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This book provides in-depth information about the ecology, diversity and applications of Actinomycetes. The book is divided into two major parts. The first part discusses the diversity, chemical biology and ecology of Actinomycetes. It also covers the discovery of natural products from soil, endophytic and marine-derived Actinomycetes. It includes natural product discovery, chemical biology, new methods for discovering secondary metabolites, structure elucidation and biosynthetic research of natural products. The chapters in this part focus on the effects of biological and chemical elicitation at molecular level on secondary metabolism in Actinomycetes. The second part of the book discusses genomic and synthetic biology approaches in Actinomycetes drug discovery. This part includes chapters focused on the application of metabolic engineering to optimize natural product synthesis and the use of omics data in engineering of regulatory genes. It covers the advanced tools of synthetic biology and metabolic engineering including cluster assembly, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction in Actinomycetes. It describes the use of bioinformatics tools for reprogramming of biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These advanced genomic and molecular tools are expected to accelerate the discovery and development of new natural products from Actinomycetes with medicinal and other industrial applications. The book is useful to researchers and students in the field of microbiology, pharmaceutical sciences and drug discovery.
This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The third of 3 volumes covering Natural product biosynthesis by microorganisms and plants. This new volume continues the legacy of this premier serial contains quality chapters authored by leaders in the field. The third of 3 volumes, it has chapters on such topics as metabolic pathways in Aspergillus oryzae, heterologous gene clusters and cyanobacteria as a source of natural products.
Heterologous expression of natural product biosynthetic pathways is of rising interest to fungal bioactive compound discovery and engineering. However, most eukaryotic genes are regulated by individual regulatory elements and transcribed as monocistronic mRNA. Therefore, regardless of plasmid transformation or chromosomal integration of DNA, reconstruction and expression of biosynthetic gene clusters in the eukaryotic recipient require improved assembling pipeline of vectors and coordinated control of expression of multiple genes. Moreover, valuable medicine biosynthesis based on heterologous expression remains an ongoing challenge owing to the strict metabolic regulation in the chosen host. To circumvent these obstacles, a specialized vector was designed, for flexible but modularized construction of multiple genes. In the scheme, TEV protease and its recognition site combined with a previously documented P2A-mediated expression system, allow expression of eukaryotic genes in a “polycistronic” mRNA by co-translational and post-translational cleavages. As another highlight, the split fluorescent protein was introduced for visualizing and rapid screening of fungal transformants. On the basis of this new tool, the austinoid biosynthetic pathway has been rewired successfully in A. nidulans. Also, it represents the first-time producing psilocybin heterologously using fermenting condition, from which the high titer 15.4 mg/g (110 mg/L) was achieved. Subsequently, the de-branching strategy was beneficial for the yield of psilocybin, which even lifted to 57 mg/g in the yeast cell-based factory. Meanwhile, the Trp auxotrophic strain has been endowed with the capability to synthesize isotope-labeled psilocybin in an atom-economic manner.
This book summarizes the basics of actinobacteria, from microbiology to synthetic biology. It focuses on diversity, NRPS, sesquiterpenes, lantipeptide, bioinformatics apparatuses, cloning, CRISPR, reverse engineering, FDA supported medications, and marine actinobacteria. It also covers the latest trends in drug discovery from actinobacteria, and introduces several recently developed bioinformatics and synthetic biology tools to explore new antibiotics from actinobacteria. Many natural products such as polyketides, isoprenoids, phenazines, peptides, indolocarbarbazoles, sterols, and others have been isolated and characterized from actinobacteria. Some products are synthesized by the non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), or other functional genes. Although genome sequencing has uncovered the differing qualities of these chemicals, recognizing new items and their biosynthetic pathways is still under examination. Cryptic metabolic pathways have been explored using molecular techniques or culture-dependent approaches. In recent years, researchers’ primary interest is to identify the specific conditions or agents that wake the cryptic antibiotics. Several bioinformatics and synthetic biology tools were developed to explore new antibiotics from actinobacteria. The book comprises 14 chapters with different aspects of application and utilization of actinomycetes from the microbiology; systems biology, pharmacology of natural products, bioinformatics, actinomycete and its diversity, CRISPR, artificial Intelligence, synthetic biology, metabolic engineering, expressional studies, and biosynthetic gene clusters. The book delivers useful information on actinomyces to researchers, novices in genome designing, specialists, clinicians, policymakers, and professionals.
Natural product biosynthesis in Streptomyces venezuelae ISP5230 was investigated in three ways. First, through precursor-directed jadomycin biosynthesis using non-proteinogenic amino acids (4-(aminomethyl)benzoic acid, 3-(aminomethyl)benzoic acid, 8-aminooctanoic acid), five novel jadomycins were isolated and characterized. Four novel jadomycins were evaluated for cytotoxic activity in drug-sensitive and drug-resistant cancer cells and a panel of six microbes. Next, jadomycin C-glycoside production was attempted by heterologous expression of urdGT2 in place of the native O-glycosyltransferase, jadS. C-glycoside production was not observed from three complementation methods pursued. Finally, a S. venezuelae mutant was prepared to evaluate a silent biosynthetic gene cluster identified through genome mining. While the changes to the biosynthetic profile were apparent in the activator overexpression mutant, the major products detected were previously identified metabolites of tryptophan, including tryptophol. The studies demonstrated the pliability of S. venezuelae ISP5230 as a producer of natural products through manipulations to growth conditions, supplementation, and genetic variation.
Actinomycetes are known as a treasure trove for bioactive compounds as they are the origin of about two-thirds of all antibiotics in clinical use. Unique habitats such as marine and unexplored habitats are considered promising sources for sampling new actinomycetes with a high potential to produce new natural products. Indonesia is one of the countries with the most extensive mangrove marine ecosystems and a vast diversity of microbial species. Thus, exploration of Indonesian samples for actinomycetes may lead to the discovery of new antibiotics. About 422 strains of actinomycetes were isolated from marine sediments of Bali and Lombok Island and soil samples from Enggano Island. The nine most bioactive strains have been prioritized for further drug screening approaches. The nine strains were cultivated on different solid and liquid media using the OSMAC cultivation strategy to assess their biosynthetic capacity to produce natural compounds. A combination of genome mining and mass spectrometry-based (MS) molecular network analysis was applied to identify potential new substances from the nine strains. Several biosynthetic products encoded by the gene clusters of the nine strains were identified, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Additionally, sixteen putative biosynthesis products and numerous biosynthetic gene clusters were found, which could not be associated with any known compound or biosynthetic gene cluster, respectively, indicating that the nine Indonesian strains have the potential to produce new secondary metabolites. Furthermore, a regulator-driven strategy was applied to activate (silent) gene clusters in the Indonesian strains. As an example, the SARP-type activator PapR2 was expressed in the Indonesian strain isolate SHP 22-7 to unlock its chemical diversity. Thereby it could be shown that PapR2 expression activates the biosynthesis of the nucleoside antibiotic plicacetin in SHP 22-7. The increased transcription of the plicacetin cluster was detected by RT-PCR analysis and production of the compound was confirmed by HPLC-MS analysis. Overall, the methodologies described here represent promising strategies for acquiring new natural products from well-known bacterial sources. In addition, the reported unidentified compounds provide a basis for the further characterization and development of these compounds as drug candidates.
Beyond being the most important natural compound source, actinomycetes are the origin of up to two-thirds of all clinically used antibiotics. Currently, new antimicrobials are urgently needed, as infections caused by antibiotic-resistant pathogens are on the rise. In the identification of new antibiotics, many scientists are currently investigating biosynthetic aspects of antibiotic production in actinomycetes. Since the emergence of next-generation sequencing technologies, the field of antibiotics research has experienced a remarkable revival. These bacteria have the potential to produce more antibiotics than previously thought possible. Some antibiotics are produced in standard media, while others require the presence of a specific signaling molecule in the medium. Others, however, are only produced when the native regulation of the biosynthesis gene cluster is overruled. This book covers topics in the field of antibiotic-producing actinomycetes. The following tops are addressed: - Approaches to access novel antibiotic producers for novel natural compounds - Omics and genome mining approaches for the discovery of novel natural compounds - Analyses and genetic engineering of antibiotic biosynthesis - Regulation of the secondary metabolism in actinomycetes
Abstract: Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods. Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound. The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes Tü6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism
This textbook describes the types of natural products, the biosynthetic pathways that enable the production of these molecules, and an update on the discovery of novel products in the post-genomic era.