Cloning and Biochemical Characterization of the Hectochlorin Biosynthetic Gene Cluster from the Marine Cyanobacteriumlyngbya Majuscula

AN ABSTRACT OF THE DISSERTATION OF

Aishwarya V. Ramaswamy for the degree of Doctor of Philosophy in Microbiology presented on June 02, 2005. Title: Cloning and Biochemical Characterization of the Hectochiorin Biosynthetic Gene Cluster from the Marine Cyanobacterium Lyngbya maluscula

Abstract approved: Redacted for privacy

William H. Gerwick

Cyanobacteria are rich in biologically active secondary metabolites, many of which have potential application as anticancer or antimicrobial drugs or as useful probes in cell biology studies. A Jamaican isolate of the marine cyanobacterium, Lyngbya majuscula was the source of a novel antifungal and cytotoxic secondary metabolite, hectochlorin. The structure of hectochiorin suggested that it was derived from a hybid PKS/NIRPS system. Unique features of hectochlorin such as the presence of a gem dichloro functionality and two 2,3-dihydroxy isovaleric acid prompted efforts to clone and characterize the gene cluster involved in hectochiorin biosynthesis. Initial attempts to isolate the hectochlorin biosynthetic gene cluster led to the identification of a mixed PKS/NRPS gene cluster, LMcryl,whose genetic architecture did not substantiate its involvement in the biosynthesis of hectochlorin. This gene cluster was designated as a cryptic gene cluster because a corresponding metabolite remains as yet unidentified. The expression of thisgene cluster was successfully demonstrated using RT-PCR and these results form the basis for characterizing the metabolite using a novel interdisciplinary approach. A 38 kb region putatively involved in the biosynthesis of hectochiorin has also been isolated and characterized. The hct gene cluster consists of eightopen reading frames (ORFs) and appears to be colinear with regard to hectoclilorin biosynthesis. An unusual feature of this gene cluster includes the presence ofa ketoreductase domain in an NRPS module and appears to be the first report of suchan occurrence in a cyanobacterial secondary metabolite gene cluster Other tailoring enzymes present in the gene cluster are two cytochrome P450 monooxygenases anda putative halogenase. The juxtaposition of two ORF's with identical modular organization suggests that this gene cluster may have resulted from a gene duplication event. Furthermore, biochemical characterization of two adenylation domains from this cluster strengthens its involvement in the biosynthesis of hectochiorin. ©Copyright by Aishwarya V. Ramaswamy June 02, 2005 All Rights Reserved Cloning and Biochemical Characterization of the Hectochiorin Biosynthetic Gene

Cluster from the Marine CyanobacteriumLyngbya majuscula

by Aishwarya V. Ramaswamy

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented June 02, 2005 Commencement June 2006 Doctor of Philosophy dissertation of Aishwarya V. Ramaswamy presented on June 02, 2005.

APPROVED: Redacted for privacy

Maj or Professor, representing Microbiology Redacted for privacy

Chair of the Department of Microbiology

Redacted for privacy

Dean of the GiIaduè School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes the release of my dissertation to any reader upon request.

Redacted for privacy

shwarya V. Ramaswamy, Author ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my major professor, Dr. William H. Gerwick, for his remarkable support and guidance throughout the course of my graduate work. I would also like to thank my committee members, Dr. Mark Zabriskie, Dr. Steve Giovannoni, Dr. George Constantine and Dr. Jeff Stone for their assistance. 11 am very grateful to Dr. Patricia Flatt and Dr. Daniel Edwards for their mentorship and invaluable guidance throughout my graduate career. I would like to acknowledge Dr. Kerry McPhail, Dr. Harald Gross and Dr. Lena Gerwick for their academic guidance. I would also like to thank Gloria Zeller for her help and friendship. Special thanks also go to former and current members of the Gerwick group for their friendship. Most importantly, I share this accomplishment with my parents and am deeply indebted to them for their love, constant encouragement, unconditional support and confidence in my abilities. Finally, I thank my husband for his love and patience which helped me immensely during these challenging years. TABLE OF CONTENTS

CHAPTER I: GENERAL INTRODUCTION...... 1 GENERAL THESIS CONTENTS...... 38 REFERENCES...... 39

CHAPTER II: ISOLATION OF A CRYPTIC GENE CLUSTER FROM THE CYANOBACTERIUM LYNGBYA MAJUSCULA JHB AND DEMONSTRATION OF ITS EXPRESSION

INTRODUCTION...... 54 RESULTS AND DISCUSSION...... 57 EXPERIMENTAL...... 85 REFERENCES...... 92

CHAPTER III: CLONING AND BIOCHEMICAL CHARACTERIZATION OF THE HECTOCHLOR1N BIOSYNTHETIC GENE CLUSTER FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA

INTRODUCTION...... 99 RESULTS AND DISCUSSION...... 102 EXPERIMENTAL...... 126 REFERENCES...... 132 TABLE OF CONTENTS (Continued)

CHAPTER IV: EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF ADENYLATION DOMAINS FROM THE CARMABIN BIOSYNTHETIC GENE CLUSTER

INTRODUCTION...... 138 RESULTS AND DISCUSSION...... 143 EXPERIMENTAL...... 148 REFERENCES...... 150

CHAPTER V: CONCLUSIONS...... 153

BIBLIOGRAPHY...... 156 LIST OF FIGURES

Figure Page 1.1 NRPS module organization...... 20

1.2 Recognition of cognate amino acid by binding pocket residues (A) and activation of amino acid by the NIRPS A-domain (B)...... 21

1.3 Post-translational modification of the PCP domain...... 22

1.4 Loading of PCP domain with activated amino acid...... 22

1.5 Condensation domains catalyze peptide bond formation...... 23

1.6 Steps involved in nonribosomal peptide biosynthesis...... 24

1.7 Reactions catalyzed by NRPS tailoring domains...... 25

1.8 PKS module organization...... 28

1.9 Recognition of substrate by the AT domain and its transfer to ACP domain...... 29

1.10 Ketide bond formation catalyzed by the KS domain...... 29

1.11 Reactions catalyzed by PKS auxiliary domains...... 30

1.12 The barbamide (53) biosynthetic gene cluster...... 35 LIST OF FIGURES (Continued)

Figure 1.13 The curacin A (14) biosynthetic gene cluster...... 35

1.14 The jamaicamide A (27) biosynthetic gene cluster...... 36

1.15 The lyngbyatoxin A (30) biosynthetic gene cluster...... 36

ILl Isolation of high molecular weight genomic DNA...... 58

11.2 Proposed biosynthetic precursors of hectochiorin...... 60

11.3 Amplification of NRPS A-domain probes...... 62

11.4 ClustalW alignment of cysteine adenylation domains...... 63

11.5 Amplification of cyteine-specific A-domain...... 64

11.6 Amplification of KS domain probe...... 65

11.7 Restriction digest analyses (A) and Southern hybridizations of cosmids 1-105 and 111-105 (B)...... 67

11.8 Genetic architecture of LMcryland modular organization of LMcryl ...... 70

11.9 Alignment of heterocyclization domains...... 71 LIST OF FIGURES (Continued)

Figure

11.10 Alignment of N-methyl transferases...... 73

11.11 Nonribosomal peptides from freshwater and terrestrial cyanobacteria...... 73

11.12 Sequence alignments of PKS domains of LMcryl with other cyanobacterial PKS's...... 75

11.13 Structure of proposed peptide encoded by hybrid NRPS/PKS encoded byLMciyl.R = unknown side chain...... 76

11.14 Reversed phase HPLC profiles for the crude extract and nine normal phase VLC fractions (A-I) of the cultured Lyngbya majusculaJHB...... 79

11.15 Schematic representation of the novel approach used to isolate and elucidate structure of putative metabolite encoded by LMcryl...... 80

11.16 Primers for RT-PCR...... 82

11.17 Results of RT-PCR...... 83

111.1 Chemical structure of hectochiorin (54) and related metabolites 101

111.2 High molecular weight genomic DNA from cultured L. majuscula JHB...... 102

111.3 Alignment of heterocyclization-adenylation domains for primer design (A) and PCR amplification of 1.8 kb heterocyclization- adenylation domain probe (B)...... 104 LIST OF FIGURES (Continued)

Figure ge 111.4 Genetic map of the hct gene cluster spanning38 kb...... 106

111.5 Alignment of barbamide BarB 1 and B2, syringomycin SyrB2, coronamic acid CmaB and PAHX with HctB...... 108

111.6 Sequence alignments of PKS domains from HctD with other cyanobacterial PKS's...... 109

111.7 Overexpression and purification of adenylation domains in HctE and Western blot analysis...... 112

111.8 Results of the ATP-PPi exhange assay for HctEAO.IVA (A) and HctEAc (B) and represent average of duplicate experiments...... 114

111.9 Overexpression and purification of HctA...... 115

111.10 The proposed biosynthesis of hectochiorin...... 116

111.11 Alignment of heterocyclization domains...... 123

IV.1 Genetic architecture of the carmabin A biosynthetic gene cluster...... 140

IV.2 Overexpression of CarG (A) and Carl (B) adenylation domains 145

IV.3 Results for the ATP-PPi exhange assay for CarG A-domain (A) and Carl A-domain (B) ...... 147 LIST OF TABLES

Table Page 1.1 List of selected marine-derived natural products in clinical and preclinicalstudies...... 3

11.1 Substrate specificity of NRPS A-domain probes...... 62

11.2 Substrate specificity of cysteine A-domain probe...... 64

11.3 Substrate specificities of A domain LMcryl and other related A- domains...... 74

111.1 Deduced functions of the proteins in the hct biosynthetic gene cluster...... 107

111.2 Substrate specificity of A-domains from the first modules in HctE andHctF...... 119

111.3 Substrate specificity of the adenylation domains in the second modules present in HctE and HctF...... 122

IV.1 Deduced functions of open reading frames in the car gene cluster...140

IV.2 Substrate specificity of A-domains in car...... 142 LIST OF ABBREVIATIONS

A Adenylation aa Amino acid ACP Acyl carrier protein AMP Adenosine monophosphate AT Acyl transferase ATP Adeno sine triphosphate BLAST Basic alignment search tool bp Base pairs C Condensation cDNA Complementary DNA CTAB Hexadecyltrimethylammonium bromide DII Dehydratase DHiv 2,3-dihydroxyisovaleric acid DIG Digoxigenin DNA Deoxyribonucleic acid DTT Dithiothreitol E Epimerase EDTA Ethylenediaminetetraacetic acid ER Enoyl reductase GHNMQC Gradient hydrogennitrogen multiple quantum coherence Hal Halogenase Het Heterocyclization domain His Histidine kb kilobase kDa kilodalton KR Ketoreductase KS Ketosynthase LIST OF ABBREVIATIONS (Continued)

mRNA messenger RNA MT Methyl transferase NCBI National Center for Biotechnology Information NMR Nuclear magnetic resonance N-Mt N-methyl transferase NRPS Nonribosomal peptide synthetase 0-IVA 2-oxo-valine 2-OH WA 2-hydroxy isovaleric acid ORE Open reading frame Ox Oxidase PAGE Polyacrylamide gel electrophoresis PCP Peptidyl carrier protein PCR Polymerase chain reaction PKS Polyketide synthase Ppant Phosphopantetheine Ppi Inorganic pyrophosphate RNA Ribonucleic acid RT Reverse transcriptase RT-PCR Reverse transcriptase-polymerase chain reaction SAM S-adenosyl methionine SDS Sodium dodecyl sulphate TE Thioesterase VLC Vacuum liquid chromatography CLONING AND BIOCHEMICAL CHARACTERIZATION OF THE HECTOCHLORIN BIOSYNTHETIC GENE CLUSTER FROM THE MARINE CYANOBACTERIUMLYNGBYA MAJUSCULA

CHAPTER I

GENERAL INTRODUCTION

Marine natural products Nature has been a source of medicinal agents for thousands of years and the use of natural products in the treatment of human diseases has been well documented in both traditional Eastern and Western medicine (Newman et al., 2000). Natural products have played a significant role in the process of drug discovery and development and about 60% of drugs in current use are derived from natural products (Newman et al., 2003). The inexorable increase in the emergence of drug-resistant microbes and parasites in recent decades has created a pressing need for the development of new therapeutics (Dougherty et al., 2002). Additionally, the discovery of new pharmaceuticals is vital for treatment of other diseases such as inflammation, immune disorders and different types of cancer. Terrestrial plants and microorganisms have been long recognized as a valuable source of bioactive compounds and serve as an important source for the discovery of new pharmaceuticals. In fact, soil actinomycetes account for the production of over 7000 bioactive compounds (Jensen et al., 2005). Lately, the marine environment, which has remained relatively unexplored, is beginning to gain much attention as a reservoir of bio active natural products. In contrast to studies on terrestrial natural products, exploration of marine natural products started 50 years ago (Bergman and Feeney, 1951) and it was only in the 1970's that an intensive search for natural products from marine plants and invertebrates was initiated. Several factors spurred this vigorous search for bioactive 2 natural products from the sea. The oceans comprise over 70% of the earth's surface and there exists a wealth of biodiversity in the marine environment. Conservative estimates suggest that there are over ten million species and a majority of these remain undescribed (Grassle and Maciolek, 1992; May, 2005). Recently, whole-genome shotgun sequencing of microbial populations from the Sargasso Sea near Bermuda revealed the presence of enormous diversity and abundance of organisms and is believed to represent only a fraction of entire population (Venter et al., 2004). The presence of such a tremendous variety of flora and fauna in the marine ecosystem along with distinct physicochemical conditions present in the ocean create an intensively competitive environment which in turn fosters the development of a wide range of survival strategies not found among terrestrial organisms. As such, natural products produced by marine organisms exhibit tremendous structural complexity and possess novel functionalities and appear to be highly evolved as potent inhibitors of physiological processes in potential predators. This is especially true for sessile invertebrates such as sponges and tunicates that produce toxic metabolites to ward off potential predators. Thus, the marine realm represents a wealth of resource for natural product chemistry and biotechnology. At present, approximately 16,000 natural products have been characterized from marine organisms (MarineLit, 2005). Marine microorganisms, phytoplankton, algae, sponges, soft corals and tunicates are among the predominant sources of novel chemical entities (Blunt et al., 2005). A considerable number of natural products isolated from marine organisms appear to possess anticancer activity and several candidates are currently in clinical studies or preclinical trials. Additionally, some marine-derived bioactive compounds are also being developed as pain-killers and anti- inflammatory substances (Donia and Hamann, 2003). Table 1.1 adapted from several recent reviews on marine-derived lead compounds (Newman and Cragg, 2004b; Newman and Cragg, 2004a; Jimeno et al., 2004; Simmons et al., 2005) provides examples of notable natural products of marine origin or their synthetic derivatives that are currently in use or in preclinical/clinical studies. 3

Table 1.1: List of selected marine-derived natural products in clinical and preclinical studies.

Name Source Target/iViechanism of action Status Ara-C (1) Synthetic analog Anticancer (acute myelocytic Clinical use of sponge leukemia) nucleosides Ara-A (2) Synthetic analog Antiviral (Infectious herpes) Clinical use of sponge nucleosides Ecteinascidin (3) Ecteinascidia Anticancer Phase III (ET743) turbinata It has been shown to bind to the minor (Rinehart et al., 1990). groove of DNA but downstream mechanisms still unclear but may interfere with DNA binding proteins and transcription. Also shown to affect nucleotide excision repair (Manzanares et al., 2001). Ziconotide (4) Conus magus Neuropathic pain Clinical use (Ferber et al., 2003) Bryostatin (5) (Pettit etBugula neritina Anticancer Phase II al., 1982). Serves as a competitive inhibitor of tumor-promoting phorbol esters by binding to identical receptor sites on protein kinase C without possessing tumor promoting activity itself (Amador et al., 2003). Aplidine (6) Aplidium Anticancer Phase II (Rinehart and albicans Blocks the secretion of angiogenic Lithgow-Bertelloni, factor vascular endothetial growth 1991). factor and may have an effect on tumor angiogenesis. The exact mechanism remains to be clarified (Taraboletti et al., 2004). Kahalalide F (7) Elysia rufescens/ Anticancer Phase II (Hamann and Scheuer,Bryopsis sp. Selective for tumors with high 1993). lysosomal activity. Found to induce cell-death probably initiated by lysosomal membrane polarization (Garcia-Rocha et al., 1996). Dolastatin 10 (8) Cyanobacterial Anticancer (Pettit et al., 1987). symbiont of Binds to tubulin and inhibits Dolabella microtubule assembly and subsequently auricularia blocks cytokinesis. Also shown to be a non-competitive inhibitor of Vinca alkaloids (Bai et al., 1990). TZT- 1027 (9) Synthetic Anticancer Phase II (Miyazaki et al., dolastatin The synthetic derivative of dolastatin 1995). 10 was designed to have enhanced antitumor activity and reduced cytotoxic effects (Kobayashi et al., 1997). Table I.! continued. Name Source Target/Mechanism of action Status Discodermolide (10) Discodermia Anticancer Phase I (Gunasekera et al., dissoluta Microtubule stabilizing agent which 1990). causes arrest cell cycle arrest at the G2IM phase. Also possesses immunosuppressive properties (Kalesse, 2000). E73 89 (11) Lissodendoryx Anticancer Phase I (Halichondrin B sp. Binds tubulin at a site distinct from the derivative) (Aicher et Vinca site and causes tubulin al., 1992). depolymerization (Towle et aL, 2001). IPL-576092 (12) Petrosia contignata Anti-inflammatory Phase I Derivative of It inhibits allergen-induced histamine contignasterol release from mast cells. Topical (Burgoyne et al., application showed that it inhibited 1992). allergen-induced plasma protein exudation (Coulson and ODonnell, 2000).

