotism during history (Bauer et al. 2017).
Although the daily exposure of consumers to various IDTs is unknown, these toxic compounds have been detected in a number of food and drink products in highly variable concen- trations, depending on the geographical location. Penitrem A and its producer, the food spoilage fungus P. crustosum have been detected in several agricultural products and foodstuff, such as beer, cheese, chestnut, meat products, vegetables, pud- ding, grape berries, honey, and sausage (El-Banna and Leistner 1988; Overy et al. 2003; Sengun et al. 2008; Tancinova and Labuda 2009; Kacaniova et al. 2012; Santini et al. 2014; Camardo Leggieri et al. 2016; Olsen et al. 2017; Prencipe et al. 2018). Of course, penitrem A is not the only IDT that can be detected in contaminated foods. Paxilline and other IDTs were found in moldy tomatoes infected with Penicillium tularense (Andersen and Frisvad 2004). Lolitrem B and epoxy- janthitrem were observed in the fat and milk of growing and lactating animals that grazed on contaminated tall fescue (Miyazaki et al. 2004; Finch et al. 2012; Finch et al. 2013; Shimada et al. 2013; Zbib et al. 2015). The maximum concen- tration of these IDTs in cow milk reached 5 ng/ml and 109 ng/ml for lolitrem B and epoxy-janthitrem, respectively (Finch et al. 2013). Miyazaki et al. (2004) measured 210 ppb lolitrem B in the perirenal fat of Japanese Black cattle feeding on contaminated ryegrass (Miyazaki et al. 2004). However, the concentrations of these IDTs decrease rapidly in vivo when the animals are removed from the contaminated pasture, thus re- ducing the prevalence of food containing these two mycotoxins and limiting the threat to human health (Miyazaki et al. 2004; Finch et al. 2013).
Today, highly sensitive HPLC-MS methods are available to facilitate the detection of mycotoxin contaminants in food and drink samples (Rundberget and Wilkins 2002). A recent sur- vey (Kalinina et al. 2018) revealed that 10% of cheese samples taken from the European single market contained penitrem A at an average concentration of 28.4 μg/kg and with a maxi- mum concentration of 429 μg/kg. Considering such observa- tions, it would be important to devise standardized analytical protocols for tremorgenic IDT mycotoxins. Even more impor- tantly, it would be highly desirable for national and interna- tional regulatory agencies to define the maximum legally per- mitted levels of tremorgenic IDTs in human food and animal feed, similar to legal limits established for other mycotoxins such as aflatoxin (Medeiros et al. 2012; Oliveira et al. 2014).
Threats for the fermentations industries
C. paspali is used to produce ergot alkaloids in the pharma- ceutical industries for the manufacture of drugs against mi- graine and for the treatment of Parkinson’s disease. IDT bio- synthesis by C. paspali during industrial fermentations or ag- ricultural production on infected grasses represents both an economic and a safety problem for the manufacturers. Precursors and cofactors that may be utilized for ergot production are depleted by IDT biosynthesis, reducing pro- ductivity. At the same time, IDTs are hazardous impurities that must be removed from ergot alkaloid products and must also be safely disposed (Kozák et al. 2018).
A. oryzae is widely used in biotechnology and the food industry. This fungus is the domesticated descendant of
A. flavus. While the genotypes of A. oryzae and A. flavus are nearly identical, these fungi can still be distinguished by their morphological and physiological characteristics, and by their secondary metabolite profiles (Frisvad et al. 2018). While
A. oryzae does not produce aflatoxin (Barbesgaard et al. 1992; Tao and Chung 2014), certain isolates of A. oryzae were demonstrated to produce tremorgenic paspaline-type IDTs such as 13-desoxypaxilline (Qiao et al. 2010; Rank et al. 2012). Production of these IDTs may represent an overlooked risk factor for fermented food products.
A possible solution may be to eliminate the production of IDT mycotoxins during various fermentations, for example by inactivating key IDT biosynthetic genes. This was demonstrat- ed by the knockout of the idtCBGF paspalitrem biosynthetic genes in the industrially important ergot producer C. paspali. This led to the complete abrogation of IDT biosynthesis during fermentations, while the production of ergot alkaloids remained undisturbed with this strain (Kozák et al. 2018).
