scholarly journals Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Bohdan Bilyk ◽  
Sora Kim ◽  
Asif Fazal ◽  
Tania A. Baker ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Rna Polymerase ◽  
Natural Product ◽  
Morphological Differentiation ◽  
Dynamic Responses ◽  
Biosynthetic Pathways ◽  
Recognition Sequence ◽  
Σ Factor

ABSTRACT The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntA in vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation. IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

scholarly journals Regulation of antimycin biosynthesis is controlled by the ClpXP protease

2019 ◽  
Author(s):  
Bohdan Bilyk ◽  
Sora Kim ◽  
Asif Fazal ◽  
Tania A. Baker ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Rna Polymerase ◽  
Morphological Differentiation ◽  
Dynamic Responses ◽  
Biosynthetic Pathways ◽  
Recognition Sequence ◽  
Data Link ◽  
Σ Factor

AbstractThe survival of any microbe relies upon its ability to respond to environmental change. Use of Extra Cytoplasmic Function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors; nearly all of which regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstitute in vitro ClpXP proteolysis of σAntA, but not a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo is enhanced by removal of the ClpXP recognition sequence, and that levels of the protein rise when cellular ClpXP protease activity is abolished. These data establish direct proteolysis as an alternative and thus far unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.ImportanceNatural products produced by Streptomyces species underpin many industrially- and medically-important compounds. However, the majority of the ~30 biosynthetic pathways harboured by an average species are not expressed in the laboratory. This undiscovered biochemical diversity is believed to comprise an untapped resource for natural products drug discovery. A major roadblock preventing the exploitation of unexpressed biosynthetic pathways is a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

scholarly journals Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways

Science ◽  
2013 ◽  
Vol 341 (6150) ◽  
pp. 1089-1094 ◽  
Author(s):  
Mark C. Walker ◽  
Benjamin W. Thuronyi ◽  
Louise K. Charkoudian ◽  
Brian Lowry ◽  
Chaitan Khosla ◽  
...  
Keyword(s):  
Natural Products ◽  
Synthetic Biology ◽  
Natural Product ◽  
Polyketide Synthase ◽  
Building Blocks ◽  
Living Systems ◽  
Synthetic Compounds ◽  
Fluorinated Building Blocks

Organofluorines represent a rapidly expanding proportion of molecules that are used in pharmaceuticals, diagnostics, agrochemicals, and materials. Despite the prevalence of fluorine in synthetic compounds, the known biological scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural-product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems, and we show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be inserted site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the production of complex fluorinated natural products using synthetic biology.

scholarly journals Inhibition of Transcription in Staphylococcus aureus by a Primary Sigma Factor-Binding Polypeptide from Phage G1

2009 ◽  
Vol 191 (12) ◽  
pp. 3763-3771 ◽  
Author(s):  
Mohammed Dehbi ◽  
Gregory Moeck ◽  
Francis F. Arhin ◽  
Pascale Bauda ◽  
Dominique Bergeron ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Growth Inhibitory ◽  
Σ Factor ◽  
Transcription In Vitro

ABSTRACT The primary sigma factor of Staphylococcus aureus, σSA, regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with σSA both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of σSA that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-σ factor may occlude the −35 element recognition domain of σSA. As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of σSA to DNA and σSA-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

scholarly journals Zebrafish as an Emerging Model for Bioassay-Guided Natural Product Drug Discovery for Neurological Disorders

Medicines ◽  
2019 ◽  
Vol 6 (2) ◽  
pp. 61 ◽  
Author(s):  
Arjun Pitchai ◽  
Rajesh Kannan Rajaretinam ◽  
Jennifer L. Freeman
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Natural Product ◽  
Neurological Disorders ◽  
Experimental Models ◽  
Bioactive Natural Products ◽  
Natural Product Research ◽  
Model Platform

Most neurodegenerative diseases are currently incurable, with large social and economic impacts. Recently, there has been renewed interest in investigating natural products in the modern drug discovery paradigm as novel, bioactive small molecules. Moreover, the discovery of potential therapies for neurological disorders is challenging and involves developing optimized animal models for drug screening. In contemporary biomedicine, the growing need to develop experimental models to obtain a detailed understanding of malady conditions and to portray pioneering treatments has resulted in the application of zebrafish to close the gap between in vitro and in vivo assays. Zebrafish in pharmacogenetics and neuropharmacology are rapidly becoming a widely used organism. Brain function, dysfunction, genetic, and pharmacological modulation considerations are enhanced by both larval and adult zebrafish. Bioassay-guided identification of natural products using zebrafish presents as an attractive strategy for generating new lead compounds. Here, we see evidence that the zebrafish’s central nervous system is suitable for modeling human neurological disease and we review and evaluate natural product research using zebrafish as a vertebrate model platform to systematically identify bioactive natural products. Finally, we review recently developed zebrafish models of neurological disorders that have the potential to be applied in this field of research.

scholarly journals Cryptosporidiosis: A Potential Anti-diarrheal Natural Product Drug Discovery Journey in Ghana, West Africa

2020 ◽  
pp. 85-93
Author(s):  
Senyo K. Botchie ◽  
Andrew G. Mtewa ◽  
Irene Ayi
Keyword(s):  
Developing Countries ◽  
Natural Products ◽  
Drug Discovery ◽  
Drug Development ◽  
Natural Product ◽  
Causative Organism ◽  
Drug Candidates ◽  
Cause Of Deaths

The overwhelming resistance to current drugs and the exhaustion of drug development interventions, as well as synthetic libraries, have compelled researchers to resort to the use of novel drug candidates derived from natural products. Cryptosporidium, the causative organism of Cryptosporidiosis, is no exception. The diarrhea-causing parasite is known to be the leading cause of deaths in children below age 5 in developing countries like Ghana and second to rotavirus as the causative agent for diarrhea in newborn calves and infants. Currently, the only FDA approved drug for the treatment of Cryptosporidiosis is Nitazoxanide. It is, therefore, needful to develop novel alternative candidates as it could aid in the decrease in child mortality and malnutrition in developing countries. Even though there have been significant limitations into anti-cryptosporidial drug development in vitro and in vivo, essential advancements are being made of which this article addresses the need for research into natural products. Some studies outlined in this paper has stated potential plant extracts showing anti-cryptosporidiosis efficacy. With the wealth of medicinal plant products and Cryptosporidium in vitro culture expertise available in our labs at Noguchi Memorial Institute for Medical research we are certain of making potential significant strides in the world of natural product Cryptosporidium drug discovery in Africa.

Natural product inhibitors of cyclooxygenase (COX) enzyme: A Review on Current status and future perspectives

2020 ◽  
Vol 27 ◽  
Author(s):  
Goutami Ambati G ◽  
Sanjay M. Jachak
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Current Status ◽  
Marine Origin ◽  
Cox Inhibitors ◽  
Potential Source ◽  
Cox 2 ◽  
Inhibition Property

Background: Several clinically used COX-1 and COX-2 inhibitor drugs were reported to possess severe side effects like GI ulcers and cardiovascular disturbances, respectively. Natural products being structurally diverse always attracted the attention of chemists/medicinal chemists as a potential source of lead molecules in drug discovery process. COX-2 inhibitory natural products also possess potential cancer chemopreventive property against various cancers including that of colon, breast, and prostate. Methods: Various in vitro, in vivo, in silico standardized methods were used to evaluate COX inhibition property of different secondary metabolites isolated from plant, microbial and marine origin. Results: We had earlier reported a detailed account of natural product inhibitors of COX reported during 1995-2005 in 2006. In the proposed review we report 158 natural product inhibitors of COX during 2006 to 2019 belonging to various secondary metabolite classes such as alkaloids, terpenoids, polyphenols as flavonoids, chromones, coumarins, lignans, anthraquinones, naphthalenes, curcuminoids, diarylheptanoids and miscellaneous compounds of plant and marine origin. Further structure activity relationship (SAR) studies of possible leads are also included in the article. Conclusion: COX inhibitors served as a potential source of lead molecules for discovery and development of anti-inflammatory drugs. Compilation of natural product and semi-synthetic inhibitors of COX may serve as valuable information to the researchers who are looking for possible lead molecules from natural source to conduct further preclinical and clinical studies.