Eleutherobin (13) Eleutherobia sp. Anticancer Preclinical (Lindel et al., 1997). Induces tubulin polymerization in vitro in a manner similar to paclitaxel. Cells exposed to eleutherobin contain multiple nuclei and arrest at mitosis (Long et al., 1998). Curacin A (14) Lyngbya ma] uscula Anticancer Preclinical (Gerwick et al., 1994). Binds to colchicine site of tubulin and induces depolymerization induced by glutamate or microtubule associated proteins (Wipfet al., 2004). Thiocoraline (15) Micromonospora Anticancer Preclinical (Perez-Baz et al., marina Thiocoralme inhibits DNA elongation 1997; Romero et al., and probably inhibits the activity of 1997). DNA polymerase-a (Erba et at., 1999). Salinosporamide A Salinospora sp. Proteosome inhibitor Preclinical (16) (Fehling et al., (salinosporamide A inhibited 2003). proteasomal chymotrypsin- like proteolytic activity) Also displayed in vitro cytotoxicity against several cancer cell lines (Fehling et al., 2003). NH2 NH2 N NN ON HOOI HO0 HO HO

OH OH Ara-C (1) Ara-A (2) cidin 743 (3)

NH2-CKGKGAKCSRLMYDC TGSCRSGKC-CONH2

Ziconotide (4)

OH 0

NH N 00 Bryostatin 1 (5) H2N NNH OCH3

Aplidine (6) O HN 0 0NH O2NH 0 H / HN 'H0 0 HO NH \ / Y H 0 O N T T ( T y Kahalalide F (7) 0 OCH OCH

Dolastatin 10 (8) NANTIrIyNO 0..- CH3OH 0 0CH

TZT 1027 (9)

OH OH NH2 Discodermoljde (10)

H2N

E7389 (11) HOJOH 0 IPL 576092 (12)

OCH3 CH3 o: O OCH3 OCH3 N4,,CH3 CuracinA (14) OH H'V'H

Eleutherobin (13)

O QoH N O ONH NO O HN sSH ci o NH0 Salinosporamide A (16)

0 Thiocoraline (15) HO- 7

The preceding section has illustrated some of the significant contributions of marine natural products especially to the field of biomedicine. Among marine organisms, cyanobacteria are emerging as one of the most prolific producers of novel bioactive secondary metabolites (Burja et al., 2001; Gerwick et al., 2001). In the following section, a brief overview of cyanobacteria will be presented along with examples of some noteworthy secondary metabolites isolated from marine cyanobacteria (especially those belonging to the genusLyngbya).A predominant theme encountered in cyanobacterial secondary metabolites is the rich integration of complex enzyme systems known as polyketide synthases and nonribosomal peptide synthetases. Details regarding these enzymes and the biosynthetic gene clusters of notable cyanobacterial metabolites will be discussed in the next section. The final section will provide a brief outline of the various chapters presented in this thesis.

Secondary metabolites from marine cyanobacteria Cyanobacteria, also known as blue-green algae, are ancient (3.5 Ma years) and morphologically the most diverse photoautotrophic prokaryotes, which inhabit a wide diversity of habitats including open oceans, tropical reefs, shallow water environments, terrestrial substrates such as rocks, aerial environments such as in trees, and fresh water ponds, streams and puddles. Most cyanobacteria are generalists and can tolerate a great range of environmental conditions (Whitton and Potts, 2000). It is presumed that cyanobacteria evolved in the Precambrain Era and caused the transition in the Earth's atmosphere from its primeval anaerobic state to its current oxygenic one (Schopf, 2005). The first global evolutionary scheme of cyanobacteria based on phylogenetic analysis of 1 6s rRNA indicated that they underwent rapid diversification within a relatively short span of time (Giovannoni et al., 1988). Cyanobacteria were regarded as algae by botanists until electron microscopy and other biochemical studies provided conclusive evidence in favor of their prokaryotic nature. Therefore, early cyanobacterial taxonomy was based on morphological features in accordance to the Botanical code. However, this approach was confronted by Stanier and collaborators who pioneered the use of physiological and genotypic features determined with axenic cultures and advocated a bacteriological approach for cyanobacterial taxonomy (Stanier et al., 1978). The basis of bacteriological taxonomy of cyanobacteriawas first proposed by Rippka (Rippka et al., 1971). This system also relieson morphology to a large extent and allows identification of strains at the generic level. A modified version of this system is described in Bergey's Manual of Systematic Bacteriology [Subsection I (Chroococcales), Subsection II (Pleurocapsales), Subsection III (Oscillatoriales), Subsection IV (Nostocales) and Subsection V (Stigonematales)] (Castenholz et al., 2001). While analyses with 16S rDNAsequences have revealed that two out of the five taxanomical groupingsare phylogenetically coherent, members belonging to Chroococcales, Pleurocapsales and Oscillatoriales do not form coherent phylogenetic lineages making phylogenetic relationship among cyanobacteria disputatious (Giovannoni et al., 1988; Wilmotte, 1994; Ishida et al., 2001; Seo and Yokota, 2003). Although 16S rDNA sequence analyses have provided valuable information on the genotypic relationships of cyanobacteria,a polyphasic approach to link this molecular data with phenotypic studies is important in order to obtaina more meaningful interpretation of these results. Cyanobacteria play a major role in global cycles of several key elements and have also been recognized as model systems for studying important physiological processes such as photosynthesis (Barry et al., 1994) and circadian organization (Iwasaki and Kondo, 2004). Cyanobacteria are becoming increasingly important economically in both positive and harmful ways. They possess a rich assortment of photosynthetic pigments such as chlorophyll a and b as well as phycoerythrin, phycocyanin and allophycocyanin. Phycoerythrin present in cyanobacteria has found application as a conjugate to antibodies that then allow visualization of cellular constituents and processes (Batard et al., 2002) and chlorophyll is being explored for its chemoprevention properties (Egner et al., 2001). Other cyanobacteria suchas Spirulina are being promoted as health food because they contain essential amino acids. On the other hand, certain freshwater bloom-forming cyanobacteria contaminate drinking water supplies by producing neurotoxins suchas saxitoxin (17) and hepatotoxins like microcystins (18) and nodularin (19) andpose significant threat to human and livestock health (Briand et al., 2003). Cyanobacterial blooms have also been shown to impact ecosystem structure and trophic interactions (Paerl, 1988).

H2NOgJH

I )=NH HN'N Nft0H OH Saxitoxin (17)

O COOH ,CH3 COOH CH3O NN CH3 NH CH2I NH H3C0 H3C, o OCH3 H3C CH3 CH3 H0NNH CH3 HN cH3 CH3 CH3 _yNy1yN(L.L.CHH H 0 COOH

COO NH HN H2N- HN=< NH NH2 Microcystin-LR (18) Nodularin (19)

Marine cyanobacteria appear to possess an additional feature, the rich elaboration of bioactive natural products that has enabled these organisms to survive in predator-rich tropical reef ecosystems. Richard Moore at the University of Hawaii pioneered the chemical investigation of marine cyanobacteria for their unique natural products. In the early 1970's his laboratory published several surveys of marine cyanobacteria from the Pacific showing that they were rich in potential anticancer and antiviral substances (Moore, 1978; Moore, 1981; Moore, 1996). Early work showed that cyanobacteria, as opposed to most other marine life forms at the time,were rich in nitrogen-containing natural products. Subsequently, freshwater cyanobacteriawere shown to have similar biosynthetic capabilities and to producean equally exciting array of biologically active lipopeptides. They are now widely recognized to be one of 10 the richest of all marine life in this regard, bothas free living organisms as well as when living in association with marine invertebrates (sponges and tunicates) (Sings and Rinehart, 1996; Gerwick et al., 2001; Piel, 2004). As a result, members of the Oscillatoriaceae, (especially the filamentous cyanobacterium Lyngbya majuscula) have proven to be exciting sources of novel natural products with therapeutic and biotechnological potential. Over 200 novel secondary metabolites have been isolated from various collections of L. majuscula (MarinLit, 2005) and accounts for about 30% of all natural products reported from marine cyanobacteria (Burja et al., 2001). Detailed experimental investigation of the ecological role of marine cyanobacterial natural products has generally shown that marine cyanobacteria are chemically defended against predation by generalist feeders (Nagle and Paul, 1999; Nagle and Paul, 1998). Interestingly the chemical composition of some marine cyanobacteria, especially Lyngbya majuscula, is remarkably diverse between different populations. For instance, at least five different chemotypeswere identified from an apparent monoculture of L. majuscula from Guam (Nagle and Paul, 1999). A comparison of morphological characteristics, secondary metabolite composition and 16S rDNA sequences revealed that morphology and secondary metabolite profile could not be correlated to genetic variation and suggest that phylogeny based on 1 6S rDNA cannot be used to predict chemical variability (Thacker and Paul, 2004). The apparent lack of correlation could bea result of the higher rate of evolution of secondary metabolite gene clustersor horizontal transfer of these clusters between organisms. Alternately, the varied chemical profiles of cyanobacteria may also be an adaptive response to the environmental conditions present at a particular location (Nagle and Paul, 1999; Thacker and Paul, 2004). A high proportion of cyanobacterial secondary metabolites appear to possess the ability to interfere with the assembly of protein polymers in eukaryotic cells, particularly, actin and tubulin. Another emerging trend is the isolation of metabolites that target mammalian ion channels like the voltage-gated sodium channel. L. majuscula is also notorious for the formation of massive blooms in recreational waters often causing contact dermatitis and other skin irritations (Osborne et al., 2001). The 11 following section recounts several prominent marine cyanobacterial products basedon their biological activity. i. Tubulin binding compounds Microtubules play many significant roles in cell biology and microtubule assembly is vital for the formation of spindle apparatus during cell replication and mitosis. Dolastatin 10 (8), a linear peptide was first isolated from the sea-hare Dolabella auricularia (Pettit et al., 1987) and subsequently re-isolated from the marine cyanobacterium Symploca sp. confirming that the true source is the cyanobacterium and not the sea-hare (Luesch et al., 2001 a). Dolastatin 10 binds to tubulin and inhibits microtubule assembly and subsequently blocks cytokinesis. It has also shown to be a non-competitive inhibitor of Vinca alkaloids (Bai et al., 1990) and is currently in Phase II clinical trials for the treatment of renal carcinoma, lymphoma and lymphocytic leukemia (Newman and Cragg, 2004a). A Caribbean strain of L. majuscula was the source of curacin A (14), a molecule that consists of an interesting thiazoline ring containing a 14-carbon alkyl chain with conjugated diene and terminal olefin, anda methyl substituted cyclopropyl moiety (Gerwick et al., 1994). Curacin A was shown to be selective for colon, renal and breast cancer cell-lines when tested in NCI's 60 cell line assay. It was later found to be a potent inducer of tubulin depolymerization(1050= 4.0 pM). Further testing revealed that curacin A binds to the colchicine site of tubulin and stimulates the uncoupled GTPase reaction that is typical of such agents. A series of semi-synthetic analogs have been produced and curacin A is currently in preclinical trials asan antiproliferative agent (Table 1.1) (Wipf et al., 2004). The curacin A biosyntheticgene cluster has been recently characterized (Chang et al., 2004). Cryptophycin A (20), a cyclic peptide was initially isolated from a terrestrial cyanobacterium Nostoc sp. for its antifungal activity (Golakoti et al., 1994). The compound exhibited strong cytotoxicity against KB (1050= 5 pglmL). Cryptophycin A was found to be an effective inhibitor of tubulin polymerization, causing aggregation of tubulin and depolymerization to linear polymers ina manner similar to Vinca 12 alkaloids and also inhibits vinbiastine binding to tubulin but not coichicine binding suggesting that their binding sites may be identical or overlapping (Kerksiek et al., 1995). Interestingly, a similar cyclic peptide, arenastatin A (21) was then isolated from the Okinawan sponge Dysidea arenaria (Kobayashi et al., 1994). Cryptophycin and its synthetic derivatives were in Phase I clinical trialsas anticancer agents but were withdrawn in 2002 (Newman and Cragg, 2004a).

° L:J HI'J

HNOLOCH3 O HNO OCH3 oi Arenastatin A (21) Cryptophycin A (20) (Cryptophycin-24)

ii. Actin-binding compounds The actin cytoskeleton is a dynamic network of filaments which plays an important role in maintaining cell shape, motility and signal transduction (Fenteany and Zhu, 2003). Lyngbyabellins A (22) and B (23)are Lyngbya majuscula-derived lipopeptides (Luesch et al., 2000a; Luesch et al., 2000b; Milligan et al., 2000) thatare structural homologs of dolabellin (24) isolated from the sea-hare, Dolabella auricularia (Sone et al., 1995). Lyngbyabellin A (22) exhibitedIC50values of 0.03 tg/mL and 0.50 ig/mL against KB cells (human nasopharyngeal carcinoma cell line) and LoVo cells (human colon adenocarcinoma cell line), respectively. Lyngbyabellin A was also shown to disrupt the microfilament network in fibroblastic A- 10 cells at concentrations between 0.0 1-5.0 tg/mL. However, when treated with a higher concentration of lyngbyabellin A many cells became binucleate, an observation which is consistent for compounds inhibiting cytokinesis. Lyngbyabellin B (23)was found to be less toxic than lyngbyabellin A(1050values of 0.10 pg!mL and 0.83 pg/mL 13 against KB and LoVo cell lines, respectively) but exerted similar cellular effectsas the latter (Luesch et al., 2000a; Luesch et al., 2000b).

CI CI N=y HNO 0NJJo 0HNQ -m S N -, N

Lyngbyabellin A (22) Lyngbyabellin B (23)

CI Th,,ONOH 0 OH

DCH3

Dolabellin (24)

iiii.Neurotoxic compounds Antillatoxin (25), a cyclic lipopeptide was first reported as a potent ichthyotoxin (Orj ala et aL, 1995). It is the most potent cyanobacterial ichthyotoxin to date with with anLD50 0.05 ug/mL. Initial pharmacological studies showed that antillatoxin was acutely neurotoxic and rapidly induced morphological changes in rat cerebellar granule neurons (CGC's), including blebbing of neurite membranes (Berman et al., 1999). Further testing showed that antillatoxin was a powerful activator of voltage-gated sodium channels and mediated this activity through a unique binding site (Li et al., 2001). 14

Antillatoxin (25)

Kalkitoxin (26), a linear lipopeptide was first isolated from a Caribbean collection ofL.majuscula and exhibited potent brine shrimp and fish toxicity (Wu, 1996). It was subsequently re-isolated in very small quantities from various Caribbean collections ofL.majuscula (Wu et al., 2000). Kalkitoxin was toxic to rat CGC's and had an LC50 =3.86 nM (Berman et al., 1999) and there is also evidence indicating that kalkitoxin is a blocker of voltage-sensitive Na channel in mouse neuro-2a cells (EC50 = 1 nM) (Wu et al., 2000).

Kalkitoxin (26)

The jamaicamides A-C (27-29) were isolated as highly functionalized lipopeptides from a Jamaican strain of L. majuscula. Jamaicamaide A (27)was particularly unique in its presence of an acetylenic bromide functionality. The jamaicamides exhibited cytotoxicity against H-460 human lung cell line and mouse neuro-2A neuroblastoma cell lines with an LC5015 pM for both cell lines and showed sodium channel blocking activity ata concentration of 5 tM (Edwards et al., 2004). The recently characterized jamaicamide A biosynthetic gene cluster has provided several powerful insights into the biosynthesis of this metabolite (Edwards et al., 2004). 15

OCH3 NO

Jamaicamide A (27) R=Br Jamaicamide B (28) R=H

OCH 'NO Jamaicaniide C (29)

iv. Other interesting metabolites Lyngbyatoxin A (30), was isolated from a Hawaiian shallow-water variety of L. majuscula and is believed to be responsible for a condition known as 'swimmer's itch' (Cardellina et al., 1979). This highly inflammatory compound had anLD100 = 0.3 mg/kg in mice which was comparable to the toxicity of the teleocidins (Takashima and Sakai, 1960). It was also found to be ichthyotoxic and exposure to 0.15 tg/mL resulted in death of fish in 30 minutes (Cardellina et al., 1979). Ensuing pharmacological studies showed that lyngbyatoxin A (30) was a potent tumor promoter in a maimer similar to that of the phorbol esters when tested in mice (Fujiki et al., 1984). The lynbyatoxin A biosynthetic gene cluster has been characterized from a Hawaiian strain of L. majuscula (Edwards et al., 2004).

Debromoaplysiatoxin (31) Lyngbyatoxin A (30) Iri

Debromoaplysiatoxin (31), another highly inflammatory compound, was isolated from an Okinawan collection of L. majuscula (Mynderse et al., 1977). Further testing showed that the purified toxin induces cutaneous inflammation and pustular folliculitis in humans and caused a severe inflammation in rabbits and hairless mice upon topical application (Solomon and Stoughton, 1978).

L. bouilloniiisolated from Apra Bay, Guam, initially mis-identified asL. majuscula, was found to produce a very potent cytotoxin, apratoxin A (32) which exhibited anIC50= 0.52 nM and 0.36 nM against KB and LoVo cells, respectively. However, its exact mode of action at present is uncharacterized (Luesch et al., 2001b). A total synthesis of aparatoxin A has been reported (Chen and Forsyth, 2004) and the synthesis of several structural analogs may provide insights into its mode of action.

OCH3

N 0

( Apratoxin A (32)

The yellow-brown pigment, scytonemin (33), accumulates in the extracellular sheath of many cyanobacteria (Nageli, 1849) but its structural determinationwas completed only recently (Proteau et al., 1993). Structurally, the scytoneman skeleton is very unique and is proposed to arise from the condensation of phenyipropanoid and tryptophan units (Garcia-Pichel and Castenholz, 1991). It has since been established that the production of scytonemin is a strategy to protect cells against the damaging effects of ultraviolet radiation (UV) (Garcia-Pichel and Castenholz, 1991; Garcia- 17

Pichel et al., 1992). The production of scytonemin is induced only by UV and increases proportionally to the flux received by the cell. This metabolite is also the first described small molecule inhibitor of human polo-like kinase and is non-toxic, making it an attractive candidate for the development ofnew anti-proliferatives (Stevenson et al., 2002a; Stevenson et al., 2002b).

Scytonemin (33)

Marine cyanobacteria, especiallyLyngbya majuscula,are clearly an extraordinary source of complex and bio active secondary metabolites, and there isa growing recognition that many of the more interesting natural products previously attributed to marine invertebrates are indeed of cyanobacterial origin. The continued discovery of new compounds from these metabolically talented organisms will provide a range of novel and bioactive natural products which maybe developedas potential therapeutics.

Nonribosomal peptide synthetase and polyketide synthases A predominant theme encountered in marine cyanobacterial metabolites is the rich integration of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) to generate a seemingly endless number of novel metabolites (Gerwick et al., 2001). PKS and NRPS are large multifunctional enzymes that share striking organizational similarities. These enzymes have a modular arrangement and specific domains within these modules are responsible for the activation, incorporation and modification of simple carboxylic acids (PKS) or amino acid (NRPS) precursors (Walsh, 2004). Each domain consists of highly conserved amino acid residues orcore motifs that enable the prediction or verification of domain function. The extensive modification of these precursors or incorporation of unusual building blocks results in unparalleled structural diversity (Cane et al., 1998; Walsh, 2004). A brief overview of both biosynthetic systems is presented here.