Future perspectives
Potential medical applications
Paspaline-derived IDTs are not in current medical use. Nevertheless, these metabolites show potent and wide- ranging bioactivities and have been considered for pharmaceu- tical development over the years. It is noteworthy that terpendole congeners were isolated in a systematic screening for acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors of microbial origin (Huang et al. 1995). ACAT inhibitors are po- tential agents for the prevention of atherosclerosis (Lee et al. 1998). The most potent inhibitor is terpendole C (IC50: 2.1 μM) but terpendole D (IC50: 3.2 μM) was considered even more promising since that congener displayed relatively low cytotox- icity against J774 macrophages (Huang et al. 1995). After the discovery of two ACAT isozymes in mammals (Anderson et al. 1998; Cases et al. 1998), isozyme selectivity became a prereq- uisite for potential anti-atherosclerotic agents (Giovannoni et al. 2003). Testing the selectivity of a number of microbial ACAT inhibitors revealed that terpendole C inhibits both ACAT iso- zymes, reducing the enthusiasm for the further clinical devel- opment of these IDTs (Ohshiro et al. 2007).
Terpendoles were re-discovered in a systematic screening for specific M phase cell cycle inhibitors where terpendole E was found to specifically inhibit the human kinesin Eg5 (Nakazawa et al. 2003), a potential target for cancer therapy (Knight and Parrish 2008; Sarli and Giannis 2008). Terpendole E does not affect microtubules directly, but in- duces the formation of monopolar mitotic spindles in the M phase, similar to monastrol (Nakazawa et al. 2003; Tarui et al. 2014; Sheff et al. 2017). To increase the production of terpendole E that is an intermediate of terpendole K biosyn- thesis and is thus produced only in low amounts, the terP gene was inactivated in T. album leading to the accumulation of terpendole E (Motoyama et al. 2012). The same terP knockout strain also produces the novel shunt product 11-ketopaspaline (Tarui et al. 2014). Later studies revealed that terpendole E and 11-ketopaspaline are both potent inhibitors of the microtubule-stimulated ATPase activity of Eg5. Importantly, these terpendoles not only inhibit the wild type Eg5, but retain excellent activity against Eg5 variants that are resistant to the known Eg5 inhibitors S-trityl-L-cysteine and GSK-1 (Tarui et al. 2014).
Penitrems show in vitro antiproliferative, anti-haptotactic (cell migration inhibitory) and anti-invasive activities against human breast cancer cell lines. Penitrem A induces G1 cell cycle arrest and upregulates the arrest protein p27 (Goda et al. 2018). The documented synergistic effects of penitrem A treat- ment with anti-HER drugs may have a significant impact on future development of breast cancer chemotherapies (Goda et al. 2018). Interestingly, the early biosynthetic intermediates paspaline and emindole SB also show noticeable antiprolifera- tive and antimigratory activities, with the anti-haptotactic activ- ity of paspaline almost equipotent to that of the more elaborate congener penitrem A (Sallam et al. 2013a; Sallam et al. 2013b). These notable activities of penitrem A are due, at least in part, to the inhibition of the Wnt/β-catenin pathway (Sallam et al. 2013a) that is a validated target of novel anticancer drugs (Lu et al. 2011). However, the BK channel (high-conductance Ca2+-activated potassium channel) inhibitory and tremorgenic activities of these IDTs present a formidable obstacle towards the development of penitrems as novel drugs (Sings and Singh 2003). Inhibition of the α-subunit of the BK channel interferes with neurotransmitter release mechanisms and neuroreceptors in the central and the peripheric nervous systems. To overcome these challenges, new penitrem analogues were synthesized. Some of these lack the BK channel inhibitory and tremorgenic activities while still repress β-catenin in human breast cancer cells (Sallam et al. 2013a). Another interesting approach is to mitigate the undesired effects of penitrems in a combination therapy with preventative agents such as astaxanthin or docosahexaenoic acid. Simultaneous application of penitrem A with these agents in rats significantly reduced toxicity and reversed the BK channel blockade associated with penitrem A alone (Goda et al. 2016).
