scholarly journals In situ activation and heterologous production of a cryptic lantibiotic from a plant-ant derived Saccharopolyspora species

2019 ◽  
Author(s):  
Eleni Vikeli ◽  
David A. Widdick ◽  
Sibyl F. Batey ◽  
Daniel Heine ◽  
Neil A. Holmes ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Genetic Manipulation ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Ecological Niches ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Structural Characterisation ◽  
In Situ Activation

AbstractMost clinical antibiotics are derived from actinomycete natural products (NPs) discovered at least 60 years ago. Repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial NPs as a source of new chemical diversity but advances in genome sequencing revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of the strain, by the application of chemical genetics, or by microbial co-cultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here we report the novel lanthipeptide antibiotic kyamicin generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatia-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium. Heterologous production and purification of kyamicin allowed its structural characterisation and bioactivity determination. Our activation strategy was also successful for the expression of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.ImportanceThe discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here we report an activation strategy that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

scholarly journals In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species

2019 ◽  
Vol 86 (3) ◽  
Author(s):  
Eleni Vikeli ◽  
David A. Widdick ◽  
Sibyl F. D. Batey ◽  
Daniel Heine ◽  
Neil A. Holmes ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Ectopic Expression ◽  
Chemical Diversity ◽  
Ecological Niches ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
In Situ Activation

ABSTRACT Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium. Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature. IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

scholarly journals Structure and biosynthesis of deoxy-polyamine in X. bovienii

2021 ◽  
Author(s):  
Sebastian L Wenski ◽  
Natalie Berghaus ◽  
Nadine Keller ◽  
Helge B Bode
Keyword(s):  
Natural Products ◽  
Gene Clusters ◽  
Mass Spectrometric ◽  
Spectrometric Analysis ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
In Trans ◽  
In Cis ◽  
Dh Domain

Abstract Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.

scholarly journals A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

2019 ◽  
Author(s):  
Asif Fazal ◽  
Divya Thankachan ◽  
Ellie Harris ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Gene Clusters ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Critical Aspect ◽  
Biosynthetic Gene Clusters ◽  
Streptomyces Albus ◽  
Microbial Natural Products ◽  
Suitable Expression

AbstractCloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign BGCs by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

scholarly journals Recapitulation of the evolution of biosynthetic gene clusters reveals hidden chemical diversity on bacterial genomes

2015 ◽  
Author(s):  
Pablo Cruz-Morales ◽  
Christian E. Martínez-Guerrero ◽  
Marco A. Morales-Escalante ◽  
Luis Yáñez-Guerra ◽  
Johannes Florian Kopp ◽  
...  
Keyword(s):  
Natural Products ◽  
Chemical Space ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Chemical Diversity ◽  
Biosynthetic Gene ◽  
Bacterial Genomes ◽  
Biosynthetic Gene Clusters

AbstractNatural products have provided humans with antibiotics for millennia. However, a decline in the pace of chemical discovery exerts pressure on human health as antibiotic resistance spreads. The empirical nature of current genome mining approaches used for natural products research limits the chemical space that is explored. By integration of evolutionary concepts related to emergence of metabolism, we have gained fundamental insights that are translated into an alternative genome mining approach, termed EvoMining. As the founding assumption of EvoMining is the evolution of enzymes, we solved two milestone problems revealing unprecedented conversions. First, we report the biosynthetic gene cluster of the ‘orphan’ metabolite leupeptin in Streptomyces roseus. Second, we discover an enzyme involved in formation of an arsenic-carbon bond in Streptomyces coelicolor and Streptomyces lividans. This work provides evidence that bacterial chemical repertoire is underexploited, as well as an approach to accelerate the discovery of novel antibiotics from bacterial genomes.

scholarly journals A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

2019 ◽  
Vol 113 (4) ◽  
pp. 511-520 ◽  
Author(s):  
Asif Fazal ◽  
Divya Thankachan ◽  
Ellie Harris ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Gene Clusters ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Critical Aspect ◽  
Biosynthetic Gene Clusters ◽  
Streptomyces Albus ◽  
Microbial Natural Products ◽  
Suitable Expression

AbstractCloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign biosynthetic gene clusters by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

scholarly journals Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiaqi Liu ◽  
Haibo Zhou ◽  
Zhiyu Yang ◽  
Xue Wang ◽  
Hanna Chen ◽  
...  
Keyword(s):  
Natural Products ◽  
Heterologous Expression ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Wild Type ◽  
Biosynthetic Gene Clusters ◽  
Gram Negative ◽  
Cell Autolysis ◽  
Rational Construction ◽  
Genomic Regions

AbstractHeterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

scholarly journals Heterologous Production of Lasso Peptide Capistruin in a Burkholderia Host

2019 ◽  
Author(s):  
Sylvia Kunakom ◽  
Alessandra Eustaquio
Keyword(s):  
Natural Products ◽  
Gene Clusters ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
E Coli ◽  
Lasso Peptide ◽  
Close Phylogenetic Relationship ◽  
Current Paradigm ◽  
Precursor Supply

<i>Burkholderia</i> bacteria are an emerging source of natural products with applications in agriculture and medicine. Heterologous expression of biosynthetic gene clusters can streamline natural product discovery. However, production yields with the commonly used <i>Escherichia coli</i> host are usually low. Following the current paradigm that one host does not fit all, we aim to develop a <i>Burkholderia</i> host in order to ultimately tap into the biosynthetic potential of <i>Burkholderia</i> genomes, which can contain up to 27 biosynthetic gene clusters per genome. Because close phylogenetic relationship is expected to improve odds of success due to compatible gene expression and precursor supply, we tested <i>Burkholderia</i> sp. FERM BP-3421, a non-pathogenic isolate previously used to produce natural products at industrial scale. We show here that FERM BP-3421 can produce the model lasso peptide capistruin in yields that are at least 65-fold, and up to 580-fold higher than the previously used <i>E. coli</i> host.

scholarly journals HEx: a heterologous expression platform for the discovery of fungal natural products

2018 ◽  
Author(s):  
Colin JB Harvey ◽  
Mancheng Tang ◽  
Ulrich Schlecht ◽  
Joe Horecka ◽  
Curt R Fischer ◽  
...  
Keyword(s):  
Natural Products ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Gene Clusters ◽  
Fungal Species ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Expression Platform ◽  
The World ◽  
Fungal Natural Products

AbstractFor decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.SummaryHere we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform.

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