DOCSLIB.ORG

Antiplasmodial and Trypanocidal Activity of Violacein and Deoxyviolacein Produced from Synthetic Operons Elizabeth Bilsland1,2,3* , Tatyana A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Antiplasmodial and Trypanocidal Activity of Violacein and Deoxyviolacein Produced from Synthetic Operons Elizabeth Bilsland1,2,3* , Tatyana A Bilsland et al. BMC Biotechnology (2018) 18:22 https://doi.org/10.1186/s12896-018-0428-z RESEARCH ARTICLE Open Access Antiplasmodial and trypanocidal activity of violacein and deoxyviolacein produced from synthetic operons Elizabeth Bilsland1,2,3* , Tatyana A. Tavella3, Renata Krogh4, Jamie E. Stokes5, Annabelle Roberts1, James Ajioka6, David R. Spring5, Adriano D. Andricopulo4, Fabio T. M. Costa3 and Stephen G. Oliver1 Abstract Background: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioAto vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. Results: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein. Conclusions: We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs. Keywords: Violacein, Deoxyviolacein, Plasmodium falciparum, Trypanosoma cruzi, Synthetic operon, Antiparasitic, Escherichia coli Background potential and may represent a chemical scaffold for the Violacein is a violet indolocarbazole pigment that is pro- developemt of clinically useful drugs. duced by bacteria such as Chromobacterium violaceum, Commercially, violacein is usually isolated from Chro- which are commonly found in water and soil throughout mobacterium [20–22]orJanthinobacterium [23, 24]; the world [1–6]. Violacein has antipyretic [7, 8], ulcer- however, this process is costly and there are reports of protective [8], antibacterial [9–11], antifungal [3, 12], rare but deadly infections caused by these bacteria [25– trypanocidal [13, 14], antileishmanial [15], antinematode 28]. Hence, there has been considerable interest in the [16], and antiviral [17] activities. It also has the potential development of safe, and efficient routes to the biosyn- of inducing apoptosis in certain cancer cells [2, 18]. Vio- thesis of this compound [1, 29–34]. lacein kills wild-type and drug-resistant strains of the The violacein biosynthetic pathway from L-tryptophan malaria parasite, Plasmodium falciparum and is thera- (Fig. 1) requires the expression of five genes: vioA, vioB, peutically against malaria in mice [19]. These character- vioC, vioD, and vioE [35–39]. It should be noted that istics suggest that violacein has considerable research VioC enzyme is involved in both the production of deoxyviolacien from protodeoxyviolaceinic acid and in the generation of violacein from protoviolaceinic acid. * Correspondence: [email protected] Several studies have shown that transforming and ex- 1Cambridge Systems Biology Centre and Department of Biochemistry, pressing a complete metabolic pathway into a different University of Cambridge, Cambridge, UK bacterial host may lead to improved production of viola- 2Department of Structural and Functional Biology, Institute of Biology, UNICAMP, Campinas, SP, Brazil cein [40–43]. For example, Rodrigues and co-workers Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bilsland et al. BMC Biotechnology (2018) 18:22 Page 2 of 8 ab c d e Fig. 1 Violacein biosynthetic pathway (a) and plasmid maps of the complete (b) and partial (c, d and e) operons for violacein biosynthesis [32–34] have successfully engineered Escherichia coli to they fine-tuned the expression of violacein-producing en- produce high yields of violacein and the side-product zymes, leading to an improvement in the production of deoxyviolacein. This was accomplished by cloning the the compound by up to 30-fold when compared to previ- complete vioABCDE and the partial vioABCE operons (re- ously reported work. spectively) from C. violaceum into pBADMycHisB, which We have generated a synthetic operon containing the allows the induction of the operon by L-arabinose [32, coding sequences of each of the five genes required for vio- 34]. These authors also metabolically engineered the host’s lacein biosynthesis, with a codon-usage optimized for E. coli tryptophan production to maximize the yield of violacein (http://parts.igem.org/Part:BBa_K274002). We also con- and deoxyviolacein [32, 34, 42]. More recently Jones and structed strains lacking vioD, to promote the accumulation co-workers [44] and Xu and co-workers [45] utilized vio- of deoxyviolacein. A similar approach was employed by Ro- lacein biosynthesis as a model for metabolic pathway bal- drigues and co-workers for the production of high yields of ancing and optimization. Employing different approaches, violacein and deoxyviolacein [32–34]. We produced and Bilsland et al. BMC Biotechnology (2018) 18:22 Page 3 of 8 purified violacein and deoxyviolacein, and characterized Violacein and deoxyviolacein purification their toxicity and antiplasmodial activity in wild-type and Deoxyviolacein drug-resistant Plasmodium falciparum strains; we also de- The acetone cell extract was evaporated to dryness. The termined their activity against Trypanosoma cruzi. crude residue was suspended in acetone and dry-loaded onto silica gel (SiO2). Deoxyviolacein was purified by column chromatography on silica gel (SiO ), first wash- Methods 2 ing with petroleum ether (boiling point = 40–60 °C) and Bacterial strains and plasmids eluting with a 1:1 solution of ethyl acetate and petrol- We constructed plasmids expressing violacein and deoxy- eum ether. Deoxyviolacein was obtained as a purple violacein by sub-cloning the synthetic violacein operon solid and was analytically pure (100%). The identity and (Part: BBa_K274002) designed by Shuna Gould for the purity of deoxyviolacein were confirmed by 1H NMR iGEM09_Cambridge project (http://parts.igem.org/Part:B- (Additional file 1: Figure S1). We calculated the purity Ba_K274002).Thesyntheticviolaceinoperoniscomprised by integrating related peaks and comparing the areas. of the 5 coding sequences specifying violacein pathway en- No other analysis was run because the data is consistent zymes (vioA, vioB, vioC, vioD and vioE), each preceded by with that from the literature [46]. The apparent purity of a ribosome-binding site. The operon was designed with a deoxyviolacein allowed its quantitation and that of viola- BamHI site in the space between vioB and vioC open cein (see below). Minor contamination by inorganic reading-frames (ORFs), a BglII site between the vioC and compounds cannot be excluded but these would need to vioD ORFs, and a BclI site between the vioD and vioE be soluble in acetone. reading-frames. Since cleavage of the BamHI, BglII, and BclI sites generate compatible cohesive ends, this facili- tated the construction of three different operons: Violacein vioABCE; vioABDE; vioABE. The synthetic operons are The acetone cell extract was evaporated to dryness. The flanked by EcoRI and PstI restriction endonuclease crude residue was suspended in acetone with sonication sites, enabling the use of these two enzymes to readily and dry-loaded onto silica gel (SiO2). Violacein was puri- subclone the entire (vioABCDE) and partial operons fied by column chromatography on silica gel (SiO2), first (vioABCE, vioABDE, vioABE)intotheEcoRI and NsiI washing with petroleum ether (boiling point = 40–60 °C) sites of pBAT4 (Fig. 1). and eluting with 4:6, 1:1, and 6:4 solutions of ethyl acet- ate and petroleum ether (boiling point = 40–60 °C). Violacein was obtained as a crude mixture with approxi- Production and purification of violacein and deoxyviolacein mately 12% deoxyviolacein (estimated from 1H NMR). We transformed E. coli BL21(DE3) (New England Bio- The identity of violacein was confirmed by 1H NMR labs) cells with plasmids expressing the synthetic (Additional file 1: Figure S1) and was consistent with the vioABCDE (for production of both violacein and deoxy- literature [46]. violaein) or vioABCE (for production of deoxyviolacein alone) operons. The leaky expression from the T7 pro- moter in these plasmids was enough to allow sufficient Plasmodium falciparum drug sensitivity assays synthesis of the enzymes in the violacein biosynthetic We cultivated Plasmodium falciparum 3D7 and W2 pathway. strains in complete RPMI (RPMI 1640, Sigma, USA), − We picked individual colonies, inoculated 50 mL cul- supplemented with 10% plasma (AB ) and 2% Haemato- tures in 2× YT (16 g/L Tryptone, 10 g/L yeast extract, crit (O+). Plasmodium
Load more

Recommended publications
  • A Genochemetic Strategy for Derivatization of the Violacein Natural Product Scaffold
    bioRxiv preprint doi: https://doi.org/10.1101/202523; this version posted December 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A GenoChemetic strategy for derivatization of the violacein natural product scaffold Hung-En Lai1#, Alan M. C. Obled4, Soo Mei Chee1,3, Rhodri M. Morgan2, Rosemary Lynch4, Sunil V. Sharma4, Simon J. Moore1^, Karen M. Polizzi5, Rebecca J. M. Goss*4, 1,3, 6 Paul S. Freemont* 1Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London, London SW7 2AZ, UK. 2Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. 3London Biofoundry, Imperial College Translation & Innovation Hub, London, W12 0BZ, UK 4School of Chemistry and Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK. 5Department of Chemical Engineering, Imperial College London, London SW7 2AZ, UK. 6UK DRI Care Research and Technology Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. *Correspondence: [email protected]; [email protected] ^current address School of Biosciences, University of Kent, Canterbury, CT7 2NJ #current address School of Biological Sciences, Victoria University of Wellington, Wellington 6011, New Zealand. Lead Contact: [email protected] Keywords Violacein, antibiotics biosynthesis, natural product analogue, rebeccamycin, bisindole, halogenase, cross-coupling, GenoChemetics Abstract Natural products and their analogues are often challenging to synthesise due to their complex scaffolds and embedded functional groups.
