Advanced techniques can accelerate the pace of natural product discovery from microbes, which has been lagging behind the drug discovery era. Therefore, the present review article discusses the various interdisciplinary and cutting-edge techniques to present a concrete strategy that enables the high-throughput screening of novel natural compounds (NCs) from known microbes. Recent bioinformatics methods revealed that the microbial genome contains a huge untapped reservoir of silent biosynthetic gene clusters (BGC). This article describes several methods to identify the microbial strains with hidden mines of silent BGCs. Moreover, antiSMASH 5.0 is a free, accurate, and highly reliable bioinformatics tool discussed in detail to identify silent BGCs in the microbial genome. Further, the latest microbial culture technique, HiTES (high-throughput elicitor screening), has been detailed for the expression of silent BGCs using 500-1000 different growth conditions at a time. Following the expression of silent BGCs, the latest mass spectrometry methods are highlighted to identify the NCs. The recently emerged LAESI-IMS (laser ablation electrospray ionization-imaging mass spectrometry) technique, which enables the rapid identification of novel NCs directly from microtiter plates, is presented in detail. Finally, various trending \'dereplication\' strategies are emphasized to increase the effectiveness of NC screening.

Cheesemaking played a pivotal role in the life of the Pyrenean villages where cheese was a most prized commodity and the subject of much local competition. In one of them (Sasa de Sobrepuerto), Mrs. Sebastiana Palacio decided in 1877 to label all the cheeses made in her household with a seal to differentiate them from those made by other local producers. The cheese seal was last used in 1936 and, since then, it has been kept under excellent storage conditions. Since well-preserved cheese seals are rare, and bacterial cells may survive desiccation for long periods, the objective of this work was to isolate and characterize any lactic acid bacteria that survived in the seal. Analysis of the milky crust material revealed the presence of sheep caseins. Culture-based analysis led to the isolation of a strain of Bacillus licheniformis and a strain of Ligilactobacillus salivarius (L. salivarius SP36). The latter was characterized in vitro for safety and dairy-related functional properties. Its genome encodes several genes involved in protein, peptide, and amino acid catabolism, and flavor. Overall, the phenotypic and genetic features of this strain support a high potential for being used as adjunct culture in cheesemaking.

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 ºC), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95% conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations.

Fungal azaphilones have attracted widespread attention due to their significant potential as sources of food pigments and pharmaceuticals. Genome mining and gene cluster activation represent powerful tools and strategies for discovering novel natural products and bioactive molecules. Here, a putative azaphilone biosynthetic gene cluster lut from the endophytic fungus Talaromyces sp. was identified through genome mining. By overexpressing the pathway-specific transcription factor LutB, five new sclerotiorin-type azaphilones (1, 6, 8, and 10-11) together with seven known analogues (2-5, 7, 9, 12) were successfully produced. Compounds 8 and 9 exhibited antibacterial activity against Bacillus subtilis with MIC values of 64 and 16 μg/mL, respectively. Compound 11 showed cytotoxic activity against HCT116 and GES-1 with IC50 values of 10.9 and 4.9 μM, respectively, while 1, 4, 5, and 7-10 showed no obvious cytotoxic activity. Gene inactivation experiments confirmed the role of the lut cluster in the production of compounds 1-12. Subsequent feeding experiments unveiled the novel functional diversity of the dual megasynthase system. Furthermore, a LutC-LutD binary oxidoreductase system was discovered, and in combination with DFT calculations, the basic biosynthetic pathway of the sclerotiorin-type azaphilones was characterized. This study provided a good example for the discovery of new azaphilones and further uncovered the biosynthesis of these compounds.

Bacterial endophytes dwelling in medicinal plants represent an as yet underexplored source of bioactive natural products with the potential to be developed into drugs against various human diseases. For the first time, several Streptomyces spp. were isolated from the rare and endangered traditional medicinal plant Leontopodium nivale ssp. alpinum, also known as Edelweiss. In the search for novel natural products, nine endophytic Streptomyces spp. from Edelweiss were investigated via genome sequencing and analysis, followed by fermentation in different media and investigation of secondary metabolomes. A total of 214 secondary metabolite biosynthetic gene clusters (BGCs), of which 35 are presumably unique, were identified by the bioinformatics tool antiSMASH in the genomes of these isolates. LC-MS analyses of the secondary metabolomes of these isolates revealed their potential to produce both known and presumably novel secondary metabolites, whereby most of the identified molecules could be linked to their cognate BGCs. This work sets the stage for further investigation of endophytic streptomycetes from Edelweiss aimed at the discovery and characterization of novel bioactive natural products.

Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.

Magnesium ions (Mg2+) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation. In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis. Notably, we determined its crystal structure and identified Mg2+ within its active site. This finding has shed light on the previously elusive question of Mg2+ binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme, including steps for its purification, crystallization, and kinetic analysis.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied.
RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin.
CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Streptomyces species are attractive sources of secondary metabolites that serve as major sources of antibiotics and other drugs. In this study, genome mining was used to determine the biosynthetic potential of Streptomyces sp. 21So2-11 isolated from Antarctic soil. 16S rRNA gene sequencing revealed that this strain is most closely related to Streptomyces drozdowiczii NBRC 101007T, with a similarity of 98.02%. Genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) showed that strain 21So2-11 represents a novel species of the genus Streptomyces. In addition to a large number of genes related to environmental adaptation and ecological function, a total of 28 putative biosynthetic gene clusters (BGCs) responsible for the biosynthesis of known and/or novel secondary metabolites, including terpenes, lantipeptides, polyketides, nonribosomal peptides, RiPPs and siderophores, were detected in the genome of strain 21So2-11. In addition, a total of 1456 BGCs were predicted to contribute to the biosynthesis of more than 300 secondary metabolites based on the genomes of 47 Streptomyces strains originating from polar regions. The results indicate the potential of Streptomyces sp. 21So2-11 for bioactive secondary metabolite production and are helpful for understanding bacterial adaptability and ecological function in cold terrestrial environments.

Lectins are proteins that bind specifically and reversibly to carbohydrates, and some of them have significant anti-tumor activities. Compared to those of lectins from land plants, there are far fewer studies on algal lectins, despite of the high biodiversity of algae. However, canonical strategies based on chromatographic feature-oriented screening cannot satisfy the requirement for algal lectin discovery. In this study, prospecting for novel OAAH family lectins throughout 358 genomes of red algae and cyanobacteria was conducted. Then 35 candidate lectins and 1843 of their simulated mutated forms were virtually screened based on predicted binding specificities to characteristic carbohydrates on cancer cells inferred by a deep learning model. A new lectin, named Siye, was discovered in Kappaphycus alvarezii genome and further verified on different cancer cells. Without causing agglutination of erythrocytes, Siye showed significant cytotoxicity to four human cancer cell lines (IC50 values ranging from 0.11 to 3.95 μg/mL), including breast adenocarcinoma HCC1937, lung carcinoma A549, liver cancer HepG2 and romyelocytic leukemia HL60. And the cytotoxicity was induced through promoting apoptosis by regulating the caspase and the p53 pathway within 24 h. This study testifies the feasibility and efficiency of the genome mining guided by evolutionary theory and artificial intelligence in the discovery of algal lectins.