UNNATURAL PRODUCTION OF NATURAL PRODUCTS: HETEROLOGOUS EXPRESSION AND COMBINATORIAL BIOSYNTHESIS OF CYANOBACTERIAL-DERIVED COMPOUNDS a dissertation submitted by ALEXANDRA A. ROBERTS B.Sc (Biotechnology Hons.) UNSW in partial fulfilment of the requirements for the award of DOCTOR OF PHILPSOPHY (Ph.D.) at the School of Biotechnology and Biomolecular Sciences The University of New South Wales Sydney, Australia MAY 2008 Roberts Alexandra Anne PhD Biotechnology and Biomolecular Sciences Science Unnatural production of natural products: Heterologous expression and combinatorial biosynthesis of cyanobacterial-derived compounds Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated by the same A-domain. This was supported by preliminary ATP-[32P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products. ii SUPERVISOR Prof. Brett Neilan The School of Biotechnology and Biomolecular Sciences The University of New South Wales iii ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ............................................................... Dated ............................................................... iv ABSTRACT Cyanobacteria produce a myriad of structurally unique secondary metabolites with useful bioactive properties. Heterologous expression of a variety of microbial natural compounds has been used to harness their diversity and facilitate their combinatorial biosynthesis. However, these genetic techniques have not been developed for secondary metabolite-producing cyanobacteria. Therefore the genetically manipulable Escherichia coli and Synechocystis sp. PCC6803 were engineered in order to develop effective heterologous hosts and promoters for the expression of cyanobacterial-derived compounds. The phosphopanthetheinyl transferase (PPT), Sppt, from Synechocystis sp. PCC6803 was characterised to determine its ability to activate carrier proteins from secondary metabolite pathways. Despite in silico evidence which suggested Sppt was able to activate a wide range of carrier proteins, biochemical analysis revealed that it is dedicated for fatty acid synthesis. Consequently, E. coli and Synechocystis sp. PCC6803 were engineered to encode a broad-range PPT, from the filamentous cyanobacteria Nodularia spumigena NSOR10, for the activation of carrier proteins from nonribosomal peptide synthesis. Cyanobacterial natural product engineering was also explored with the characterisation of two relaxed specificity adenylation domains (A-domains) from the biosynthetic pathway of the toxin microcystin. The wide variety of microcystin compounds produced by cyanobacterial species suggests that multiple amino acids can be activated 32 by the same A-domain. This was supported by preliminary ATP-[ P]PPi exchange assays and was subsequently harnessed in the production of a variety of dipeptides using two reconstituted modules in vitro. Transposition was investigated as a potential mechanism for the transfer of nonribosomal peptide synthetase gene clusters to heterologous hosts. This was performed via the characterisation of the putative transposase, Mat, physically linked with the microcystin synthetase gene cluster (mcyS). PCR screening, in silico analysis v and nitrocellulose filter binding assays indicated that this transposase may have mediated mcyS gene cluster rearrangements but not entire gene cluster mobilisation between species. The potential role of transposases in the natural combinatorial biosynthesis of microcystin has evolutionary implications for the dynamic nature of cyanobacterial genomes and applications for use in the engineering of novel bioactive compounds. Therefore, the results from this study may provide a biotechnological platform for the transfer, expression and combinatorial biosynthesis of novel cyanobacterial-derived natural products. vi ACKNOWLEDGEMENTS This thesis would not have been possible without the invaluable support of many people: Thankyou to the funding bodies who financially supported this work including the Australian Research Council, the CRC for Water Quality and Treatment and Diagnostic Technology Pty Ltd. I would like to especially thank the CRCWQT whose support enabled me to travel to two overseas conferences and several domestic conferences during my PhD. The School of Biotechnology and Biomolecular Sciences, UNSW, also assisted with travel support as did the NSW Branch of the Australian Society for Microbiology who generously supported my attendance of the 2006 ASM conference. I would like to especially thank Brett Neilan for allowing me to undertake my PhD in his lab. Thankyou for your inspiration, scientific guidance and friendship over the past five years. Thankyou also for giving me such an interesting, although often infuriating, project. I am also very grateful for Brendan Burns’ and Mark Tanaka’s help and advice throughout my PhD. Mark Raftery, Anne Poljak and Linda Ly at the Bioanalytical Molecular Spectrometry Facility, UNSW, assisted me with electrospray ionisation mass spectrometry. Thankyou for your expertise and assistance which has been invaluable to this research. Several people generously provided DNA, strains and vectors that were used throughout my PhD. Thankyou to Susan Golden from Texas A&M University, USA who provided the Synechococcus sp. PCC7942 DNA; Vitor Vasconcelos from the University of Porto, Portugal who provided the non-toxigenic Microcystis spp. DNA and Tony George from the University of Technology, Sydney who provided the pPOW vector. I would like to thank everyone in the BGGM lab for their friendship and support (and all the laughs) over the last five years. In particular, I would like to thank Janine Copp who taught me so much during the early stages of my PhD and collaborated with me on vii the Synechocystis and Nodularia PPT sections. Thankyou to Leanne Pearson who performed the PPi release assays and co-wrote the nonribosomal peptides book chapter. I would also like to acknowledge Naomi Roue who carried out the preliminary work on the adenylation domain chapter. I am greatly indebted to the people who assisted in editing this thesis: Brett Neilan, Janine Copp, Leanne Pearson, Ralitza Alexova, David Roberts and Hannah Ginn. Thankyou for your much-appreciated comments on this manuscript. I would never have made it this far without the love and support of my family and friends. In particular, I would like to thank