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University of Groningen Bacterial natural products Ceniceros, Ana IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ceniceros, A. (2017). Bacterial natural products: Prediction, regulation and characterization of biosynthetic gene clusters in Actinobacteria. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 08-10-2021 Bacterial natural products Prediction, regulation and characterization of biosynthetic gene clusters in Actinobacteria Ana Ceniceros Microbial Physiology University of Groningen The work described in this thesis was carried out in the Microbial Physiology group of the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) of the University of Groningen, The Netherlands. The author gratefully acknowledges the financial support of University of Groningen and the GBB for printing this thesis This research was supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs, and by the University of Groningen. Cover design: Javier Alonso-Olalla Printed by: Ipskamp Drukkers, Enschede ISBN: 978-90-367-9506-7 ISBN: 978-90-367-9505-0 (electronic version) Bacterial natural products Prediction, regulation and characterization of biosynthetic gene clusters in Actinobacteria PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Friday 17 February 2017 at 11.00 hours by Ana Ceniceros born on 23 March 1986 in Pamplona, Spain Supervisor Prof. L. Dijkhuizen Co-supervisor Dr. J.M. Petrusma Assessment Committee Prof. O.P. Kuipers Prof. A.J.M. Driessen Prof. H.A.B. Wosten A mis padres Contents Chapter 1: General introduction .............................................................. 1 Natural products and their physiological roles..................................... 2 Human uses of natural products ....................................................... 10 Potential of bacterial natural product synthesis ................................ 12 Scope of this thesis ............................................................................. 23 Chapter 2: Characterization of the Streptomyces clavuligerus indigoidine synthetase and its associated tautomerase ........................................... 35 Chapter 3: Identification and characterization of the tunicamycin-like antibiotics (MM 19290) of Streptomyces clavuligerus ........................... 65 Chapter 4: Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus ..................................................... 97 Chapter 5: Molecular characterization of a Rhodococcus jostii RHA1 γ-butyrolactone-like signalling molecule and its main biosynthesis gene gblA ....................................................................................................... 141 Chapter 6.1: Summary and concluding remarks .................................. 171 Hoofdstuk 6.2: Samenvatting en conclusies ........................................ 181 Capítulo 6.3: Resumen y conclusiones ................................................ 193 Acknowledgements ............................................................................. 209 CHAPTER 1 General introduction CHAPTER 1 Natural products and their physiological roles Natural products are compounds produced by living organisms. They can be essential compounds such as vitamins, or secondary metabolites which are not essential for an organism but may provide an advantage in their natural environment. These metabolites are therefore also referred to as specialized compounds. Latex is an example of a natural product 2 obtained from plants. It is formed by a mixture of different chemicals that include alkaloids and rubber, and proteins such as proteases or chitinases. This mixture acts as defence mechanism against insects by trapping them or intoxicating them. It is also thought to be a way to excrete waste compounds, coverage of damaged tissue and defence against pathogens 1. Natural products can also be chelators, molecules that improve the availability of essential metals that are normally present in low amounts in the environment. Iron chelators (siderophores) are of great importance since iron is usually available in limiting amounts. In pathogenic bacteria like Mycobacterium tuberculosis, living as an intracellular parasite, siderophores are essential for their survival since the iron is sequestered by the host cell 2. Pigments are another important type of natural product. They can function as photoreceptors in photosynthesis, light protector, antioxidants or can be involved in virulence, as is the case for carotenes 3. Antibiotics are thought to play a role as defence mechanism by eliminating competing organisms from their habitat. But in many cases the physiological role of secondary metabolites is not fully understood. Some antimicrobial compounds are produced (under laboratory conditions) in too low amounts to be able to inhibit the growth of surrounding organisms and therefore are thought to have a different function in the producer strain, possibly as signalling molecules or involved in motility 4. Many secondary metabolites are highly valued by humankind in a wide range of applications. The most relevant producers of secondary metabolites are plants, fungi and bacteria. Depending on the chemical nature of the precursor, they are classified in 5 major classes: saccharides, terpenes, alkaloids, peptides or GENERAL INTRODUCTION polyketides. The latter are synthesized by specialized enzymes named polyketide synthases (PKS). Peptide metabolites include ribosomally and nonribosomally synthesized ones. The last are synthesized by dedicated enzymes called nonribosomal peptide synthetases (NRPS) 5. In many cases these are mixed types of metabolites synthesized from hybrid gene clusters, e.g. NRPS plus PKS 6, 7. Saccharides 3 Saccharides, also known as carbohydrates, are a large group of molecules categorized by the number of monomers that form them as monosaccharides, oligosaccharides and polysaccharides. Saccharides may have very diverse functions and act either as primary or secondary metabolites. Polysaccharides for instance are a main component in biofilms, important for survival and colonization of surfaces 8. These biofilms support survival of microorganisms in their environment. Biofilms are especially problematic in public health since under these conditions microorganisms are more resistant to antimicrobial agents and they are difficult to remove from contaminated instruments 9. The best-known example of biofilm formation is dental plaque. But biofilms are especially dangerous when involving colonization by Staphylococcus spp. of surgical instruments in hospitals which results in many deaths every year 10 (Figure 1). Saccharides also form the O-antigen from lipopolysaccharides in the outer membrane of Gram-negative bacteria, which results in the different serotypes and are important for their pathogenicity and detection by the adaptive immune system 11, 12. Furthermore, saccharides can also be bioactive molecules. This class of molecules has not been explored for bioactivity as much as other classes. Bioinformatics analysis has shown that they constitute a big proportion of the gene clusters present in Actinobacteria, the main bacterial source of secondary metabolites 13, 14. This class of molecules is highly interesting and may hold a great number of novel compounds with bioactive properties. CHAPTER 1 4 Figure 1 Representation of the structure of the intercellular polysaccharide adhesin (PIA) I from Staphylococcus epidermidis which is part of its biofilm and formed by β-1,6-linked 2-acetamido-2- deoxy-D-glucopyranosyl residues (GlcNAc). In some cases, they are not acetylated and therefore + 15 have a positive charge (GlcNH3 ). Adapted from Rhode et al. Terpenes Terpenes are one of the biggest chemical families of secondary metabolites. Their properties vary tremendously, from fragrances or pigments to hormones and bioactive compounds 16, 17. This type of molecules are formed by terpene synthases which contain characteristic domains which have improved their annotation
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