Terpenes are the most abundant class of natural product that exist in nature. They possess a myriad of industrial applications including pharmaceutical, perfumery and flavors, bulk chemicals, and fuel. Intriguingly, until today, the vast majority of characterized terpenoids have been isolated from plants and fungi, and only in recent years bacteria were found to generate a representative reservoir of terpenoids molecules. Mining Streptomyces sp. CBMAI 2042 genome data has revealed the presence of five terpene cyclase genes. Chemical analysis of mycelium extract of this bacteria strain has unveiled at least 28 volatile terpenes molecules, where three encoding sesquiterpene cyclase (STC) genes are apparently responsible for their biosynthesis. The cyclic products obtained by incubation of these three purified recombinant STCs with farnesyl diphosphate (FPP) were analyzed by gas chromatography-mass spectrometry (GC-MS) and identified using the Van den Dool and Kratz equation.

The genus Streptomyces represents one of the largest producers of molecules with antibiotic activity. The whole genome sequencing of the endophytic microorganism Streptomyces sp. CBMAI 2042 revealed 35 gene clusters encoding for secondary metabolism including 3 non-ribosomal peptide synthetases (NRPS) and 7 NRPS-hybrids. Combining genome mining and cultivation profile analysis, the depsipeptide ionophore valinomycin was identified as one of the main metabolites produced by this strain. To better understand the metabolic machinery codified in CBMAI 2042 genome an adenylation domain from a hybrid NRPS cluster was deleted through a double crossing knockout experiment. Though the deletion was not plentiful to elucidate the encoded NRP metabolite related to the adenilation domain, an astounding increase of 10.5-fold in valinomycin production was observed in the mutant. These results suggest a metabolic flux redistribution of common substrates as an outcome of a gene target deletion.

Natural products continue to inspire scientists on the search for biological active structures. Recent advances in synthetic biology allow modifying genes to extend structural diversity present on natural products by changing important functional groups guided through genetic information. In this context, polyketides synthases (PKS) represent a class of enzymes from where the first applications and well succeed modifications were achieved. During the last two decades PKSs have been extensively studied, sequenced and deposited in data banks available to scientific community around the world. This newly wide information associated to different methodologies accessible to modify the PKSs genetic circuits including genetic insertion, gene deletion and gene replacement leads to discovery of new natural products. In this review, the recent advances in combinatorial biosynthesis, as well as modern techniques in synthetic biology to promote structural diversity on natural products will be discussed.

The actinobacteria Streptomyces wadayamensis A23, an endophitic strain, was recently sequenced and previous work showed qualitatively that the strain inhibits the growth of some pathogens. Herein we report the genome analysis of S. wadayamensis which reveals several antibiotic biosynthetic pathways. Using mass spectrometry, we were able to identify desferoxamines, several antimycins and candicidin, as predicted. Additionally, it was possible to confirm that the biosynthetic machinery of the strain when compared to identified known metabolites is far underestimated. As suggested by biochemical qualitative tests, genome encoded information reveals that the strain A23 has high capability to produce antibiotics.

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains.

Um ensaio enzimático miniaturizado com sondas fluorescentes para triagem rápida de microrganismos produtores de esterases foi implementado e aplicado a 64 linhagens de bactérias de solo. Os melhores resultados foram validados por métodos convencionais e substratos não fluorogênicos (feniletanol acetilado e propanoilado) e confirmaram os resultados obtidos nas triagens rápidas. Os microrganismos que apresentaram atividades enzimáticas mais relevantes (razão enantiomérica E > 30) na hidrólise de ésteres e na obtenção de boas razões enantioméricas foram identificados como pertencentes ao gênero Bacillus. Parte dos microrganismos se encontra preservada em glicerol e/ou liofilizada no IQ/Unicamp e três linhagens serão depositadas na CBMAI do CPQBA/Unicamp como depósitos abertos.

A miniaturized enzymatic assay using fluorescent probes to reveal esterase producing microorganisms was optimized and applied to screen 64 soil bacterial strains. The best results were validated using traditional non-fluorogenic assays with acetyl and propanoyl phenylethanol to confirm the miniaturized results. The most active microorganisms belong to the genus Bacillus showing esterase activity and good enantiomeric ratios for the resolution of phenylethanol derivatives (E > 30). Part of the microorganisms are kept in our laboratory in glycerol or freeze-dried and the best microorganisms will be deposited in the CBMAI/CPQBA/Unicamp culture collection.

This review describes the endeavors that led to the total synthesis of a novel class of antibiotic compounds: the crocacins A-D. Other aspects such as isolation, structural elucidation as well as the biological activities are also presented.