    [Show full text]
  • High Crude Violacein Production from Glucose by Escherichia Coli
    Fang et al. Microbial Cell Factories (2015) 14:8 DOI 10.1186/s12934-015-0192-x RESEARCH Open Access High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway Ming-Yue Fang1, Chong Zhang1*,SongYang2, Jin-Yu Cui2, Pei-Xia Jiang3, Kai Lou4, Masaaki Wachi5 and Xin-Hui Xing1* Abstract Background: As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process. Results: Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr/trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED + pVio was realized with a titer of 0.6 ± 0.01 g L−1 in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED + pVio exhibited a crude violacein titer of 1.75 g L−1 and a productivity of 36 mg L−1 h−1, which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.
    [Show full text]
  • Engineering Oleaginous Yeast Yarrowia Lipolytica for Violacein Production: Extraction, Quantitative Measurement and Culture Optimization
    bioRxiv preprint doi: https://doi.org/10.1101/687012; this version posted July 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Engineering oleaginous yeast Yarrowia lipolytica for violacein production: extraction, quantitative measurement and culture optimization Yingjia Tong1, 2, Jingwen Zhou1, Liang Zhang1*, and Peng Xu2,* 1 School of Biotechnology, Jiangnan University, Wuxi, China 2 Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD, USA * Corresponding author Tel: +1(410)-455-2474; fax: +1(410)-455-1049. E-mail address: [email protected] (PX) and [email protected] (LZ) 1 bioRxiv preprint doi: https://doi.org/10.1101/687012; this version posted July 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Violacein is a naturally occurring anticancer therapeutic with deep purple color. Yeast fermentation represents an alternative approach to efficiently manufacturing violacein from inexpensive feedstocks. In this work, we optimized the extraction protocol to improve violacein recovery ratio and purity from yeast culture, including the variations of organic solvents, the choice of mechanical shear stress, incubation time and the use of cell wall-degrading enzymes. We also established the quantitative correlation between HPLC and microplate reader method. We demonstrated that both HPLC and microplate reader are technically equivalent to measure violacein from yeast culture. Furthermore, we optimized the yeast cultivation conditions, including carbon/nitrogen ratio and pH conditions.
    [Show full text]
  • Chromobacterium Violaceum ATCC31532 Produces
    bioRxiv preprint doi: https://doi.org/10.1101/589192; this version posted March 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A chemical counterpunch: Chromobacterium violaceum ATCC31532 produces 2 violacein in response to translation-inhibiting antibiotics 3 4 Gabriel L. Lozano1,2, Changhui Guan2, Yanzhuan Cao2, Bradley R. Borlee3, Nichole A. 5 Broderick4, Eric V. Stabb5, Jo Handelsman1,2 6 7 1 Wisconsin Institute for Discovery and Department of Plant Pathology, University of 8 Wisconsin-Madison 9 2 Department of Molecular, Cellular and Developmental Biology, Yale University 10 3 Department of Microbiology, Immunology and Pathology, Colorado State University 11 4 Department of Molecular and Cell Biology, University of Connecticut 12 5 Department of Microbiology, University of Georgia 13 14 *To whom correspondence should be addressed [email protected] 15 16 1 bioRxiv preprint doi: https://doi.org/10.1101/589192; this version posted March 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 17 ABSTRACT 18 Bacterially produced antibiotics play important roles in microbial interactions and 19 competition. Antibiosis can induce resistance mechanisms in target organisms and may 20 induce other countermeasures as well. Here, we show that hygromycin A from 21 Streptomyces sp.
    [Show full text]
  • Marine Natural Products from Tunicates and Their Associated Microbes
    marine drugs Review Marine Natural Products from Tunicates and Their Associated Microbes Chatragadda Ramesh 1,2,*, Bhushan Rao Tulasi 3, Mohanraju Raju 2, Narsinh Thakur 4 and Laurent Dufossé 5,* 1 Biological Oceanography Division (BOD), CSIR-National Institute of Oceanography (CSIR-NIO), Dona Paula 403004, India 2 Department of Ocean Studies and Marine Biology, Pondicherry Central University, Brookshabad Campus, Port Blair 744102, India; [email protected] 3 Zoology Division, Sri Gurajada Appa Rao Government Degree College, Yellamanchili 531055, India; [email protected] 4 Chemical Oceanography Division (COD), CSIR-National Institute of Oceanography (CSIR-NIO), Dona Paula 403004, India; [email protected] 5 Laboratoire de Chimie et Biotechnologie des Produits Naturels (CHEMBIOPRO), Université de La Réunion, ESIROI Agroalimentaire, 15 Avenue René Cassin, CS 92003, CEDEX 9, F-97744 Saint-Denis, Ile de La Réunion, France * Correspondence: [email protected] (C.R.); [email protected] (L.D.); Tel.: +91-(0)-832-2450636 (C.R.); +33-668-731-906 (L.D.) Abstract: Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisi- tion of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, “tambjamines”, produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily Citation: Ramesh, C.; Tulasi, B.R.; originated from their bacterial symbionts, which are involved in their chemical defense function, indi- Raju, M.; Thakur, N.; Dufossé, L. cating the ecological role of symbiotic microbial association with tunicates. This review has garnered Marine Natural Products from comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts.
    [Show full text]
  • Chromobacterium Violaceum: a Review of Pharmacological and Industiral Perspectives
    Critical Reviews in Microbiology, 27(3):201–222 (2001) Chromobacterium violaceum: A Review of Pharmacological and Industiral Perspectives Nelson Durán1,3* and Carlos F. M. Menck 2 1Instituto de Química, Biological Chemistry Laboratory, Universidade Estadual de Campinas, Campinas, C.P. 6154, CEP 13083-970, S.P., Brazil and 2Instituto de Ciências Biomédicas, Departamento de Microbiologia, Universidade de São Paulo, S.P., Brazil, and 3Núcleo de Ciências Ambientais, Universidade de Mogi das Cruzes, Mogi das Cruzes, S.P., Brazil * Corresponding author: Prof. Nelson Durán, E-mail: [email protected] FOREWORD This article presents the historical and actual importance of the Chromobacterium violaceum and focuses on the biotechnological and pharmacological importance of their metabolites. Although many groups in the world are working with this bacterium, very few reviews have been written in the last 40 years.39,45,69 ABSTRACT: Violet-pigmented bacteria, which have been described since the end of the 19th century, are occasionally the causative agent of septicemia and sometimes cause fatal infection in human and animals. Bacteria, producing violet colonies due to the production of a nondiffus- ible pigment violacein, were classified as a redefined genus Chromobacterium. Chromobacte- rium violaceum is Gram-negative, and saprophyte from soil and water is normally considered nonpathogenic to human, but is an opportunistic pathogen of extreme virulence for human and animals. The biosynthesis and biological activities of violacein and the diverse effects of this pigment have been studied. Besides violacein, C. violaceum produces other antibiotics, such as aerocyanidin and aerocavin, which exhibit in vitro activity against both Gram-negative and Gram-positive bacteria.
    [Show full text]
  • High-Level Production of Violacein by the Newly Isolated Duganella Violaceinigra Str. NI28 and Its Impact on Staphylococcus Aureus
    www.nature.com/scientificreports OPEN High-level production of violacein by the newly isolated Duganella violaceinigra str. NI28 and its Received: 19 February 2015 Accepted: 23 September 2015 impact on Staphylococcus aureus Published: 22 October 2015 Seong Yeol Choi1, Sooyeon Kim1, Sungsoo Lyuck1, Seung Bum Kim2 & Robert J. Mitchell1 A violacein-producing bacterial strain was isolated and identified as a relative of Duganella violaceinigra YIM 31327 based upon phylogenetic analyses using the 16S rRNA, gyrB and vioA gene sequences and a fatty acid methyl ester (FAME) analysis. This new strain was designated D. violaceinigra str. NI28. Although these two strains appear related based upon these analyses, the new isolate was phenotypically different from the type strain as it grew 25% faster on nutrient media and produced 45-fold more violacein. When compared with several other violacein producing strains, including Janthinobacterium lividum, D. violaceinigra str. NI28 was the best violacein producer. For instance, the crude violacein yield with D. violaceinigra str. NI28 was 6.0 mg/OD at 24 hours, a value that was more than two-fold higher than all the other strains. Finally, the antibacterial activity of D. violaceinigra str. NI28 crude violacein was assayed using several multidrug resistant Staphylococcus aureus. Addition of 30 μM crude violacein led to a 96% loss in the initial S. aureus population while the minimum inhibitory concentration was 1.8 μM. Consequently, this novel isolate represents a phenotypic variant of D. violaceinigra capable of producing much greater quantities of crude violacein, an antibiotic effective against multidrug resistant S. aureus. Violacein is a bisindole derived from the condensation of two molecules of tryptophan1.
