scholarly journals Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces

2021 ◽  
Vol 11 (4) ◽  
pp. 1851
Author(s):  
Van Thuy Thi Pham ◽  
Chung Thanh Nguyen ◽  
Dipesh Dhakal ◽  
Hue Thi Nguyen ◽  
Tae-Su Kim ◽  
...  
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Heterologous Expression ◽  
Antitumor Agents ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Heterologous Biosynthesis ◽  
Promoter Replacement ◽  
Production Platform

Streptomyces is a significant source of natural products that are used as therapeutic antibiotics, anticancer and antitumor agents, pesticides, and dyes. Recently, with the advances in metabolite analysis, many new secondary metabolites have been characterized. Moreover, genome mining approaches demonstrate that many silent and cryptic biosynthetic gene clusters (BGCs) and many secondary metabolites are produced in very low amounts under laboratory conditions. One strain many compounds (OSMAC), overexpression/deletion of regulatory genes, ribosome engineering, and promoter replacement have been utilized to activate or enhance the production titer of target compounds. Hence, the heterologous expression of BGCs by transferring to a suitable production platform has been successfully employed for the detection, characterization, and yield quantity production of many secondary metabolites. In this review, we introduce the systematic approach for the heterologous production of secondary metabolites from Streptomyces in Streptomyces and other hosts, the genome analysis tools, the host selection, and the development of genetic control elements for heterologous expression and the production of secondary metabolites.

scholarly journals Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059

2021 ◽  
Author(s):  
Xiyan Wang ◽  
Thomas Isbrandt ◽  
Emil Ørsted Christensen ◽  
Jette Melchiorsen ◽  
Thomas Ostenfeld Larsen ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Pseudoalteromonas Rubra

Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression.

scholarly journals Characterization and Engineering of Streptomyces griseofuscus DSM 40191 as a Potential Host for Heterologous Expression of Biosynthetic Gene Clusters

2020 ◽  
Author(s):  
Tetiana Gren ◽  
Christopher M. Whitford ◽  
Omkar S. Mohite ◽  
Tue S. Jørgensen ◽  
Eftychia E. Kontou ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Phosphorus Sources ◽  
Further Development ◽  
Core Genes ◽  
Target Effects

AbstractStreptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Using CRISPR-BEST technology, core genes of 4 biosynthetic gene clusters (BGCs) that are situated on the chromosome arms were inactivated and the outcomes of the inactivations were tested. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5,19% genome reduction. Sequencing confirmed that DEL2 does not bear any crucial off-target effects or rearrangements in its genome. It can be characterized by faster growth and inability to produce three main native metabolites of S. griseofuscus: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to confirm the production of actinorhodin by both S. griseofuscus wild type and DEL2. We believe that this strain will serve as a good chassis for heterologous expression of BGCs.ImportanceThe rise of antibacterial resistance calls on the development of the next generation of antibiotics, majority of which are derived from natural compounds, produced by actinomycetes. The manipulation, refactoring and expression of BGCs coding for such natural products is a promising approach in secondary metabolite discovery. Thus, the development of a versatile panel of heterologous hosts for the expression of BGCs is essential. We believe that first-to-date systematic, detailed characterisation of S. griseofuscus, a highly promising chassis strain, will not only facilitate the further development of this particular strain, but also will set a blueprint for characterisation of other potential hosts.

Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

2019 ◽  
Author(s):  
Patrick Videau ◽  
Kaitlyn Wells ◽  
Arun Singh ◽  
Jessie Eiting ◽  
Philip Proteau ◽  
...  
Keyword(s):  
Natural Products ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Test Case ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Cyanobacterium Anabaena ◽  
Anabaena Sp ◽  
Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

scholarly journals Phytoplankton trigger the production of cryptic metabolites in the marine actinobacteria Salinispora tropica

2020 ◽  
Author(s):  
Audam Chhun ◽  
Despoina Sousoni ◽  
Maria del Mar Aguiló-Ferretjans ◽  
Lijiang Song ◽  
Christophe Corre ◽  
...  
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Antibiotic Production ◽  
Antimicrobial Effect ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Marine Actinobacteria ◽  
Salinispora Tropica ◽  
Eukaryotic Phytoplankton

AbstractBacteria from the Actinomycete family are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacteria Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide A but half of its putative BGCs are still orphan. Although previous studies have looked into using marine heterotrophs to induce orphan BGCs in Salinispora, the potential impact of co-culturing marine phototrophs with Salinispora has yet to be investigated. Following the observation of clear antimicrobial phenotype of the actinobacterium on a range of phytoplanktonic organisms, we here report the discovery of novel cryptic secondary metabolites produced by S. tropica in response to its co-culture with photosynthetic primary producers. An approach combining metabolomics and proteomics revealed that the photosynthate released by phytoplankton influences the biosynthetic capacities of S. tropica with both production of new molecules and the activation of orphan BGCs. Our work pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from actinobacteria.ImportanceThe alarming increase of antimicrobial resistance has generated an enormous interest in the discovery of novel active compounds. The isolation of new microbes to untap novel natural products is currently hampered because most biosynthetic gene clusters (BGC) encoded by these microorganisms are not expressed under standard laboratory conditions, i.e. mono-cultures. Here we show that co-culturing can be an easy way for triggering silent BGC. By combining state-of-the-art metabolomics and high-throughput proteomics, we characterized the activation of cryptic metabolites and silent biosynthetic gene clusters in the marine actinobacteria Salinispora tropica by the presence of phytoplankton photosynthate. We further suggest a mechanistic understanding of the antimicrobial effect this actinobacterium has on a broad range of prokaryotic and eukaryotic phytoplankton species and reveal a promising candidate for antibiotic production.

scholarly journals New Approaches to Detect Biosynthetic Gene Clusters in the Environment

Medicines ◽  
2019 ◽  
Vol 6 (1) ◽  
pp. 32 ◽  
Author(s):  
Ray Chen ◽  
Hon Wong ◽  
Brendan Burns
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Gene Clusters ◽  
Screening Methods ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Diverse Range ◽  
Bioinformatic Tools ◽  
Culture Independent ◽  
Traditional Approaches

Microorganisms in the environment can produce a diverse range of secondary metabolites (SM), which are also known as natural products. Bioactive SMs have been crucial in the development of antibiotics and can also act as useful compounds in the biotechnology industry. These natural products are encoded by an extensive range of biosynthetic gene clusters (BGCs). The developments in omics technologies and bioinformatic tools are contributing to a paradigm shift from traditional culturing and screening methods to bioinformatic tools and genomics to uncover BGCs that were previously unknown or transcriptionally silent. Natural product discovery using bioinformatics and omics workflow in the environment has demonstrated an extensive distribution of BGCs in various environments, such as soil, aquatic ecosystems and host microbiome environments. Computational tools provide a feasible and culture-independent route to find new secondary metabolites where traditional approaches cannot. This review will highlight some of the advances in the approaches, primarily bioinformatic, in identifying new BGCs, especially in environments where microorganisms are rarely cultured. This has allowed us to tap into the huge potential of microbial dark matter.

scholarly journals Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

2016 ◽  
Vol 82 (19) ◽  
pp. 5795-5805 ◽  
Author(s):  
Min Xu ◽  
Yemin Wang ◽  
Zhilong Zhao ◽  
Guixi Gao ◽  
Sheng-Xiong Huang ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Genome Mining ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Functional Screening ◽  
Biosynthetic Pathways ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type ◽  
Bac Libraries

ABSTRACTGenome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries inStreptomycesspp. We demonstrate mining from a strain ofStreptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate hostStreptomyces lividansSBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic fromS. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways.IMPORTANCEMicrobial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites fromStreptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic fromStreptomyces rocheiSal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery.

scholarly journals Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

2017 ◽  
Vol 20 (4) ◽  
pp. 1103-1113 ◽  
Author(s):  
Kai Blin ◽  
Hyun Uk Kim ◽  
Marnix H Medema ◽  
Tilmann Weber
Keyword(s):  
Natural Products ◽  
Small Molecules ◽  
Sequence Similarity ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Rule Based ◽  
Chemical Structures ◽  
Annotation Quality

