Periodic Reporting for period 1 - CyanoAsterisMETAB (Exploration of the biosynthetic potential of the secondary metabolome of the newly identified fungal endophyte Cyanodermella asteris)
Reporting period: 2023-01-01 to 2025-01-31
Traditionally, identification of bioactive compounds from different natural sources (e.g. plants, fungi and bacteria) has always been an important avenue for pharmaceutical discovery efforts. Due to these endeavors, various classes of pharmaceuticals, such as antibiotics (e.g. -lactams, tetracycline, macrolides), antiparasitics (e.g. avermectin), antiprotozoals (e.g. artemisinin), immunosuppressive drugs (e.g. rapamycins) and antineoplastic agents (e.g. paclitaxel, doxorubicin) have been discovered and proved to be highly relevant for clinical application. In this regard, The Fungal Kingdom has played a key role. Especially after the groundbreaking discovery of penicillin, the world experienced an enormous interest to explore various fungal sources aiming to identify new antimicrobial compounds and other medically useful agents, such as the lipid-lowering agents (e.g. lovastatin) and immunosuppressive drugs (e.g. cyclosporine). Nowadays, an increasing interest for fungal natural products (NPs) has been encountered. This is mainly triggered by the availability of whole-genome information for different fungal species and by the tremendous progress in bioinformatics, genetic manipulation techniques and analytical technologies. Information gained from whole-genome sequences of different fungal species through powerful bioinformatic tools made us understand that the amount of NPs so far isolated from the fungal world is just a drop in the ocean compared to the great potential of NPs waiting to be explored. Hence, all these multidisciplinary advancements coupled with the urgent need for new antibiotics to treat life-threatening infections from resistant microorganisms, have once again made fungal-based NPs an attractive source for drug leads. Within the fungal world, endophytic fungi comprise an important source of species with multiple implications. An endophyte is a microorganism (fungi or bacteria) that lives within the plant tissues without causing apparent harmful health effects for the plant itself. Interestingly, in several cases it was demonstrated that fungal endophytes are responsible for the production of bioactive compounds which were originally reported as products of the respective fungal host plants. To illustrate this concept, currently it was demonstrated the direct implication of fungal endophytes in the biosynthesis of the anti-tumor/immunosuppressive astins which were originally reported to be produced by the traditional Chinese medicinal plant Aster tataricus L. The fungal endophyte involved in astins biosynthesis was isolated from flowering parts of A. tataricus L. and named Cyanodermella asteris. Since the majority of biosynthetic gene clusters (BGCs) from C. asteris seem to be silent or expressed in very low yields under laboratory conditions, we hypothesize that C. asteris possesses a substantial hidden potential for novel and possibly bioactive natural products (NPs). In this regard, we explored several in silico-predicted BGCs, which have been examined within the framework of this project. Hence, the main aim of this project was to elucidate the chemical products of the selected BGCs by cloning, heterologous expression as well as via metabolome mining and secondary metabolomics of the wild-type species C. asteris.
The main objectives of the intended project are:
- Objective 1: Cloning and vector construction of the selected BGCs. The aim of this objective was that bioinformatically selected BGCs to be cloned in suitable vectors for fungal transformation.
- Objective 2: Heterologous expression of selected BGCs in suitable fungal hosts. The purpose of this objective was that constructed vectors harboring BGCs of interest to be transformed via protoplastation into heterologous fungal hosts such as Aspergillus oryzae.
- Objective 3: HRMS-based metabolomics and dereplication of comparative metabolomes. The purpose of this objective was annotation of metabolites making the difference between transformed and native (isogenic) fungal hosts via global untargeted metabolomics comparison. In case of biomarker annotation, dereplication strategies using in-house and comprehensive external databases (e.g. Antibase) as well as online tandem MS-based infrastructures (e.g. GNPS) will be used. Metabolome mining of the wild-type was also explored in detail.