While paspaline-derived IDTs are associated with tremorgenic activities, emindole SB-derived IDTs such as nodulisporic acids display highly potent insecticidal activities without observable adverse effects in mammals (Shoop et al. 2001). Thus, non-tremorgenic IDT analogues may find appli- cations as pesticides in agriculture or even as anti-parasitic agents against insects feeding on humans.
Conversely, the potent BK channel inhibitory activity of penitrems may also be utilized in the future. The standard inhib- itor for BK channels is iberiotoxin whose high price and mem- brane impermeability makes its use less than ideal for studies in organ models or whole animals. The higher membrane perme- ability, potency and efficacy of penitrem A may recommend this IDT as a good alternative to iberiotoxin for studying BK chan- nels in vitro and in vivo (Stewart et al. 2012; Asano et al. 2012; Kyle et al. 2013; Needham et al. 2014).
Invasive fungal infections are an important medical prob- lem, particularly in immunocompromised patients. However, treatment of invasive candidiasis is restricted to only a few drug families with a limited number of mechanisms of action (Tkacz and DiDomenico 2001). The toxicity of amphotericin B, and the emergence of resistance in the clinically relevant Candida albicans species against the azoles and the candins presents a clear demand for the development of new therapeu- tic strategies (Kathiravan et al. 2012). Importantly, shearinines D and E block the formation of biofilms by C. albicans (You et al. 2013). Biofilm formation makes the treatment of fungal infections very problematic, because biofilms constitute a bar- rier that can prevent antifungal drugs from reaching the fungal cells (Douglas 2003; Nett et al. 2010; Nett et al. 2011).
C. albicans treated with shearinine D forms an irregular, sparse layer instead of a well-developed biofilm. Co- application of shearinine D or E synergistically enhanced the potency of amphotericin B against clinical Candida isolates (You et al. 2013).
Systematic screening for natural compounds active against the influenza A virus subtype H1N1 led to the isolation and charac- terization of an array of IDT congeners from P. camemberti. Three novel and six known paspaline- and paxilline-derived IDT analogues showed significant antiviral activity (IC50 17.7–
73.3 μM) (Fan et al. 2013), raising hopes that novel antiviral agents may be developed from these IDTs in the future.
Industrial scale production of IDTs using filamentous fungi
The industrial-scale production of paspaline-derived IDTs for future medical and agricultural applications requires careful consideration of the potential producer strains. For example,
P. paxilli has been extensively used to clarify the individual steps of IDT production (Young et al. 2001). However, the pharmaceutical industry has yet to invest in the strain devel- opment and fermentation process optimization of this strain. Similarly, N. lolii may be difficult to adopt for the large-scale production of secondary metabolites due to its fastidious growth habits, genetic instability, and its requirement for a plant host for IDT production (Wiewióra et al. 2015).
Only a few studies investigated the production of IDTs in laboratory scale fermentations. Thus, P. nigricans produced 60 mg/L penitrem in a 60-L stirred fermentor in 5 days (Mantle et al. 1984). Engineered T. album accumulated 36 mg/L terpendole E, an intermediate of terpendole K bio- synthesis, in a 30 L fermentor in 2 days (Motoyama et al. 2012). Kalinina and coworkers demonstrated that the spec- trum of penitrems produced by P. crustosum can also be con- trolled by varying the chemical and physical conditions of the fermentation (Kalinina et al. 2017). Encouragingly, C. paspali is currently used for ergot alkaloid production in the pharma- ceutical industry. Although these fermentations have not been optimized for IDT production, the significant knowledge base available for the safe and economical industrial scale fermen- tation and the genetic manipulation of this fungus (Arcamone et al. 1960; Tudzynski et al. 2001; Kozák et al. 2018) may recommend C. paspali as a useful candidate to produce IDT congeners in the future.
