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| Author(s): |
Lamonielli Faga Michaliski
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT) |
| Defense date: | 2024-06-10 |
| Examining board members: |
Roberto Gomes de Souza Berlinck;
Hector Henrique Ferreira Koolen;
Edson Rodrigues Filho
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| Advisor: | Roberto Gomes de Souza Berlinck |
| Abstract | |
Microorganisms from isolated environments can exhibit significantly differentiated metabolic potential, producing bioactive substances in culture media. This project aimed to investigate the secondary metabolism of endophytic and Antarctic microorganisms, aiming at the discovery of bioactive natural products. A strain of endophytic actinomycete was selected for cultivation on large scale. The chemical investigation of the secondary metabolites produced in culture by this strain led to the isolation of a macrocyclic polyketide, of which the planar structure and relative configuration were determined by NMR experiments. Its partial absolute configuration was proposed by genomic analysis of this bacterial strain. The project also aimed to investigate the biosynthesis of α-aminopyrones, a class of uncommon bioactive substances produced only by fungi of the Aspergillus genus. The skeleton of α-aminopyrones indicates that they are of a mixed biosynthetic origin, PKS-NRPS type. The genome (gDNA) of the wild-type strain of Aspergillus sp. DLM38 was sequenced. Bioinformatics analyses prioritized two biosynthetic gene clusters (BGCs) that may be responsible for the biosynthesis of α-aminopyrones produced by this strain, isopyrophen and naphthoquinoneimine. Using genetic engineering tools to inactivate these BGCs, the objective was to verify if the production of α-aminopyrones is interrupted in their absence. Mutants were constructed via homologous recombination and evaluated by diagnostic PCR. The chemical profiles produced in culture by the wild-type strain and the mutant strains were compared by UPLC-HRMS with the previously isolated isopyrophen and naphthoquinoneimine standards. Production of the targeted α-aminopyrones was completely abolished in the mutant strains, indicating that the BGCs studied are involved in the biosynthesis of these compounds by Aspergillus sp. DLM38. In a BEPE internship abroad linked to this Ph.D. project, we proposed to further investigate the biosynthesis of phomactins produced by the marine fungal strain Biatriospora sp. CBMAI 1333. After sequencing the complete gDNA of this strain, we identified four putative diterpene cyclases (DTCs) and two geranylgeranyl diphosphate synthases (GGPPSs) that could be involved in the biosynthesis of phomactins. The four putative DTCs were expressed in Aspergillus nidulans together with one of the two identified GGPPSs, to supplement the GGPP precursor for the DTCs. The production of metabolites by the transformed strains was investigated by GC-MS analysis, and four transformant strains, DTC1-4+GGPPS1, were investigated on a larger scale. The diterpenes produced by DTC1, DTC2, and DTC4 were isolated, and identified by NMR analyses. The product of DTC3 was identified by GC-MS. Additionally, an advanced biosynthetic product of the DTC2 cluster was identified through the expression of its additional modification enzymes, oxidoreductases. The identified compounds belong to the family of pimarane diterpenes. The advanced biosynthetic product encoded by the DTC2 cluster has not yet been reported from fungal cultures. (AU) | |
| FAPESP's process: | 19/07894-3 - Discovery of bioactive secondary metabolites produced by microbial strains from isolated environments and study of the biosynthesis of alpha-aminopyrones produced by Aspergillus sp. DLM38 |
| Grantee: | Lamonielli Fagá Michaliski |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |