- Email: [email protected]
Recent advances on the molecular aspects of ochratoxin A biosynthesis
Accepted Manuscript Title: Recent advances on the molecular aspects of ochratoxin A biosynthesis Author: Antonia Gallo Massimo Ferrara Giancarlo Perrone PII: DOI: Reference:
S2214-7993(17)30088-7 https://doi.org/doi:10.1016/j.cofs.2017.09.011 COFS 275
To appear in: Received date: Revised date: Accepted date:
19-6-2017 21-9-2017 25-9-2017
Please cite this article as: Gallo, A., Ferrara, M., Perrone, G.,Recent advances on the molecular aspects of ochratoxin A biosynthesis, COFS (2017), https://doi.org/10.1016/j.cofs.2017.09.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights • An update on OTA producing species is presented • Three key genes are involved in OTA production pks, nrps and hal.
Ac ce pt e
d
M
an
us
cr
• Regulatory elements act at different level on OTA biosynthesis
ip t
• Possible involvement of two different pks genes in OTA production is discussed
1 Page 1 of 16
Recent advances on the molecular aspects of ochratoxin A biosynthesis Antonia Galloa, Massimo Ferrarab and Giancarlo Perroneb a Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Prov.le Lecce-Monteroni, Lecce 73100, Italy b Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Amendola 122/O, Bari 70126, Italy
Corresponding author: Antonia Gallo ([email protected])
cr
ip t
E-mail Authors: Antonia Gallo: [email protected] Massimo Ferrara: [email protected] Giancarlo Perrone: [email protected]
us
Recently, studies on the molecular aspects of ochratoxin A biosynthesis have significantly advanced. Differently from other mycotoxins, the genes and the enzymatic stages involved in biosynthesis pathway of
an
ochratoxin A have remained long unknown. New ecological data have led to the definition of new producing species, responsible of ochratoxin A contamination in several food and feed. In parallel, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key
M
steps of the mycotoxin biosynthesis. Further studies are still needed to completely clarify the regulatory mechanisms underlying the activation of the production of ochratoxin A. Previous findings on fungal secondary metabolism biosynthesis and the availability of new data from different omics approaches will
Ac ce pt e
Introduction
d
permit to fill the gap of knowledge in the near future.
Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, beer and wine, and meats); it is also carcinogenic, neurotoxic teratogenic and immunotoxic [1, 2]. This mycotoxin is essentially produced by species of genus Aspergillus and Penicillium.
In particular, P. verrucosum and P. nordicum are the two most important Penicillium OTA producing species in temperate regions, with the former predominantly contaminating grains while the latter has been mainly found in cheese and fermented meats. The genus Aspergillus counts more than twenty ochratoxigenic species diffusely spread worldwide, mainly in warmer region than Penicillia like Mediterranean, tropical and subtropical area. Various ecophysiological studies have been made to identify conditions (incubation temperature, water activity, pH, different substrates) favoring growth, sporulation and toxin production by potential ochratoxigenic species. The Aspergillus OTA producers belong to Sections Circumdati and Nigri, with only two minor OTA producing species in Sec. Flavi. In Sec. Circumdati, eleven species produce large amounts of OTA with the most important being A. ochraceus, A. westerdijkiae and A. steynii, mainly responsible for OTA contamination of coffee, rice, cocoa, beverages and other foodstuffs [3]. In Sec. Nigri, the most important 2 Page 2 of 16
OTA producing species is A. carbonarius, mainly responsible for the contamination of wine, grape derived products and raisins; followed by A. niger and its cryptic sister species A. welwitschiae [4]. Among the two OTA producing species of the Flavi group, only A. alliaceus has been rarely isolated in figs and tree nuts in California [5]. OTA is a hybrid molecule composed of a polyketide dihydroisocoumarin mojety linked via amide bond to the amino acid phenylalanine. From the pentaketide structure, it has been supposed that a PKS enzyme is
ip t
required for the formation of the isocoumarin pentaketide group from acetate and malonate. Then, a NRPS is essential for the link between the dihydroisocoumarin and the phenylalanine synthesized via the shikimic acid pathway. At the end, the halogenation step is required to add the chlorine atom to the molecule. Likely,
cr
other enzymes are involved such as oxidase, esterase, transporters and transcription factors.
Differently from other mycotoxins, the genes involved in biosynthesis of OTA have been long unknown.
us
Hence, also the mechanism of the biosynthetic pathway has remained not completely clarified until recent findings. The studies based on labeled precursors feeding experiments had suggested a reliable scheme of the
an
biosynthesis of OTA [6,7,8], however doubts on the sequence of some enzymatic steps had remained unresolved. The successive studies on the molecular aspects of the pathway allowed to establish the involved genes and their role in the biosynthesis. The fact that biosynthetic genes of secondary metabolism in fungal
M
genome are usually clusterized, has considerably facilitate the study and the identification of most of the genes responsible of OTA biosynthesis reactions. In addition, in the last decade, the considerable increase of
clusters in several species.
d
the genome sequencing projects regarding fungal microorganisms has led to identify the ochratoxin A
Ac ce pt e
In this review we briefly summarize the recent history and the latest advances on OTA biosynthesis at molecular level, and on the regulatory aspects, which have not yet properly investigated but on which great interest has been focused lately.
OTA producing fungi: an update
The genus Aspergillus includes the major number of OTA producing species (27, in Table 1); in Penicillium three species are confirmed as OTA producer, namely: P. nordicum, P. verrucosum and the recently described P. thymicola [3, 9,10].
Penicillium thymicola, evolutionary related to P. nordicum and P. verrucosum, has been isolated from dried thyme, soil and sorghum grain, while the specific strain studied for OTA production has been isolated from cheddar cheese. Recently, the revision in Circumdati section evidenced that A. westerdijkiae and A. steynii are considered more important for OTA contamination of food products with respect to the closely related species A. ochraceus [11]. In 2017, the production of OTA was claimed from new species of Penicillium (P. commune and P. rubens), Aspergillus (A. aculeatus) and Talaromyces (T. rugulosus) [12]; but these data were commented and needed further confirmation that has not yet been published [13, 14]. Updating the knowledge on potential ochratoxigenic species is of great importance in the vision of potential new scenarios 3 Page 3 of 16
due to climate change. In this context, preliminary studies showed emerging risk related to the spread of Aspergillus species also in cold temperate zone, evidencing the possible linkage of carbon dioxide increase in stimulating/reducing OTA biosynthesis in Aspergilli [15].
OTA biosynthesis genes
ip t
Recently, the otapksPV gene has been identified and characterized as the gene encoding the PKS required for OTA biosynthesis in P. verrucosum BT 22713, through gene disruption approach [16]. In previous work of Geisen and coauthors [17], it was shown that the OTA biosynthetic pathways in P. nordicum BFE487 and P.
cr
verrucosum BFE808 were highly homologous for the putative genes otanpsPN, otatraPN and otachlPN but not for the pks gene. The phylogenetic analysis positioned otapksPV and otapksPN in two different clades;
us
the two proteins show similar domains with the additional methyl transferase domain (MT) in the former, but disposed in a different order. The genome walking strategy led to the identification of two other ORFs next
an
to the pks gene in P. verrucosum: otaT and otaE, encoding a transporter and an oxidoreductase, respectively. They appeared to be coordinately expressed with the otapksPV gene, suggesting a possible role in OTA biosynthesis. The homology analysis of the protein sequences of these genes revealed the best match score
M
with proteins involved in citrinin production in Monoascus anka, which displays the same sequential order of OTA cluster in P. verrucosum, able to produce citrinin as well. In 2015, the genome sequencing and assembly of P. verrucosum BFE808 has been released (Stoll et al.,
d
conference abstract). From a preliminary analysis, a truncated gene cluster for OTA biosynthesis and a
Ac ce pt e
complete gene cluster for citrinin biosynthesis were identified. The PKS of the citrinin cluster seemed to be identical to the otapksPV described by O'Callaghan et al. [16]. Future analyses could give a deeper insight in the OTA biosynthesis cluster to better explain the homology and differences between P. nordicum and P. verrucosum.
The genome of Penicillium thymicola strain was sequenced and analyzed for the presence of OTA biosynthesis cluster [10]. No proteins homologous to known OTA genes from P. nordicum and P. verrucosum have been identified. However, a gene cluster was identified containing a pks and a hybrid pksnrps genes with high degree of similarity to OTA genes of A. carbonarius and A. ochraceus. The analysis revealed also the presence of a regulatory and a transporter gene, but not a halogenase or chloroperoxidase encoding gene. Also due to differences in the structure of multidomain enzymes involved in the biosynthesis of OTA, authors suggested that OTA genes could not be clustered in a single locus in P. thymicola. Aspergillus ochraceus and A. westerdijkiae, belonging to section Circumdati, initially were assigned to the same A. ochraceus taxon and only recently have been separated. In the study of Bacha et al., it was reported that two PKSs could be involved in the biosynthesis of OTA in A. westerdijkiae [18]. The gene aolc35-12, orthologous of the pks gene found to be responsible of OTA biosynthesis in A. ochraceus [19], appeared to differentially control the expression of aoks1, the OTA PKS previously identified in A. westerdijikiae [20]. The two PKS showed an homology of about 34%. 4 Page 4 of 16
The whole genome sequence of A. westerdijkiae CBS 112803 was released in 2016 [21]. The functional annotation of the genome has led to the identification of two putative OTA-related gene clusters [22]. One of these (cluster 37) includes 17 genes among which the pks gene corresponding to the above mentioned aolc35-12. Other genes of the cluster 37 encode a NRPS, a cytochrome P450 monoxygenase, a bZIP transcription regulator and a halogenase, and in addition a Zn2Cys6 transcription regulator and a sugar transporter. The second identified putative cluster (cluster 69) comprises the pks gene identical to the aoks1
ip t
previously characterized, and also a cytochrome P450 monoxygenase, two transporters and an Acyl-CoA synthetase, which could be related to the putative action of Acetyl-CoA as precursor of OTA synthesis. Furthermore in this latter cluster, two genes encoding enzymes involved in sucrose and glucose metabolism
cr
have been annotated. The authors suggested that they might have important influences on OTA production in various substrates, in agreement to the high ability of A. westerdijkiae in producing OTA in media containing
us
high quantity of sucrose and glucose.
Likewise, also in A. ochraceus the annotation and analysis of the sequenced genome led to the identification
an
of two pks genes, AoOTApks-1 and AoOTApks-2 [23]. The obtained deletion mutants for the respective genes produced no and lesser OTA, respectively, demonstrating their involvement in OTA biosynthesis. The two predicted proteins show 26 % identity at amino acid level between them, but no indication was reported
M
about their homology to the two OTA pks genes of A. westerdijkiae, likely because only partial sequences of A. westerdijkiae PKSs were available at the time. Anyway, from the two different works, it can be deduced that AoOTApks-1 from A. ochraceus and aolc35-12 from A. westerdijkiae are both highly reducing PKS
d
(HR-PKS), due to the presence of reducing domains, while AoOTApks-2 of A. ochraceus and aoks1 of A.
structure.
Ac ce pt e
westerdijkiae are both partially reducing PKS (PR-PKS) for the absence of the ER domain in their modular
As for A. westerdijkiae, also in A. ochraceus the expression profile during OTA production suggested that AoOTApks-1 gene was directly involved in OTA biosynthesis while AoOTApks-2 might complement the expression of the former gene and be involved in the biosynthesis indirectly. Other fungal secondary metabolites are reported to require two different PKSs for the biosynthesis of a single polyketide such as lovastatin, compactin and the mycotoxins zearaleone and T-toxin. Same evidence of the involvement of two PKS for OTA biosynthesis could derive also from the study that our group carried out on A. carbonarius. Initially, we identified the ACpks gene that showed an expression profile correlated to the production of OTA; however, this gene was not inactivated for the confirmation of its direct role in the biosynthesis [24]. Then, the whole genome sequencing of A. carbonarius ITEM 5010 permitted the identification of an OTA biosynthesis cluster based on comparative analysis in silico. Hence, firstly we demonstrated through gene inactivation that AcOTAnrps gene was responsible of the linking of phenylalanine to the polyketide dihydroisocoumarin [25]. Furthermore, chemical analysis on the presence of the principal secondary metabolites involved in OTA biosynthesis, such as OTB, OTC, OTβ and OTα, clarified the order of reaction of OTA biosynthesis pathway, confirming the hypothesis that the enzymatic step adding phenylalanine to OTβ, the dihydroisocoumarin based structure, precedes the chlorination 5 Page 5 of 16
reaction to form OTA, and that OTα may be a product of hydrolysis of OTA in A. carbonarius. Carrying on with the analysis of the genomic region identified as putative OTA cluster, the adjacent gene coding for a PKS, different from the previously identified ACpks, was inactivated and its role in the biosynthetic pathway was confirmed [26]. The designated AcOTApks gene encodes for a HR-PKS containing a putative methyl transferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. Finally, we demonstrated that a flavin-halogenase is implicated in the biosynthesis of OTA in A.
ip t
carbonarius. The encoding gene AcOTAhal is contiguous to the above described biosynthetic genes, resulting as part of the cluster [27]. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway, as confirmed by the accumulation of
cr
OTB, the metabolite formed by the action of the AcOTAnrps enzyme, simultaneously to the loss of OTA production, when AcOTAhal was inactivated. In Fig.1 a schematic representation of the order of reactions in
us
OTA biosynthesis pathway is depicted. In addition, during the same study, the expression profile of two other genes located in the assumed biosynthetic cluster correlated to the OTA production kinetics similarly to
an
OTA genes. These two genes encode a cytochrome P450 oxidase (AcOTAp450) and a bZIP transcription factor (AcOTAbZIP), putatively involved in OTA biosynthesis. Other genes were annotated in the OTA cluster in A. carbonarius that need further investigation to establish their role in OTA biosynthetic pathway,
M
such as a predicted transporter protein and another fungal specific transcription factor. Aspergillus niger is the OTA producing fungus phylogenetically most related to A. carbonarius. Genome sequencing of A .niger CBS 513.88 revealed the presence of a putative OTA cluster [28] on the basis of
d
sequence homology to A. ochraceus. From in silico analyses, the genomic regions corresponding to OTA
Ac ce pt e
clusters in A. carbonarius and A. niger harbor the putative and already characterized OTA biosynthetic genes organized in the same order and direction of transcription [29]. Only recently, the A. niger An15g07920 pks gene of the putative OTA cluster was demonstrated experimentally to be a crucial player in the biosynthesis of OTA [30].
Lately, numerous studies have been carried out on molecular aspects of OTA biosynthesis, but still the length and composition of OTA cluster remain not completely defined. The key enzymes- such as PKS, NRPS, halogenase-and their role have been identified in several OTA producing species. Other genes, included in the interested genomic region or external to it, have to be investigated to fully elucidate the mechanism of the biosynthetic pathway and to better clarify the molecular regulation at the basis of OTA production. In fungal species only recently recognized as OTA producing, comparative and homology analyses led to the identification of OTA genes as in A. affinis with regards to a pks gene putatively involved in the first step of OTA biosynthesis [31]. In A. steynii a genomic fragment with three contiguous genes has been identified encoding a cytochrome P450 monoxygenase (p450ste), a NRPS (nrpsste) and a PKS (pksste), whose expression patterns were coordinated and appeared to be related to OTA production [32]. In Fig. 2, the OTA clusters of different producing species are represented. Finally, from the results obtained until now, some divergences in the structure of putative clusters and in domain composition of key enzymes (PKS and NRPS) have been observed in fungi belonging to different 6 Page 6 of 16
sections and genera. In a short time, a huge amount of new genomic data will be available and will enable to explain the phylogenetic relationship and the evolutionary events occurred among OTA producing species.
Regulation of OTA biosynthesis Numerous studies have reported the influence of biotic and abiotic stressors on OTA biosynthesis in different fungal species. The regulation of secondary metabolism is very complex and acts at different regulatory
ip t
levels. It is a mechanism that may utilize regulator elements specific of the biosynthesis pathway by controlling expression of the corresponding biosynthesis genes. At a superior level, global regulators and multiprotein complexes transmit environmental cues to biosynthesis mechanism; these regulatory processes
cr
are often interconnected and overlapping. Other regulation mechanisms based on signaling pathways or epigenetic control could be responsible of the activation of biosynthetic genes. Studies on the regulation of
us
OTA biosynthesis has intensified in the last few years and more significant results are expected from future investigations. At present, most of the regulatory aspects underlying the OTA production remain unclear. It
an
is likely that some of them are based on functioning models already identified for the production of other mycotoxins.
Usually, secondary metabolite biosynthesis cluster contain cluster specific transcription factors. In the OTA
M
cluster of A. carbonarius, between AcOTAp450 and AcOTAhal genes, the AcOTAbZIP gene, encoding a basic leucine zipper transcription factor, was identified. Its expression profile showed correlation with the expression profile of other OTA genes and with the kinetics of OTA accumulation [27]. Many fungal bZIPs,
d
have been characterized as stress response transcription factors, responding to a variety of environmental
Ac ce pt e
stresses and linking them to secondary metabolite production [33]. Among these, a bZIP transcription factor, Aoyap1, was found in A. ochraceus to mediate signal perception in the regulation of OTA synthesis under oxidative stress [34].
Towards the edges of the OTA genomic region in A. carbonarius, other two genes have been annotated encoding transcription factors containing a Cys-rich motif (http://jgi.doe.gov/carbonarius/). This structure characterizes the zinc-cluster proteins (Zn2Cys6 ), which are the most common as cluster regulators of fungal secondary metabolites, among which is the AflR regulator of aflatoxin/sterigmatocystin production. More definitive conclusions about their role in the regulation of OTA biosynthesis can be provided by knocking out experiments.
Among the global regulators, the heterotrimeric velvet complex has been the most studied in OTA producing fungi to clarify the link of the light-dependent fungal morphology and sexual development to secondary metabolism. This complex consists of proteins LaeA, VeA and VelB and is fully functional in the nucleus under dark conditions [33]. The deletion of laeA and veA in A. carbonarius led to a strong reduction in conidial production and a drastic decrease of OTA production, which was correlated to a down regulation of the AcOTAnrps gene [35]. These results confirmed the role of the two proteins in regulating conidiation and OTA biosynthesis in response to light likely to other fungi. Recently, laeA has been reported to have also a regulatory effect on production of citric acid and cellulolytic enzymes in A. carbonarius [36]. 7 Page 7 of 16
In the study of El Khoury et al. [37], essential oils from different plant origin were observed to induce reduction of OTA production in the same fungus. Some of them such as fennel, chamomile and rosemary essential oils were able to downregulate laeA and veA genes and consequently the expression of OTA biosynthesis genes was reduced; others, such as celery and anise essential oils, did not have significant effect on laeA and veA expression and the observed downregulation of OTA genes was suggested to be due to different mode of regulation.
ip t
Besides the velvet protein complex, other global regulators have been demonstrated to be involved in regulation of secondary metabolism genes in fungi [38]. Among them, the most studied have been PacC, the key factor for pH regulation; CBC, which regulates the response to redox status and iron stresses; CreA,
cr
involved in the regulation of carbon metabolism; AreA and AreB , involved in nitrogen metabolism.
Many studies have been reported that OTA production is responsive to general environmental factors, such
us
as carbon and nitrogen factors, temperature, light and pH; despite of this, very little of the role of the global regulators in converting the environmental cues in activation or inhibition of OTA biosynthesis genes has
an
been described. During a preliminary study we carried out on the promoter regions of OTA biosynthesis genes in A. carbonarius, a number of conserved motifs known to act as binding sites of transcription factors and specific of the most common global regulators, have been identified (unpublished).
M
External stimuli are also transmitted to the genetic level by signal transduction pathways that translate the external signal to transcriptional level by activating positive or negative transcription factors, which then regulate downstream genes. The production of OTA was suggested to contribute to the adaptation of P.
d
nordicum and P. verrucosum to NaCl rich environment by ensuring chloride homeostasis in the cell, through
Ac ce pt e
the excretion of OTA that carries a chlorine in its molecule [39]. The correlation between this environment stress and OTA biosynthesis was demonstrated to be mediated by the HOG MAP kinase signal cascade pathway [40], through the phosphorylation of the kinase HOG, the last protein of the cascade, that subsequently activates the downstream transcription factors. In the same study, a different behavior was observed for A. carbonarius, in which the increased levels of NaCl lead to the increased phosphorylation status of HOG but not to an increase in OTA biosynthesis, inferring that A. carbonarius lacks the mechanism to cope with NaCl stress, which reduces also the ability to grow of A. carbonarius. Interestingly, it was found that the biosynthesis of citrinin and OTA is mutually regulated in P. verrucosum, with increasing concentration of NaCl shifting production from citrinin to OTA and the oxidative stress increasing citrinin biosynthesis at the expense of the other mycotoxin, by means of a different signaling pathway [41]. Further explanation are expected by proteomics and transcriptomics studies performed in producing species grown under different permissive and not permissive conditions for OTA production. Crespo-Sempere et al. studied two strains of A. carbonarius, very dissimilar for their OTA production ability, by suppression subtractive -hybridization technique, identifying several differentially ESTs, 26% of which were potentially involved in OTA regulation processes [42]. Some of them were related to the Target of Rapamycin (TOR) that concerns a number of cellular signalling pathways in response to nutrients, hormones, and stresses; and to the MAP kinase-dependent signal transduction. Other proteomics and transcriptomic analyses have been 8 Page 8 of 16
carried out under different growth condition by using weak and strong producing species and even not producing species, leading to the accumulation of data about genes and proteins differentially expressed that could provides a deeper understanding of the regulatory mechanism in the future studies [43-45].
Conclusions
ip t
The contamination of OTA in different food and feed is an important issue for the health and safety hazards that it implies. Climate, agronomic and global market changes could cause the emergency of novel risks. Therefore, the knowledge about new producing species and matrices that could be contaminated is essential
cr
to face in an appropriate way the emerging exposure. To this aim, the genomic data of an increasing number of fungal species is a powerful means that allow to identify a biosynthesis cluster in silico and to determine
us
the possibility of producing by a fungus. In the same way, the understanding of the genetic pathway involved in the biosynthesis and the molecular regulators that activate or inhibit the biosynthetic genes, could provide
an
instruments for the development of prevention strategies, diagnostic methods and remediation measures.
M
References and recommended readings
Ac ce pt e
d
1. Barlow S, Bolger M, Pitt JI, Verger P: Safety Evaluation of Certain Contaminants in Food In Ochratoxin A (addendum) WHO Food Additives Series 59, World Health Organization, Geneva; 2008: 357–429. 2. IARC (International Agency for Research on Cancer): Ochratoxin A IARC monographs on the evaluation of carcinogenic risks to humans. IARC Press. 1993, 56: 489–521. 3. Visagie CM, Varga J, Houbraken J, Meijer M, Kocsubé S, Yilmaz N, Fotedar R, Seifert KA, Frisvad JC, Samson RA : Ochratoxin production and taxonomy of the yellow aspergilli (Aspergillus section Circumdati). Stud Mycol 2014, 78: 1-61. 4. Perrone G, Susca A, Cozzi G, Ehrlich K, Varga J, Frisvad JC, Meijer M, Noonim P, Mahakarnchanakul W, Samson RA: Biodiversity of Aspergillus species in some important agricultural products. Stud Mycol 2007, 59: 53-66. 5. Bayman P, Baker JL, Doster MA, Michailides TJ, Mahoney NE: Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus. Appl Environ Microbiol 2002, 68: 2326-2329. 6. Huff WE, Hamilton PB: Mycotoxins—their biosynthesis in fungi: ochratoxins—metabolites of combined pathways. J Food Prot 1979, 42:815–820. 7. Ruhland M, Engelhardt G, Wallnöter PR: Production of 14C-ochratoxin a by Penicillium verrucosum sp. 1761 in liquid culture. Mycotox Res 1996, 12: 7–13. 8. Harris JP, Mantle PG: Biosynthesis of ochratoxins by Aspergillus ochraceus. Phytochemistry 2001, 558:709 –716. 9 Page 9 of 16
9. Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, Samson R.A: New and revisited species in Aspergillus section Nigri. Stud Mycol 2011, 69: 1-17. 10. Nguyen HD, McMullin DR, Ponomareva E, Riley R, Pomraning KR, Baker SE, Seifert KA: (). Ochratoxin A production by Penicillium thymicola. Fungal Biol 2016, 120(8): 1041-1049. * In this paper the production of ochratoxin A from a new species of Penicillium is reported together with the genome analysis. Interestingly, this OTA cluster is more related to Aspergillus species than to its sister species P. nordicum and P. verrucosum.
cr
ip t
11. Gil-Serna J, Vázquez C, Sardiñas N, González-Jaén MT, Patiño B: Revision of ochratoxin A production capacity by the main species of Aspergillus section Circumdati. Aspergillus steynii revealed as the main risk of OTA contamination. Food Control 2011, 22: 343–345.
us
12. Zhang X, Li Y, Wang H, Gu X, Zheng X, Wang Y, Diao J, Peng Y, Zhang H: Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes. Toxins 2016, 8(11): 333.
an
13. Perrone G., Logrieco A F, Frisvad JC: Comments on “Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes". Toxins 2016, 8: 333—In Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces. Toxins 2017, 9(2): 65.
