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| Author(s): |
Renata Sigrist
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Química |
| Defense date: | 2019-07-30 |
| Examining board members: |
Luciana Gonzaga de Oliveira;
Daniela Barretto Barbosa Trivella;
Mônica Tallarico Pupo;
Paulo Cezar Vieira;
Anita Jocelyne Marsaioli
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| Advisor: | Luciana Gonzaga de Oliveira |
| Abstract | |
Actinobacteria is a phylum of Gram-positive terrestrial or aquatic bacteria of significant importance for academic research and pharmaceutical industries due to their great potential to biosynthesize different types of natural products. Polyketides and nonribosomal peptides are produced by enzymes known as polyketide synthases and nonribosomal synthetase and stand out by combining high structural complexity with a wide range of therapeutic activities, such as antibiotics, antitumor and immunosuppressor. In order to accelerate identification of new therapeutic compounds, the biosynthetic genes encoding to these and other class of enzymes can be mapped by genome sequence and mining of different Streptomyces strains. The endophytic strain Streptomyces sp. CBMAI 2042, isolated from Citrus sinensis, was sequenced and in silico analysis endorsed identification of the orphan biosynthetic genes comprising a type I PKS, trans-AT PKS and NRPS. This study aimed to obtain this biosynthetic gene cluster from genomic libraries constructed in BAC and ESAC vectors and by recombination methodologies l- RED, Gibson Assembly® and TAR- cloning, followed by heterologous expression in S. coelicolor. The orphan cluster obtainen by TAR-cloning and transferred to S. coelicolor M1146 was associated to alpiniamide biosynthesis. Deletion of adenylation domain confirmed the biosynthetic origin of isolated molecule and experiments with labeled precursors embased the proposed biosynthetic steps for assembly of alpiniamide. Additionally, the gene cluster related to biosynthesis of the already described valinomycin was identified from genome annotation and also screened for clones in the genomic library, as it is the major metabolite produced by CBMAI 2042. Cloning and heterologous expression in S. coelicolor M1146 resulted in 3 times increase in production of valinomycin and association with molecular networking allowed the identification of at least six analogues, including montanastatin, related for the first time to the same biosynthetic pathway (AU) | |
| FAPESP's process: | 15/01013-4 - Mining a t1PKS-transAT PKS-NRPS in Streptomyces sp. genome |
| Grantee: | Renata Sigrist |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |