scholarly journals Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Yousra Ahmed ◽  
Yuriy Rebets ◽  
Marta Rodríguez Estévez ◽  
Josef Zapp ◽  
Maksym Myronovskyi ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Secondary Metabolite ◽  
Parental Strain ◽  
Genomic Library ◽  
Gene Clusters ◽  
Genetically Engineered ◽  
Biologically Active ◽  
Endogenous Gene ◽  
Site Specific Integration

Abstract Background Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. Results In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. Conclusion The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

2021 ◽  
Vol 85 (3) ◽  
pp. 714-721
Author(s):  
Risa Takao ◽  
Katsuyuki Sakai ◽  
Hiroyuki Koshino ◽  
Hiroyuki Osada ◽  
Shunji Takahashi
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Secondary Metabolite ◽  
Response Regulator ◽  
Bac Library ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Gene Organization ◽  
Biosynthetic Gene ◽  
Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

scholarly journals Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

2021 ◽  
Author(s):  
◽  
Luke Stevenson
Keyword(s):  
Natural Products ◽  
New Zealand ◽  
Heterologous Expression ◽  
Gene Clusters ◽  
Bioinformatic Analysis ◽  
Coli Strain ◽  
Functional Screening ◽  
Functional Difference ◽  
Biosynthetic Pathways ◽  
New Members

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose.  Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies.  Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity.  The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class.  The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

scholarly journals Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Juan Pablo Gomez-Escribano ◽  
Jean Franco Castro ◽  
Valeria Razmilic ◽  
Scott A. Jarmusch ◽  
Gerhard Saalbach ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Bioinformatic Analysis ◽  
Growth Media ◽  
Content Type ◽  
Lasso Peptide ◽  
Lasso Peptides ◽  
New Family

ABSTRACT Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides. IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

scholarly journals Extremophilic Fungi from Marine Environments: Underexplored Sources of Antitumor, Anti-Infective and Other Biologically Active Agents

Marine Drugs ◽  
2022 ◽  
Vol 20 (1) ◽  
pp. 62
Author(s):  
Lesley-Ann Giddings ◽  
David J. Newman
Keyword(s):  
Natural Products ◽  
Antitumor Agents ◽  
Marine Fungi ◽  
Gene Clusters ◽  
Biologically Active ◽  
Bioactive Metabolites ◽  
Marine Environments ◽  
Environmental Pressures ◽  
Symbiotic Associations ◽  
Extremophilic Fungi

Marine environments are underexplored terrains containing fungi that produce a diversity of natural products given unique environmental pressures and nutrients. While bacteria are commonly the most studied microorganism for natural products in the marine world, marine fungi are also abundant but remain an untapped source of bioactive metabolites. Given that their terrestrial counterparts have been a source of many blockbuster antitumor agents and anti-infectives, including camptothecin, the penicillins, and cyclosporin A, marine fungi also have the potential to produce new chemical scaffolds as leads to potential drugs. Fungi are more phylogenetically diverse than bacteria and have larger genomes that contain many silent biosynthetic gene clusters involved in making bioactive compounds. However, less than 5% of all known fungi have been cultivated under standard laboratory conditions. While the number of reported natural products from marine fungi is steadily increasing, their number is still significantly lower compared to those reported from their bacterial counterparts. Herein, we discuss many varied cytotoxic and anti-infective fungal metabolites isolated from extreme marine environments, including symbiotic associations as well as extreme pressures, temperatures, salinity, and light. We also discuss cultivation strategies that can be used to produce new bioactive metabolites or increase their production. This review presents a large number of reported structures though, at times, only a few of a large number of related structures are shown.

scholarly journals A Pseudoalteromonas clade with remarkable biosynthetic potential

2021 ◽  
Author(s):  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Jesse Cain ◽  
John A. Kalaitzis ◽  
Brett A. Neilan
Keyword(s):  
Natural Products ◽  
Gene Cluster ◽  
Genome Mining ◽  
Gene Clusters ◽  
Average Density ◽  
Biologically Active ◽  
Biosynthesis Gene Cluster ◽  
Diverse Range ◽  
Siderophore Biosynthesis ◽  
Biosynthesis Gene

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here we report the biochemical and genomic analysis of Pseudoalteromonas sp. HM-SA03, isolated from the blue-ringed octopus, Hapalochalaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides and four hitherto novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of ten biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the under-explored potential of Pseudoalteromonas as a source of new natural products. Importance This study demonstrates that the Pseudoalteromonas strain, HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We have identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we have demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

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.

scholarly journals Loseolamycins: A Group of New Bioactive Alkylresorcinols Produced after Heterologous Expression of a Type III PKS from Micromonospora endolithica

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4594
Author(s):  
Constanze Lasch ◽  
Nils Gummerlich ◽  
Maksym Myronovskyi ◽  
Anja Palusczak ◽  
Josef Zapp ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Transcriptional Activation ◽  
Polyketide Synthase ◽  
Biologically Active ◽  
Aliphatic Chain ◽  
Biosynthetic Pathways ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Potential Applications

Natural products are a valuable source of biologically active compounds with potential applications in medicine and agriculture. Unprecedented scaffold diversity of natural products and biocatalysts from their biosynthetic pathways are of fundamental importance. Heterologous expression and refactoring of natural product biosynthetic pathways are generally regarded as a promising approach to discover new secondary metabolites of microbial origin. Here, we present the identification of a new group of alkylresorcinols after transcriptional activation and heterologous expression of the type III polyketide synthase of Micromonospora endolithica. The most abundant compounds loseolamycins A1 and A2 have been purified and their structures were elucidated by NMR. Loseolamycins contain an unusual branched hydroxylated aliphatic chain which is provided by the host metabolism and is incorporated as a starter fatty acid unit. The isolated loseolamycins show activity against gram-positive bacteria and inhibit the growth of the monocot weed Agrostis stolonifera in a germination assay. The biosynthetic pathway leading to the production of loseolamycins is proposed in this paper.

scholarly journals Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophileThermobifida fusca

2008 ◽  
Vol 105 (40) ◽  
pp. 15311-15316 ◽  
Author(s):  
Eric J. Dimise ◽  
Paul F. Widboom ◽  
Steven D. Bruner
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Gene Cluster ◽  
Structure Elucidation ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Biologically Active ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomyceteThermobifida fuscarevealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product.T. fuscais a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced byT. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

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