Exploitation of a novel heterologous expression system for characterisation of fungal secondary metabolites

Aspergillus species have a great impact on economy and human health. These filamentous ascomycetes are known to cause food and feed spoilage and can act as pathogens of plants and humans. Besides these detrimental effects, several Aspergillus species have been exploited for food production and as producers of bioactive metabolites, organic acids and proteins. The study presented here focusses on Aspergillus terreus, which combines most of the features described above. A. terreus is used for production of the primary metabolite itaconic acid and the natural product lovastatin. It has been described as cause of disease on potato plants and is an emerging pathogen of humans. Investigation of the interaction of A. terreus with immune cells showed that acidification of phagolysosomes in macrophages is not inhibited by A. terreus as observed with other Aspergillus species. Since inhibition of acidification has been attributed to dihydroxynaphthalene (DHN)-melanin in Aspergillus conidia, a different type of melanin in A. terreus conidia was expected. A search for the origin of the pigment in A. terreus conidia resulted in the serendipitous identification of the biosynthesis gene cluster responsible for the production of terrein with the polyketide synthase TerA as key biosynthetic enzyme. Further characterisation revealed that terrein production is induced under environmental conditions like those found in the rhizosphere. Combined with its biological activities, terrein can be assumed to increase the fitness of A. terreus in the environment. Terrein is produced in large quantities and production depends on the activity of the transcriptional regulator TerR. Binding sites for TerR were found in all promoter regions of the terrein biosynthesis gene cluster, which led to the generation of heterologous expression systems in Aspergillus niger and Aspergillus oryzae using genetic elements from the A. terreus terrein biosynthesis gene cluster. Expression platform strains were generated that contained the terR gene either under a sugar- or doxycycline-inducible promoter. Then a gene of interest cloned under control of the terA promoter is highly transcribed by binding of TerR. Thereby, the doxycycline-dependent TetOn-terR system provides fine-tunable gene expression, which makes it suitable for the production of metabolites toxic to the producer. Furthermore, the use of viral self-splicing peptide sequences was successfully used to express fungal secondary metabolite biosynthesis genes from a polycistronic messenger. Therefore, the heterologous expression systems were subsequently exploited to study secondary metabolism in A. terreus and related species. This system essentially contributed to the identification of the true origin of the pigment in A. terreus conidia. This new type of melanin called Asp-melanin derives from the metabolite aspulvinone E that is produced by a non-ribosomal peptide synthetase- (NRPS)-like enzyme and is oxidised and activated for polymerisation by a tyrosinase. The pathway was successfully reconstituted in the heterologous expression system and, furthermore, under in vitro conditions. This Asp-melanin protects conidia from UV-radiation and reduces the attraction of soil amoeba, resulting in reduced phagocytosis rates. Subsequent studies revealed that Asp-melanin is common to species from section Terrei and is a discriminator from other Aspergillus sections. However, species less closely related to A. terreus show either no pigment biosynthesis pathway, a combination of the DHN-melanin and Asp-melanin pathway or a DHN-melanin pathway that has lost its transcriptional activation. Therefore, the lack of a functional DHN-melanin pathway accompanied by the evolution of the Asp-melanin pathway appears to describe a specific environmental adaptation of species from Terrei. The discovery of the contribution of an NRPS-like enzyme to pigment formation in A. terreus resulted in further interest in this class of enzymes. NRPS-like enzymes with a domain structure of adenylation, thiolation and thioesterase domain generally use two identical aromatic α-keto acids as substrates that get condensed under the formation of different interconnecting core structures such as bis-indolylquinones, terphenylquinones, dioxolanones or furanones with different substitution patterns. While it is known that core