Nonribosomalpeptide synthetases Nonribosomal peptide biosynthesis is widespread in several bacteria and fungi and plays an important role in the production of several biologically relevant products (Schwarzer et al., 2003). These include antibiotics such as gramicidin S (34) (Kratzschmar et al., 1989; Hon et al., 1989), bacitracin (35) (Gaidenko and Khaikinson, 1988), and the penicillin precursor ACV-tripeptide (36) (Maccabe et al., 1991); immunosuppressive agents suchas cyclosporine (37) (Weber et al., 1994); and siderophores such as enterobactin (38) (Liu et al., 1989) and myxochelin A (39) (Silakowski et al., 2000).

NH2

NH o NH2 0 SH HN o ACV-Tripeptide (36)

Gramicidin (34)

o NHNH o NH s o HN 0 NH2 ON HO 0 NH L) Bacitracin:35) o,_N)i0H

HO 19

hN-

Cyclosporin (37)

HOi )OH0 OH ONH HN

0 0 0 0 OH HNONH HON)OH 0 QOHOH Moche1in A (39) OH OH Enterobactin (38)

Three domains, the adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domain are involved in substrate recognition and activation, its transfer to various catalytic centers and formation of peptide bond, respectively. These three domains are integral parts of all NRPS modules and are often referred to as the minimum module (Marahiel et al., 1997) (Figure 1.1). Nonribosomal peptides are structurally more diverse than those that are ribosomally synthesized and this is achieved by the incorporation of proteinogenic amino acids as wellas "unusual" amino acids such as a-hydroxy acids and carboxylic acids that may undergo further modification such as N-, C-, or 0-methylation, heterocyclization, epimerization and glycosylations mediated by associated tailoring enzymes (Finking and Marahiel, 2004; Sieber and Marahiel, 2005). 20

1'eptidvl carrier Condensation Adenviation teim Thioesterase

Cl C2 Al A2 A3 A4A5 A6A7A8A9 AlO E I\ Required ZIZ2C3 Z3Z4Z5Z6Z7 El E2 E3E4E5E6E7 MI M2 M3 Heterocyclization Rl R2 R3R4R5R6 R7 N-Methylation Epimerization Reductase Optional domains Figure I.!: NRPS module organization. A minimum module is composed of the three required domains, adenylation, peptidyl carrier protein and condensation. Conserved amino acid residues within each domain are represented as bold lines (Marahiel et al., 1997).

The adenylation domain (550 amino acids in size) is involved in the selection of the cognate amino (carboxy) acid substrate and its activation as the aminoacyl adenylate in a manner akin to t-RNA synthetases. But despite their catalytic similarity to t-RNA synthetases, crystal structure of the phenylalanine activating A-domain from gramicidin S synthetase revealed that its tertiary structure was more similar to firefly luciferases (Conti et al., 1997). The crystal structure of this representative A- domain along with sequence comparisons with other A-domains revealed that conserved amino acid residues, most of which lie between the cores A4 and AS, played an important role in determining substrate specificity (Stachelhaus et al., 1999). Furthermore, these signature amino acid residues, referred to "codons", enable reliable prediction of substrate specificity of newly identified A-domains (Figure I.2A) (Challis et al., 2000; Stachelhaus et al., 1999). Selection of the cognate amino acid is then followed by its activation as a highly reactive aminoacyl adenylate in a reaction that requires ATP and Mg2 (Figure I.2B) (Finking and Marahiel, 2004). 21 A

NH NH2 B

R R 0 0 0 9 H II H + 0P0P0P0 AH2N H2N(O I I I O __kOri 0 0- 0- PPi 0 Mg2 Mg2 OH OH OH OH

Figure 1.2: Recognition of cognate amino acid by binding pocket residues (A) [figure adapted from (Challis et al.,2000)] and activation of amino acid by the NRPS A-domain (B).

In the next step, the activated amino acid is transferred to the post- translationally modified PCP domain. This modification involves the transfer ofa phosphopantetheinyl co-factor (Ppant) onto a conserved serine residue in the PCP domain by a 4'-phosphopantetheinyl transferase (PPTase) to convert them to their functionally active holo-form (Figure 1.3) (Finking and Marahiel, 2004). The Ppant co-factor acts as a flexible arm and facilitates the transfer of the bound aminoacyl and peptidyl substrates between various catalytic domains. Activated amino acidsare covalently tethered onto to the thiol group of the Ppant co-factor (Figure 1.4) (Schwarzer et al., 2003). 22

NH2

H H N HS' N 0 H H OH 0 N N -.HS' 0 OH 4-PPtase 0 0E-0

0 holo-PCP

apo-PCP

Figure 1.3: Post-translational modification of the PCP domain. Conversion ofapo- PCP to holo-PCP is achieved through the action of a dedicated 4'- phosphopanteteinyl transferase which transfers a phosphopanteteinyl co-factor

R R H2N(AMP . HN £ -AMP

Figure 1.4: Loading of PCP domain with activated amino acid (Schwarzer et al., 2003).

The condensation domain of NRPS's (450 amino acids) is responsible for peptide bond formation and chain translocation along the assembly line. Condensation domains are localized between each consecutive A-PCP domains and catalyze peptide bond formation between the electrophilic upstream peptidyl-S-PCP and the free amine group of the downstream aminoacyl-S-PCP (Figure 1.5). Additionally, studies have also revealed that the condensation domains may havean editing function and are able to discriminate against amino acids of opposite stereochemistry or larger side chains (Beishaw et al., 1999; Ehmann et al., 2000). 23

H 0 RNH 0

d a Ri R2

SH SH

Figure 15: Condensation domains catalyze peptide bond formation. PCP domains of adjacent modules are loaded with activated cognate amino acids. The electrophilic acceptor amino acid enters the donor site (d) and the nucleophilic donor amino acid is located on the acceptor site (a) of the C-domain. After peptide bond formation, the resulting dipeptide is transferred to donor site of the adjacent C-domain. Figure adapted from (Finking and Marahiel, 2004).

Termination of nonribosomal peptide synthesis is catalyzed by a thioesterase domain (TE) located at the C-terminal end of the last module (Finking and Marahiel, 2004). Chain release is occurs when the nascent peptidyl chain is transferred toan active site serine present in the TE domain to generate an acyl-O-TE-enzyme intermediate which is then attacked by a peptide nucleophile or water to forma macrocycle or linear peptide product, respectively (Kohli and Walsh, 2003; Sieber and Marahiel, 2003). Although macrocyclic release appears to be the preferred mechanism, the mode of termination varies significantly between systems (Kohli et al., 2001; Sieber and Marahiel, 2005). Module 1 Module 2 Module 3 Module 1 Module 2 Module 3 Priming

R10 Cyclization/ LH LH Release HN Loading

R3 0 Module 1 Module 2 Module 3 Mode1Module2Module3

H s 1-0 SH H2NK. H2NK H2N R1N R1 R2 R3 NH \ H 8 Initiation

H Termination /1

3 Elongation

R1< H NH 0=<

H2N

Figure 1.6: Steps involved in nonribosomal peptide biosynthesis. Figure adapted from (Schwarzer et al., 2003)

In addition to the required domains involved in building the peptide backbone, structural diversity is also introduced by the modification of the growing peptide chain and is catalyzed by a variety of embedded tailoring domains such as epimerization (E), N-methylation (N-Mt), oxidation (Ox), reduction (R). These auxiliary domains can act in cis or in trans during the biosynthetic process (Finking and Marahiel, 2004). The reactions of some of these tailoring domains are summarized in Figure 1.7. 25

Epimerization N-methyl transferase domain domain R R R R

+H3N( S-adenosyl H3N( HN methonine 0 o (SAM) +H3Nri CH3 0

Heterocyclization domain R S ..,,. +H3N +H3N( -2H0 OãHS R

Oxidase Reductase domain domain 0 0

HJLN R2 N +H3N - +H3N - H*LH ..7.__&:)L..ir0 +NADPH FMN R R FL j 0 R £

Figure 1.7: Reactions catalyzed byNRPStailoring domains. Figure adapted from (Schwarzer et al., 2003). Polyketide synthases Polyketides represent another important group of structurally diverse natural products and include important antibiotics such as erythromycin A (40) (Cortes et al., 1990; Donadio et al., 1991), immunosuppressive agents such as rapamycin (41) (Schwecke et aL, 1995) and FK506 (42) (Motamedi et al., 1997) and antiparasitics such as avermectin (43) (MacNeil et al., 1992). Polyketide synthases (PKS)are multifunctional enzymes that catalyze the formation of complex polyketides by repetitive addition of extender units derived from malonateor methyl malonate to an activated carboxylic acid starter unit in a manner that is similar to fatty acid synthases (Weissman, 2004). On the basis of their structure and biochemistry, three different types of polyketide synthases have been described (Staunton and Weissman, 2001). The type I PKS ' s are modular enzymes similar to NRPS 's in architecture and consist of distinct domains that catalyze incorporation of simple precursors. The type I PKS involved in the biosynthesis of 6-deoxyerythronlide B, the non-glycosylatedprecursor of erythromycin A (40) is one of the best examples of such systems (Khosla, 1997; McDaniel et al., 2005). A brief overview of modular type I PKS's is presented in the following sections. In contrast, type II PKS's are composed of iteratively acting individual proteins that generate poly-3-keto intermediates and associated ketoreductases, cyclases and aromatases catalyze the formation of multicyclic aromatics like actinorhodin (44) (Staunton and Weissman, 2001). The type III PKS's in bacteria are a newly characterized group andare similar to chalcone synthases of higher plants. They are distinct from type I and type II PKS'5in that they use free CoA thioesters as substrates without the involvement of 4'-phosphopantheteineco- factor (Moore and Hopke, 2001; Moore et al., 2002). The recently characterized enterocin (45) biosynthetic gene cluster from Streptomyces maritimus isa good example of a type III polyketide synthase-derived product (Piel et al., 2000). 27

OMe

Erythromycin A (40) Rapamycin (41)

FK506 (42)

Avermectjn Bib (4

I Ii COOH OH 0

Actinorhodjn (44)

Enterocjn (45) Modular type I PKS's are architecturally similar to NRPS's andpossess mandatory or core domains along with additional tailoring domains. In thecase of PKS, a minimum module is composed of the ketosynthase (KS), acyl transferase (AT), acyl carrier protein (ACP) and thioesterase domains. A variable number of ancillary domains such as the ketoreductase domain, enoyl reductase domain and dehydratase domains are also associated with PKS's (Cane and Walsh, 1999; Staunton and Weissman, 2001).

Carrier Protein Thioeste:::

Optional domains

Figure 1.8: PKS module organization. The ketosynthase, acyl transferase and acyl carrier protein are the required domains and compose the minimum module. Additionally, auxiliary domains such as the ketoreductase, enoyl reductase and dehydratase may also be present.

The first step in polyketide biosynthesis is mediated by the AT domain which transfers activated precursors to the corresponding modified ACP domain and is similar to NRPS A-domains in this regard (Figure 1.9). However, while A-domains also catalyze the activation of amino acid substrates the AT domains receive substrates in the activated form (malonyl-CoA or methylmalonyl-CoA). It has been established that substrate specificity of the AT-domain for malonyl-CoA or methylmalonyl-CoA is determined by the presence of certain conserved amino acid residues (Haydock Ct al., 1995). Modification of the ACP domain involves the posttranslational attachment of the phospantetheinyl co-factor onto a conserved serine residue in amanner similar to NRPS PCP domains. Although they have similar functions, ACP and PCP domains 29

do not reveal any obvious sequence similarities except for the conserved serine residue (Finking and Marahiel, 2004).

0 0 0 0 -OS-CoAi R R

Figure 1.9: Recognition of substrate by the AT domain and its transfer to ACP domain.

The KS domain mediates the fundamental chain elongation reaction and catalyzes C-C bond formation through a Claisen-type condensation. The KS domain first decarboxylates the malonyl or methylmalonyl extender unit bound to the ACP domain. The resulting nucleophile then reacts with the thio ester group of the polyketide chain attached to the KS domain and ultimately this decarboxylative condensation results in the extension of the growing ketide chain by two carbons (Figure 1.10) (Katz, 1997).

R)(L L

Figure 1.10: Ketide bond formation catalyzed by the KS domain. Ii]

The ketoreductase domain (KR), dehydratase domain (DH) and the enoyl reductase domain (ER) are auxiliary domains that catalyze three consecutive reductive reactions. Before transfer of the elongated polyketide chain to the KS domain of the adjoining module, the 13-keto group can be reduced to a hydroxyl by the KR domain and requires NADPH as a co-factor. The dehydratase domain catalyzes the dehydration of the J3-hydroxyl group resulting in the formation of a double bond. Finally, the enoyl reductase uses an NADPH co-factor to form a saturated bond in the ketide chain (Figure 1.11) (Katz, 1997). After the final extension, the nascent ketide chain is transferred to the TE domain which is responsible for product release and cyclization.

OH 0 KR OH ER RJUJLNHRJ1JL RJ1

Figure 1.11: Reactions catalyzed by PKS auxiliary domains. The ketoreductase domain (KR) catalyzes reduction of the 13-keto function, the dehydratase domain (DH) mediates dehydration of the-hydroxyl function and further reduction to form a saturated bond is catalyzed by the enoyl reductase domain (ER).

Although modular type I PKS's and NRPS's employ a similar biosynthetic logic, they exhibit significant differences in quaternary structure. In the double-helical model proposed for modular type I PKS's, each subunit consists of identical polypeptides twisted around each other in a helix. At the core of the helix are the KS, AT and ACP domains, while the reductive domains are accommodated at the periphery (Staunton et al., 1996). On the other hand, NRPS's are monomeric (Sieber et al., 2002) and in hybrid systems it is believed that the dimeric PKS forms a core, with NRPS loops (Weissman, 2004). Hybrid PKS/NRPS clusters combine both 31 strategies for the production of structurally complex and therapeutically active molecules such as epothiolone (46) from Sorangium cellulosum (Molnar et al., 2000), myxothiazol (47) from Stigmatella auriantica DW 4/3-1 (Silakowski et al., 1999), yersiniabactin (48) from Yersinia pestis (Gehring et al., 1998), and bleomycin (49) from Streptomyces verticillus (Du et aL, 2000).

Myxothiazol (47)

Epothiolone (46) H2NO H2 H H OH OH S NN 0 Xl'oH H ii 0 HN, Yersiniabactin (48)

OH I \\ N Bleomycin A2 (49) / H ni-i

The modularity of these natural product assembly lines and permissivity of tailoring enzymes offer exciting prospects for the generation of entirelynew or modified products with optimal therapeutic potential through combinatorial biosynthesis (Cane et al., 1998; Walsh, 2004). Despite several daunting challenges, steady progress is being made in the field of combinatorial biosynthesis (McDaniel et al., 2005; Sieber and Marahiel, 2005). Advances have been made in understanding crucial interactions between different modules in PKS and NRPS systems (Hahn and 32

Stacheihaus, 2004; Wu et al., 2001). Several PKS/NRPS gene clusters suchas those encoding DEBS and yersniabactin have been successfully expressed in E. coil (Pfeifer and Khosla, 2001; Pfeifer et al., 2001; Pfeifer et al., 2003). Similarly, the production of artificial nonribosomal peptide products inan engineeredE.coil host has also been reported (Gruenewald et al., 2004). Recent work to exploit the chemoenzymatic potential of TE domains has also demonstrated its ability to catalyze the robust cyclization of linear peptide and mixed peptide/polyketide metabolites to their final macrocyclic forms (Trauger et al., 2001; Kohli et al., 2002b; Kohli et al., 2002a; Watanabe et al., 2003; Boddy et al., 2003; Sieber et al., 2003). Another strategy by which biosynthetic pathways can be utilized to generate natural productsor natural product-like compounds is by partial pathway expression andprecursor directed biosynthesis. For example, in the case of the epothilone biosynthesis from the myxobacterium Sorangium cellulosum, anE.coil strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosyntheticenzymes was obtained by chemical synthesis. Under high-density cell culture conditions, this engineeredE. coil strain processed exogenously fed synthetic substrate into epothilone C (Boddy et al., 2004).

Secondary metabolite biosynthetic gene clusters from marine cyanobacteria The rich integration of PKS/NRPS systems is the predominant biogenetic theme in marine cyanobacterial metabolites. Although theseenzymes are similar to those present in terrestrial organisms, many of the metabolites from marine organisms possess unique functional groups that are not encountered in the former. The unique structural features and bio active properties ofmany cyanobacterial metabolites have prompted efforts to study the biosynthesis of these compounds at the molecular genetics level. The overarching goal of current research in thisarea is to functionally express cyanobacterial secondary metabolite gene clusters in a heterologous host. Not only will such technologies enable increased production of compounds of biomedical importance, but also will aid in the development of strategies aimed at combinatorial 33 biosynthesis of these metabolites. Analysis of structural types presented in Table 1.1 indicates that a majority of these compoundsare derived from modular PKSINRPS's (Salomon et al., 2004). Additionally, there is growing recognition that microbial symbionts are often important contributors to the biosynthesis of biologically active compounds (Piel, 2004). Therefore, knowledge gleaned from in-depth analysis of these gene clusters can led to the development of genetic systems capable of overexpressing these metabolites in order to provide an unlimited supply for clinical evaluation, thus circumventing the supply issue. The increasing inventory of PKS/NRPS biosyntheticgene clusters available in public databases has facilitated the use of these sequences to design targeted cloning strategies to isolate new biosynthetic gene clusters from marine organisms. Additionally, the isolation and characterization of several freshwater and terrestrial cyanobacterial gene clusters such as those involved in the biosynthesis of microcystin (18) (Tillett et aL, 2000), nodularin (19) (Moffitt and Neilan, 2004), nostopeptolides (50) (Hoffmann et al., 2003), nostocyclopeptide (51) (Becker et al., 2004) and anabaenopeptilide (52) (Rouhiainen et al., 2000) have provided the foundation for further pursuit of similar gene clusters from their marine counterparts.

CH3 H

0 0 ONH HN HO' HNO 0 0 O HHN° ONH2 H ss" 0 Nostopeptolide A (50) H0NyNostocyclopeptide Al (51) 34

OH HO OCH3CCI3 HN 1y4Ny=&kH LJ 0NH N"S Barbamide (53) - OH 1

90B (52) rAnabaenopeptilide

Inherent difficulties of culturing marine organisms, such as cyanobacteria and the lack of effective techniques to genetically manipulate these organisms have hindered research efforts in this area. However, the ability to culture certain marine cyanobacteria like Lyngbya majuscula along with improved methods to isolate DNA and generate genomic libraries has made it possible to initiate molecular genetic studies with these organisms. Only recently have the genetic architectures of several cyanobacterial biosynthetic gene clusters been determined, and studies to understand and exploit this biosynthetic machinery present an exciting newarea of research. At present, four secondary metabolite biosynthetic gene clusters have been described in the literature from different strains of L. majuscula. The barbamide (53) biosynthetic gene cluster (bar) was the first marine cyanobacterial gene cluster to be elucidated (Chang et al., 2002). It isa mixed PKS/NRPS system with unusual features that include a stand-alone peptidyl carrier (PCP) and unusual A-domains, as well as a PKS module that is encodedon two separate ORFs. The biosynthetic system also encodes several tailoring enzymes involved in the chlorination, a-oxidation, and decarboxylation of leucine to forma trichioroisovaleric acid moiety, and the oxidative decarboxylation of the cysteine residue at the end of the assembly line to form the terminal thiazole ring (Figure 1.12) (Chang et al., 2002). 35

Put. L-amino PCPhaIogenase NRPS NRPS-PKS PKS NRPS Decarboxylase oxidase barBil barA B2 barC barD barE barF barG barH bar/-K D DDDE'EDEF______4 26kb

Figure 1.12: The barbamide (53) biosynthetic gene cluster.

In addition to the barbamide biosynthesic gene cluster, the curacin A (14) biosynthetic gene cluster was also isolated from this same strain of L. majuscula (Chang et al., 2004). A particularly notable feature of the pathway involvesa unique biochemical mechanism for cyclopropyl ring formation mediated viaa novel enzyme that bears homology with 3-hydroxy-3 -methyl glutaryl CoA (HMG-CoA) synthases (Figure 1.13) (Chang et al., 2004).

HMG NRPS PKS cassette - Pics PKS PKS PKS PKS PKS PKS PKS TEHyd curA curB-E curF curG curH curl curJ curK curL curM N I >DDDD1 X )_____ )I l l ___)D

64kb

Figure 1.13: The curacin A (14) biosynthetic gene cluster.

The 58 kb jamaicamide A (27) biosynthetic gene cluster (jam) is a remarkable example of a co-linear pathway for the assembly ofa complex lipopeptide and represents a highly integrated mixed PKS/NRPS pathway (Figure 1.14) (Edwards et aL, 2004). The jamaicamide pathway possessesa range of genetic elements that encode for enzymes catalyzing several biochemical transformations includinga novel alkynyl PKS starter unit, an HMG-CoA synthase containing gene cassette for pendent vinyl or vinyl chloride formation, incorporation ofa decarboxylase domain into JamL NRPS module, and chain termination resulting in pyrrolinone ring formation. HMC PKS- PKS cassette PKS PKS NRPS PKS tIRPS PKS Con jamA-D famE jamF-I jamJ jamK jamL M N 0 P Q DDDDI DDDiX DE XE a

58kb

Figure 1.14: The jamaicamide A (27) biosynthetic gene cluster.

The lyngbyatoxin biosynthetic gene cluster was cloned and characterized from a Hawaiian strain of L. majuscula (Edwards and Gerwick, 2004). The11 kb cluster consists of two NRPS modules that catalyze the formation the L-N-Me-Val-L- tryptophanol which is modified to form the indolactam-V by a P450monooxygenase. A prenyl transferase then catalyzes the attachment of the geranylgroup to form lyngbyatoxin A (Figure 1.15) (30).

P450 Prenyl Reductasel NRPS monooxygenase transferase Oxidase

ItxA ItxB ItxC ItxD

11.3 kb

Figure 1.15: The lyngbyatoxin A (30) biosynthetic gene cluster.

The study of marine cyanobacterial natural product chemistry has revealeda number of metabolic themes as well as novel and re-occurring structural motifs. An overriding theme is the rich integration of polyketide/peptide units. Othercommon themes include the incorporation of novel starter units (e.g. tertiary-butyl, terminal alkene, alkynes, and even a bromo-alkyne in 27),a number of diverse halogenations, heterocylces, the incorporation of functionalized pendent carbons from the action of 37 an HMGCoA synthase gene cassette, and the incorporation of diverse amino acid units (e.g. j3-amino acids, N-methylated and 0-methylated amino acids). The genetic features that encode for these functional units in marine cyanobacteria will providea rich toolbox for the creation of structural diversity through pathway engineering and chemoenzymatic synthesis. GENERAL THESIS CONTENTS Marine cyanobacteria are an extraordinary source of complex and bioactive secondary metabolites that are derived from hybrid PKS/NRPS systems. Along with the search for new and bioactive metabolites from marine cyanobacteria,our laboratory has also undertaken studies to understand the biosynthesis of these metabolites at the molecular genetic level. A Jamaican strain of the cyanobacteriumL. majuscula was the source of the novel cytotoxic and antifungal metabolite, hectochiorin. The unique biological properties and presence of intriguing structural features in hectochiorin, such as thegem dichioro group and two 2,3-dihydroxy isovaleric acid units, prompted us to clone and characterize its biosyntheticgene cluster to obtain details regarding its biosynthesis at the molecular genetic level. Initial attempts to isolate the hectochiorin biosynthetic gene cluster led to the serendipitous discovery of a cryptic gene cluster from thegenome of the organism. Details regarding the cloning, sequence characterization and demonstration of this cryptic cluster expression are reported in chapter two. A novel interdisciplinary approach designed to identify the metabolite produced by this cryptic cluster is also discussed. The isolation, sequence characterization and partial biochemical characterization of the hectochlorin biosynthetic gene cluster is discussed in chapter three. The 38 kb gene cluster possesses several unusual features suchas the presence of KR domains in two peptide synthetase modules which are predicted to be involved in the formation of the two 2,3-dihydroxy isovaleric acid (DHiv) units and appears to be the first report of such an arrangement in a cyanobacterial secondary metabolite gene cluster. Also present are two cytochrome P450 monooxygenases and a putative halogenase. The gene cluster encoding for the biosynthesis of carmabin was elucidated from a CaribbeanL.majuscula (Z. Chang, D. Sherman, W.H. Gerwick, unpublished) and chapter four provides details regarding the expression and biochemical characterization of two adenylation domains from this gene cluster. The concluding chapter will provide a brief summary and perspective of the preceding chapters. 39

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CHAPTER II

ISOLATION OF A CRYPTIC GENE CLUSTER FROM THE CYANOBACTERIUM LYNGBYA MAJUSCULA JHB AND DEMONSTRATION OF ITS EXPRESSION

INTRODUCTION

Marine cyanobacteria have proven to be one of the most versatile producers of secondary metabolites and many of these metabolites demonstrate antiproliferative, cytotoxic or neurotoxic activity, making them invaluableas potential therapeutic leads or pharmacological tools. Hectochiorin (54) and jamaicamides A-C (27-29) were isolated as major components of a Jamaican collection of the cyanobacterium, Lyngbya majuscula (designated as L. majuscula strain JHB) (Edwards et al., 2004; Marquez et al., 2002). Hectochlorin (54) possesses structurally intriguing features such as the presence of gem-dichioro group and two dihydroxyisovaleric acid units. It was also found to be possess potent antifungal and cytotoxic properties (Marquez et al., 2002). Ptk2 cells (derived from the rat kangaroo, Potorous tridactylus) treated with hectochiorin showed an increase in the number of binucleated cellsas a result of the arrest of cytokinesis, a finding typical of compounds that cause hyperpolymerization of actin. The unique biological properties and presence of intriguing structural features in hectochiorin, such as the gem-dichloro group and two 2,3- dihydroxyisovaleric acid units, promptedus to attempt the cloning and characterization its biosynthetic gene cluster to obtain details regarding its biosynthesis at the molecular genetic level. 55

O 16..J 27 O 0 0 1"O -21)j__8

24 HO S2O 25 Hectochlorin (54)

HfNJLLOCH3 R=BrJaniaicamide A(27) N o R=H=3amaicamide B (28) HNAykOCH3 N O Jamaicade C (29)

The structure of hectochiorin (54) suggested that it was derived from a PKS/NRPS pathway, a characteristic pathway for metabolites derived from L. majuscula. In order to isolate the hectochiorin biosynthetic gene cluster,we decided to employ a general PCR method (Turgay and Marahiel, 1994) and amplify NRPS genes from L. majuscula JHB genomic DNA using degenerate primers based on core motifs (Neilan et al., 1999). However, this approach led to the identification ofan approximately 20 kb NRPS/PKS gene cluster whose genetic architecture suggested its involvement in the biosynthesis of a metabolite unrelated to hectochiorin (54). Because the metabolite for which this cluster codes the biosynthesis remains as yet uncharacterized, it was designated as a "cryptic cluster" (Bentley et al., 2002; Omura et al., 2001; Zazopoulos et al., 2003). Herein, the cloning and sequence characterization of the cryptic gene cluster along with demonstration of its expression from the cultured L. majuscula JHB is described. A novel interdisciplinary approach, designed to identify the metabolite produced by this cryptic cluster, is also discussed. Genomics guided approaches, such as the one discussed here, will become increasingly important in natural products research as a complement to the traditional methods for the isolation of novel natural products. 57

RESULTS AND DISCUSSION

Isolation of genonuc DNA from Lyngbya majuscula JHB The first step towards generating a genomic library of Lyngbya majuscula JHB is the isolation and purification of high molecular weight genomic DNA. Wewere also interested in surveying several DNA isolation procedures in order to optimizea simplified protocol for the isolation of high molecular weight DNA from samples immediately after their collection in the field. In addition to L. majuscula JHB, three other strains of L. majuscula (L. majuscula l9L, Lyngbya NPI 03 and Lyngbya MNT 01) and a Phormidium sp. were used in the various methods that were explored. L. majuscula JHB, 19L and Phormidium were maintained as unialgal cultures inour laboratory whereas Lyngbya NPI and Lyngbya MNT were field collected samples stored at -20°C in a mixture of isopropanol and water for future chemical extraction purposes (Experimental). Several methods were explored in order to optimize a protocol that consistently yielded intact high molecular weight DNA in large concentrations and ina form suitable for subsequent manipulations and analysis. The first method used for the isolation of genomic DNA was designed for filamentous fungi (M. Cone, personal communication). This method resulted in low recovery of genomic DNA from L. majuscula JHB, L. majuscula 19L and Phormidium sp., and genomic DNA obtained from Lyngbya MNT 01 and NPI 03 appeared to be completely degraded (Figure II.1A). DNA obtained by a second method using CTAB-buffer resulted in isolation of low molecular weight genomic DNA. Moreover, this method could not be scaled-up for higher yields of genomic DNA. Finally for library construction, genomic DNA from L. majuscula JHB was isolated using a previously described protocol (Sambrook et al., 1989) (Figure II.1B). However, a major drawback with this method was carryover contamination of phenol in DNA samples. The presence of phenol was undesirable because it is known to interfere with downstream enzymatic manipulation of DNA. Therefore, this method was modified further to includea purification step after phenol extraction using Qiagen's 100IG Genomic Tip DNA Kit. The only disadvantage with this method was lowrecovery of DNA and therefore the need for large amounts of DNA at the start of the procedure(- 5 pjg). Nonetheless, DNA obtained after this purification step was high molecular weight and free of phenol.

XHindlll 2 XHindlll 2 Genomic DNA 1740kb 1 2 3 45 AMarker B Marker (L.majuscula JHB) control 40kb-

Genomic DNA kHindIII 2 C L.majuscula JHB Marker

22kb

Figure 11.1: Isolation of high molecular weight genomic DNA. A. High molecular weight isolated by using CTAB-buffer from Lyngyba MNTO1 (1), Lyngbya NPIO3 (2), L. majusucla JHB (3) Phormidium (4) and L. majuscula 1 9L (5). B. High molecular weight genomic DNA was isolated from L. majuscula JHB by extraction with phenol followed by further purification using Qiagen's G/100 Genome Tip Kit. C. L. majuscula JHB genomic DNA isolated using DNeasy Plant Kit.

DNA isolation using several commercially available kits namely, DNeasy Plant Mini Kit (Qiagen), DNAzol (Gibco Life Sciences) and Wizard DNA Purification Kit (Promega) was also investigated. DNA isolation using DNeasy Plant kit hadsome distinct advantages because the DNeasy shredder column supplied with the kitwas 59 designed to remove cellular debris, particularly polysaccharides froma viscous lysate, making recovery of genomic DNA more efficient. Although this method resulted in the isolation of good quality genomic DNA, it was not of high molecular weight(< 22 kb) and was therefore unsuitable for library construction; however, itwas very efficient for use as template for PCR (Figure II.1C). The Wizard kitwas also fairly effective but the DNAzo1 reagent proved to be inefficient in recovering genomic DNA from all samples used in the study. Isolation of genomic DNA from the various cyanobacterial strains proved to be challenging. The presence of extracellular mucilage, pigments and filamentous morphology of some cyanobacteria are among the reasons forpoor recovery of DNA from these organisms (Tillett and Neilan, 2000). The method adapted from Sambrook et al. (1986) (Method III, Experimental) proved to be most efficient for the isolation of intact high molecular weight DNA. Lysis with liquid nitrogen along with prolonged digestion with proteinase K at 50°C probably facilitated efficient lysis for therecovery of high molecular weight intact genomic DNA whichwas ultimately used for library construction. There were slight variations with regard to yield of genomic DNA from the various strains and higher concentrationswere obtained from L. majuscula JHB and L. majuscula 1 9L when compared to the Phormidium sp. Thepresence of copious amounts of mucilaginous sheath material and bioflim-flim like colony morphology of some Phormidium sp. are believed to hinder cell lysis and purification of nucleic acids (Fiore et al., 2000). Genomic DNA obtained from the two preserved strains was completely degraded and this suggested that the mode of tissue preservation hasa marked effect on the quality of nucleic acids. A simple method without the use of liquid nitrogen or organic solvents could not be established for isolation of genomic DNA during field collections. However, we discovered that storage of samples in RNA LaterTM (Ambion) for transport to the laboratory preserved the quality of DNA without affecting yield thus obviating the need for sucha protocol. PCR amplification of probes The structure of hectochlorin suggested that it was derived froma hybrid PKS/NRPS pathway. While Cl -C8 were hypothesized to be derived from acetate units assembled by PKS's, the other two units were proposed to be derived from the amino acid cysteine and 2,3-dihydrdxyisovaleric acid (DHiv),a precursor in valine biosynthesis (Figure 11.2). In order to isolate the hectochiorin biosyntheticgene cluster, we decided to employ a general PCR method (Turgay and Marahiel, 1994) and amplify NRPS/PKS genes from L. majuscula JHB genomic DNA using degenerate primers based on core motifs. Our goal was then to select adenylation domains that were specific for valine and cysteine and use them as probes to screen the library.

II 20 0

Acetate OH

Figure 11.2: Proposed biosynthetic precursors of hectochiorin. Precursor feeding studies have demonstrated that the C1-C8 polyketide and C26-C27 derive from acetate, while the pendant methyl C9 is derived from SAM. Biosynthetic feeding studies to investigate the origins of the two DHiv units were inconclusive (Nogle, 2002). 61

The degenerate primers used for this purpose were designed basedon conserved peptide synthetase motifs from several bacteria and fungi. Additionally, they were designed to favor codon bias of cyanobacterial genes (Neilan et al., 1999). The primers A2-Forward based on the adenylation domain AGGAVYPcore motif I and A8-Reverse based on the QVKIRG adenylation domain core motif V (Experimental) were used to amplify an approximately 1.2 kb fragment from L. majusculagenomic DNA (Figure 11.3). This PCR product was purified and cloned into pGEM-T Easy vector and clones were screened by PCR using SP6 and T7-P primers. PCR products were digested with the frequent cutting enzymeTaqI,and based on their restriction digest pattern; clones were grouped into four distinctgroups, I-IV. BLAST analysis of representatives of each group revealed that theywere all peptide synthetases with highest homology to nostopeptolide NosD (54% identity) (Hoffmann et al., 2003) and anabaenopeptilide ApdA (53% identity) (Rouhiainen et al., 2000). In order to determine the substrate specificity of the various clones,a bioinformatics-based approach developed by Stacheihaus et al. and Challis et al. was used in which clones from each group were translated and key amino acids involved in substrate recognition and binding were identified by alignment with GrsA-Phe adenylation domain (Stachelhaus et al., 1999; Challis et al., 2000). Basedon this analysis, group I clones were predicted to activate valine, clones from group II hada predicted specificity for proline, group III for asparagine and IV for leucine (Table 11.1). A single clone (Val-Aden-20) from Group 1 was selected asa probe and was labeled with dioxigenin (DIG) for library screening. 62

Figure 11.3: Amplification of NRPS A-domain probe. Degenerate primers based on A2 and A8 adenylation domain core motifs were used to amplify an approximately 1.2 kb fragment fromL. majusculaJHB genomic DNA.

Table 11.1: Substrate specificity of NRPS A-domain probes. The amino acid residues involved in substrate binding and recognition were determined using the method of Challis et al. (2000) for representatives from each group of unique clones digested withTaqI.

Group Position of binding pocket amino acids Predicted amino acid 235 236 239 278 299 301 322 330 substrate

I D A L W L G G T valine

II D F Q F I A H V proline III D L T K I G H V asparagine

IV D A W F L G N V leucine 63

In order to focus on an adenylation domain probe with predicted specificity for cysteine, a pair of primers was designed based on alignments of several previously characterized cysteine adenylation domains. Thus, HMWP from Yersinia pestis (Gehring et al., 1998), bacitracin BacA from Bacillus lichenformis (Konz et al., 1997) epothilone EpoB from Sorangium cellulosum (Molnar et al., 2000), and pyochelin PchE and PchF from Pseudomonas aeruginosa (Quadn et al., 1999) were aligned to design primers 720F and 840R. (Chang et al., 2002) (Figure 11.4).

HMWP2 752 DRVLALSALHFDLSVYDI ------HWRSIPYGFPLTNQRY 896 BacA 708 DRVLALSSLSFDLSVYDV ------SWASIPYGNPLRNQTF 852 EpoB 708 DRVLALSSLSFDLSVYDV ------SWASIPYGRPLRNQTF 852 PchE 694 DRVLGLAELSFDLSVYDF ------ELASIPYGRALRGQSV 839 PchF 731 DRLLAVSALDFDLSVFDT ------HWRSIPYGRALPGQAY 874 720F 4840R

Figure 11.4: ClustaiW alignment of cysteine adenylation domains. High molecular weight 2 (HMWP), bacitracin BacA, epothiolone (EpoP), pyochelin PchE and PchF (Quadri et al., 1999). Conserved amino acids for cysteine adenylationwere used to design primers 720F and 840R.

PCR amplification using this primer pair 720F and 840R resulted in the amplification of an approximately 400 bp fragment which was purified and cloned into pGEM-T Easy vector for sequencing (Figure 11.5). Several cloneswere sequenced and these results revealed that all the clones were identical which led to the conclusion that this primer pair favored the amplification of a single adenylation domain due to PCR bias. BLAST analysis revealed that it was most similar to EpoB (57% identity) (Molnar et al., 2000). The substrate specificity determined by aligning translated sequences with GrsA-Phe adenylation domain also revealed that itwas predicted to activate cysteine (Table II. 2) (Challis et al., 2000). Basedon these results, a single clone Cys-Aden-9 was purified and labeled with DIG for library screening. 64

720F/840R Marker product

Figure 11.5: Amplification of cyteine-specific A-domain. PCR amplification using the degenerate primers, 720F and 840R resulted in the amplification of an approximately 400 bp product from L. majuscula JHB genomic DNA.

Table 11.2: Substrate specificity of cysteine A-domain probe. The amino acid residues involved in substrate binding and recognition were determined using the method of Challis et al. (2000) for the adenylation domain fragment amplified using 720F/840R.

Group Position of binding pocket amino acids Predicted 235 236 239 278 299 301 322 330 amino acid substrate Cys-Aden D L Y N I G I V cysteine Polyketide synthase probes spanning a small region of the ketosynthase domain (KS) were amplified using previously described primers (Beyer et al., 1999). The approximately 700 bp product was cloned into pGEM-T Easy and sequenced. All sequences showed high homology to known polyketide synthases such as epothilone EpoF (58% identity) (Molnar et al., 2000) myxothiazol MtaE (55%) (Silakowski et al., 1999) and microcystin McyD (53% identity) (Tillett et al., 2000). A "mixed" pool of KS fragments was used for probe generation and was obtained by purification of the PCR product from genomic DNA. The PCR product was purified and labeled with DIG.

Figure 11.6: Amplification of KS domain probe. PCR amplification using the degenerate primers, KSUp and KSD1 resulted in the amplification ofa700 bp fragment of the 3-ketosynthase domain from L. majuscula JHB genomic DNA. Construction and screening of a genomic DNA library Once a method for the isolation of good quality genomic DNA was established, our next goal was the construction of a library that offered fullcoverage of the L. majuscula genome whose sizewas maximally estimated to be around 10 Mb based on the genome sizes ofNostocpunctiforme (Meeks et al., 2001) and Anabaena PCC712O (Kaneko et al., 2001). Therefore, with an average insert size of about 40 kb we aimed to screen at least 2500 clones in order to obtain adequate coverage of the genome (approximately 8-fold). Several vectors for library construction were considered and our initial choice was the cosmid vector pOJ446 (Bierman et al., 1992) because it has been used for the isolation of the barbamide and curacin A biosynthetic gene clusters (Chang et al., 2002; Chang et al., 2004). However, this vector was found to be unstable in E. coli and could not be purified from glycerol stocks (P. Flatt, personal communication). Therefore, we decided to use the pWEB::TNC cosmid cloning kit from Epicentre Technology whichwas designed to hold inserts of approximately 40 kb. Next, isolated and purified genomic DNA was subjected to size selection in a 0.7 % agarose gel. However, recovering sufficient DNA from the gel after size selection proved to be a challenge, and several concentrations of the Gelase®enzyme and buffer had to be tested in order to maximize recovery of end-repaired genomic DNA from the gel. We found that equilibration of the gel slice inexcess Gelase buffer (5 volumes) prior to overnight digestion with the Gelase® enzyme resulted in increased recovery of DNA. The genomic DNA was then ligated into cosmid pWEB::TNC for construction of the library. The titer of the library generated was approximately 2.2 xiO3which was screened with the three DIG-labeled probes were discussed previously by colony hybridization techniques. This resulted in the identification of 53 cosmid clones which probed positive with the KS-domain probe and 30 clones which probed positive with both of the NRPS probes. Of these 83 clones, only eleven probed positive with both PKS and NRPS probes and thesewere selected for further analysis by restriction mapping and Southern hybridization. Southern analysis of the eleven cosmids led to the identification of two, designatedas 67

1-105 and 111-105, which hybridized strongly to all three probes. Restriction digest of

the two cosmids with EcoRI,HindIlI,BamHI, NcoI and PstI revealed that they had similar restriction patterns and were likely overlapping DNA fragments. This ledus to speculate that these two cosmids might harbor different regions of the putative hectochiorin biosynthetic gene cluster and thus theywere selected for shot-gun sequencing. The total size of each cosmidwas estimated to be approximately 41 kb (1-105) and 49 kb (111-105).

EcoRI HindIil BarnHI NcoI PsS DIG-lobeled A lAndiS A BA B A BA B A B 2,narker

EcoR HirtdIII BamHI NcoI Psti EcoRl HindIIIBamHI NcoI PsLI Cys-eden KS positive DIG-lobeled DIG-lebeled A B A B A B A B A B control yorker merirer A B A B A B A B A B

21 kb b 21 kb

5 I kb 35 b 5 1 kL 35kb

2 0kb - *0 20k'

Figure 11.7: Restriction digest analyses (A) and Southern hybridizations of cosmids 1-105 and 111-105 (B). A. Restriction digest of cosmids 1-105 (A) and 111-105 (B) show that they are overlapping fragments. B. Southern analysis using ketosynthase domain probe (left) and cysteine adenylation domain probe confirmed that theywere overlapping cosmids (right). LSI

In order to verify the accuracy of Southern analyses, a PCR screen of the two cosmids employing primers, KS-Up/KS-Dl and 720F/840R,was performed. The resulting amplicons were cloned into pGEM-T vector and sequenced. Most of the clones obtained with the 720F/840R primers were most similar to EpoB (57% identity) (Molnar et al., 2000) and confirmed the presence ofa domain likely involved in the incorporation of cysteine in both cosmids. Results with the ketosynthase domain primer products were mixed in thata large fraction of clones sequenced were similar to a putative L-glutamine: D-fructose aminotranferase from fromShigella flexneri(70%) (NP_709542) and only one clone was similar to MtaD (46% identity) (Silakowski et al., 1999). Based on this data, cosmids 1-105 and 111-105 were chosen as candidates for sequencing. However, during subsequent purification of the two cosmids for shotgun cloning, we observed that cosmid 111-105 had undergonea rearrangement and was missing several EcoRI fragments that were initially observed. Thereare several reasons for cosmid instability including genotype of the host strain, the presence of short tandem repeats (Song et al., 2001) andcopy number of the vector. It has been observed that cosmid stability is improved inrecAmutant host strains (Silby and Mahanty, 2000). Because theE. coli strain EPI100 used in this study isarecAmutant, the high copy number of the cosmid is likely the cause for the observed instability.

Shot-gun cloning and sequencing of cosmid 1-105 For sequencing of cosmid 1-105, two main sub-cloning approacheswere explored. In the first method, cosmid DNA was sheared by sonication and fragments ranging from 0.75 kb-i .5 kb were size selected, end-repaired and ligated into the prepared pGEM3Zf (+) vector. The second approach consisted of digesting the cosmid partially with a frequent cutting enzyme, like AluI and Sau3Al, followed by selection of fragments in the right size range and ligation into pGEM3Zf (+)or pBluescript, respectively. Upon sequencing the sub-clones, we observed thata large fraction of clones obtained by the first method had insert sizes < 750 bp whichwas not conducive for the assembly of large contiguous stretches of DNA. However, subclones obtained by the latter method contained fragments that were larger and more suitable for assembly.

Sequence analysis of gene cluster The DNA sequence of cosmid 1-105 was determined by shotgun cloning and assembled into a roughly 20 kb contiguous stretch of DNA. The overall G+C content in this region is approximately 45% which is consistent with the G+C content encountered in cyanobacterial genomes (www.kazusa.or.ip/cyano) and other secondary metabolite gene clusters from L. majuscula (Chang et al., 2002; Chang et al., 2004; Edwards and Gerwick, 2004; Edwards et al., 2004). Analysis of the 20 kb region revealed that it consists of three ORF'5.Preliminary analysis of the largest ORF revealed that it was a mixed NRPS/PKS gene cluster and that it contained several features that suggested its involvement in the biosynthesis ofa metabolite unrelated to hectochlorin (54) (Figure II.8B). Previous chemical investigations revealed the presence of only four major compounds in this strain of Lyngbya majuscula (Edwards et aL, 2004; Marquez et al., 2002) and none of these is consistent with the gene cluster present on 1-105. Therefore, this cluster was designated as "cryptic gene cluster", which has been defined as a gene cluster for whicha corresponding metabolite remains as yet unidentified. A survey of all literature reporting the presenceor isolation of cryptic gene clusters from several organisms revealed thata uniform system of nomenclature of these clusters does not exist (Bentley et al., 2002; Challis and Ravel, 2000; Omura et al., 2001; Zazopoulos et al., 2003; McAlpine et al., 2005). Therefore, we propose here a system that will identify these clusters with first letter of genus and first letter of species followed by cry (cryptic gene cluster). Hereafter, according to the proposed nomenclature, this cryptic gene cluster will be referred toas LMcryl (ngbyarnajusculayptic gene cluster). 70

ORF LMcryl II III >c

20kb

B LMcryl

NRPS NRPS NRPS F;S Module I Module 2 Module 3 Module 4

Figure 11.8: Genetic architecture ofLMcryland modular organization of LMcryl. A 20 kb contiguous region sequenced from cosmid 1-105 was shown to contain three open reading frames,LMciyl,ORF II and ORF III. (A). Modular organization of LMcryl which was found to encode a mixed PKS/NRPS pathway. Three NRPS modules (1,2 and 3) precede a single PKS module with an embedded TE (4) (B).

LMcryl encodes a large protein (-j 6500 amino acids) that contains three NRPS modules followed by a PKS module which has an embedded thioesterase. The first module possesses a Cy-A-PCP domain organization required for the activation and heterocyclization of cysteine and is most similar to curacin CurF (51% identity) (Chang et al., 2004) and barbamide BarG (50% identity) (Chang et al., 2002). The condensation domain shows the presence of conserved motifs required for the heterocyclization of cysteine when compared with the heterocyclization domains from these two proteins (Figure 11.9). Analysis of the adenylation domain identified the binding pocket amino acid residues that are in consensus for the activation of cysteine (Table 11.3) (Challis et al., 2000). The absence of an embedded oxidase domain leads 71

us to speculate that heterocyclization results in the formation of a thiazoline ring as opposed to a thiazole ring (Du et al., 2000; Schneider et al., 2003).

Cyl Cy2 C3 Cy3

CYCCUrF FPLNDIQQAYWIGR- - - -RMVVLADGNQQ- - - -DIFDAWS -- LPPAPEIP CycBarG FPLTEIQQAYWLGR- - - -RMVILPNGQQ- -- -DALII4DAWS - LPPAPELP CycLMcryl FPLTDMQQAFWVGS- - - -RAVVLPDGQQK- - - -NPLILDTWS- - -FPESPELP Cons. FPLTxxQxAYxxGR----RxVxLPxGxQ ----- DxxxxDxxS-- --LPxxPExP

Cy4 Cy5 Cy6 Cy7

CycCurF TPSGVLLSFASVLNYW- - -GDFTS- - - -GVVFTSTL- - -QVLLDHIVTEEKGALAFSWN CycBarG TPSGVLLAAFADAYW- - - -GDFTS - - - -GVVFTSTL- - -QVWLDNSVEQNGALLLIWH CycLMcryl TPSG?LAAAFAEVLTRW- - - -GNFTS - - - -PVLFTSTL- - -RVWIDHQVYEENEALKFHWD Cons. TPxGxxxxxxxxVxxW- - - -GDFTS- - - -PVVFTSxL- - -QVxLDxQxxxxxxxxxxxWD

Figure 11.9: Alignment of heterocyclization domains. Barbamide BarG and curacin A CurF aligned with heterocyclization domain from LMcryl show the presence of consensus core motifs.

The second module consisted of a standard C-A-PCP structure and was found to be most similar to module 2 in microcystin McyB (39% identity) (Tillett et al., 2000). The adenylation domain was predicted to be specific for glutamine when analyzed by the method of Challis et al (Table 11.3). However, comparison with other cyanobacterial A-domains known to activate glutamine revealed considerable differences in the binding pocket amino acids. An invariant Gln278 residue believed to be involved in hydrogen bonding with substrate glutamine is replaced by Asn at this position. However, this Asn residue could be involved in a similar interaction with the substrate (Challis et al., 2000). We also compared the A-domain with asparagine activating A-domain NosC3 from the nostopeptolide gene cluster (Hoffmann et al., 2003) which revealed that the highly conserved G1u322 residue that is selective for Asn was missing from this A-domain. Therefore, asparagine was eliminated as a possible substrate (Stachelhaus et al., 1999). Similarly, conserved residues believed to 72

play an important role in the selection of aspartate (Thr239, His322) and glutamate (Asp278) were also missing in this A-domain making it difficult to predict a putative substrate for module 2. Although most binding pockets lend themselves easily to chemical interpretation of their cognate amino acid substrate specificity, others clearly do not. Additional biochemical characterization of this A-domain will be important in determining its substrate specificity. The presence of acidic amino acid residues in marine cyanobacterial metabolites is relatively rare (Gerwick et al., 2001). This is in sharp contrast with peptidic natural products of freshwater and terrestrial cyanobacteria, suchas microcystin (18) and nodularin (19), which contain acidic amino acid residues (Figure 11.11) (Carmichael et al., 1988; Krishnamurthy et al., 1986; Fujii et al., 1996). Next, module three consists of a C-A-NM-PCP domain architecture and is similar to module 2 in tubulysin TubC (37% identity) which also possesses identical architecture (C-A-NM-A-PCP) (Sandmann et al., 2004). Analysis of the binding pocket amino acids revealed that the A-domain was specific for valine and showed good correlation with other valine-activating domains (Table 11.3). An N-methyl transferase is embedded between the A9-A1 0 core motifs and shows the presence of conserved residues and shows all the conserved residues when aligned with other N- methyl tranferases from tubulysin TubC (Sandmann et al., 2004), microcystin McyA (Tillett et al., 2000) and lyngbyatoxin LtxA (Edwards and Gerwick, 2004) (Figure 11.10). 73

Ml M2 M3 N-MtLMcryl IVELGCGTG- -HNELNRFRYDV- - - IAYCRVPNARLMGEE N-MtTubC ILZVGCGTG- - -PNEMARERYNA---LGVAGIPNRVLPAV N-MtMcyA VLEIGCGTG- -HNELTQFRYNV- - -LRVIGVPNSRLFRPL N-HtLtxPi ILEIGVGTG- - -HNEMSQY?YNV- - -LKLVNVPNCRVTPAL

Figure 11.10: Alignment of N-methyl transferases from tubulysin TubC, microcystin McyA, lyngbyatoxin LtxA with the LMcryl N-MT shows the presence of conserved residues.

D-Gtu

D-GIu COON 0 OCH3

O CHJ

0-Me-Asp

Nodularin (19) NI-I COON D-Me-Asp HN N H2 Microcystin (18)

Figure 11.11: Nonribosomal peptides from freshwater and terrestrial cyanobacteria. Chemical structures show the presence of acidic amino acid residues such as glutamate (Glu),and aspartate (Asp). 74

Table 11.3: Substrate specificities of A domain LMcryl and other related A-domains. BarG (Chang et al., 2002), CurF (Chang et al., 2004), NcpA3 (Becker et al., 2004), NosAl and NosC3 (Hoffmann et al., 2003), LtxAl (Edwards and Gerwick, 2004) and TubC2 (Sandmann et al., 2004). The binding pocket amino acid residues correspond to Grs-APhe A domain binding (Challis et al., 2000).

A-domain Binding pocket amino acids Substrate 235236239278299301322330

LMcrylModAl D L Y N I G I V cysteine

BarGc D L Y N L S L I cysteine

CurFc D L Y N V D L I cysteine

LMcrylModA2 D A M N L L G V glutamine

NcpA3 D A W Q F G L I glutamine

ApdAl D A W Q F G L I glutamine

NosC3 D A T K V G E V asparagine

LMcrylModA3 D A L W L G G T valine

NosAl D A F F L G V T valine

LtxAl D I Y W F G G T valine

TubC2 D A F F L G G T valine

The PKS module reveals a KS-AT-CM-KR-ACP domain organization. The incorporation of malonate versus methylmalonate extender units by a PKS is determined by the AT-domain. In this case, the AT-domain in this module is predicted to be specific for malonyl-CoA (Figure 11.12) (Haydock et al., 1995), although a conserved valine is replaced by an isoleucine. The C-MT possesses a signature motif (LExGxGxG) that is also present in the C-MT's from the jamaicamide A and curacin A biosynthetic gene clusters (Figure 11.12) (Chang et al., 2004; Edwards et al., 2004). An embedded type I thioesterase with the conserved GxSxG motif is present at the C-terminal of this ORF and may likely catalyze the hydrolysis and release of the product. 75

A B KS-JamK AVDTACSSSL---YLB.AI3GT----GRTEGAA A-Jam1c YTQPCLFLEYLCQL---MGHSVGE KS-JamL AIDTACSASL---YVEAEGT---IGHLEAA AT-JamL YTQPALFAIEYALYKL---MGHSVGE KS-CurF AVEAACSSSL---YLEABGT---XGNAATAA AT-CUrF YTQPLFAIEThLWKL---MGHSVGE KS-CurG TVQTACSTSL- - -YVEABGT- - -VGHLADP.A AT-CurG YTQPM.FAIEYALFKL- - -NGHSVGE KS-LMcrylAVQTACSTSL---YIETHGT---XGRLDAAA AT-LMcryl FAQPALFAIEThLNQL---LGHSIGE Con. DtaCS5SL B H Cons. YTQ E A GHSV (inalonylC0A)

C-MT-JamJ LLEIGAGTGGTT C-14T-CurJ LLEIAGTGGTT C-MT-LMcryl ILEIGAGNGLLT Cons. ILHxGxGxGXxT Figure 11.12: Sequence alignments of PKS domains of LMcryl with other cyanobacterial PKS's (jamaicamide A and curacin A PKS genes). KS domain alignment (A); AT-domain specific for malonyl-CoA (B); C-methyl transferase domain alignment (C).