Heterologous biosynthesis of IDTs
Domesticated microbial hosts for the heterologous expression of biosynthetic pathways are becoming increasingly important for the characterization of biosynthetic pathways and the clar- ification of reaction mechanisms. As a prominent example, the Oikawa group has been developing a heterologous production system for secondary metabolites. In this system, biosynthetic genes are cloned into multiple expression vectors and co- integrated into the genome of the domesticated filamentous fungus A. oryzae. In an influential set of publications, Oikawa and coworkers have used this expression system for the reconstitution of the biosynthesis of various IDTs to clarify the individual biosynthetic steps (Oikawa et al. 2016). Thus, the biosynthesis of paspaline was reconstituted in 2012 using
A. oryzae transformants producing the P. paxilli IDT biosyn- thetic enzymes PaxG, PaxC, PaxM and PaxB (Liu et al. 2015). Adding the paxP and paxQ genes yielded paxilline in
A. oryzae. In 2014, seven genes from the aflatrem biosynthetic locus of A. flavus were expressed in the A. oryzae host, leading to the heterologous production of aflatrem A and β-aflatrem in addition to paspaline and paspalinine (Tagami et al. 2014). In 2015, the same A. oryzae chassis was utilized to dissect the biosynthesis of penitrems by functionally analyzing 13 of the 17 genes encoded in the biosynthetic gene cluster of
P. simplicissimum (Liu et al. 2015). The same A. oryzae het- erologous expression system was also used to clarify the bio- synthesis of shearinines from P. janthinellum (Liu et al. 2016). Other hosts have also been used to express IDT biosynthet-
ic gene clusters. For example, a versatile multigene expression system termed MIDAS was developed to express biosynthetic gene clusters in a P. paxilli strain with the entire paxilline biosynthetic cluster deleted (van Dolleweerd et al. 2018). This system was used to express key biosynthetic genes from Hypoxylon pulicicidum to produce nodulisporic acid interme- diates and congeners (Van de Bittner et al. 2018). Although nodulisporic acid is not a paspaline-derived IDT and is thus not the subject of this review, the work still shows that strains derived from IDT producers can be useful hosts to produce other bioactive molecules. Meanwhile, nodulisporic acid de- rivatives are potent insecticides that lack observable adverse effects in mammals, including the tremorgenic activities asso- ciated with the paspaline-derived IDT core.
Combinatorial biosynthesis of novel, unnatural IDTs in synthetic biological platforms is another promising applica- tion. To design such a platform, the Tang group has used Saccharomyces cerevisiae as the chassis for the heterologous production of epoxy-geranylgeranyl indole (Tang et al. 2015). Co-expression of various IDT cyclases, some with previously unknown product specificities yielded not only paspaline (1.5 mg/L), but also various seco-IDTs and aflavinine- or anominine-type Markovnikov-derived cyclic scaffolds. This work demonstrates that diversity-oriented combinatorial bio- synthesis with enzymes coopted from distinct and even or- thogonal IDT biosynthetic pathways may be used to generate diverse, drug-like chemical matter towards the discovery of future medications. Presumably, such synthetic biological platforms will also be useful in the future for the large-scale and economical production of IDT congeners for medical or agricultural applications.
Funding information This work was supported by the European Union and the European Social Fund through the project EFOP-3.6.1-16-2016- 00022 (to I. P.), the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary (Biotechnology thematic program to I. P. and I. M.) and the U.S. National Institutes of Health (NIGMS 5R01GM114418 to I. M.).
Compliance with ethical standards
Conflict of interest I. P. declares no conflict of interests. I. M. has disclosed financial interests in Teva Pharmaceuticals Works Ltd., Hungary, and DSM Nutritional Products, LLC, USA, which are unrelated to the subject of the research presented here. L. K., Z. S., and L. T. are employees of Teva Pharmaceutical Works Ltd., Hungary. Responsibility for the conclusions drawn, and the opinions expressed in this article are solely those of the authors and are not shared by Teva Pharmaceutical Works Ltd.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the Authors.
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