structure formation is specifically catalysed by the thioesterase domain, product-predictive sequence patterns had not been identified. Studies were undertaken to convert a furanone forming aspulvinone E synthetase into a quinone forming atromentin synthetase but approaches by site-directed mutagenesis failed. This indicated that the exchange of individual amino acids is not sufficient to re-direct the chemistry of a thioesterase domain. Domain-swapping experiments successfully converted an aspulvinone E synthetase into an atromentin synthetase, but only when the donor thioesterase domain derived from a phylogenetic closely related species. The reason for this was later identified by a detailed phylogenetic analysis of atromentin synthetases from basidio- and ascomycetes which showed that the phylogenetic origin of a species results in greater sequence differences than caused by differences in the chemistry of the thioesterase domain of an NRPS-like enzyme. Therefore, it can be assumed that a prototype of an NRPS-like enzyme may have been present in a common ancestor of basidio- and ascomycetes, but evolution of NRPS-like enzymes forming a specific metabolite occurred independent in the two fungal lineages. Unexpectedly, expression of NRPS-like enzymes producing quinone core structures resulted in different metabolites when expressed in A. niger compared to A. oryzae. Detailed analyses on the expression of atromentin synthetases in A. niger revealed that the formation of the quinone structure of atromentin is re-directed towards the formation of atrofuranic acid with a furanic acid core. Further analyses showed that this cross-chemistry enforced by the physiology of the producer is not limited to A. niger, but also observed in the black fungus Aspergillus brasiliensis from section Nigri that contains an intrinsic atromentin/atrofuranic acid synthetase. As this cross-chemistry seems to apply to all quinone core structures that were attempted to be produced in A. niger, host physiology may significantly influence product formation. While this broadens the spectrum of metabolites that can be obtained from NRPS-like enzymes, it may be recommended to use at least two different expression platforms with different physiology when investigating the metabolites produced from previously uncharacterised secondary metabolite producing enzymes. Further genome analyses from Aspergillus species from section Terrei revealed that a large number of NRPS-like enzymes with a C-terminal thioesterase domain is present in this section. Therefore, heterologous expression systems were used to obtain insights into the spectrum of metabolites produced in this section. NRPS-like enzymes were grouped into families and individual members selected for heterologous gene expression and product analysis. These analyses confirmed the broad spectrum of NRPS-like-derived metabolites in this section, but also revealed that attributing individual enzymes to specific families and to define structure-predicting sequences in the thioesterase domain requires the characterisation of additional enzymes, especially of those from outside of the genus Aspergillus. However, this study identified a putative phenylbutyrolactone IIa and a polyporic acid synthetase. Both enzymes had not previously been described from ascomycetes. It will be interesting to see whether phenylbutyrolactone IIa has similar quorum sensing effects on the producer Aspergillus ambiguus as observed for butyrolactone I on A. terreus. Furthermore, polyporic acid, a metabolite related to atromentin, has previously only been known from basidiomycetes. This study indicates that polyporic acid might also be produced by ascomycetes and provides the first example of a putative polyporic acid synthetase from any fungal source. Finally, it was of interest to obtain ideas on how these secondary metabolites could be further exploited. Therefore, an in silico target fishing approach was tested using atromentin as a model compound. This in silico modelling proposed a weak estrogenic activity on human estrogen receptors and a strong inhibitory function on 17-β-hydroxysteroid dehydrogenase as potential targets. Subsequent in vitro experiments were able to confirm the estrogenic activity of atromentin, which indicates that an in silico target fishing approach accompanied with in vitro experiments is suitable for target prediction and further exploitation of metabolites deriving from NRPS-like enzymes