ORF II, is located about 700 bp downstream of LMcryl and encodes a putative transposase. BLAST analysis of this gene showed that it was most similar to transposase encoded by an 1S1136 type insertion sequence element from Clostridium perfringens (33% identity) (Shimizu et al., 2002). Next, ORF III shows 52% identity to a Natdriven multi-drug efflux pump from Nostoc punctiforme PCC73 102 (ZP_00 108972). The presence of these two proteins suggests that the TE domain in the C-terminal end of LMcryl is the terminus of the gene cluster. Additional sequencing will be required in order to determine the presence of other amino acid/ketide incorporating domains or any tailoring enzymes that may be associated with this gene cluster. While sequence information upstream of ORF I remains to be determined, it was apparent that this gene cluster was not involved in the biosynthesis of hectochlorin (54). The presence of a module involved in the activation of an acidic amino acid residue, an embedded N-methyl transferase in module 3 and the probable termination of the cluster with a PKS segment eliminate its involvement in the biosynthesis of hectochiorin (54). The gene cluster is most likely involved in the biosynthesis of a metabolite whose deduced structure is represented in Figure 11.13. ,I1

NH2

Figure 11.13: Structure of proposed peptide encoded by hybrid NRPS/PKS LMcryl. R unknown side chain.

A curious insight resulting from sequence analysis of cosmid 1-105 is the presence of short regions dispersed over an 8 kb fragment that show similarity to cyanobacterial transposases such as those from Nostoc punctforme PCC73 102 (ZP_00 106214), Anabaena variabilis ATCC294 13 (ZP_00 162592) and Crocosphaera watsonii (ZP_00 1779422). The presence of such interdigitated putative transposases has also been reported in Synechocystis PCC6803 (Mahillon et al., 1999). These may have resulted from the insertion of several IS elements that may be rendered inactive over time as a result of mutations (Meeks et al., 2001). Our strategy which involved the use of general adenylation domain probes proved to be unsuccessful in isolating the hectochlorin biosynthetic gene cluster. The homology-based approach (Turgay and Marahiel, 1994) was not feasible for organisms known to harbor multiple PKS/NRPS gene clusters in their genomes and lately there has been a shift towards the use of highly specific approaches to isolate secondary metabolite gene clusters from organisms known to possess multiple PKS/NRPS gene (Cheng et al., 2002; Sandmann et al., 2004; Edwards et al., 2004). Sequence analyses of several bacterial genomes have revealed that there is often discrepancy between the number of secondary metabolite gene clusters harbored in the genome of an organism and the number of natural products thatcan be isolated. 77

For example, genome sequencing of Streptomyces avermitilis revealed the presence of twenty five secondary metabolite gene clusters that accounted for 6.4% of the total genome size and putative products encoded by five PKS and eight NRPS clusters have not yet been identified (Omura et al., 2001). Similarly, Streptomyces coelicolor shows the presence of about twenty gene clusters involved in the biosynthesis of secondary metabolites, but the corresponding metabolites formed from several of these remain unidentified (Bentley et al., 2002). Mxyobacteria such as Stigmatella aurantiaca and Sorangium cellulosum So ce9O have also been shown to harbor multiple hybrid PKS/NRPS gene clusters (Pradella et al., 2002; Silakowski et al., 2001). Cyanobacteria are emerging as prolific producers of secondary metabolites and filamentous cyanobacteria such as Nostoc punctforme, Anabaena sp, Lyngbya majuscula tend to harbor a number of gene clusters involved in secondary metabolite bioynthesis. Analysis of the recently annotated genome of the terrestrial cyanobacterium, Nostocpunctforme has revealed the presence of at least 16 secondary metabolite gene clusters and for many of these, a corresponding secondary metabolite has not yet been isolated (Salomon et al., 2004). The genome of filamentous cyanobacterium Anabaena PCC 7120 also shows the presence of several biosynthetic gene clusters involved in the production of natural products of PKS/NRPS origin (http://www.kazusa.or.jp/cyano/Anabaenalindex.html). While genome sequence data for Lyngbya majuscula is currently unavailable, strains like L. majuscula 19L have been shown to possess multiple gene clusters of which three are completely characterized: barbamide (Chang et al., 2002) curacin (Chang et al., 2004) and carmabin (Chang et al., unpublished). Re-screening of a L. majuscula JRB genomic library concurrent to this work also resulted in the identification of multiple PKS/NRPS gene clusters (D.J. Edwards, unpublished). Among organisms that possess multiple PKS/NRPS gene clusters, a critical question that is often arises is whether the organism expresses all of these clusters to produce secondary metabolites under normal culture conditions. In the case of marine cyanobacteria whose natural habitat is a complex ecosystem, a delicate balance of physical and nutrient conditions along with other habitat interactions influence growth I3

and production of secondary metabolites (Patterson et al., 1994). The fact that certain cyanobacterial strains are more amenable to laboratory cultivation does not necessarily mean that they retain all of their biosynthetic capabilities to produce secondary metabolites. Indeed, frequent loss, alteration or decreased metabolite production is encountered in cultured cyanobacteria (Moore et al., 1988). Nevertheless, several groups have succeeded in the isolation of numerous bioactive metabolites from cultured cyanobacteria (Gerwick et al., 1994; Patterson et al., 1991; Patterson et al., 1993; Patterson et al., 1994; Rossi et al., 1997; Schwartz et al., 1990). A Caribbean collection of L. majuscula which produces the antitubulin agent, curacin A, was successfully adapted to laboratory culture. Furthermore, even after extended culturing, production of curacin A in the cultured strain was comparable to that found in wild material (Rossi et al., 1997). Intensive culture efforts have also led to the identification of an axenic strain of the cyanobacterium, Scytonema ocellatum, which produces twice as much tolytoxin as the parental strain (Patterson et al., 1994). Therefore, it is interesting to establish whether the putative metabolite encoded by the cryptic cluster, LMcryl is produced by cultures L. majuscula JHB. This prompted us to re-examine the organism's secondary metabolite profile. The crude organic extract (CH2Cl2-MeOH, 2:1) of the cultured organism was fractionated usinga standard VLC protocol (Marquez et al., 2002) to give nine fractions (A-I). Reversed phase HPLC profiling of these fractions revealed three major components that were present in large amounts (Figure 11.14) and corresponded to four previously characterized metabolites, hectochiorin (54) and jamaicamides A-C (27-29)(Edwards et al., 2004; Marquez et al., 2002). Although fractions B, C and E contained several small peaks, these proved to be in sub-milligram quantities (<0.2 mg) of mostly impure materials. Consequently, no structure characterization by NMRor other spectral methods was attempted (K. L. McPhail, unpublished). Jamaicamide A (27) Hectochlorin (54) Jamaicamide B (28) A Jamaicamide C (29) A A - ft crude .1 ______

.1 ____ ) ii Hectochlorin (54) Jamaicamide A (27)

Jamaicamide B (28)

AU m:mideC(29)

Hectochlorin (54) Jamaicamide A (27)

Jamaicamide B (28)

jrnidec(29) Jamaicamide A (27) Jamaicamide B (28)

:boni54 JJamaicamideC (29) -I

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.0 Minutes Figure 11.14: Reversed phase HPLC profiles for the crude extract and nine normal phase VLC fractions (A-I) of the culturedLyngbya majusculaJHB. In each case 10 pL (0.25 mg) of sample was injected onto an analytical colunm (Varian Microsorb-MV 100-5C18)and eluted over 50 mm, firstly with a gradient of 50% acetonitrile/water through 100% acetonitrile (40 mm) and then continued elution with 100 % acetonitrile (10 mm). All samples were monitored at thesame 5 wavelengths (211, 230, 254, 270, 320 nm) using a Waters 996 Photodiode Array detector. Results are plotted at exactly the same vertical scale in eachcase. 80

Therefore, because the crude extract ofL.majuscula JHB contained several candidate metabolites of limited mass, we decided to explore a more targeted and novel strategy that combined aspects of molecular biology and natural products chemistry. The first step in this approach is demonstration ofLMcrylexpression using reverse transcriptase-PCR (RT-PCR) or Northern blot analysis. In the next step, bioinformatics prediction of the adenylation domain specificities is used to select

doubly labeled 13C-15N amino acidprecursors that are be fed to a culture ofL. majuscula JHB. Finally, selective 13C-'5N 2D NMR methods, such as the gradient hydrogennitrogen multiple quantum coherence (GHNMQC) experiment, will be used in an "NMR-guided" fractionation of the labeled extract to isolate the putative metabolite, which can then be characterized by routine 2D NMR spectroscopy and mass spectrometry (Figure 11.15).

Isolation of cryptic gene cluster

( Bioinformatics prediction of Demonstration of gene A-domain specificities/ expression Experimenatal determination of J Lsubstrate specificity

[Labeled amino acid precursor feeding experiments based on A-domain specificity

rIsolation and structural elucidation of corresponding bmetabolite using select 2D-NMR methods

Figure 11.15: Schematic representation of the novel approach used to isolate and elucidate structure of putative metabolite encoded byLMcryl. 81

Demonstration ofLMcrylexpression A number of investigations have been carried out to study the influence of environmental factors on secondary metabolite production in cyanobacteria (Patterson and Bolis, 1995; Rapala et al., 1997; Repka et al., 2004; Watanabe and Oishi, 1985). However, there is very little understanding of cyanobacterial secondary metabolite regulation at the genetic level and thus far, expression analysis has been reported only for the microcystin biosynthetic gene cluster (Kaebernick et al., 2000; Kaebernick et al., 2002). Various methods are available for the detection and monitoring of bacterial gene expression including Northern hybridization, RNase protection assay and different forms of RT-PCR. Northern hybridization has a major advantage in that it is the only method which provides information regarding the size of the transcript (Sharkey et al., 2004). However, Northern blot analyses could not detect expression of the microcystin gene cluster. Low transcript levels and potential length of the transcripts were believed to be responsible for this observation (Kaebernick et al., 2002). Therefore, we decided to use the RT-PCR approach. The first step in RT-PCR is the production of cDNA from mRNA using retroviral reverse transcriptase. The cDNA then serves as a template for exponential amplification by PCR. RT-PCR has become an indispensable tool for examining gene expression and can even be used to detect transcripts present in very low abundance. Specific primers were designed to amplify approximately 500 bp regions along the length of theLMcrylas follows: primers F 1 fRi amplified a region in the first module, F2/R2 amplified a region within the second module and F3/R3 were designed to amplify a small region at the end of module three (Figure 11.16). A small region of thejamBgene from jamaicamide A biosynthetic gene cluster (Edwards et al., 2004) was selected as a positive control and specific primers for its amplification were also designed. 82

Fl/Ri F2/R2 F3/R3

I Modei I Module 2 I Module 3 I Module 4 LMciyl

Figure 11.16: Primers for RT-PCR. Primers Fl/Ri amplified a 520 bp region within module 1, primers F2/R2 amplified a 500 bp region within module 2 and F3/R3 amplified a 500 bp region towards the end of module 3.

Total RNA was isolated from cultures of L. majuscula JHB using RNAwiz (Ambion) at early (2-week old culture) and late (5-week old culture) logarithmic growth stages. The isolated total RNA was treated with DNase to remove any contaminating DNA before it was used as template for RT-PCR. Concentration and purity were determined using a spectrophotometer. Generally, RT-PCR can be performed in a one-step format or two-step format. In the former, reverse transcription and PCR take place in a single tube which reduces the risk of carryover contamination, whereas in the latter format, each step is performed in separate tubes and 10% of the reverse transcription reaction is used as template for PCR (Sharkey et al., 2004). Initially, we used the one-step format but this method proved unreliable and failed to produce consistent results. We then tested the two-step format which was highly successful and could be replicated readily. Total RNA was converted to cDNA using the gene-specific reverse primers, Ri, R2, R3 and jamB-R, and cDNA obtained was used as template for subsequent PCR reactions. Amplicons were obtained for Fl/Ri, F2/R2 from cultures in both early and late logarithmic growth stages, thus demonstrating expression of the LMcryl in the cultured L. majuscula JHB (Figure 11.17). The presence of transcripts in the early and late logarithmic cultures was also consistent with the observation that cyanobacteria, unlike Streptomyces sp., appear to produce secondary metabolites throughout the growth-phase (Orr and Jones, 1998; Repka et al., 2004; Rossi et al., 1997). However, primer pair F3/R3 failed to amplify the corresponding fragment from both culture samples. A likely explanation for this observation may be the location of these two primers at the 3'-end of the transcript which is more susceptible to degradation by RNases (Kennell, 2002; Kushner, 2002; Steege, 2000). Amplification ofjamB positive control from both cultures was also successful. Negative control experiments in which RNA was used as template for PCR did not result in amplification, thus indicating the complete absence of contaminating DNA in the total RNA sample (Figure 11.17). All PCR products were subcloned into pGEM-T Easy vector and theirsequences were verified as deriving from the LMcry 1 cluster.

DNA template p. * RNA template 0 Marker Fl/Ri F2/R2 F3/R3 NegPos Fl/Ri F2/R2 F31R3 NegPos con. con. con. con.

4 DNA template 0 4 RNA template + Marker Fl/Ri F2/R2 F31R3 Neg Pos Fl/Ri F2/R2 F31R3 Neg Pos con. con. con. con.

Figure 11.17: Results of RT-PCR. RT-PCR analysis using primers Fl/Ri, F2/R2, F31R3 and jamaicamide jamB as positive control confirms expression of LMcryl characterized from cultures of L. majuscula JHB in early (A) and late (B) logarithmic phases. Negative control experiments in which RNAwas used as template revealed complete absence of contaminating DNA. Thus expression ofLMcrylin culturedL. majusculaJHB was successfully demonstrated using RT-PCR. Precursor feeding studies with labeled amino acid are currently underway and will hopefully lead to the identification of the corresponding peptide natural product. With the identification and characterization of over 150 hybrid PKSINRPS gene clusters from several organisms (Salomon et al., 2004), our understanding of these systems has advanced to the point where we can use sequence information to assign structures with reasonable accuracy. Natural products continue to be a major source of molecules for drug discovery; however, development of new methodologies is vital for the identification of novel chemical structures and methods for their production. As the number of gene clusters encoding unknown PKS/NRPS metabolites increases with the rapidly accumulating genome sequence data from several organisms, the utility of genomics in conjunction with standard spectroscopic techniques will become invaluable for the isolation and structural elucidation of novel metabolites. EXPERIMENTAL

General All standard DNA manipulations such as restriction digests, ligations and transformations were carried out using standard methods (Sambrook Ct al., 1989). Restriction digest enzymes were purchased from several vendors and used according to manufacturer's recommendations.

Bacterial strains and culture conditions Escherichia coli strain DH1OB was used as a host for DNA cloning and sequencing, while E. coli EPI 100 was used as a vector for construction of cosmid library. E. coli DH1OB cells harboring pGEM-T Easy, pGEM3ZF(+) or pBluescript was grown at 37°C overnight in Luria-Bertani medium (LB) with a final concentration of ampicillin at 100 tg/mL. E. coli EPI 100 cells with cosmid pWEB::TNCwere grown at 37°C in LB medium supplemented with 100 jtg/mL ampicillin. Lyngbya majuscula strain JHB, Lyngbya majuscula strain 19L and Phormidiumsp were grown under previously described conditions (Marquez et al., 2002; Rossi et al., 1997; Williamson et al., 2002). Genomic DNA for library construction was isolated from a four-week old culture of Lyngbya majuscula JHB. Lyngbya MNT 01 was collected near Nosy Tanikely, Madagascar (April17th2000) and Lyngbya NPI 03 was collected in Curacao (May11th1996) and preserved in isopropanol/seawater mixture (60:40). Both preserved samples were stored at -20°C.

Isolation of genomic DNA Method I (Asper.-illus prep): About one gram of each cyanobacteria was ground in liquid nitrogen and treated with 0.6 mL of extraction buffer [0.1 M Na2(S03),0.1 M Tris-Cl pH 7.5, 0.05 EDTA, and 1% (w/v) SDS} and incubated at 65°C for 20 minutes. The lysate was then treated with an equal volume of chloroform-isoamyl alcohol and incubated on ice for 30 minutes. After centrifugation at 12000rpm for 30 minutes, the aqueous layer was transferred to a fresh tube and genomic DNAwas precipitated by the addition of isopropanol. It was then washed 2x times with 70% ethanol and resuspended in 50 p.L water and incubated at 4°C to aid resuspension. Method II (CTAB buffer): About one gram of cyanobacteria was ground to a fine powder using liquid nitrogen and treated with 5 mL of TE25S buffer (0.025 M Tris- HC1 [pH 8], 0.025 M EDTA [pH 8], 0.3 M sucrose) with 2 mg!mL lysozyme and incubated for one hour at 37°C. Proteinase K and SDS at a final concentration of 0.18 mg!mL and 0.5%, respectively were then added and incubated for 1 hour at 55°C. Next, the lysate was treated with 0.5 M NaCl and extracted twice with 10% CTAB (hydroxyacetyl trimethyl ammonium bromide)! 0.7 M NaCI followed by 24:1 chloroform: isoamyl alcohol. Genomic DNA was finally precipitated with isopropanol and washed twice with 70% ethanol. Method III : Two grams (wet weight) of L. majuscula JHB, L. majuscula 19L or Phormidium sp was harvested and rinsed three times with 20 mL of sterile Instant Ocean BG1 1 medium, followed by 20 mL of 10 mM Tris (p11=8) in a Buchner funnel. The tissue was then flash-frozen in liquid nitrogen and homogenized in a pre-chilled mortar and pestle using liquid nitrogen. After evaporation of excess liquid nitrogen, the resulting fine powder was resuspended in 20 mL of lysis buffer (10 mM Tris [pH=8], 0.1 mM EDTA [pH=8J, 0.5% (w/v) SDS and 20 .tg/mL DNase free RNase A and incubated for 1 hour at 37°C. During incubation, the lysate was gently mixed by inversion every 20 minutes. Proteinase K (20 mg/mL) was added to the lysate at a final concentration of 100 p.g!mL and incubated at 50°C for 1 hour. The solution was cooled to room temperature before adding 20 mL of phenol equilibrated with 0.1 M Tris [pH=8]. The two phases were gently mixed for 10 minutes on a platform shaker and the phases were separated by centrifugation at 25°C for 10 minutes at 4000 rpm. The viscous aqueous phase was transferred by careful pipetting to a clean tube using a wide-bore tip (0.3 cm orifice diameter). The extraction with phenol was repeated two more times and the aqueous extracts were pooled. The pooled aqueous extracts were treated with 0.2 volumes of 10 M ammonium acetate and 2 volumes of absolute ethanol at room temperature. After gentle mixing, the DNA was removed from the aqueous extract by using a sterile Pasteur pipette and transferred to a fresh tube. The DNA was then washed with 70 % ethanol three times and re-suspended in 500 .t1 of low TE (2 mM Tris [pH=8] and 0.2 mM EDTA) and incubated at 4°C for 12-24 hours. After re-suspension genomic DNA was subjected to further purification using Qiagen's Genomic Tip 100 according to instructions provided with the kit. Concentration and purity (A260/A280) of genomic DNA were measured using a Beckman DU600 spectrophotometer. Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen), DNAzo1 (Gibco Life Sciences) and Wizard DNA Purification Kit (Promega) according manufacturer's protocols. The DNeasy kit was used for the isolation of genomic DNA for PCR amplification of probes.

