Streptomyces as Microbial Chassis for Heterologous Protein Expression

Heterologous production of recombinant proteins is gaining increasing interest in biotechnology with respect to productivity, scalability, and wide applicability. The members of genus Streptomyces have been proposed as remarkable hosts for heterologous production due to their versatile nature of expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes. However, there are several issues that limit their use, including low yield, difficulty in genetic manipulation, and their complex cellular features. In this review, we summarize rational engineering approaches to optimizing the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species in terms of genetic tool development and chassis construction. Further perspectives on the development of optimal Streptomyces chassis by the design-build-test-learn cycle in systems are suggested, which may increase the availability of secondary metabolites and recombinant proteins.

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Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/462049 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 18

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.

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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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IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites

mBio ◽

10.1128/mbio.00932-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 66

Author(s):

Michalis Hadjithomas ◽

I-Min Amy Chen ◽

Ken Chu ◽

Anna Ratner ◽

Krishna Palaniappan ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Genomic Data ◽

Gene Clusters ◽

Metagenomic Data ◽

Integrated Analysis ◽

Analysis Tool ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Analysis Tools

ABSTRACTIn the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time inAlphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.IMPORTANCEIMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

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New Approaches to Detect Biosynthetic Gene Clusters in the Environment

Medicines ◽

10.3390/medicines6010032 ◽

2019 ◽

Vol 6 (1) ◽

pp. 32 ◽

Cited By ~ 7

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.

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Use of Permanent Wall-Deficient Cells as a System for the Discovery of New-to-Nature Metabolites

Microorganisms ◽

10.3390/microorganisms8121897 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1897

Author(s):

Shraddha Shitut ◽

Güniz Özer Bergman ◽

Alexander Kros ◽

Daniel E. Rozen ◽

Dennis Claessen

Keyword(s):

Cell Wall ◽

Secondary Metabolites ◽

Gene Clusters ◽

Chemical Diversity ◽

Biosynthetic Gene Clusters ◽

Microbial Cell Factories ◽

Cell Factories ◽

Genome Recombination ◽

Antibiotic Discovery ◽

Insight Into

Filamentous actinobacteria are widely used as microbial cell factories to produce valuable secondary metabolites, including the vast majority of clinically relevant antimicrobial compounds. Secondary metabolites are typically encoded by large biosynthetic gene clusters, which allow for a modular approach to generating diverse compounds through recombination. Protoplast fusion is a popular method for whole genome recombination that uses fusion of cells that are transiently wall-deficient. This process has been applied for both inter- and intraspecies recombination. An important limiting step in obtaining diverse recombinants from fused protoplasts is regeneration of the cell wall, because this forces the chromosomes from different parental lines to segregate, thereby preventing further recombination. Recently, several labs have gained insight into wall-deficient bacteria that have the ability to proliferate without their cell wall, known as L-forms. Unlike protoplasts, L-forms can stably maintain multiple chromosomes over many division cycles. Fusion of such L-forms would potentially allow cells to express genes from both parental genomes while also extending the time for recombination, both of which can contribute to an increased chemical diversity. Here, we present a perspective on how L-form fusion has the potential to become a platform for novel compound discovery and may thus help to overcome the antibiotic discovery void.

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Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽

10.3390/biom10050734 ◽

2020 ◽

Vol 10 (5) ◽

pp. 734 ◽

Cited By ~ 2

Author(s):

Yawei Zhao ◽

Guoquan Li ◽

Yunliang Chen ◽

Yinhua Lu

Keyword(s):

Natural Product ◽

Genome Editing ◽

Genetic Manipulation ◽

Chemical Compounds ◽

Gene Clusters ◽

Future Research ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Research Focus ◽

Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

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Draft Genome Sequence of Geobacillus sp. LEMMY01, a Thermophilic Bacterium Isolated from the Site of a Burning Grass Pile

Genome Announcements ◽

10.1128/genomea.00200-17 ◽

2017 ◽

Vol 5 (19) ◽

Cited By ~ 2

Author(s):

Yuri Pinheiro Alves de Souza ◽

Fábio Faria da Mota ◽

Alexandre Soares Rosado

Keyword(s):

Secondary Metabolites ◽

Genome Sequence ◽

Draft Genome ◽

Axenic Culture ◽

Gene Clusters ◽

Thermophilic Bacterium ◽

Draft Genome Sequence ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Biotechnological Potential

ABSTRACT We report here the 3,586,065-bp draft genome of Geobacillus sp. LEMMY01, which was isolated (axenic culture) from a thermophilic chemolitoautotrophic consortium obtained from the site of a burning grass pile. The genome contains biosynthetic gene clusters coding for secondary metabolites, such as terpene and lantipeptide, confirming the biotechnological potential of this strain.

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