M
14. Zhang X, Li, Y., Wang, H, Gu X, Zheng X, Wang Y, Diao J, Peng Y, Zhang H: Reply to Comment on “Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes”. Toxins 2016, 8, 333”–in Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces. Toxins 2017, 9(2): 66.
Ac ce pt e
d
15. Akbar A, Medina A, Magan N: Impact of interacting climate change factors on growth and ochratoxin A production by Aspergillus section Circumdati and Nigri species on coffee. World Mycotoxin J 2016, 9(5): 863-874. 16. O'Callaghan J, Coghlan A, Abbas A, Garcia-Estrada C,Martin JF, Dobson ADW: Functional characterization of the polyketide synthase gene required for ochratoxin A biosynthesis in Penicillium verrucosum. Int J Food Microbiol 2013, 161: 172–181. 17. Geisen R, Schmidt-Heydt M, Karolewiez A: A gene cluster of the ochratoxin A biosynthetic genes in Penicillium. Mycotox Res 2006, 22: 134–141. 18. Bacha N, Mathieu F, Liboz T, Lebrihi A: Polyketide synthase gene aolc35-12 controls the differential expression of ochratoxin A gene aoks1 in Aspergillus westerdijkiae. World Mycotoxin J 2012, 5: 177186. 19. O’Callaghan J, Caddick MX, Dobson ADW: A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus. Microbiology 2003, 149: 3485–3491. 20. Bacha N, Atoui A, Mathieu F, Liboz T, Lebrihi A: Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A. Fungal Genet Biol 2009, 46:77– 84. 21. Chakrabortti A, Li J, Lianga Z-X: Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A. Genome Announc 2016, 4(5): pii: e00982-16.
10 Page 10 of 16
22. Han X, Chakrabortti A, Zhu J, Liang Z-X, Li J: Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae. BMC Genomics 2016, 17: 633. *This paper reports the identification in the sequence genome of A. westerdijkiae of two clusters putatively involved in OTA biosynthesis, including a HR-PKS and a PR-PKS encoding genes, respectively
ip t
23. Wang L, Wang Y, Wang Q, Liu F, Selvaraj JN, Liu L, Xing F, Zhao Y, Zhou L, Liu Y: Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1. Toxins 2015, 7(8): 2723-2738.
cr
*This paper provides experimental evidence of the involvement of two different psk genes in the production of OTA
us
24. Gallo A, Perrone G, Solfrizzo M, Epifani F, Abbas A, Dobson AD, Mulè G: Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus carbonarius. Int J Food Microbiol 2009, 129: 8–15
an
25. Gallo A, Bruno KS, Solfrizzo M, Perrone G,Mulè G, Visconti A, Baker SE: New insight into the ochratoxin A biosynthetic pathway through deletion of a nonribosomal peptide synthetase gene in Aspergillus carbonarius. Appl Environ Microbiol 2012, 78: 8208–8218.
M
26. Gallo A, Knox BP, Bruno KB, Solfrizzo M, Baker SE, Perrone G: Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius. Int J Food Microbiol 2014, 179: 10–11.
Ac ce pt e
d
27. Ferrara M, Perrone G, Gambacorta L, Epifani F, Solfrizzo M, Gallo A: Identification of a halogenase involved in the biosynthesis of ochratoxin A in Aspergillus carbonarius. Appl Environ Microbiol 2016, 82 (18): 5631-5641. * This work describes for the first time the identification and characterization of an halogenase gene responsible of the chlorination of OTA molecule in the biosynthesis pathway. In addition, the correlation between a transcription factor in OTA cluster and the mycotoxin accumulation was evidenced. 28. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K et al: Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 2007, 25:221–231. 29. Ferracin LM, Fier CB, Vieira ML, Monteiro-Vitorello CB, Varani A, Rossi MM, Müller-Santos M, Taniwaki MH, Iamanaka BT, Fungaro MH: Strain-specific polyketide synthase genes of Aspergillus niger. Int J Food Microbiol 2012, 155:137–145. 30. Zhang J, Zhu L, Chen H, Li M, Zhu X, Gao Q, Wang D, Zhang Y: A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger. J Agric Food Chem 2016, 64(51): 9680-9688. 31. Davolos D, Pietrangeli B: A molecular and bioinformatic study on the ochratoxin A (OTA)producing Aspergillus affinis (section Circumdati). Mycotoxin Res 2014, 30(2): 113-122.
11 Page 11 of 16
32. Gil-Serna J, Vázquez C, González-Jaén MT, Patiño B: Clustered array of ochratoxin A biosynthetic genes in Aspergillus steynii and their expression patterns in permissive conditions. Int J Food Microbiol 2015, 214:102-108. 33. Knox BP, Keller NP: Key Players in the Regulation of Fungal Secondary Metabolism. In Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites, Volume 2. Edited by Zeilinger S, Martín J-F, García-Estrada C. Springer; 2015: 13-28
ip t
34. Reverberi M, Gazzetti K, Punelli F, Scarpari M, Zjalic S, Ricelli A, Fabbri AA, Fanelli C: Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus. Appl Microbiol Biotechnol 2012, 95(5):1293-1304.
cr
35. Crespo-Sempere A, Marin S, Sanchis V, Ramos AJ: VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius. Int J Food Microbiol 2013, 166: 479–486.
us
36. Linde T, Zoglowek M, Lübeck M, Frisvad JC, Lübeck PS: The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius. J Ind Microbiol Biotechnol 2016, 43(8):1139-1147.
M
an
37. El Khoury R, Atoui A, Verheecke C, Maroun R, El Khoury A, Mathieu F: Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius. Toxins 2016, 8(8): pii: E242. 38. Macheleidt J, Mattern DJ, Fischer J, Netzker T, Weber J, Schroeckh V, Valiante V, Brakhage AA: Regulation and Role of Fungal Secondary Metabolites. Annu Rev Genet 2016, 50:371-392.
Ac ce pt e
d
39. Schmidt-Heydt M, Graf E, Stoll D, Geisen R: The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods. Food Microbiol 2012, 29: 233–241. 40. Stoll D, Schmidt-Heydt M, Geisen R: Differences in the regulation of ochratoxin A by the HOG pathway in Penicillium and Aspergillus in response to high osmolar environments. Toxins 2013, 5: 1282–1298. 41. Schmidt-Heydt M, Stoll D, Schütz P, Geisen R: Oxidative stress induces the biosynthesis of citrinin by Penicillium verrucosum at the expense of ochratoxin. Int J Food Microbiol 2015, 192: 1–6. * In this paper authors reported that in P. verrucosum production of ochratoxin A and citrinin is shifted towards one of them at the expense of the other depending on environmental conditions by means of a signaling pathway 42. Crespo-Sempere A, González-Candelas L, Martínez-Culebras PV: Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions. Int J Food Microbiol 2010, 142(1-2): 170-179. 43. Crespo-Sempere A, Gil JV, Martínez-Culebras PV: Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions. Int J Food Microbiol 2011 , 147(3): 162-169. 44. Sartori D, Massi FP, Ferranti LS, Fungaro MHP: Identification of Genes Differentially Expressed Between Ochratoxin-Producing and Non-Producing Strains of Aspergillus westerdijkiae. Indian J Microbiol 2014, 54(1): 41–45. 45. Gerin D, De Miccolis Angelini RM, Pollastro S, Faretra F: RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius. PLoS One 2016, 11(1): e0147089 12 Page 12 of 16
ip t
us
M
an
Sec. Circumdati (Weak OTA producers) A. melleus A. ostianus A. persii A. salwaensis A. sclerotiorum A. sesamicola A. subramanianii A. westlandensis
Ac ce pt e
Sec. FLAVI A. albertensis A. alliaceus
Sec. NIGRI A. carbonarius A. lacticoffeatus A. niger A. sclerotioniger A. welwitschiae
d
Sec. CIRCUMDATI A. affinis A. cretensis A. flocculosus A. fresenii A. muricatus A. occultus A. ochraceus A. pseudoelegans A. pulvericola A. roseoglobulosus A. steynii A. westerdijkiae
cr
Table 1 Aspergillus species producing ochratoxin A (OTA)
13 Page 13 of 16
Fig.1 Schematic representation of the pathway of OTA biosynthesis in which key enzymatic steps are shown
Fig.2 Organization of OTA clusters in different Aspergillus and Penicillium species. Highlighted in bold are
ip t
the genes that have been knocked out. In parentheses are the references from which cluster structures
Ac ce pt e
d
M
an
us
cr
were taken. (* Stool et al., 2015. conference abstract)
14 Page 14 of 16
an M ed Ac ce pt Page 15 of 16
e pt ce Ac
Page 16 of 16
S2214-7993(17)30088-7 https://doi.org/doi:10.1016/j.cofs.2017.09.011 COFS 275
To appear in: Received date: Revised date: Accepted date:
19-6-2017 21-9-2017 25-9-2017
Please cite this article as: Gallo, A., Ferrara, M., Perrone, G.,Recent advances on the molecular aspects of ochratoxin A biosynthesis, COFS (2017), https://doi.org/10.1016/j.cofs.2017.09.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights • An update on OTA producing species is presented • Three key genes are involved in OTA production pks, nrps and hal.
Ac ce pt e
d
M
an
us
cr
• Regulatory elements act at different level on OTA biosynthesis
ip t
• Possible involvement of two different pks genes in OTA production is discussed
1 Page 1 of 16
Recent advances on the molecular aspects of ochratoxin A biosynthesis Antonia Galloa, Massimo Ferrarab and Giancarlo Perroneb a Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Prov.le Lecce-Monteroni, Lecce 73100, Italy b Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via Amendola 122/O, Bari 70126, Italy
Corresponding author: Antonia Gallo ([email protected])
cr
ip t
E-mail Authors: Antonia Gallo: [email protected] Massimo Ferrara: [email protected] Giancarlo Perrone: [email protected]
us
Recently, studies on the molecular aspects of ochratoxin A biosynthesis have significantly advanced. Differently from other mycotoxins, the genes and the enzymatic stages involved in biosynthesis pathway of
an
ochratoxin A have remained long unknown. New ecological data have led to the definition of new producing species, responsible of ochratoxin A contamination in several food and feed. In parallel, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key
M
steps of the mycotoxin biosynthesis. Further studies are still needed to completely clarify the regulatory mechanisms underlying the activation of the production of ochratoxin A. Previous findings on fungal secondary metabolism biosynthesis and the availability of new data from different omics approaches will
Ac ce pt e
Introduction
d
permit to fill the gap of knowledge in the near future.
Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, beer and wine, and meats); it is also carcinogenic, neurotoxic teratogenic and immunotoxic [1, 2]. This mycotoxin is essentially produced by species of genus Aspergillus and Penicillium.
In particular, P. verrucosum and P. nordicum are the two most important Penicillium OTA producing species in temperate regions, with the former predominantly contaminating grains while the latter has been mainly found in cheese and fermented meats. The genus Aspergillus counts more than twenty ochratoxigenic species diffusely spread worldwide, mainly in warmer region than Penicillia like Mediterranean, tropical and subtropical area. Various ecophysiological studies have been made to identify conditions (incubation temperature, water activity, pH, different substrates) favoring growth, sporulation and toxin production by potential ochratoxigenic species. The Aspergillus OTA producers belong to Sections Circumdati and Nigri, with only two minor OTA producing species in Sec. Flavi. In Sec. Circumdati, eleven species produce large amounts of OTA with the most important being A. ochraceus, A. westerdijkiae and A. steynii, mainly responsible for OTA contamination of coffee, rice, cocoa, beverages and other foodstuffs [3]. In Sec. Nigri, the most important 2 Page 2 of 16
OTA producing species is A. carbonarius, mainly responsible for the contamination of wine, grape derived products and raisins; followed by A. niger and its cryptic sister species A. welwitschiae [4]. Among the two OTA producing species of the Flavi group, only A. alliaceus has been rarely isolated in figs and tree nuts in California [5]. OTA is a hybrid molecule composed of a polyketide dihydroisocoumarin mojety linked via amide bond to the amino acid phenylalanine. From the pentaketide structure, it has been supposed that a PKS enzyme is
ip t
required for the formation of the isocoumarin pentaketide group from acetate and malonate. Then, a NRPS is essential for the link between the dihydroisocoumarin and the phenylalanine synthesized via the shikimic acid pathway. At the end, the halogenation step is required to add the chlorine atom to the molecule. Likely,
cr
other enzymes are involved such as oxidase, esterase, transporters and transcription factors.
Differently from other mycotoxins, the genes involved in biosynthesis of OTA have been long unknown.
us
Hence, also the mechanism of the biosynthetic pathway has remained not completely clarified until recent findings. The studies based on labeled precursors feeding experiments had suggested a reliable scheme of the
an
biosynthesis of OTA [6,7,8], however doubts on the sequence of some enzymatic steps had remained unresolved. The successive studies on the molecular aspects of the pathway allowed to establish the involved genes and their role in the biosynthesis. The fact that biosynthetic genes of secondary metabolism in fungal
M
genome are usually clusterized, has considerably facilitate the study and the identification of most of the genes responsible of OTA biosynthesis reactions. In addition, in the last decade, the considerable increase of
clusters in several species.
d
the genome sequencing projects regarding fungal microorganisms has led to identify the ochratoxin A
Ac ce pt e
In this review we briefly summarize the recent history and the latest advances on OTA biosynthesis at molecular level, and on the regulatory aspects, which have not yet properly investigated but on which great interest has been focused lately.
OTA producing fungi: an update
The genus Aspergillus includes the major number of OTA producing species (27, in Table 1); in Penicillium three species are confirmed as OTA producer, namely: P. nordicum, P. verrucosum and the recently described P. thymicola [3, 9,10].
Penicillium thymicola, evolutionary related to P. nordicum and P. verrucosum, has been isolated from dried thyme, soil and sorghum grain, while the specific strain studied for OTA production has been isolated from cheddar cheese. Recently, the revision in Circumdati section evidenced that A. westerdijkiae and A. steynii are considered more important for OTA contamination of food products with respect to the closely related species A. ochraceus [11]. In 2017, the production of OTA was claimed from new species of Penicillium (P. commune and P. rubens), Aspergillus (A. aculeatus) and Talaromyces (T. rugulosus) [12]; but these data were commented and needed further confirmation that has not yet been published [13, 14]. Updating the knowledge on potential ochratoxigenic species is of great importance in the vision of potential new scenarios 3 Page 3 of 16
due to climate change. In this context, preliminary studies showed emerging risk related to the spread of Aspergillus species also in cold temperate zone, evidencing the possible linkage of carbon dioxide increase in stimulating/reducing OTA biosynthesis in Aspergilli [15].
OTA biosynthesis genes
ip t
Recently, the otapksPV gene has been identified and characterized as the gene encoding the PKS required for OTA biosynthesis in P. verrucosum BT 22713, through gene disruption approach [16]. In previous work of Geisen and coauthors [17], it was shown that the OTA biosynthetic pathways in P. nordicum BFE487 and P.
cr
verrucosum BFE808 were highly homologous for the putative genes otanpsPN, otatraPN and otachlPN but not for the pks gene. The phylogenetic analysis positioned otapksPV and otapksPN in two different clades;
us
the two proteins show similar domains with the additional methyl transferase domain (MT) in the former, but disposed in a different order. The genome walking strategy led to the identification of two other ORFs next
an
to the pks gene in P. verrucosum: otaT and otaE, encoding a transporter and an oxidoreductase, respectively. They appeared to be coordinately expressed with the otapksPV gene, suggesting a possible role in OTA biosynthesis. The homology analysis of the protein sequences of these genes revealed the best match score
M
with proteins involved in citrinin production in Monoascus anka, which displays the same sequential order of OTA cluster in P. verrucosum, able to produce citrinin as well. In 2015, the genome sequencing and assembly of P. verrucosum BFE808 has been released (Stoll et al.,
d
conference abstract). From a preliminary analysis, a truncated gene cluster for OTA biosynthesis and a
Ac ce pt e
complete gene cluster for citrinin biosynthesis were identified. The PKS of the citrinin cluster seemed to be identical to the otapksPV described by O'Callaghan et al. [16]. Future analyses could give a deeper insight in the OTA biosynthesis cluster to better explain the homology and differences between P. nordicum and P. verrucosum.
