Solid phase microextraction as a powerful alternative for screening of secondary metabolites in actinomycetes.

Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non-exhaustive, minimally invasive, and non-destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro-scale and direct immersion SPME (DI-SPME) for screening of secondary metabolites, describes the optimization of the developed DI-SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI-SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro-scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI-SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME-LC-MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI-SPME is herein presented as an alternative to micro-scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI-IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI-SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium.

Bioguided isolation, characterization and media optimization for production of Lysolipins by actinomycete as antimicrobial compound against Xanthomonas citri subsp. citri.

Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8 µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.

Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI-MS).

The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract ᅟ.