- Objective 4: Isolation and structure elucidation of novel natural products from selected BGCs. The aim of the last objective was to perform classical compound purification and subsequent structural elucidation in the case of unsuccessful dereplication due to structural novelty.
The main objectives of the intended project are:
- Objective 1: Cloning and vector construction of the selected BGCs. The aim of this objective was that bioinformatically selected BGCs to be cloned in suitable vectors for fungal transformation.
- Objective 2: Heterologous expression of selected BGCs in suitable fungal hosts. The purpose of this objective was that constructed vectors harboring BGCs of interest to be transformed via protoplastation into heterologous fungal hosts such as Aspergillus oryzae.
- Objective 3: HRMS-based metabolomics and dereplication of comparative metabolomes. The purpose of this objective was annotation of metabolites making the difference between transformed and native (isogenic) fungal hosts via global untargeted metabolomics comparison. In case of biomarker annotation, dereplication strategies using in-house and comprehensive external databases (e.g. Antibase) as well as online tandem MS-based infrastructures (e.g. GNPS) will be used. Metabolome mining of the wild-type was also explored in detail.
- Objective 4: Isolation and structure elucidation of novel natural products from selected BGCs. The aim of the last objective was to perform classical compound purification and subsequent structural elucidation in the case of unsuccessful dereplication due to structural novelty.
The work carried out in the framework of this project has shed new light in understanding the biosynthetic and metabolic behavior of the fungal endophyte Cyanodermella asteris and subsequently has increased our general vision towards exploitation of endophytic microorganisms for novel natural product pharmacophores. Milestones were achieved for each work package. The genome of C. asteris was carefully inspected via genome mining employing different bioinformatic tools (e.g. fungiSMASH). After detailed genome mining, the following polyketide synthase (PKS) biosynthetic gene clusters (BGCs), including PKS1.7 PKS3.6 PKS4.3 PKS 5.2 PKS6.3 PKS9.2 and PKS13.5 were selected and cloned in suitable vectors (WP1). BGC selection and cloning was followed by heterologous expression in the fungal host Aspergillus oryzae NSAR1 (WP2). Untargeted metabolomics profiling of the heterologously transformed strains as well as wild-type strains was performed via different multi-variate analysis and dimension-reduction techniques. In this connection, Global Natural Product Social (GNPS) molecular networking in combination with detailed interpretation of MSMS spectra was employed successfully and demonstrated to be a valuable tool in understanding the complexity of fungal metabolomes (WP3). The successful implementation of the analytical strategy based on high resolution mass spectrometry (HRMS) and molecular networking has resulted in a manuscript explaining the chemodiversity of halogenated cyclopentapeptides (astins and cyclochlorotines) in nature. Isolation and characterisation of novel natural products was successful in the case of cyclochlorotines (WP4) and is currently being finalized which will be part of the publication in a respected journal of the field. The dissemination of results from this project and the overall engagement of the fellow researcher at the Gulder lab occurred in several publications, both already published and in preparation (WP5), in addition to attending both national and international conferences.
All selected core and tailoring genes from C.asteris were successfully cloned in adequate vectors. Positive E. coli colonies harbouring the plasmid with our insert were selected via colony PCR. Afterwards, the successful integration of the insert to the respective plasmid was confirmed through restriction digest of the final vector as well as via Sanger sequencing. Constructed vectors were successfully transformed in A. oryzae. Although the heterologous transformation was implemented, unfortunately these genes were not expressed in the metabolite level. This could be due to incorrect processing of C. asteris introns by A. oryzae. Aiming to address this issue, we extracted mRNA from C. asteris, however the amplification of genes of interest (generation of cDNA) from the extracted RNA was not successful most likely due to the possibility of silent BGCs under standard laboratory conditions. Another explanation for the unsuccessful expression could be differences in codon usage between C. asteris and A. oryzae, hence future research in C. asteris BGCs should seriously consider codon optimization before heterologous expression.