Cross-chemistry leads to product diversity from atromentin synthetases in Aspergilli from section Nigri

Nonribosomal peptide synthetase (NRPS)-like enzymes catalyse the non-oxidative homodimerisation of aromatic α-keto acids, but the exact reaction mechanism is unknown. The furanone-forming thioesterase domain of the Aspergillus terreus aspulvinone E synthetase MelA displays a predicted quinone-forming motif, whereby its catalytic triad contains an essential cysteine indicating an unusual thioester intermediate. To convert MelA into a quinone-forming atromentin synthetase its thioesterase domain was replaced with that from a Paxillus involutus or A. terreus atromentin synthetase. Phylogenetic proximity of donor and acceptor seems important as only replacement with the A. terreus thioesterase was functional. Heterologous expression of atromentin synthetases in Aspergillus niger and Aspergillus oryzae revealed host-dependent product formation whereby cross-chemistry directed atromentin biosynthesis in A. niger towards atrofuranic acid. Screening of aspergilli from section Nigri identified an atromentin synthetase in Aspergillus brasiliensis that produced atrofuranic acid in the homologous host. Therefore, cross-chemistry on quinone cores appears common to section Nigri

Terrein Biosynthesis in Aspergillus terreus and Its Impact on Phytotoxicity

Terrein is a fungal metabolite with ecological, antimicrobial, antiproliferative, and antioxidative activities. Although it is produced by Aspergillus terreus as one of its major secondary metabolites, not much is known about its biosynthetic pathway. Here, we describe an unexpected discovery of the terrein biosynthesis gene locus made while we were looking for a PKS gene involved in production of conidia coloration pigments common for Aspergilli. The gene, ATEG_00145, here named terA, is essential for terrein biosynthesis and heterologous production of TerA in Aspergillus niger revealed an unusual plasticity in the products formed, yielding a mixture of 4-hydroxy-6-methylpyranone, orsellinic acid, and 6,7-dihydroxymellein. Biochemical and molecular genetic analyses indicate a low extension cycle specificity of TerA. Furthermore, 6-hydroxymellein was identified as a key intermediate in terrein biosynthesis. We find that terrein production is highly induced on plant-derived media, that terrein has phytotoxic activity on plant growth, and induces lesions on fruit surfaces

Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

Background: Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Results: Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. Conclusion: This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin

Hybrid in silico/in vitro target fishing to assign function to “orphan” compounds of food origin – The case of the fungal metabolite atromentin

Many small molecules of food origin may effect human health but lack an adequate description of their biological activity. To fill this knowledge gap, a first-line workflow is needed to assign putative functions, rank the endpoints for testing and guide wet-lab experiments. In this framework, the identification of potential biological targets can be used to probe the activity of orphan compounds using a so-called “target fishing” approach. Here, we present a proof of concept study using an in silico/in vitro target fishing approach on the fungal secondary metabolite atromentin. The procedure relies on a computational screening for activity identification coupled with experimental trials for dose-response characterization. Computational results identified estrogen receptors and 17-β-hydroxysteroid dehydrogenase as potential targets. Experiments confirmed a weak estrogenic activity, supporting the reliability of the procedure. Despite limited estrogenicity of atromentin, the proposed inhibition of 17-β-hydroxysteroid dehydrogenase should be considered as a source for endocrine disruptive effects

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and Interactions with plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.Institut de Recherche pour le Developpement (IRD); French Agence Nationale pour la Recherche [ANR-11-BSV7-009-01]; European Community [242095, 223601]info:eu-repo/semantics/publishedVersio

Sex difference and intra-operative tidal volume: Insights from the LAS VEGAS study

BACKGROUND: One key element of lung-protective ventilation is the use of a low tidal volume (VT). A sex difference in use of low tidal volume ventilation (LTVV) has been described in critically ill ICU patients.OBJECTIVES: The aim of this study was to determine whether a sex difference in use of LTVV also exists in operating room patients, and if present what factors drive this difference.DESIGN, PATIENTS AND SETTING: This is a posthoc analysis of LAS VEGAS, a 1-week worldwide observational study in adults requiring intra-operative ventilation during general anaesthesia for surgery in 146 hospitals in 29 countries.MAIN OUTCOME MEASURES: Women and men were compared with respect to use of LTVV, defined as VT of 8 ml kg-1 or less predicted bodyweight (PBW). A VT was deemed 'default' if the set VT was a round number. A mediation analysis assessed which factors may explain the sex difference in use of LTVV during intra-operative ventilation.RESULTS: This analysis includes 9864 patients, of whom 5425 (55%) were women. A default VT was often set, both in women and men; mode VT was 500 ml. Median [IQR] VT was higher in women than in men (8.6 [7.7 to 9.6] vs. 7.6 [6.8 to 8.4] ml kg-1 PBW, P < 0.001). Compared with men, women were twice as likely not to receive LTVV [68.8 vs. 36.0%; relative risk ratio 2.1 (95% CI 1.9 to 2.1), P < 0.001]. In the mediation analysis, patients' height and actual body weight (ABW) explained 81 and 18% of the sex difference in use of LTVV, respectively; it was not explained by the use of a default VT.CONCLUSION: In this worldwide cohort of patients receiving intra-operative ventilation during general anaesthesia for surgery, women received a higher VT than men during intra-operative ventilation. The risk for a female not to receive LTVV during surgery was double that of males. Height and ABW were the two mediators of the sex difference in use of LTVV.TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov, NCT01601223

