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Geib, Elena (2018) Exploitation of a novel heterologous expression system for characterisation of fungal secondary metabolites. PhD thesis, University of Nottingham.

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Abstract

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.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Brock, Matthias Avery, Simon
Keywords:	Aspergillus terreus; Inhibition of acidification; Melanin; Secondary metabolites; Atromentin; NRPS-like enzymes
Subjects:	Q Science > QR Microbiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	55449
Depositing User:	Geib, Elena
Date Deposited:	10 Dec 2020 08:19
Last Modified:	25 Jul 2024 14:26
URI:	https://eprints.nottingham.ac.uk/id/eprint/55449

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