Bioinformatics and biogeography to mine natural products in metagenomes

Author(s)

Ulysses Amâncio de Frias

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-12-14

Examining board members

Monica Tallarico Pupo; Hosana Maria Debonsi; Luciana Gonzaga de Oliveira; Nelilma Correia Romeiro; José Freire da Silva Neto

• Advisor: Monica Tallarico Pupo

Abstract

Microbial natural products (NP) have proven to be invaluable starting points in the discovery and development of many drugs approved by FDA. The traditional approach to identify microbial natural products requires the culturing in the laboratory. Unfortunately, conventional culture-based methods have been deemphasized due to high rediscovery rates. Culture-independent methods applying microbial (meta)genome sequencing suggest the occurrence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters (BGCs) in the environment. Here we have used a PCR-based approach and barcoding ampliconsequencing derived from important families of microbial natural products such as nonribosomal peptides (NRP), polyketides (PK), 3-amino-5-hydroxybenzoic acidcontaining NPs (AHBA), tryptophan dimmers (TD), aminoglycosides, phosphonocontaining NPs and others. We have written an internal script called SecMetPrimer that allowed us to bioinformatically design sets of primers containing a range of degeneracy to amplify these genes. At the total, we designed 165 different sets of primers. The amplicons were obtained by standard PCR containing double-barcodedtarget primers and sequenced by Illumina MiSeq platform. The validation process was conducted using eDNA from metagenomic libraries containing a 223 millions of clones. The rarefaction and diversity analyses were assigned, and the best-hit primer for each family was chosen. We have re-amplified the nonribosomal peptide adenylation domains and polyketide ketosynthase domains, using as substrate environmental DNA isolated from 25 different samples collected in Atlantic Forest, Cerrado and marine environment. Our data indicate a correlation between geographic distance and biome-type, and the biosynthetic diversity found in these environments. Thus, by assigning reads to known BGCs against taxonomic and metabolic profiles, we have identified the hotspots of relevant biosynthetic diversity among the analyzed samples. (AU)

• FAPESP's process: 12/16139-5 - Search of microbial natural products using metagenomics
• Grantee: Ulysses Amancio de Frias
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)