Although we did not identify meaningful metabolites for the mutant strains, we successfully applied untargeted metabolic profiling in wild-type strains. Metabolic profiling and HRMS data unveiled a huge chemical diversity among astin and cyclochlorotine pool of pentapeptides. In the case of astins, the chemodiversity shows to be higher in A. tataricus extracts as compared to its endophytic fungus. C. asteris appears to find a better fungal fitness in planta which results in a more prolific secondary metabolome. The genetic homology of both non-ribosomal peptide synthetase (NRPS) systems is also reflected in the metabolome level since homologous compounds are produced by both fungal species, C. asteris and Talaromyces islandicus. Moreover, two identical compounds were identified in both NRPS systems which further supports the idea for a shared logic of biosynthesis. Moreover several cyclochlorotine derivatives were isolated and their structure was unambiguously characterized by NMR and Marfey’s analysis. The scientific findings from this project will truly assist in solving the biosynthetic enigma of astins. More research is needed to shed new light into astins/cyclochlorotines biochemistry which consequently would encapsulate our understanding regarding the enzymology of the intriguing DUF3328 proteins. In this context, focus should be drawn to the DUF3328-based halogenases (e.g. AstS) which seem to catalyze chlorination in non-activated carbons in a novel mechanism of halogenation.
Exploitation of the results will be achieved through the publication of open access scientific publications and scientific conferences. Already published publications as well as upcoming ones have been disseminated to the community via online repositories including LinkedIn, ResearchGate, X and BlueSky. The impact of the analytical methodology based on HRMS, multivariate statistics, molecular networking and MSMS fragmentation data implemented by the fellow researcher in the entire group of the Gulder lab was significant. Exploitation of this methodology has occurred through successful one-to-one or group teaching with Master/PhD students. This will surely facilitate their workflow especially in terms of dereplicating the already known natural products and avoiding the need for the time-consuming compound isolation and NMR analysis. Most of the PhD students are now utilizing this methodology for their own projects. This is likely to result in additional publications and future collaborations for the fellow researcher.
Although we did not identify meaningful metabolites for the mutant strains, we successfully applied untargeted metabolic profiling in wild-type strains. Metabolic profiling and HRMS data unveiled a huge chemical diversity among astin and cyclochlorotine pool of pentapeptides. In the case of astins, the chemodiversity shows to be higher in A. tataricus extracts as compared to its endophytic fungus. C. asteris appears to find a better fungal fitness in planta which results in a more prolific secondary metabolome. The genetic homology of both non-ribosomal peptide synthetase (NRPS) systems is also reflected in the metabolome level since homologous compounds are produced by both fungal species, C. asteris and Talaromyces islandicus. Moreover, two identical compounds were identified in both NRPS systems which further supports the idea for a shared logic of biosynthesis. Moreover several cyclochlorotine derivatives were isolated and their structure was unambiguously characterized by NMR and Marfey’s analysis. The scientific findings from this project will truly assist in solving the biosynthetic enigma of astins. More research is needed to shed new light into astins/cyclochlorotines biochemistry which consequently would encapsulate our understanding regarding the enzymology of the intriguing DUF3328 proteins. In this context, focus should be drawn to the DUF3328-based halogenases (e.g. AstS) which seem to catalyze chlorination in non-activated carbons in a novel mechanism of halogenation.
Exploitation of the results will be achieved through the publication of open access scientific publications and scientific conferences. Already published publications as well as upcoming ones have been disseminated to the community via online repositories including LinkedIn, ResearchGate, X and BlueSky. The impact of the analytical methodology based on HRMS, multivariate statistics, molecular networking and MSMS fragmentation data implemented by the fellow researcher in the entire group of the Gulder lab was significant. Exploitation of this methodology has occurred through successful one-to-one or group teaching with Master/PhD students. This will surely facilitate their workflow especially in terms of dereplicating the already known natural products and avoiding the need for the time-consuming compound isolation and NMR analysis. Most of the PhD students are now utilizing this methodology for their own projects. This is likely to result in additional publications and future collaborations for the fellow researcher.