RESUMEN
Streptomyces sp. BRA-346 is an Actinobacteria isolated from the Brazilian endemic tunicate Euherdmania sp. We have reported that this strain produces epoxyketone peptides, as dihydroeponemycin (DHE) and structurally related analogs. This cocktail of epoxyketone peptides inhibits the proteasome chymotrypsin-like activity and shows high cytotoxicity to glioma cells. However, low yields and poor reproducibility of epoxyketone peptides production by BRA-346 under laboratory cultivation have limited the isolation of epoxyketone peptides for additional studies. Here, we evaluated several cultivation methods using different culture media and chemical elicitors to increase the repertoire of peptide epoxyketone production by this bacterium. Furthermore, BRA-346 genome was sequenced, revealing its broad genetic potential, which is mostly hidden under laboratory conditions. By using specific growth conditions, we were able to evidence different classes of secondary metabolites produced by BRA-346. In addition, by combining genome mining with untargeted metabolomics, we could link the metabolites produced by BRA-346 to its genetic capacity and potential regulators. A single biosynthetic gene cluster (BGC) was related to the production of the target epoxyketone peptides by BRA-346. The candidate BGC displays conserved biosynthetic enzymes with the reported eponemycin (EPN) and TMC-86A (TMC) BGCs. The core of the putative epoxyketone peptide BGC (ORFs A-L), in which ORF A is a LuxR-like transcription factor, was cloned into a heterologous host. The recombinant organism was capable to produce TMC and EPN natural products, along with the biosynthetic intermediates DH-TMC and DHE, and additional congeners. A phylogenetic analysis of the epn/tmc BGC revealed related BGCs in public databases. Most of them carry a proteasome beta-subunit, however, lacking an assigned specialized metabolite. The retrieved BGCs also display a diversity of regulatory genes and TTA codons, indicating tight regulation of this BGC at the transcription and translational levels. These results demonstrate the plasticity of the epn/tmc BGC of BRA-346 in producing epoxyketone peptides and the feasibility of their production in a heterologous host. This work also highlights the capacity of BRA-346 to tightly regulate its secondary metabolism and shed light on how to awake silent gene clusters of Streptomyces sp. BRA-346 to allow the production of pharmacologically important biosynthetic products.
RESUMEN
Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.
RESUMEN
Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.
RESUMEN
Technologies based on synthetic biology to produce bacterial natural carotenoids depend on information regarding their biosynthesis. Although the biosynthetic pathway of common carotenoids is known, there are carotenoids whose pathways are not completely described. This work aimed to mine the genome of the deep-sea bacterium Erythrobacter citreus LAMA 915, an uncommon bacterium that forms yellow colonies under cultivation. This work further explores the potential application of the carotenoids found and low-cost substrates for bacterial growth. A combined approach of genome mining and untargeted metabolomics analysis was applied. The carotenoid erythroxanthin sulfate was detected in E. citreus LAMA 915 cell extract. A proposal for carotenoid biosynthesis by this bacterium is provided, involving the genes crtBIYZWG. These are responsible for the biosynthesis of carotenoids from the zeaxanthin pathway and their 2,2'-hydroxylated derivatives. E. citreus LAMA 915 extracts showed antioxidant and sun protection effects. Based on the high content of proteases and lipases, it was possible to rationally select substrates for bacterial growth, with residual oil from fish processing the best low-cost substrate selected. This work advances in the understanding of carotenoid biosynthesis and provides a genetic basis that can be further explored as a biotechnological route for carotenoid production.