The genome of Penicillium thymicola strain was sequenced and analyzed for the presence of OTA biosynthesis cluster [10]. No proteins homologous to known OTA genes from P. nordicum and P. verrucosum have been identified. However, a gene cluster was identified containing a pks and a hybrid pksnrps genes with high degree of similarity to OTA genes of A. carbonarius and A. ochraceus. The analysis revealed also the presence of a regulatory and a transporter gene, but not a halogenase or chloroperoxidase encoding gene. Also due to differences in the structure of multidomain enzymes involved in the biosynthesis of OTA, authors suggested that OTA genes could not be clustered in a single locus in P. thymicola. Aspergillus ochraceus and A. westerdijkiae, belonging to section Circumdati, initially were assigned to the same A. ochraceus taxon and only recently have been separated. In the study of Bacha et al., it was reported that two PKSs could be involved in the biosynthesis of OTA in A. westerdijkiae [18]. The gene aolc35-12, orthologous of the pks gene found to be responsible of OTA biosynthesis in A. ochraceus [19], appeared to differentially control the expression of aoks1, the OTA PKS previously identified in A. westerdijikiae [20]. The two PKS showed an homology of about 34%. 4 Page 4 of 16
The whole genome sequence of A. westerdijkiae CBS 112803 was released in 2016 [21]. The functional annotation of the genome has led to the identification of two putative OTA-related gene clusters [22]. One of these (cluster 37) includes 17 genes among which the pks gene corresponding to the above mentioned aolc35-12. Other genes of the cluster 37 encode a NRPS, a cytochrome P450 monoxygenase, a bZIP transcription regulator and a halogenase, and in addition a Zn2Cys6 transcription regulator and a sugar transporter. The second identified putative cluster (cluster 69) comprises the pks gene identical to the aoks1
ip t
previously characterized, and also a cytochrome P450 monoxygenase, two transporters and an Acyl-CoA synthetase, which could be related to the putative action of Acetyl-CoA as precursor of OTA synthesis. Furthermore in this latter cluster, two genes encoding enzymes involved in sucrose and glucose metabolism
cr
have been annotated. The authors suggested that they might have important influences on OTA production in various substrates, in agreement to the high ability of A. westerdijkiae in producing OTA in media containing
us
high quantity of sucrose and glucose.
Likewise, also in A. ochraceus the annotation and analysis of the sequenced genome led to the identification
an
of two pks genes, AoOTApks-1 and AoOTApks-2 [23]. The obtained deletion mutants for the respective genes produced no and lesser OTA, respectively, demonstrating their involvement in OTA biosynthesis. The two predicted proteins show 26 % identity at amino acid level between them, but no indication was reported
M
about their homology to the two OTA pks genes of A. westerdijkiae, likely because only partial sequences of A. westerdijkiae PKSs were available at the time. Anyway, from the two different works, it can be deduced that AoOTApks-1 from A. ochraceus and aolc35-12 from A. westerdijkiae are both highly reducing PKS
d
(HR-PKS), due to the presence of reducing domains, while AoOTApks-2 of A. ochraceus and aoks1 of A.
structure.
Ac ce pt e
westerdijkiae are both partially reducing PKS (PR-PKS) for the absence of the ER domain in their modular
As for A. westerdijkiae, also in A. ochraceus the expression profile during OTA production suggested that AoOTApks-1 gene was directly involved in OTA biosynthesis while AoOTApks-2 might complement the expression of the former gene and be involved in the biosynthesis indirectly. Other fungal secondary metabolites are reported to require two different PKSs for the biosynthesis of a single polyketide such as lovastatin, compactin and the mycotoxins zearaleone and T-toxin. Same evidence of the involvement of two PKS for OTA biosynthesis could derive also from the study that our group carried out on A. carbonarius. Initially, we identified the ACpks gene that showed an expression profile correlated to the production of OTA; however, this gene was not inactivated for the confirmation of its direct role in the biosynthesis [24]. Then, the whole genome sequencing of A. carbonarius ITEM 5010 permitted the identification of an OTA biosynthesis cluster based on comparative analysis in silico. Hence, firstly we demonstrated through gene inactivation that AcOTAnrps gene was responsible of the linking of phenylalanine to the polyketide dihydroisocoumarin [25]. Furthermore, chemical analysis on the presence of the principal secondary metabolites involved in OTA biosynthesis, such as OTB, OTC, OTβ and OTα, clarified the order of reaction of OTA biosynthesis pathway, confirming the hypothesis that the enzymatic step adding phenylalanine to OTβ, the dihydroisocoumarin based structure, precedes the chlorination 5 Page 5 of 16
reaction to form OTA, and that OTα may be a product of hydrolysis of OTA in A. carbonarius. Carrying on with the analysis of the genomic region identified as putative OTA cluster, the adjacent gene coding for a PKS, different from the previously identified ACpks, was inactivated and its role in the biosynthetic pathway was confirmed [26]. The designated AcOTApks gene encodes for a HR-PKS containing a putative methyl transferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. Finally, we demonstrated that a flavin-halogenase is implicated in the biosynthesis of OTA in A.
ip t
carbonarius. The encoding gene AcOTAhal is contiguous to the above described biosynthetic genes, resulting as part of the cluster [27]. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway, as confirmed by the accumulation of
cr
OTB, the metabolite formed by the action of the AcOTAnrps enzyme, simultaneously to the loss of OTA production, when AcOTAhal was inactivated. In Fig.1 a schematic representation of the order of reactions in
us
OTA biosynthesis pathway is depicted. In addition, during the same study, the expression profile of two other genes located in the assumed biosynthetic cluster correlated to the OTA production kinetics similarly to
an
OTA genes. These two genes encode a cytochrome P450 oxidase (AcOTAp450) and a bZIP transcription factor (AcOTAbZIP), putatively involved in OTA biosynthesis. Other genes were annotated in the OTA cluster in A. carbonarius that need further investigation to establish their role in OTA biosynthetic pathway,
M
such as a predicted transporter protein and another fungal specific transcription factor. Aspergillus niger is the OTA producing fungus phylogenetically most related to A. carbonarius. Genome sequencing of A .niger CBS 513.88 revealed the presence of a putative OTA cluster [28] on the basis of
d
sequence homology to A. ochraceus. From in silico analyses, the genomic regions corresponding to OTA
Ac ce pt e
clusters in A. carbonarius and A. niger harbor the putative and already characterized OTA biosynthetic genes organized in the same order and direction of transcription [29]. Only recently, the A. niger An15g07920 pks gene of the putative OTA cluster was demonstrated experimentally to be a crucial player in the biosynthesis of OTA [30].
Lately, numerous studies have been carried out on molecular aspects of OTA biosynthesis, but still the length and composition of OTA cluster remain not completely defined. The key enzymes- such as PKS, NRPS, halogenase-and their role have been identified in several OTA producing species. Other genes, included in the interested genomic region or external to it, have to be investigated to fully elucidate the mechanism of the biosynthetic pathway and to better clarify the molecular regulation at the basis of OTA production. In fungal species only recently recognized as OTA producing, comparative and homology analyses led to the identification of OTA genes as in A. affinis with regards to a pks gene putatively involved in the first step of OTA biosynthesis [31]. In A. steynii a genomic fragment with three contiguous genes has been identified encoding a cytochrome P450 monoxygenase (p450ste), a NRPS (nrpsste) and a PKS (pksste), whose expression patterns were coordinated and appeared to be related to OTA production [32]. In Fig. 2, the OTA clusters of different producing species are represented. Finally, from the results obtained until now, some divergences in the structure of putative clusters and in domain composition of key enzymes (PKS and NRPS) have been observed in fungi belonging to different 6 Page 6 of 16
sections and genera. In a short time, a huge amount of new genomic data will be available and will enable to explain the phylogenetic relationship and the evolutionary events occurred among OTA producing species.
Regulation of OTA biosynthesis Numerous studies have reported the influence of biotic and abiotic stressors on OTA biosynthesis in different fungal species. The regulation of secondary metabolism is very complex and acts at different regulatory
ip t
levels. It is a mechanism that may utilize regulator elements specific of the biosynthesis pathway by controlling expression of the corresponding biosynthesis genes. At a superior level, global regulators and multiprotein complexes transmit environmental cues to biosynthesis mechanism; these regulatory processes
cr
are often interconnected and overlapping. Other regulation mechanisms based on signaling pathways or epigenetic control could be responsible of the activation of biosynthetic genes. Studies on the regulation of
us
OTA biosynthesis has intensified in the last few years and more significant results are expected from future investigations. At present, most of the regulatory aspects underlying the OTA production remain unclear. It
an
is likely that some of them are based on functioning models already identified for the production of other mycotoxins.
Usually, secondary metabolite biosynthesis cluster contain cluster specific transcription factors. In the OTA
M
cluster of A. carbonarius, between AcOTAp450 and AcOTAhal genes, the AcOTAbZIP gene, encoding a basic leucine zipper transcription factor, was identified. Its expression profile showed correlation with the expression profile of other OTA genes and with the kinetics of OTA accumulation [27]. Many fungal bZIPs,
d
have been characterized as stress response transcription factors, responding to a variety of environmental
Ac ce pt e
stresses and linking them to secondary metabolite production [33]. Among these, a bZIP transcription factor, Aoyap1, was found in A. ochraceus to mediate signal perception in the regulation of OTA synthesis under oxidative stress [34].
