Downloaded from orbit.dtu.dk on: Oct 05, 2021 Discovery of Bioactive Natural Products from Marine Bacteria Månsson, Maria Publication date: 2011 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Månsson, M. (2011). Discovery of Bioactive Natural Products from Marine Bacteria. Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal 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. This thesis is submitted to the Technical University of Denmark in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Chemistry. The work was carried out from February 1st 2008 to February 1st 2011, at the Center for Microbial Biotechnology at the Department of Systems Biology under the main supervision of associate professor Thomas Ostenfeld Larsen (CMB) with associate professors Kristian Fog Nielsen (CMB) and Charlotte Held Gotfredsen (Department of Chemistry, DTU) as co-supervisors. The project was funded by the Programme Committee for Food, Health, and Welfare under the Danish Strategic Research Council. First and foremost, I would like to thank my three supervisors for their scientific advice, constructive criticism, and support that extended beyond the confines of work. Thomas, your endless enthusiasm has always inspired and motivated me to stretch beyond my own expectations. Kristian and Charlotte, both with indispensable areas of expertise, have guided my work and provided pep talks at critical turns of the path. A special thanks to professor Lone Gram (National Food Institute, DTU) for mentoring me and always keeping her door open for invaluable discussions. I am also grateful to; Laboratory technician Hanne Jacobsen for running a seemingly endless amount of MS samples for me and teaching me the ways of the TOF; Laboratory technician Jette Melchiorsen for cultivating massive batches of bacteria and screening hundreds of samples without batting an eye; My friend Mette Lyngaae Rasmussen, who worked at CMB for 6 months, keeping track of me and my samples. A special thank you to Dr. Richard Kerry Phipps for always knowing (…42!), and to all my other wonderful colleagues for providing me with food for thought, helpful ideas, and good laughs in equal measure! I would also like to thank a number of productive students, especially Louise Kjærulff who worked hard on isolating compounds from Vibrio. In addition, I would like to extend my gratitude to the other Galathea 3 partners at the National Food Institute, KU-Life, and KU-Health for an interesting and educational collaboration. I greatly appreciate the opportunity to be involved in such a significant and fascinating project. To my fellow PhD students on the project, Matthias (thanks for sharing the dissertation journey with me!), Nikolaj, Anita, Kristina, and Helle: It has been a pleasure to share results, frustrations, and the excitement. During my PhD I had the privilege to spend three months in the Marine Chemistry Group with professors John Blunt and Murray Munro at the University of Canterbury, New Zealand. Despite being on the brink of retirement, they took time to teach me all about CapNMR and share their wealth of experience. My stay in Christchurch was an experience for life in more than one way, and I still dream of the UoC campus coffee. Funding from Oticon, Otto Mønsted, and Knud Højgaards Foundation to finance my stay is greatly acknowledged. I also had the great pleasure to participate in a number of international conferences, which have been a noteworthy source of inspiration and a great chance to present my own work. The marine natural products community is in many ways like a family, always ready to share ‘tricks of the trade’ with a young PhD student. I am very grateful to have had this opportunity to learn and develop friendships across oceans. Credit is also due to the Carlsberg laboratories for allowing me to use their 800 MHz NMR spectrometer. It has been necessary! Finally, I owe my gratitude to my family and friends for their boundless enthusiasm on my behalf and confidence in my ability to achieve my goals. To my husband Dan for his patience through my many travels, his encouragement, and his love that gives me the strength to walk on water. I could not have done this without you. Kgs. Lyngby, February 1st, 2011 Maria Månsson Bacteria produce a vast array of low-molecular-weight compounds endowed with a multitude of biological effects. As these compounds have developed in a biological context, it is hypothesized that bacteria exploit them to gain a competitive advantage for example through antagonistic interactions. It is speculated that these compounds can be used as antibiotics against biomedically important pathogens. The oceans, covering over 70% of the Earth’s surface, constitute a unique ecosystem that promotes bacterial adaptations to the marine environment and provides a driving force for chemical diversity. Thus, marine bacteria represent a promising source of new chemical classes with antibiotic properties to counter the evolution of drug-resistant pathogens. The aim of this PhD study was to explore a global collection of over 500 marine bacteria for their ability to produce bioactive natural products. This included both compounds with antibacterial activities and compounds interfering with virulent phenotypes of pathogens such as Staphyloccocus aureus and Pseudomonas aeruginosa. A primary goal was to provide a robust setup for chemical evaluation of the collected bacteria and develop methods that facilitate rapid dereplication of the associated extracts. In order to efficiently evaluate the large collection of bacteria in terms of their biosynthetic potential and their ability to produce novel compounds, a combination of chemical profiling and dereplication techniques was applied. Profiling of the metabolites produced by members of Pseudoalteromonas and Vibrionaceae by LC-UV/MS was successful in differentiating strains at species and sub-species level. Within our collection, production of secondary metabolites was found to be independent of sample location, with ‘strain siblings’ from distant geographical locations producing the same combination of metabolites. However, significant intraspecies differences were still observed. In Pseudoalteromonas luteoviolacea and Vibrio coralliilyticus some of these differences were related to the production of antibacterial compounds. In this thesis, it is speculated that this contributes to specific niche-adaptation. The potential ecological role of antibiotics in V. coralliilyticus was further corroborated by studies showing that the antibiotic, andrimid, was produced under conditions mimicking the natural environment of this bacterium. During the course of this PhD, three known antibacterial compounds were isolated: Indolmycin (Pseudoalteromonas luteoviolacea), andrimid (Vibrio coralliilyticus), and holomycin (Photobacterium halotolerans). All three are examples of cosmopolitan antibiotics found in distantly related taxa. Genomic promiscuity among bacteria stresses the need for efficient and dynamic dereplication methods to avoid redundancy in isolation of bioactives. To extend our means of dereplication, an explorative solid-phase extraction (E-SPE) method based on three different ion-exchangers and a size-exclusion column was developed. E-SPE exploits the sensitivity of the biological assays to obtain information about the charge, polarity, and size of active components in a crude extract. This can be used to discriminate between possible candidates during dereplication and allows detailed mapping of bioactives. This thesis provides a complete overview of known bioactive metabolites produced by members of the Vibrionaceae family, which constitutes an under-explored source of chemistry. Among the compounds reported so far, there is a prevalence of non-ribosomal peptides or hybrids hereof, with examples of N-containing compounds produced by NRPS-independent pathways. Compounds isolated from Vibrio and Photobacterium during this PhD corroborated this general trend. From Photobacterium halotolerans, we isolated two new cyclic tetradepsipeptides, designated solonamides A and B, as potent inhibitors of the agr QS system involved in virulence expression in Staphyloccocus aureus. Of special interest was a pronounced effect against a highly virulent, community-acquired, methicillin-resistant S. aureus strain (USA300). In conclusion, this PhD study adds to the knowledge of marine bacteria as competent producers of secondary metabolites, some of which possess biological activities attractive for alternative strategies in antibacterial therapy. Bakterier er i stand til at producere en bred vifte af små molekyler beriget med biologisk aktivitet. Da disse stoffer er udviklet i en naturlig kontekst, er forventningen, at bakterierne bruger stofferne som