Abstract Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats such as rule-based BGC detection, sequence and annotation quality and cluster boundary prediction, which all have to be considered while planning for, performing and analyzing the results of genome mining studies.

scholarly journals Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Rosa Alduina ◽  
Giuseppe Gallo
Keyword(s):  
Secondary Metabolites ◽  
Antitumor Agents ◽  
Genetic Manipulation ◽  
Gene Clusters ◽  
Complex Compounds ◽  
Biologically Active ◽  
Biosynthetic Gene Clusters ◽  
Artificial Chromosomes ◽  
Pharmacokinetic Properties ◽  
New Strategies

Actinomycetes are an important source of biologically active compounds, like antibiotics, antitumor agents, and immunosuppressors. Genome sequencing is revealing that this class of microorganisms has larger genomes relative to other bacteria and uses a considerable fraction of its coding capacity (5–10%) for the production of mostly cryptic secondary metabolites. To access actinomycetes biosynthetic capabilities or to improve the pharmacokinetic properties and production yields of these chemically complex compounds, genetic manipulation of the producer strains can be performed. Heterologous expression in amenable hosts can be useful to exploit and to explore the genetic potential of actinomycetes and not cultivable but interesting bacteria. Artificial chromosomes that can be stably integrated into theStreptomycesgenome were constructed and demonstrated to be effective for transferring entire biosynthetic gene clusters from intractable actinomycetes into more suitable hosts. In this paper, the construction of several shuttleEscherichia coli-Streptomycesartificial chromosomes is discussed together with old and new strategies applied to improve heterologous production of secondary metabolites.

scholarly journals Understanding the evolutionary origins of bacterial natural products with immunosuppressant properties

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Secondary Metabolite ◽  
Human Body ◽  
Immune Cells ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Drug Candidates

Actinobacteria and streptomyces are known to produce a variety of natural products, some of which confer antibiotic or immunosuppressive activities. While it is understandable that microbes develop the ability to synthesize molecules such as antibiotics that attack other competing microbes, but why would a secondary metabolite (natural product) synthesized by a microbe confer immunosuppressive activities? Was the capability to synthesize such a molecule endowed by evolution in the context of enabling microbes to develop resistance to immune cells of the human body? Or did the capability come from the need to colonize human body surfaces or gut to gain a survival niche for the microbe? Given that actinobacteria and streptomyces are soil microbes not usually associated with human body surfaces, could their biosynthetic capability for particular immunosuppressants arise from horizontal gene transfer from bacteria that colonize human body surfaces and subsequently develop the ability to synthesize the pertinent compounds through evolution? An alternate line of thinking on this issue touches on the possibility that microbes could encounter analogs of immuno-active molecules in their natural environment. Such molecules might elicit undesired physiological effects on the microbes, which place a selection pressure on microbes to develop countermeasures to the immuno-active molecules through mutations. Hence, through evolution, microbes could have developed the capability to synthesize secondary metabolites able to bind analogs of immuno-active molecules and help sequester them or quench their bioactivity. Subsequent profiling of such secondary metabolites in drug discovery efforts could have uncovered compounds with immunosuppressant activity which are originally developed for counteracting analogs of immuno-active molecules in the environment. It has to be recognized that analogs of immuno-active compounds remain somewhat dissimilar to immune compounds secreted by human immune cells, but they likely share common motifs for protein-secondary metabolite interactions. Direct evidence of the evolution of natural products with immunosuppressant activities could only be obtained from challenging suitable bacterial species with immuno-active molecules. Long cultivation experiments with multiple generations may result in the evolution of biosynthetic gene clusters for the synthesis of natural products able to sequester or quench immuno-active molecules. But, on the another hand, understanding relative binding affinities between a library of natural products and immuno-active molecules from humans would suggest drug candidates and their biosynthetic gene clusters. Subsequent phylogenetic analysis of cluster genes with their homologs from other species may yield insights into the evolution of genes and their putative function.

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