Amplification of PKS and NRPS probes PCR was carried out using Eppendorf Mastercycler in 50 j.tL reaction volumes containing 40 ng of genomic DNA template, 5 tL of PCR buffer (Invitrogen), 50 mM MgC12, 10 mM dNTP's, 10 jtM primers and 2.5 units of Taq DNA polymerase (Invitrogen). Degenerate primers KS1Up 5' MGI GAR GCI HWI SMI ATG GAY CCI CAR CAT MG 3' and KSD1 5' GGR TCI CCI ARI SWI GTI CCR TG 3' were used for the amplification of KS domains from PKS (Beyer et al., 1999). Primers A2Forward 5' GCN GGY GGYGCN TAY GTN CC 3' and A8Reverse 5' CCN CGD ATY TTN ACY TG 3' were used for the amplification of NRPS adenylation domain probes (Neilan et al., 1999). The following thermocycler conditions were used for amplification: denaturation at 94°C for 30 s, annealing at 48°C for 30s, extension at 72°C for 1 mm for 30 cycles. The amplification of cysteine-specific adenylation domain primers were conducted using primers, 720F 5' TTY GAY YTI WSI GTI TAY GAY ITI TTY GG 3' and 840R 5' TGR TTI CCI ART CSI CYI CCI TAI GGI AT 3' using the following thermocycler conditions: denaturation at 94°C for 5 mins, annealing at 45°C for 30 s, extension at 72°C for 1 mm for 30 cycles. All primers were obtained from Invitrogen (Carlsbad). The PCR products were purified using Qiagen's PCR purification kit and cloned into pGEM-T Easy for sequencing. Unique NRPS clones were identified by digestion with TaqI for 1 hour and representative clones from eachgroup were sequenced. Sequencing reactions were performed at the Central Services Lab, Center for Gene Research and Biotechnology at Oregon State University and Davis Sequencing (Davis, California). Sequence analysis was carried out using National Center for Biotechnology Information's (NCBI) BLAST tool (Altschul et al., 1990) and ClustalW (Thompson et al., 1994).

Construction of cosmid library High molecular weight genomic DNA (-.. 40 kb) was end-repaired and size selected by electrophoresis on a 0.7% low-melt agarose gel. The gel slice was equilibrated in 5 volumes of Gelase buffer three times and subjected to overnight digest with 5 units of Gelase®. Library construction was conducted using Epicentre's pWEB::TNC cosmid cloning kit. Purified genomic DNA was then ligated into pWEB::TNC vector and packaged into MaxPlax phage extracts according to instructions accompanying the kit. The packaged phage was then used to infect E. coli

EPI 100 cells and an average titer of 2.2 xiocfulmL was obtained.

Screening of library Library clones were inoculated in a numbered grid on 150 mm Petri dishes containing LB agar with 100 jtg/mL ampicillin. All clones were inoculated in duplicates. Colonies were transferred to nylon membranes (Hybond N, Amersham Pharmaecia Biosciences) and were processed for colony hybridization using standard procedures (Sambrook et al., 1989). The DNA was fixed to the nylon membrane by cross-linking in a CL1 000 Ultraviolet Crosslinker. The amplified PKS and NRPS probes were purified prior to labeling. Probes were labeled with dioxygenin using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Molecular Biosciences) and 40 ng/mL of labeled probe was used for all colony hybridizations. Colony hybridization was carried out according to instructions provided with the DIG High Prime Kit. Prehybridization of nylon membranes was carried out at 42°C, hybridization at 42°C and stringency washes were performed at 50°C. The addition of CSPD (Disodium 3-(4-meth-oxyspiro {l,2- dioxetane-3,2'-(5'-chloro) tricyclo [3.3.l.l'J decan}-4-yl) phenyl phosphate) provided with the kit serves as a substrate for the detection of DIG labeled positive colonies by chemiluminescence.

Southern analysis

Purified cosmid DNA ('-1 pjg) was digested withHindIlI,EcoRI, BamHI, PstI and NcoI and separated on a 1% agarose gel. After pre-treatment of gel according to standard protocols (Sambrook et al., 1989), prehybridization, hybridization, washing were performed as described for the colony lifts. Labeled probes were used at a concentration of 20 ng/mL. Detection was also carried out as described for colony hybridizations.

Sub-cloning of cosmid 1-105

Cosmid DNA (-1tg) was dissolved in 300 i.tL of deionized water for sonication using XL2000 MicrosonTM Ultrasound homogenizer at 100W for 20 s with a 1/8" probe. The fragmented DNA was end-repaired using End-It® from Epicentre Technologies and separated on a 1% gel. Fragments within the desired size range (1.5-2 kb) were purified and ligated into prepared pGEM3Zf(+) vector. Cosmid DNA (-1 jig) was subjected to partial digest using A1uI or Sau3AI (0.15-0.20 units/jig of cosmid DNA for 1 hour). The partially digested cosmid DNA was then separated on a 1% agarose gel and fragments of size 1-2.5 kb were selected. After gel purification, the A/uI fragments were ligated into pGEM3Zf(+) vector treated with SmaIICTP (Calf Intestinal Phosphatase). Similarly, Sau3Al fragmentswere ligared into pBluescript that was digested with BamHI /CIP. Random sub-cloneswere selected for sequencing. Sequencing was carried out using Big Dye III terminator cycle sequencing (PE Biosystems) at the Central Services Lab, Center for Gene Research and Biotechnology, Oregon State University. Resulting gaps were filled using primer walking with sequence specific primers. The sequences were edited and assembled using Vector NTI's Contig Assembly software program (InforMax, Bethesda, MD). Analyses of DNA and protein sequences were carried out using the NCBI's BLAST server(Altschul et al., 1990). All alignments were conducted using ClustalW (Thompson et al., 1994).

Isolation of RNA-Gen era! Prior to isolation of total RNA, all equipment and accessories were meticulously cleaned to eliminate any contaminating RNases. Microfuge tubes and pipette tips were autoclaved for 30 minutes and all glassware was autoclaved twice for 30 minute each. Pipettes, gel apparatus, microcentrifuge and general work area were wiped with RNase Zap (Ambion, Austin,TX). During the RNA isolation process, gloves were periodically changed, in order to prevent any carry-over contamination resulting from contact with gloves. Water was treated with 1% (v/v) DEPC for 18 hours and autoclaved for 30 minutes. All reagents were made in DEPC-treated water and autoclaved before use.

Isolation of total RNA fromL. majusculaJHB For isolation of total RNA L. majuscula JHB was grown under previously described conditions (Marquez et al., 2002). Total RNA was isolated using RNAwiz (Ambion, Autin, TX). About 1.82 g (wet weight) of a 2-week old culture and 2.08g (wet weight) of a 5-week old culture was harvested and homogenized in liquid nitrogen using a mortar and pestle. The finely ground tissue was resuspended in an appropriate volume of RNAwiz and isolation of RNA were carried out according to manufacturer's protocol. Total RNA was resuspended in 50 jiL of DEPC-treated water. In order to ensure complete removal of DNA, the isolated total RNA was treated with 1 iL of Turbo DNase (Ambion, Austin,TX) for 30 minutes at 37 °C. The DNase was inactivated by the addition of DNase Inactivation reagent and incubating for 2 minutes at room temperature. Total RNA obtained by this method was used for the generation of cDNA. Total RNA was quantified using a Beckman DU600 spectrophotometer 91

Generation of cDNA and RT-PCR Reverse transcription of total RNA to generate cDNA was performed using instructions accompanying SuperScriptTMJJJ First-Strand Synthesis System (Invitrogen, Carlsbad, CA) for RT-PCR. Briefly, 10 1.tL of total RNA templatewas incubated with 1tL of gene-specific reverse primer (10 j.M), 1 xL dNTP, appropriate buffers and 1 tL of SS-Reverse transcriptase for 1 hour at 55°C. After inactivation of the reverse transcriptase, cDNA was used as a template for RT-PCR. Primers used for RT-PCR were as follows: F 1-5' GCA AAA GGC TAT TGG CGC 3' and R1-5' GTT CCT CAA TGG AAA TGC G 3'; F2-5' GTT GTA GAT TGG AAT CAG 3' and R2-5' GGT CAG AAC CAC GCC TTT 3'; F3-5' GAA CTG TAT GTA GCA GGA GA 3' and R3-5' TCC CAA TTC CAC GAT GCG 3'; jamB-F 5' ATG TCA ATG CCA ATG GAT GT 3' and jamB-R 5' GGT CTT TAC TAT CGA AAG GGT 3'. The following PCR conditions were used: denaturation at 94°C for 1 miannealing at 55°C for 45 s and extension at 72°C for 1 mm for 30 cycles. PCR products were purified using Qiagen's PCR purification kit and cloned into pGEM-T Easy vector for sequencing. 92

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Williamson, R. T., Boulanger, A., Vulpanovici, A., Roberts, M. A., and Gerwick, W. H. (2002). Structure and absolute stereochemistry of phormidolide, a new toxic metabolite from marine cyanobacterium Phormidium sp. Journal of Organic Chemistry 67, 7927-7936.

Zazopoulos, E., Huang, K., Staffa, A., Liu, W., Bachmann, B. 0., Nonaka, K., Ahiert, J., Thorson, J. S., Shen, B., and Famet, C. M. (2003). A genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nature Biotechnology 21, 187-190. CHAPTER III

CLONING AND BIOCHEMICAL CHARACTERIZATION OF THE HECTOCHLORIN BIOSYNTHETIC GENE CLUSTER FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCULA

INTRODUCTION

The marine envirorn-nent continues to offer a vast array of biologically active natural products and constitutes a major source of molecules for drug discovery and development (Newman et al., 2003). Among these organisms, marine cyanobacteria have emerged as one of the richest producers of new natural product chemotypes with potent biological properties (Burja et al., 2001; Gerwick et al., 2001). A predominant metabolic theme in cyanobacterial metabolites is the rich integration of polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) (Gerwick et al., 2001). PKS's and NRPS's represent a large group of multifunctional enzymes with a modular organization that assemble complex molecules using acetate or amino acids as building blocks, respectively. Additionally, these secondary metabolites exhibit tremendous structural diversity due to the presence of numerous tailoring enzymes that catalyze oxidation, reduction, methylation, different types of halogenation and epimerization (Finking and Marahiel, 2004; Weissman, 2004). A complete understanding of the biosynthesis of these compounds at the molecular genetic level will provide key information for harnessing these gene clusters to produce novel molecules through combinatorial genetic engineering. Hectochlorin (54) was first isolated from a Jamaican isolate of the cyanobacterium Lyngbya majuscula (designated as L. majuscula strain JHB). Itwas subsequently re-isolated from L. majuscula collected at Bocas del Toro in Panama. The planar structure of hectochiorin was elucidated by NMR methods andmass 100

spectral analyses, and its absolute stereochemistry was deduced by X-ray crystallography (Marquez et al., 2002). Structurally, hectochlorin is related to dolabellin (24) isolated from the sea-hare Dolabella auricularia (Sone et al., 1995) and lyngbyabellin A (22) (Luesch et al., 2000b) and lyngbyabellin B (23) (Luesch et

al., 2000a; Milligan et al., 2000) fromL.majuscula (Figure 111.1). Initial antimicrobial assays indicated that hectochiorin was a potent antifungal agent. Its fungicidal activity and potency against a number of plant pathogens initiated synthetic efforts and a total synthesis of hectochiorin has been reported (Cetusic et al., 2002). Further testing revealed that Ptk2 cells (derived from the rat kangaroo, Potorous tridactylus) treated with hectochiorin showed an increase in the number of binucleated cells as a result of arrest of cytokinesis, a result typical of compounds that cause hyperpolymerization of actin. Hectochiorin was also evaluated against the in vitro panel of 60 different cancer cell lines at the National Cancer Institute and showed potent activity towards cell lines in the colon, melanoma, ovarian and renal sub-panels. It yielded a flat dose-response curve against most cell lines, a characteristic of compounds that are antiproliferative but not directly cytotoxic (Marquez et al., 2002). The unique biological properties and presence of intriguing structural features in hectochiorin, such as the gem dichloro group and two Dliv units, prompted us to clone and characterize its biosynthetic gene cluster. In this study, we report the cloning, sequencing and partial biochemical characterization of the hectochlorin biosynthetic pathway fromL.majuscula ifiB. 101

S CI N 16J N OH 9 iO 0 0 0 o OH

24 S 20 H7\ 25 Hectochlorin (54) Dolabellin (24)

S I %O

HN 0 HNXO HN HN H/TJHN

Lyngbyabellin B (23) Lyngbyabellin A (22)

OCH3

R=BrJamaicamide A(27) NO RHjamaicamide B (28) HN)LjOCH3 "NO Jamaicamide C (29) c)O

Figure 111.1: Chemical structure of hectochiorin (54) and related metabolites, dolabellin (24), lyngbyabellin A (22) and lyngbyabellin B (23). Jamaicamides A-C (27-29) are additional major metabolites isolated from L. ma] uscula JHB. 102

RESULTS AND DISCUSSION

Construction of L. majuscula JHB genomic library High molecular weight DNA for library construction was obtainedas described in Chapter II. The resulting high molecular weight DNA(40-45kb)

(Figure 111.2) was used to generate a genomic DNA library using Epicentre's pCCFOS 1 fosmid cloning vector kit. The fosmid vector hada major advantage over the cosmid in that it was maintained asa single copy within the cell and therefore was more stable.

High molecular weight High 40 kb T7 Genomic DNA from molecular control DNA L. majusculaJHB weight

40kb

Figure 111.2: High molecular weight genomic DNA from culturedL. majuscula JHB. Genomic DNA was isolated by phenol extraction followed by purification with 100/G Genome purification kit (Qiagen). The resulting DNA(- 40kb) was used for library construction. 103

PCR amplification of probes and screening of library The structure of hectochiorin (54) suggested that it was derived from a mixed PKS/NRPS pathway. C1-C8 were proposed to arise from intact acetate units assembled by a PKS with the pendant methyl group (C9) added from S-adenosyl methionine (SAM). Portions of the thiazole rings, C10-C12 and C18-C20 plus the heteroatoms, were proposed to originate from cysteine (Chang et al., 2004; Sitachitta et al., 1998). DHiv, an intermediate of valine biosynthesis, was proposed to form Cl 3-C 17 and C2 1 -C25 units. Therefore, our initial strategy to isolate the hectochiorin biosynthetic gene cluster involved the use of more general adenylation domain probes based on A2 and A8 conserved motifs (Neilan et al., 1999). Unfortunately, this strategy proved unsuccessful in isolating the hct gene cluster and instead led to the identification of an approximately 20 kb cluster whose genetic architecture indicated its involvement in the biosynthesis of an unrelated metabolite (A.V. Ramaswamy and W.H. Gerwick, Chapter II). Although chemical analysis of L. majuscula JHB has yielded the jamaicamides A-C (27-29) (Edwards et al., 2004), hectochlorin (54) and 23-deoxyhectochlorin (K.L. McPhail and W.H.Gerwick, unpublished data) as major metabolites, the presence of multiple PKS/NRPS pathways was detected during library screening (D.J. Edwards A.V. Ramaswamy and W. H. Gerwick, unpublished). Therefore, a more specific approach was used, which focused on the modules involved in cysteine incorporation, and specifically on the heterocyclization domains within these modules. Degenerate primers spanning the heterocyclization domain and conserved region of the adenylation domain (between core motifs A4 and A5) were designed basedon alignments of previously characterized heterocyclization domains (Figure III.3A). These primers were used to amplify a 1.8 kb fragment which was cloned into pGEM-T Easy vector (Figure III.3B). Several clones were sequenced and yielded three distinct beterocyclization-adenylation domain products, thus indicating that there were three different gene clusters or three distinct modules involved in the activation and incorporation of cysteine in this L. majuscula genome. 104

90 BarG FPITEQQAYWLGRNSHFDLGNITTHGYLELDCEN-LALDRLSQAWQQVIDHHDMLRMVI-- CurF FPLNDIQQAYWIGRNQI FDLGNIATHIYIEVDCEN-LNLESLHQAWRRLI DI-IHDMLRMVV- Mt aC FPLTDIQETYFVGRGTDFGI SGMSCHLYHELDTRG-MDVARLSRAWQRLIEQHVMLRSVV- MtaD FPLTDIQNAYWVGRQGAFDAGGVAAJisYLELAFDT-LDLPRLES ILQRLIEYHDMLRMVV-- EpoB FPL'rDIQGSYWLGRTGAFTvp- SGIHAYREYDCTD-LDVARLSRAFRKVVARHDMLRAHT- Bim-Cys FPLTDVQRAYYVGREGGFALGGVSTHAYLEIEAPR-IDVARFTGALRGVIARHPMLRAVI-- PchF FPLTPVQAAYVLGRQAAFDYGGNACQLYAEYDWPADTDPARLEAAWIThNVERHPMLRAVI HMWP2 FPLTPVQHAYLTGRMPGQTLGGVGCHLYQE FEGHC-LTASQLEQAITTLLQRHPMLHIAF--

IletForward 760 BarG --ALNFDLSVYDI FGLLAAGGTLVMPTPEAAKDPVHWVELMTTHQVTLWNTVPALMQMLVEY CurF --ALNFDLSVYDIFGVLGAGGAIVMPPPKAPKDPACWRELI IAHEVTLWNSVPALMQMLVEH MtaC --SLGFDLSVYDI FGSLAGAAIVLPQPEVTRDPAHWLQVLRQQGVTIWNSVPTLMEMLVDQ MtaD -- SLS FDLSVYDLFGVLAAGGAIVMPEPGT SRDPGRWQVLLEKTGVT IWNSVPALMDMLVEF EpoB --SLSFDLSVYDVFGILAAGGTIVVPDASKLRDPAHWAALIEREKVTVWNSVPALMRMLVEH Bim-Cys --SPSFDLAVYDLFGVLAAGGTVVvPAHDRRRDPGHWAELIRRERVTLWNSVPALGTLLTEY PchF --ELSFDLSVYDFFGATAAGAQVVLPDPARGSDPSHWAELLERHAITLWNSVPAQGQMLI DY HMWP2 --ALHFDLSVYDI FGVLRAGGALVMVMENQRRDPHAWCELIQRHQVTLWNSVPALFDMLLTW AdenReverse

Figure III.3A: Alignment of heterocyclization-adenylation domains for primer design. Barbamide (BarG) and curacin A (CurF) from L. majuscula (Chang et al., 2002; Chang Ct al., 2004), mxyothiazol (MtaC and MtaD) from Stigmatella auriantica (Silakowski et al., 1999), epothilone (EpoB) from Sorangium cellulosum (Molnar et al., 2000), bleomycin (Bim-cys) from Streptomyces verticillus (Du Ct al., 2000b); pyochelin (PchF) from Pseudomonas aeruginosa (Quadri et al., 1999) and high molecular weight protein 2 (HMWP2) from Yersinia pestis (Gehring et al., 1998). Conserved residues used for primer design are highlighted. Primers were designed using the CODEHOP strategy (Rose et al., 1998).