    [Show full text]
  • CHAPTER 2 Cloning and Heterologous Expression of a Gene
    Kochi University of Technology Academic Resource Repository � Violacein biosynthesis and its regulation in Pse Title udoalteromonas sp. 520P1 Author(s) ZHANG, Xi Citation 高知工科大学, 博士論文. Date of issue 2010-09 URL http://hdl.handle.net/10173/603 Rights Text version author � � Kochi, JAPAN http://kutarr.lib.kochi-tech.ac.jp/dspace/ Violacein Biosynthesis and its Regulation in Pseudoalteromonas sp. 520P1 XI ZHANG Kochi University of Technology DISSERTATION Submitted for the degree of Doctor of Philosophy Graduate School of Engineering Environmental Systems Engineering Kochi University of Technology, 2010 Kochi, Japan i © All rights reserved by Xi Zhang 2010 ii 高知工科大学博士論文 ABSTRACT ABSTRACT Violacein Biosynthesis and its Regulation in Pseudoalteromonas sp. 520P1 ZHANG Xi Violacein is a violet pigment with several significant properties, such as antitrypanosomal and antitumoural activities. These bioactive properties provide a possibility to use violacein for pharmacological purpose or as a bio-dye due to its purple color. The violacein’s broad pharmaceutical prospects have attracted increased interests in industrial applications. Violacein can be produced by several Gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, Pseudoalteromonas luteoviolacea, Duganella, Collimonas sp., Pseudoalteromonas sp. 520P1 and 710P1. Studies on the violacein biosynthetic pathway have been carried out to reveal the enzymes involved in the biosynthesis and their functions. It has been identified that five enzymes, VioA to VioE, is responsible for the violacein biosynthesis in C. violaceum. These five enzymes are coded by a gene cluster consisted of five enzyme genes, vioA to vioE. The enzymes catalyze the modification and condensation of two molecules of L-tryptophan to form violacein. VioE, the recently reported enzyme, is considered to play a key role to construct the molecular skeleton of violacein.
    [Show full text]
  • A Biosynthetic Platform for Antimalarial Drug Discovery
    1 A biosynthetic platform for antimalarial drug discovery 2 Mark D. Wilkinson1, Hung-En Lai2, Paul S. Freemont2 and Jake Baum3 3 1Department of Chemistry, Imperial College London, UK 4 2Department of Infectious Diseases, Faculty of Medicine, Imperial College London, UK 5 3Department of Life Sciences, Imperial College London, UK 6 7 Correspondence to: 8 Jake Baum ([email protected]), Department of Life Sciences, Imperial College 9 London, Exhibition Road, South Kensington, London SW7 2AZ, United Kingdom 10 Paul Freemont ([email protected]), Department of Infectious Diseases, Imperial 11 College London, Exhibition Road, South Kensington, London SW7 2AZ, United Kingdom 12 13 Running Title: Biosynthesis of violacein as an antimalarial 14 ABSTRACT 15 Advances in synthetic biology have enabled production of a variety of compounds using 16 bacteria as a vehicle for complex compound biosynthesis. Violacein, a naturally occurring 17 indole pigment with antibiotic properties, can be biosynthetically engineered in Escherichia 18 coli expressing its non-native synthesis pathway. To explore whether this synthetic 19 biosynthesis platform could be used for drug discovery, here we have screened bacterially- 20 derived violacein against the main causative agent of human malaria, Plasmodium falciparum. 21 We show the antiparasitic activity of bacterially-derived violacein against the P. falciparum 22 3D7 laboratory reference strain as well as drug-sensitive and resistant patient isolates, 23 confirming the potential utility of this drug as an antimalarial. We then screen a biosynthetic 24 series of violacein derivatives against P. falciparum growth. The demonstrated varied activity 25 of each derivative against asexual parasite growth points to potential for further development 26 of violacein as an antimalarial.