Towards the edges of the OTA genomic region in A. carbonarius, other two genes have been annotated encoding transcription factors containing a Cys-rich motif (http://jgi.doe.gov/carbonarius/). This structure characterizes the zinc-cluster proteins (Zn2Cys6 ), which are the most common as cluster regulators of fungal secondary metabolites, among which is the AflR regulator of aflatoxin/sterigmatocystin production. More definitive conclusions about their role in the regulation of OTA biosynthesis can be provided by knocking out experiments.
Among the global regulators, the heterotrimeric velvet complex has been the most studied in OTA producing fungi to clarify the link of the light-dependent fungal morphology and sexual development to secondary metabolism. This complex consists of proteins LaeA, VeA and VelB and is fully functional in the nucleus under dark conditions [33]. The deletion of laeA and veA in A. carbonarius led to a strong reduction in conidial production and a drastic decrease of OTA production, which was correlated to a down regulation of the AcOTAnrps gene [35]. These results confirmed the role of the two proteins in regulating conidiation and OTA biosynthesis in response to light likely to other fungi. Recently, laeA has been reported to have also a regulatory effect on production of citric acid and cellulolytic enzymes in A. carbonarius [36]. 7 Page 7 of 16
In the study of El Khoury et al. [37], essential oils from different plant origin were observed to induce reduction of OTA production in the same fungus. Some of them such as fennel, chamomile and rosemary essential oils were able to downregulate laeA and veA genes and consequently the expression of OTA biosynthesis genes was reduced; others, such as celery and anise essential oils, did not have significant effect on laeA and veA expression and the observed downregulation of OTA genes was suggested to be due to different mode of regulation.
ip t
Besides the velvet protein complex, other global regulators have been demonstrated to be involved in regulation of secondary metabolism genes in fungi [38]. Among them, the most studied have been PacC, the key factor for pH regulation; CBC, which regulates the response to redox status and iron stresses; CreA,
cr
involved in the regulation of carbon metabolism; AreA and AreB , involved in nitrogen metabolism.
Many studies have been reported that OTA production is responsive to general environmental factors, such
us
as carbon and nitrogen factors, temperature, light and pH; despite of this, very little of the role of the global regulators in converting the environmental cues in activation or inhibition of OTA biosynthesis genes has
an
been described. During a preliminary study we carried out on the promoter regions of OTA biosynthesis genes in A. carbonarius, a number of conserved motifs known to act as binding sites of transcription factors and specific of the most common global regulators, have been identified (unpublished).
M
External stimuli are also transmitted to the genetic level by signal transduction pathways that translate the external signal to transcriptional level by activating positive or negative transcription factors, which then regulate downstream genes. The production of OTA was suggested to contribute to the adaptation of P.
d
nordicum and P. verrucosum to NaCl rich environment by ensuring chloride homeostasis in the cell, through
Ac ce pt e
the excretion of OTA that carries a chlorine in its molecule [39]. The correlation between this environment stress and OTA biosynthesis was demonstrated to be mediated by the HOG MAP kinase signal cascade pathway [40], through the phosphorylation of the kinase HOG, the last protein of the cascade, that subsequently activates the downstream transcription factors. In the same study, a different behavior was observed for A. carbonarius, in which the increased levels of NaCl lead to the increased phosphorylation status of HOG but not to an increase in OTA biosynthesis, inferring that A. carbonarius lacks the mechanism to cope with NaCl stress, which reduces also the ability to grow of A. carbonarius. Interestingly, it was found that the biosynthesis of citrinin and OTA is mutually regulated in P. verrucosum, with increasing concentration of NaCl shifting production from citrinin to OTA and the oxidative stress increasing citrinin biosynthesis at the expense of the other mycotoxin, by means of a different signaling pathway [41]. Further explanation are expected by proteomics and transcriptomics studies performed in producing species grown under different permissive and not permissive conditions for OTA production. Crespo-Sempere et al. studied two strains of A. carbonarius, very dissimilar for their OTA production ability, by suppression subtractive -hybridization technique, identifying several differentially ESTs, 26% of which were potentially involved in OTA regulation processes [42]. Some of them were related to the Target of Rapamycin (TOR) that concerns a number of cellular signalling pathways in response to nutrients, hormones, and stresses; and to the MAP kinase-dependent signal transduction. Other proteomics and transcriptomic analyses have been 8 Page 8 of 16
carried out under different growth condition by using weak and strong producing species and even not producing species, leading to the accumulation of data about genes and proteins differentially expressed that could provides a deeper understanding of the regulatory mechanism in the future studies [43-45].
Conclusions
ip t
The contamination of OTA in different food and feed is an important issue for the health and safety hazards that it implies. Climate, agronomic and global market changes could cause the emergency of novel risks. Therefore, the knowledge about new producing species and matrices that could be contaminated is essential
cr
to face in an appropriate way the emerging exposure. To this aim, the genomic data of an increasing number of fungal species is a powerful means that allow to identify a biosynthesis cluster in silico and to determine
us
the possibility of producing by a fungus. In the same way, the understanding of the genetic pathway involved in the biosynthesis and the molecular regulators that activate or inhibit the biosynthetic genes, could provide
an
instruments for the development of prevention strategies, diagnostic methods and remediation measures.
M
References and recommended readings
Ac ce pt e
d
1. Barlow S, Bolger M, Pitt JI, Verger P: Safety Evaluation of Certain Contaminants in Food In Ochratoxin A (addendum) WHO Food Additives Series 59, World Health Organization, Geneva; 2008: 357–429. 2. IARC (International Agency for Research on Cancer): Ochratoxin A IARC monographs on the evaluation of carcinogenic risks to humans. IARC Press. 1993, 56: 489–521. 3. Visagie CM, Varga J, Houbraken J, Meijer M, Kocsubé S, Yilmaz N, Fotedar R, Seifert KA, Frisvad JC, Samson RA : Ochratoxin production and taxonomy of the yellow aspergilli (Aspergillus section Circumdati). Stud Mycol 2014, 78: 1-61. 4. Perrone G, Susca A, Cozzi G, Ehrlich K, Varga J, Frisvad JC, Meijer M, Noonim P, Mahakarnchanakul W, Samson RA: Biodiversity of Aspergillus species in some important agricultural products. Stud Mycol 2007, 59: 53-66. 5. Bayman P, Baker JL, Doster MA, Michailides TJ, Mahoney NE: Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus. Appl Environ Microbiol 2002, 68: 2326-2329. 6. Huff WE, Hamilton PB: Mycotoxins—their biosynthesis in fungi: ochratoxins—metabolites of combined pathways. J Food Prot 1979, 42:815–820. 7. Ruhland M, Engelhardt G, Wallnöter PR: Production of 14C-ochratoxin a by Penicillium verrucosum sp. 1761 in liquid culture. Mycotox Res 1996, 12: 7–13. 8. Harris JP, Mantle PG: Biosynthesis of ochratoxins by Aspergillus ochraceus. Phytochemistry 2001, 558:709 –716. 9 Page 9 of 16
9. Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, Samson R.A: New and revisited species in Aspergillus section Nigri. Stud Mycol 2011, 69: 1-17. 10. Nguyen HD, McMullin DR, Ponomareva E, Riley R, Pomraning KR, Baker SE, Seifert KA: (). Ochratoxin A production by Penicillium thymicola. Fungal Biol 2016, 120(8): 1041-1049. * In this paper the production of ochratoxin A from a new species of Penicillium is reported together with the genome analysis. Interestingly, this OTA cluster is more related to Aspergillus species than to its sister species P. nordicum and P. verrucosum.
cr
ip t
11. Gil-Serna J, Vázquez C, Sardiñas N, González-Jaén MT, Patiño B: Revision of ochratoxin A production capacity by the main species of Aspergillus section Circumdati. Aspergillus steynii revealed as the main risk of OTA contamination. Food Control 2011, 22: 343–345.
us
12. Zhang X, Li Y, Wang H, Gu X, Zheng X, Wang Y, Diao J, Peng Y, Zhang H: Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes. Toxins 2016, 8(11): 333.
an
13. Perrone G., Logrieco A F, Frisvad JC: Comments on “Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes". Toxins 2016, 8: 333—In Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces. Toxins 2017, 9(2): 65.
M
14. Zhang X, Li, Y., Wang, H, Gu X, Zheng X, Wang Y, Diao J, Peng Y, Zhang H: Reply to Comment on “Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes”. Toxins 2016, 8, 333”–in Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces. Toxins 2017, 9(2): 66.