Manipulating nature's sugar biosynthetic machineries for glycodiversification of macrolides: Recent advances and future prospects

2007 ◽  
Vol 79 (4) ◽  
pp. 785-799 ◽  
Author(s):  
Christopher J. Thibodeaux ◽  
Hung-wen Liu
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Biosynthetic Pathway ◽  
Effective Means ◽  
Future Prospects ◽  
Research Groups ◽  
Targeted Disruption ◽  
In Vitro Methods

Changing the sugar structures and glycosylation patterns of natural products is an effective means of altering the biological activity of clinically useful drugs. Several recent strategies have provided researchers with the opportunity to manipulate sugar structures and to change the sugar moieties attached to these natural products via a biosynthetic approach. In this review, we explore the utility of contemporary in vivo and in vitro methods to achieve natural product glycodiversification. This study will focus on recent progress from our laboratory in elucidating the biosynthesis of D-desosamine, a deoxysugar component of many macrolide antibiotics, and will highlight how we have engineered the D-desosamine biosynthetic pathway in Streptomyces venezuelae through targeted disruption and heterologous expression of the sugar biosynthetic genes to generate a variety of new glycoforms. The in vitro exploitation of the substrate flexibility of the endogenous D-desosamine glycosyltransferase (GT) to generate many non-natural glycoforms will also be discussed. These experiments are compared with recent work from other research groups on the same topics. Finally, the significance of these studies for the future prospects of natural product glycodiversification is discussed.

scholarly journals Role of Natural Products in Aging Skin

2019 ◽  
Vol 10 (SPL1) ◽  
Author(s):  
Mahendran Sekar
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Plant Extracts ◽  
Skin Aging ◽  
In Vivo Models ◽  
Medicinal Plant Extracts ◽  
Wide Range ◽  
Recent Developments

Aging is an unavoidable progression in everyone's life and influenced by lifestyle, genetic as well as environmental factors. Herbal and plant extracts are used as antiaging since ancient times, but the evidence are still limited. Recent developments in antiaging investigation anticipated the use of natural products as the main ingredient in the formulations. Hence, this presentation focused to highlight the importance of twelve most popular medicinal plant extracts that have reported to have skin aging prevention potential. All these natural product extracts have a capacity to scavenge free radicals and defend skin matrix over the inhibition of enzymatic degradation. Some of the extracts promotes collagen synthesis in the skin and also affect the skin tightness and elasticity. However, the use of natural product extracts as an antiaging and anti-wrinkling it should be further explored using a wide range of in-vitro and in-vivo models to confirm its safety and efficacy before proceeding into the development of cosmetic products.  

scholarly journals Properties of Bacillus subtilis σA Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

2004 ◽  
Vol 186 (8) ◽  
pp. 2366-2375 ◽  
Author(s):  
Hsin-Hsien Hsu ◽  
Wei-Cheng Huang ◽  
Jia-Perng Chen ◽  
Liang-Yin Huang ◽  
Chai-Fong Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Activity ◽  
Transcription Activity ◽  
N Terminus ◽  
Σ Factor

ABSTRACT σ factors in the σ70 family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis σA, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σA and greatly reduced the transcription activity of the truncated σA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σA in RNA polymerase.

scholarly journals The σ54 System Directly Regulates Bacterial Natural Product Genes

2020 ◽  
Author(s):  
Muqing Ma ◽  
Roy Welch ◽  
Anthony Garza
Keyword(s):  
Rna Polymerase ◽  
Natural Product ◽  
Gene Clusters ◽  
Regulatory Machinery ◽  
Vitro Binding ◽  
Intergenic Sequences ◽  
Bona Fide ◽  
Polymerase Binding

Abstract Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences.

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