Characterization and engineering of Streptomyces griseofuscus DSM 40191 as a potential host for heterologous expression of biosynthetic gene clusters

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.

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An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

Antibiotics ◽

10.3390/antibiotics9080494 ◽

2020 ◽

Vol 9 (8) ◽

pp. 494

Author(s):

Lena Mitousis ◽

Yvonne Thoma ◽

Ewa M. Musiol-Kroll

Keyword(s):

Genetic Engineering ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Molecular Tools ◽

Biosynthetic Gene Clusters ◽

Streptomyces Antibioticus ◽

The Past ◽

Bioactive Agents ◽

Valuable Compounds

The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.

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Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods

Molecules ◽

10.3390/molecules26216580 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6580

Author(s):

Charlotte Beck ◽

Tetiana Gren ◽

Francisco Javier Ortiz-López ◽

Tue Sparholt Jørgensen ◽

Daniel Carretero-Molina ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Genome Mining ◽

Gene Clusters ◽

Transcriptional Regulators ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Heterologous Host ◽

Streptomyces Sp ◽

Identification And Characterization

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

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Genome mining of biosynthetic gene clusters intended for secondary metabolites conservation in actinobacteria

Microbial Pathogenesis ◽

10.1016/j.micpath.2021.105252 ◽

2021 ◽

pp. 105252

Author(s):

Karthick Raja Arulprakasam ◽

Dhanasekaran Dharumadurai

Keyword(s):

Secondary Metabolites ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters

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

Microbiology Spectrum ◽

10.1128/spectrum.01171-21 ◽

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.

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Resistance Gene-Directed Genome Mining of 50 Aspergillus species

10.1101/457903 ◽

2018 ◽

Author(s):

Inge Kjærbølling ◽

Tammi Vesth ◽

Mikael R. Andersen

Keyword(s):

Secondary Metabolites ◽

Resistance Gene ◽

Genome Sequencing ◽

Genome Mining ◽

Gene Clusters ◽

Rational Approach ◽

Whole Genome ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Valuable Compounds

AbstractFungal secondary metabolites are a rich source of valuable natural products. Genome sequencing have revealed an enormous potential from predicted biosynthetic gene clusters. It is however currently a time consuming task and an unfeasible task to characterize all biosynthetic gene cluster and to identify possible uses of the compounds. A rational approach is needed to identify promising gene clusters responsible for producing valuable compounds. Several valuable bioactive clusters have been shown to include a resistance gene which is a paralog of the target gene inhibited by the compound. This mechanism can be used to design a rational approach selecting those clusters.We have developed a pipeline FRIGG (Fungal Resistance Gene-directed Genome mining) identifying putative resistance genes found in biosynthetic gene clusters based on homology patterns of the cluster genes. The FRIGG pipeline has been run using 51 Aspergillus and Penicillium genomes, identifying 72 unique protein families with putative resistance genes using various settings in the pipeline. The pipeline was also able to identify the characterized resistance gene inpE from the Fellutamide B cluster thereby validating the approach.We have successfully developed an approach identifying putative valuable bio-active clusters based on a specific resistance mechanism. This approach will be highly useful as an ever increasing amount of genomic data becomes available — the art of identifying and selecting clusters producing novel valuable compounds will only become more crucial.ImportanceSpecies belonging to the Aspergillus genus are known to produce a large number of secondary metabolites, some of these compounds are bioactive and used as pharmaceuticals such as penicillin, cyclosporin and statin. With whole genome sequencing it became apparent that the genetic potential for secondary metabolite production is much bigger than expected. As an increasing number of species are whole genome sequenced an immense number of secondary metabolite genes are predicted and the question of how to selectively identify novel bioactive compounds from this information arises. To address this question, we have created a pipeline identifying genes likely involved in the production of bioactive compounds based on a resistance gene hypothesis approach.

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

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

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.

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Global biogeographic sampling of bacterial secondary metabolism

eLife ◽

10.7554/elife.05048 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 74

Author(s):

Zachary Charlop-Powers ◽

Jeremy G Owen ◽

Boojala Vijay B Reddy ◽

Melinda A Ternei ◽

Denise O Guimarães ◽

...

Keyword(s):

Secondary Metabolites ◽

Genome Sequencing ◽

Geographic Distance ◽

Gene Clusters ◽

Natural World ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Road Map ◽

Two Factors ◽

Natural Products Discovery

Recent bacterial (meta)genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment. Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ketosynthase domains to compare biosynthetic diversity in soil microbiomes from around the globe. We see large differences in domain populations from all except the most proximal and biome-similar samples, suggesting that most microbiomes will encode largely distinct collections of bacterial secondary metabolites. Our data indicate a correlation between two factors, geographic distance and biome-type, and the biosynthetic diversity found in soil environments. By assigning reads to known gene clusters we identify hotspots of biomedically relevant biosynthetic diversity. These observations not only provide new insights into the natural world, they also provide a road map for guiding future natural products discovery efforts.

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

10.1101/2020.05.18.103358 ◽

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.

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kat Steinke ◽

Omkar S. Mohite ◽

Tilmann Weber ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Species Complex ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Frameshift Mutations ◽

Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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