Exploitation of a novel heterologous expression system for characterisation of fungal secondary metabolites

Aspergillus species have a great impact on economy and human health. These filamentous ascomycetes are known to cause food and feed spoilage and can act as pathogens of plants and humans. Besides these detrimental effects, several Aspergillus species have been exploited for food production and as producers of bioactive metabolites, organic acids and proteins. The study presented here focusses on Aspergillus terreus, which combines most of the features described above. A. terreus is used for production of the primary metabolite itaconic acid and the natural product lovastatin. It has been described as cause of disease on potato plants and is an emerging pathogen of humans. Investigation of the interaction of A. terreus with immune cells showed that acidification of phagolysosomes in macrophages is not inhibited by A. terreus as observed with other Aspergillus species. Since inhibition of acidification has been attributed to dihydroxynaphthalene (DHN)-melanin in Aspergillus conidia, a different type of melanin in A. terreus conidia was expected. A search for the origin of the pigment in A. terreus conidia resulted in the serendipitous identification of the biosynthesis gene cluster responsible for the production of terrein with the polyketide synthase TerA as key biosynthetic enzyme. Further characterisation revealed that terrein production is induced under environmental conditions like those found in the rhizosphere. Combined with its biological activities, terrein can be assumed to increase the fitness of A. terreus in the environment. Terrein is produced in large quantities and production depends on the activity of the transcriptional regulator TerR. Binding sites for TerR were found in all promoter regions of the terrein biosynthesis gene cluster, which led to the generation of heterologous expression systems in Aspergillus niger and Aspergillus oryzae using genetic elements from the A. terreus terrein biosynthesis gene cluster. Expression platform strains were generated that contained the terR gene either under a sugar- or doxycycline-inducible promoter. Then a gene of interest cloned under control of the terA promoter is highly transcribed by binding of TerR. Thereby, the doxycycline-dependent TetOn-terR system provides fine-tunable gene expression, which makes it suitable for the production of metabolites toxic to the producer. Furthermore, the use of viral self-splicing peptide sequences was successfully used to express fungal secondary metabolite biosynthesis genes from a polycistronic messenger. Therefore, the heterologous expression systems were subsequently exploited to study secondary metabolism in A. terreus and related species. This system essentially contributed to the identification of the true origin of the pigment in A. terreus conidia. This new type of melanin called Asp-melanin derives from the metabolite aspulvinone E that is produced by a non-ribosomal peptide synthetase- (NRPS)-like enzyme and is oxidised and activated for polymerisation by a tyrosinase. The pathway was successfully reconstituted in the heterologous expression system and, furthermore, under in vitro conditions. This Asp-melanin protects conidia from UV-radiation and reduces the attraction of soil amoeba, resulting in reduced phagocytosis rates. Subsequent studies revealed that Asp-melanin is common to species from section Terrei and is a discriminator from other Aspergillus sections. However, species less closely related to A. terreus show either no pigment biosynthesis pathway, a combination of the DHN-melanin and Asp-melanin pathway or a DHN-melanin pathway that has lost its transcriptional activation. Therefore, the lack of a functional DHN-melanin pathway accompanied by the evolution of the Asp-melanin pathway appears to describe a specific environmental adaptation of species from Terrei. The discovery of the contribution of an NRPS-like enzyme to pigment formation in A. terreus resulted in further interest in this class of enzymes. NRPS-like enzymes with a domain structure of adenylation, thiolation and thioesterase domain generally use two identical aromatic α-keto acids as substrates that get condensed under the formation of different interconnecting core structures such as bis-indolylquinones, terphenylquinones, dioxolanones or furanones with different substitution patterns. While it is known that core structure formation is specifically catalysed by the thioesterase domain, product-predictive sequence patterns had not been identified. Studies were undertaken to convert a furanone forming aspulvinone E synthetase into a quinone forming atromentin synthetase but approaches by site-directed mutagenesis failed. This indicated that the exchange of individual amino acids is not sufficient to re-direct the chemistry of a thioesterase domain. Domain-swapping experiments successfully converted an aspulvinone E synthetase into an atromentin synthetase, but only when the donor thioesterase domain derived from a phylogenetic closely related species. The reason for this was later identified by a detailed phylogenetic analysis of atromentin synthetases from basidio- and ascomycetes which showed that the phylogenetic origin of a species results in greater sequence differences than caused by differences in the chemistry of the thioesterase domain of an NRPS-like enzyme. Therefore, it can be assumed that a prototype of an NRPS-like enzyme may have been present in a common ancestor of basidio- and ascomycetes, but evolution of NRPS-like enzymes forming a specific metabolite occurred independent in the two fungal lineages. Unexpectedly, expression of NRPS-like enzymes producing quinone core structures resulted in different metabolites when expressed in A. niger compared to A. oryzae. Detailed analyses on the expression of atromentin synthetases in A. niger revealed that the formation of the quinone structure of atromentin is re-directed towards the formation of atrofuranic acid with a furanic acid core. Further analyses showed that this cross-chemistry enforced by the physiology of the producer is not limited to A. niger, but also observed in the black fungus Aspergillus brasiliensis from section Nigri that contains an intrinsic atromentin/atrofuranic acid synthetase. As this cross-chemistry seems to apply to all quinone core structures that were attempted to be produced in A. niger, host physiology may significantly influence product formation. While this broadens the spectrum of metabolites that can be obtained from NRPS-like enzymes, it may be recommended to use at least two different expression platforms with different physiology when investigating the metabolites produced from previously uncharacterised secondary metabolite producing enzymes. Further genome analyses from Aspergillus species from section Terrei revealed that a large number of NRPS-like enzymes with a C-terminal thioesterase domain is present in this section. Therefore, heterologous expression systems were used to obtain insights into the spectrum of metabolites produced in this section. NRPS-like enzymes were grouped into families and individual members selected for heterologous gene expression and product analysis. These analyses confirmed the broad spectrum of NRPS-like-derived metabolites in this section, but also revealed that attributing individual enzymes to specific families and to define structure-predicting sequences in the thioesterase domain requires the characterisation of additional enzymes, especially of those from outside of the genus Aspergillus. However, this study identified a putative phenylbutyrolactone IIa and a polyporic acid synthetase. Both enzymes had not previously been described from ascomycetes. It will be interesting to see whether phenylbutyrolactone IIa has similar quorum sensing effects on the producer Aspergillus ambiguus as observed for butyrolactone I on A. terreus. Furthermore, polyporic acid, a metabolite related to atromentin, has previously only been known from basidiomycetes. This study indicates that polyporic acid might also be produced by ascomycetes and provides the first example of a putative polyporic acid synthetase from any fungal source. Finally, it was of interest to obtain ideas on how these secondary metabolites could be further exploited. Therefore, an in silico target fishing approach was tested using atromentin as a model compound. This in silico modelling proposed a weak estrogenic activity on human estrogen receptors and a strong inhibitory function on 17-β-hydroxysteroid dehydrogenase as potential targets. Subsequent in vitro experiments were able to confirm the estrogenic activity of atromentin, which indicates that an in silico target fishing approach accompanied with in vitro experiments is suitable for target prediction and further exploitation of metabolites deriving from NRPS-like enzymes

Comment on: “Melanisation of Aspergillus terreus—Is Butyrolactone I Involved in the Regulation of Both DOPA and DHN Types of Pigments in Submerged Culture? Microorganisms 2017, 5, 22”

A recent article by Palonen et al. describes the effect of butyrolactone I on the expression of a secondary metabolite biosynthesis gene cluster from Aspergillus terreus that shows similarities to fusarubin biosynthesis gene clusters from Fusarium species. The authors claim that two different types of pigments are formed in Aspergillus terreus conidia, whereby one pigment is termed a DOPA-type melanin and the second a DHN-type melanin. Unfortunately, the terminology of the classification of melanin-types requires revision as Asp-melanin present in A. terreus conidia is clearly distinct from DOPA-melanins. In addition, some hypotheses in this manuscript are based on questionable data published previously, resulting in incorrect conclusions. Finally, as biochemical data are lacking and metabolite production is only deduced from bioinformatics and transcriptomic data, the production of a second pigment type in A. terreus conidia appears highly speculative

MOESM5 of ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger

Additional file 5: Table 1. Oligonucleotides used in this study