    [Show full text]
  • Studies of Captive Western Toad (Anaxyrus Boreas) Skin
    STUDIES OF CAPTIVE WESTERN TOAD (ANAXYRUS BOREAS) SKIN MICROBIOTA FOR POTENTIAL USE IN CHYTRIDIOMYCOSIS BIOCONTROL A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Marina E. De León 2017 SIGNATURE PAGE THESIS: STUDIES OF CAPTIVE WESTERN TOAD (ANAXYRUS BOREAS) SKIN MICROBIOTA FOR POTENTIAL USE IN CHYTRIDIOMYCOSIS BIOCONTROL AUTHOR: Marina E. De León DATE SUBMITTED: Summer 2017 Biological Sciences Department Dr. Wei-Jen Lin Thesis Committee Chair Biological Sciences Dr. Jill P. Adler-Moore Biological Sciences Dr. A. Kristopher Lappin Biological Sciences ii ACKNOWLEDGEMENTS I would like to extend my deepest appreciation for my advisor, Dr. Wei-Jen Lin, who chose to take a chance with me, and with this idea. Dr. Lin’s patience, kindness, and mentoring have meant everything to me. I am very grateful for my advisory committee, Dr. Jill Adler-Moore and Dr. Kristopher Lappin, who have given me intellectual contributions and unwavering support for this research. I also thank Jon Olson not just for his lab, but also his guidance and open-mindedness. Dr. Derek Sarovich provided so much more than his plasmids; he shared his knowledge as a true scientist, and I am indebted to him for always being available to answer all of my questions. Writing this thesis would not have been possible without the incredible support of my cat and turtle dad, Jimy Hu, Esq. I also need to thank my lab mates, who were always patient and taught me every microbiology technique I know- Ann Nasongkla, Liana Ab Samad, Justin Lee, Ashley Magin, and Danielle Valencia.
    [Show full text]
  • Violacein: Properties and Production of a Versatile Bacterial Pigment
    Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 465056, 8 pages http://dx.doi.org/10.1155/2015/465056 Review Article Violacein: Properties and Production of a Versatile Bacterial Pigment Seong Yeol Choi,1 Kyoung-hye Yoon,2 Jin Il Lee,2 and Robert J. Mitchell1 1 School of Life Sciences, Ulsan National Institute of Science and Technology, 50 UNIST-gil, Eonyang-eup, Ulsan 689-798, Republic of Korea 2Division of Biological Science and Technology, College of Science and Technology, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon-do 220-710, Republic of Korea Correspondence should be addressed to Jin Il Lee; [email protected] and Robert J. Mitchell; [email protected] Received 19 September 2014; Accepted 18 December 2014 Academic Editor: Flavia Marinelli Copyright © 2015 Seong Yeol Choi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein’s function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits.
    [Show full text]
  • Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin Seong Yeol Choi1†, Sungbin Lim1†, Kyoung-Hye Yoon2*, Jin I
    Choi et al. Journal of Biological Engineering (2021) 15:10 https://doi.org/10.1186/s13036-021-00262-9 REVIEW Open Access Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin Seong Yeol Choi1†, Sungbin Lim1†, Kyoung-hye Yoon2*, Jin I. Lee3* and Robert J. Mitchell1* Abstract In this review, we discuss violacein and prodigiosin, two chromogenic bacterial secondary metabolites that have diverse biological activities. Although both compounds were “discovered” more than seven decades ago, interest into their biological applications has grown in the last two decades, particularly driven by their antimicrobial and anticancer properties. These topics will be discussed in the first half of this review. The latter half delves into the current efforts of groups to produce these two compounds. This includes in both their native bacterial hosts and heterogeneously in other bacterial hosts, including discussing some of the caveats related to the yields reported in the literature, and some of the synthetic biology techniques employed in this pursuit. Keywords: Prodigiosin, Violacein, Antibacterial, Anticancer, Secondary Metabolite, Production, Synthetic Biology Introduction compound is their cost, which range from $360 to $760 Bacterial strains are capable of producing many different per milligram [4]. Within this review, therefore, discus- secondary metabolites, including anti-cancer and anti- sion will be given primarily to the biological activities of biotic drugs. Here, we discuss two such compounds that these compounds, focusing on ecological and medical are gaining interest due to their diverse biological activ- considerations of both violacein and prodigiosin, as well ities, namely violacein and prodigiosin. Both of these as current methods to over-produce these remarkable compounds are synthesized by Gram-negative hosts and compounds.
    [Show full text]