Ac ce pt e
d
15. Akbar A, Medina A, Magan N: Impact of interacting climate change factors on growth and ochratoxin A production by Aspergillus section Circumdati and Nigri species on coffee. World Mycotoxin J 2016, 9(5): 863-874. 16. O'Callaghan J, Coghlan A, Abbas A, Garcia-Estrada C,Martin JF, Dobson ADW: Functional characterization of the polyketide synthase gene required for ochratoxin A biosynthesis in Penicillium verrucosum. Int J Food Microbiol 2013, 161: 172–181. 17. Geisen R, Schmidt-Heydt M, Karolewiez A: A gene cluster of the ochratoxin A biosynthetic genes in Penicillium. Mycotox Res 2006, 22: 134–141. 18. Bacha N, Mathieu F, Liboz T, Lebrihi A: Polyketide synthase gene aolc35-12 controls the differential expression of ochratoxin A gene aoks1 in Aspergillus westerdijkiae. World Mycotoxin J 2012, 5: 177186. 19. O’Callaghan J, Caddick MX, Dobson ADW: A polyketide synthase gene required for ochratoxin A biosynthesis in Aspergillus ochraceus. Microbiology 2003, 149: 3485–3491. 20. Bacha N, Atoui A, Mathieu F, Liboz T, Lebrihi A: Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A. Fungal Genet Biol 2009, 46:77– 84. 21. Chakrabortti A, Li J, Lianga Z-X: Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A. Genome Announc 2016, 4(5): pii: e00982-16.
10 Page 10 of 16
22. Han X, Chakrabortti A, Zhu J, Liang Z-X, Li J: Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae. BMC Genomics 2016, 17: 633. *This paper reports the identification in the sequence genome of A. westerdijkiae of two clusters putatively involved in OTA biosynthesis, including a HR-PKS and a PR-PKS encoding genes, respectively
ip t
23. Wang L, Wang Y, Wang Q, Liu F, Selvaraj JN, Liu L, Xing F, Zhao Y, Zhou L, Liu Y: Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1. Toxins 2015, 7(8): 2723-2738.
cr
*This paper provides experimental evidence of the involvement of two different psk genes in the production of OTA
us
24. Gallo A, Perrone G, Solfrizzo M, Epifani F, Abbas A, Dobson AD, Mulè G: Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus carbonarius. Int J Food Microbiol 2009, 129: 8–15
an
25. Gallo A, Bruno KS, Solfrizzo M, Perrone G,Mulè G, Visconti A, Baker SE: New insight into the ochratoxin A biosynthetic pathway through deletion of a nonribosomal peptide synthetase gene in Aspergillus carbonarius. Appl Environ Microbiol 2012, 78: 8208–8218.
M
26. Gallo A, Knox BP, Bruno KB, Solfrizzo M, Baker SE, Perrone G: Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius. Int J Food Microbiol 2014, 179: 10–11.
Ac ce pt e
d
27. Ferrara M, Perrone G, Gambacorta L, Epifani F, Solfrizzo M, Gallo A: Identification of a halogenase involved in the biosynthesis of ochratoxin A in Aspergillus carbonarius. Appl Environ Microbiol 2016, 82 (18): 5631-5641. * This work describes for the first time the identification and characterization of an halogenase gene responsible of the chlorination of OTA molecule in the biosynthesis pathway. In addition, the correlation between a transcription factor in OTA cluster and the mycotoxin accumulation was evidenced. 28. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K et al: Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 2007, 25:221–231. 29. Ferracin LM, Fier CB, Vieira ML, Monteiro-Vitorello CB, Varani A, Rossi MM, Müller-Santos M, Taniwaki MH, Iamanaka BT, Fungaro MH: Strain-specific polyketide synthase genes of Aspergillus niger. Int J Food Microbiol 2012, 155:137–145. 30. Zhang J, Zhu L, Chen H, Li M, Zhu X, Gao Q, Wang D, Zhang Y: A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger. J Agric Food Chem 2016, 64(51): 9680-9688. 31. Davolos D, Pietrangeli B: A molecular and bioinformatic study on the ochratoxin A (OTA)producing Aspergillus affinis (section Circumdati). Mycotoxin Res 2014, 30(2): 113-122.
11 Page 11 of 16
32. Gil-Serna J, Vázquez C, González-Jaén MT, Patiño B: Clustered array of ochratoxin A biosynthetic genes in Aspergillus steynii and their expression patterns in permissive conditions. Int J Food Microbiol 2015, 214:102-108. 33. Knox BP, Keller NP: Key Players in the Regulation of Fungal Secondary Metabolism. In Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites, Volume 2. Edited by Zeilinger S, Martín J-F, García-Estrada C. Springer; 2015: 13-28
ip t
34. Reverberi M, Gazzetti K, Punelli F, Scarpari M, Zjalic S, Ricelli A, Fabbri AA, Fanelli C: Aoyap1 regulates OTA synthesis by controlling cell redox balance in Aspergillus ochraceus. Appl Microbiol Biotechnol 2012, 95(5):1293-1304.
cr
35. Crespo-Sempere A, Marin S, Sanchis V, Ramos AJ: VeA and LaeA transcriptional factors regulate ochratoxin A biosynthesis in Aspergillus carbonarius. Int J Food Microbiol 2013, 166: 479–486.
us
36. Linde T, Zoglowek M, Lübeck M, Frisvad JC, Lübeck PS: The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius. J Ind Microbiol Biotechnol 2016, 43(8):1139-1147.
M
an
37. El Khoury R, Atoui A, Verheecke C, Maroun R, El Khoury A, Mathieu F: Essential Oils Modulate Gene Expression and Ochratoxin A Production in Aspergillus carbonarius. Toxins 2016, 8(8): pii: E242. 38. Macheleidt J, Mattern DJ, Fischer J, Netzker T, Weber J, Schroeckh V, Valiante V, Brakhage AA: Regulation and Role of Fungal Secondary Metabolites. Annu Rev Genet 2016, 50:371-392.
Ac ce pt e
d
39. Schmidt-Heydt M, Graf E, Stoll D, Geisen R: The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods. Food Microbiol 2012, 29: 233–241. 40. Stoll D, Schmidt-Heydt M, Geisen R: Differences in the regulation of ochratoxin A by the HOG pathway in Penicillium and Aspergillus in response to high osmolar environments. Toxins 2013, 5: 1282–1298. 41. Schmidt-Heydt M, Stoll D, Schütz P, Geisen R: Oxidative stress induces the biosynthesis of citrinin by Penicillium verrucosum at the expense of ochratoxin. Int J Food Microbiol 2015, 192: 1–6. * In this paper authors reported that in P. verrucosum production of ochratoxin A and citrinin is shifted towards one of them at the expense of the other depending on environmental conditions by means of a signaling pathway 42. Crespo-Sempere A, González-Candelas L, Martínez-Culebras PV: Genes differentially expressed by Aspergillus carbonarius strains under ochratoxin A producing conditions. Int J Food Microbiol 2010, 142(1-2): 170-179. 43. Crespo-Sempere A, Gil JV, Martínez-Culebras PV: Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions. Int J Food Microbiol 2011 , 147(3): 162-169. 44. Sartori D, Massi FP, Ferranti LS, Fungaro MHP: Identification of Genes Differentially Expressed Between Ochratoxin-Producing and Non-Producing Strains of Aspergillus westerdijkiae. Indian J Microbiol 2014, 54(1): 41–45. 45. Gerin D, De Miccolis Angelini RM, Pollastro S, Faretra F: RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius. PLoS One 2016, 11(1): e0147089 12 Page 12 of 16
ip t
us
M
an
Sec. Circumdati (Weak OTA producers) A. melleus A. ostianus A. persii A. salwaensis A. sclerotiorum A. sesamicola A. subramanianii A. westlandensis
Ac ce pt e
Sec. FLAVI A. albertensis A. alliaceus
Sec. NIGRI A. carbonarius A. lacticoffeatus A. niger A. sclerotioniger A. welwitschiae
d
Sec. CIRCUMDATI A. affinis A. cretensis A. flocculosus A. fresenii A. muricatus A. occultus A. ochraceus A. pseudoelegans A. pulvericola A. roseoglobulosus A. steynii A. westerdijkiae
cr
Table 1 Aspergillus species producing ochratoxin A (OTA)
13 Page 13 of 16
Fig.1 Schematic representation of the pathway of OTA biosynthesis in which key enzymatic steps are shown
Fig.2 Organization of OTA clusters in different Aspergillus and Penicillium species. Highlighted in bold are
ip t
the genes that have been knocked out. In parentheses are the references from which cluster structures
Ac ce pt e
d
M
an
us
cr
were taken. (* Stool et al., 2015. conference abstract)
14 Page 14 of 16
an M ed Ac ce pt Page 15 of 16
e pt ce Ac
Page 16 of 16