1.8kb +

Figure III.3B: PCR amplification of heterocyclization domain probe with Hetforward and Adenreverse resulted in the amplification of a 1.8 kb fragment. 105

An equal concentration of these three DIG-labeled probes was used to screena library of approximately 2800 clones, which identified 12 different fosmids at the

colony hybridization level. Restriction digest mapping withHindliland EcoRI enzymes and Southern hybridization analyses using the heterocyclization-adenylation domain probe led to the identification of two overlapping fosmids, pHctl and pHctIIas candidates for the hct gene cluster. To determine if these fosmids contained PKS elements, a pair of degenerate primers based on the-ketosynthase (KS) domain were used to amplify a 0.7 kb fragment from this domain (Beyer et al., 1999). Sequence analysis of the PCR products indicated that the two fosmids contained the same KS domain. These results were consistent with our findings that the starter unit in the jamaicamide A (5) pathway was a hexynoic acid that becomes extended by a single acetate derived extension (Edwards et al., 2004). Therefore, we predicted that the starter unit in hectochiorin biosynthesis might be a hexanoic acid that undergoes a single ketide extension. Additionally, end-sequencing of these two fosmids revealed that 3'-end of fosmid pHctl encoded a heterocyclization domain and 5'-end of fosmid pHctII contained a putative halo genase related to BarB I from barbamide gene cluster (Chang et al., 2002). Because, this genetic architecture was consistent with our predictions for hectochiorin biosynthesis, fosmids pHctl and pHctII were selected for further sequence analysis. 106

DNA sequencing and analysis of the hectochiorin biosynthetic gene cluster The DNA sequences of the two overlapping fosmids, pHctl and pHctII, were determined by shotgun cloning and assembled into a 42 kb contiguous region, which contained the 38 kb hct gene cluster (Figure 111.4). Flanking the hct cluster at the 5'- end are three small ORFs (I, II and III). ORF I encodes a 238 amino acid reverse transcriptase, which is 58% identical to a similar protein from Trichodesmium erythraeum, ORF II encodes a putative endonuclease with 50% identity to a similar protein from Anabaena variabilis and ORF III is similar to a hypothetical protein from Nostocpunctiforme PCC73102. At the 3'-end are two small ORF's (IV, V) that also encode hypothetical proteins from Trichodesmium erythraeum and helped to confirm the outer boundaries of hectochlorin biosynthetic gene cluster. The gene cluster

exhibits a relatively low overall G+C content(-S42%), which is comparable to the G+C content encountered in cyanobacterial genomes (http://www.kazusa.or.jp/cyano) and other secondary metabolite gene clusters from L. majuscula (Chang et al., 2002; Chang et al., 2004; Edwards and Gerwick, 2004; Edwards et al., 2004).

ORFs hcf 11111 A B C D E F G H Ivv

pHctl

DHCtII 3 kb 42kb

Figure 111.4: Genetic map of the hct gene cluster spanning38 kb. Inserts from two overlapping fosmids, pHctl and pHctII were sequenced to map the hct cluster. 107

Table III.!: Deduced functions of the proteins in the hct biosynthetic gene cluster

Protein Size (amioProposed function Sequence similarity Identity! Reference acids) (protein, organism) Similarity ORF I -238 reverse transcriptase Trichodesmium 58%, 73% erythaeum ORF II 119 HNH endonuclease Anabaena variabilis 50%, 65% ATCC 29413 ORF III 51 hypothetical protein Nostocpunct?forme 60%, 73% ATCC 73102 HctA 606 acyl-ACP synthetase' JarnA 60%, 78% Edwards et L. majuscula JHB al., 2004 Hct B 484 putative halogenase, BarBi 29%, 47% Chang et al., acyl carrier protein L. majuscula 19L 2002 JamC 35%, 54% Edwards et L. mafuscula JHB at., 2004 HctC 408 transposase TnpB 33%, 55% Cesini et al., Helicobacterpylori 1996 HctD 1924 polyketide synthase JamL 56%, 72% Edwards et L.majuscula JHB al., 2004 HctE 3356 nonribosomal peptideBarE/BarG 46%, 63% Chang et at., synthetase L.majuscula 19L 57%, 71% 2002 HctF 3945 nonribosomal peptideBarEfBarG 45%, 62% Chang et al., synthetase L.majuscula 19L 60%, 72% 2002 HctG 447 cytochrome P450 TaH (P450 36%, 54% Paitan et al., monooxygenase hydroxylase) 1996 Myxococcus xanthus; 35%, 56% spirangiene (P450 monooxygenase) biosynthesis, Polyangium cellulosum Accession no. AJ505006 I-IctH 444 cytochrome P450 (P450 monooxygenase) 36%, 57% spirangiene monooxygenase Polyangium cellulosum biosynthesis, TaH (P450 34%, Accession no. hydroxylase AJ505006 Myxococcux xanthus Paitan et al., 1996

ORF IV90 hypothetical protein Trichodesmium 70%, 83% erythraeum

ORF V 222 hypothetical protein Trichodesmium 58%,75% eiythraeum 108

Thehctgene cluster consists of eight ORFs with the first ORFhctAencoding a protein that is similar to acyl-ACP synthetases (Table 111.1). Sequence analysis revealed a 59% identity to JamA in the jamaicamide biosyntheticgene cluster (Edwards et al., 2004). Next, the product ofhctBhas two distinct domains. The N- terminal domain shows closest similarity to barbamide BarB 1 (29% identity), (Chang et al., 2002) coronamic acid CmaB (33% identity) (Ullrich and Bender, 1994) and syringomycin SyrB2 (33% identity) (Guenzi et al., 1998; Zhang et al., 1995). It also shows weak homology to Fe2/ 2-oxoglutarate-dependent hydroxylases like phytanoyl Co-A hydroxylases (PAHX) and possesses all of the conserved amino acidsnecessary for co-factor binding (Figure 111.5) (Mukherji et al., 2001). The C-terminal domain is 35% identical to JamC, an acyl carrier protein in the j amaicamidegene cluster (Edwards et al., 2004).

121 150

BarBl . .. SYDR HLDIQALSDI ISHPKIAHRV

PAUX . .. MITKVQD FQEDKELFRY CTLPEILKYV

BarB2 . .. NKMNYDR RLDIKALNDI ISHPKVVDRV SyrB2 GT.NIAUYDR HLDDDFLASH ICRPEICDRV

CmaB . .. . ITNYDR ULDVDLLTSH VFRXEIVHRV HctBVLNU4TSRDL HLFHQPIAEM FKNNKIVRVL

171 200 BarBi GDEG. TDWHQ AESFVEFEGK SK. .. LVPTE PAHX DSGXXTSRHP LHQDLHYFPF RP ..... S.. BarB2 GDEG. TDWHQ AUSFVEFEGE SK. .. LVPTE SyrB2 GDEG. TDWHQ ADTFISGX PQ. .. IIWPE CmaB GNEG.TDWHQ ADTFAHAPAS RN. . .WCAR. HctB GEGE. IKWHQ VYIDSYDPSAY DPQKPALLYP

301 330 BarBl FTSRGSE PNTSGSS ER YGWSTRPVPT PAHXFHPLLIHGS GQNKTQGFR KAISCHFASA BarB2 FTSRCMHGSE PNTSESS . IR YGWSTRPVST SyrB2 FWSTLMHASY PHSGESQEMR MGFASRYVPS CmaBFGSMLZ.PSSL PNSTTDK ...... TAWP.4S HctBFTERVMHSSP PNKSKQQ ER LGINGRYVPP

Figure 111.5: Alignment of barbamide BarBi and B2, syringomycin SyrB2, coronamic acid CmaB and PAHX with HctB. Allenzymes share putative conserved residues (highlighted) involved in binding iron (II) and 2-oxoglutarate cofactors. 109

The hctC gene is similar to insertion sequence (IS) elements from other bacteria and encodes for a putative transposase. BLAST analysis of the transposase

showed that it was most similar(P31% identity) to TnpB encoded by an 1S605 type element from Helicobacter pylon (Censini et al., 1996), and transposases encoded by other IS elements such as IS 1341 from the thermophile PS3 (Murai et al., 1995), 1S891 from the cyanobacterium Anabaena M- 131 (Bancroft and Wolk, 1989), and 1S1136, from the erythromycin gene cluster in Saccharopolyspora erythraea (Donadio and Stayer, 1993). About 50 bp downstream from hctC is located hctD, which encodes a type I PKS module with KS-AT-CM-KR-ACP domain organization. The hctDgene shows highest similarity to PKS genes from the jamaicamide A and curacin A biosynthetic pathways (Chang et al., 2004; Edwards et al., 2004) and all five domains show typical conserved core motifs when aligned with PKS genes from these twogene clusters (Figure 111.6).

A B KS-HctD LHGPSLAIDTACSSSL- --YVEAEGT---IGBLEAAA AT-HctD YTQPALFALEYALCQL- - - IGHSVGE KS-JamJ LRGPVMTVETASA3L- - -YVEAHGT-- -IGHASTAA AT-Jainj YTQPVLFAIEYALCQL-- -MGHSVGE KS-JamK LIGPNLAVDTACSSSL- - -YLF.AfiGT- - -IGH'rEGAA AT-JamK YTQPCLFALEYALCQL- - -MGHSVGE KS-CurF LKGPVVAVEAACSSSL- -YLEAHGT- - -IGNAATAA AT-CurF YTQPALFAIEYALFKL- - -MGHSVGE KS-CurG LTGPAITVQTACSTSL- -YV.?.BGT- - -VGHLDAAA AT-CurG YTQPALFATEThLFKL- - -MGHSVGE Cons. CHSVC (malonyl CoA) C C-MT-HctD LLEIGAGGGTT D C-MT-JamJ LLEIGGTGTT YLITGGLGAL- - C-MT-CurJ LLEIGAGTGGTT KR-CurH KR-JamJ KR-CurG YLITGGLGYL- -- -AVD--- KR-HctD YLITGGTGFL- - -ASD- KR-Hct YLISGGLGGI----DVD--- KR-HctFYLISGGLGGI----AVD--- KR-Ces YVVTGGLGGI----DGD--- KR-JamPYLITGGLGGI----CAD---

Figure 111.6: Sequence alignments of PKS domains from HctD with other cyanobacterial PKS's (jamaicamide A (5) and curacin A PKS genes). KS domain alignment (A), AT-domain specific for malonyl-CoA (B), alignment of C-methyl transferase (C), alignment of KR domains from jamaicamide A, curacin A, cereulide and hectochlorin biosynthetic gene clusters (D). 110

The stop site ofhctDoverlaps with the start site of the next ORFhctE,which encodes for a bimodular NRPS. Initial analysis of the first module revealed the presence of a KR domain embedded between the A and PCP domains, which is unprecedented in NRPS modules. The A-domain is most similar to the A-domain from BarE of the barbamide biosynthetic pathway (46% identity) (Chang et al., 2002) and also shows similarity to A-domains from the microcystin McyG and nodularin NdaC clusters (41% identity) (Tillett et al., 2000; Moffitt and Neilan, 2004). A notable feature of the residues that contribute to substrate specificity in the A-domain is substitution of the conserved aspartate residue (Asp23 5) involved in interactions with the amino group of the cognate amino acid by valine (Va1235) at this position. BLAST analysis of the KR domain revealed it had weak similarity to KR domains from PKS (23% identity), but higher similarity toa KR from the partially characterized cereulide biosynthetic gene cluster (31% identity) (Ehling-Schulz et al., 2005). The second module in HctE contained all of the required domains for the adenylation and heterocyclization of cysteine and is most similar to BarG from the barbamide biosynthetic gene cluster (57% identity) (Chang et al., 2002) and CurF from the curacin A biosynthetic pathway (58% identity) (Chang et al., 2004). This module also contains an FMN-dependent oxidase that is embedded between the A9 and AlO conserved motifs of the adenylation domain, which is likely involved in the fonnation of thiazole ring (Du et al., 2000a). 111

Next in the cluster is hctF, a bimodular NRPS whose domain architecture is identical to that of HctE. The predicted HctE and HctF proteins sharea 76% overall identity. The hctE and hctF genes also share overlapping start and stop sites, suggesting that hctDEF are transcribed as an operon. A thioesterase domain is located at the C-terminal end of HctF and is proposed to catalyze hydrolysis from the biosynthetic assembly line and cyclization. The next ORF, hctG, begins about 600 bp downstream from the end of hctF and encodes for a putative P450 monooxygenase. The hctG gene is followed by another ORF, hctH, whose product also shows similarity to putative P450 monooxygenases. While HctG and HctH are most similar to each other (47% identity) they also show similarity to a P450 monooxygenase involved in spirangiene biosynthesis (unpublished, accession number: AJ505006) (HctG 35% identity, HctH 36% identity) and TaH, a P450 hydroxylase from the TA biosynthetic gene cluster (HctG 36% identity, HctH 34% identity) (Paitan et al., 1999a). 112

Cloning, expression and functional analysis of adenylation domains from the hectochiorin biosynthetic gene cluster In order to obtain biochemical confirmation of the predicted adenylation domain specificities, both A-domains from HctE were overexpressedas 6x His-fusion constructs, purified and analyzed using the ATP-PPi exchange assay (Stachelhaus and Marahiel, 1995). Constructs for protein expression were based on the start and stop sites of BarE and BarG constructs which have been expressed successfully (Chang et al., 2002). The adenylation domains were PCR amplified and cloned into the pET2Ob(+) expression vector. When E. coli BL2 1 (DE3) cells harboring these pET2Ob(+) constructs were grown at temperatures above 20°C, only insoluble protein was obtained. However, soluble protein could be generated when cells were grown at 18°C for 12-18 hours without IPTG induction (Figure III.7A). These A-domainswere designated as HCtEOIVA and HctE, respectively. Western blot analysis using a-His antibodies was performed to confirm heterologous protein expression (Figure III.7B).

HctEQIVA

Figure 111.7: Overexpression and purification of adenylation domains in HctE and Western blot analysis. HctEAOIVA (66.9 kDa) and HctEAc (56 kDa) were overexpressed as 6x His-fusion proteins and purified (A). Western blot analysis using a-His antibodies (B). 113

The relative activities of the various amino acid substrates in the ATP-PPi exchange assay are displayed in Figures III.8A and III.8B. Thisassay is based on the reversible nature of the adenylation reaction (Figure 1.2, Chapter I). The recognition of a cognate amino acid by the adenylation domain results in the formation of corresponding acyladenylate and inorganic pyrophosphate (Figure 1.2). The reaction is reversible and the acyladenylate can undergo conversion to form the amino acid and ATP. The use of radioactive pyrophosphate results in its incorporation into the resulting ATP (AT32P) which is bound to charcoal and radioactivity isa measure of the ability of an adenylation domain to activatea particular amino acid. The first adenylation domain in HctE was found to activate 2-oxo isovaleric (0-NA) acid to the greatest extent; however, it also activated 2-hydroxy isovaleric acid (2-NA), 2-oxo-3-hydroxy isovaleric acid and DHiv toan appreciable level, thus indicating that it possesses fairly relaxed substrate specificity (Figure III.8A). Several other standard amino acid substrates were activated toa much less degree. These results are consistent with the arrangement of amino acid residues in the binding pocket of the A-domain in which the conserved Asp235 involved in interaction with the amino group of the amino acid substrate is replaced by Va1235,a substitution typical for a keto-acid or hydroxy-acid substrate (Stacheihaus et al., 1999). Because 2-OH WA and 2,3-DHiv were racemic mixtures in which each enantiomerwas represented at half concentration, the results of these experiments presented aboveare not an accurate representation of adenylation domain specificity. Experiments using R- and S-enantiomers of 2-OH NA and 2,3 DHivare currently underway in order to further determine the specificity of the adenylation towards these enantiomers. In the case of the second adenylation domain, L-cysteinewas activated to the highest level (100%) and L-serinewas activated up to 40%, while activation of L- glycine, L-tryptophan, L-histidinewere equivalent to background (Figure III.8B). 114

A a

HO'f r 0 120

100

0 2-oxo2-oxo-3- 2-OH 2,3 di- Val 2-oxo Leu Ala His Gin Trp IVA OH IVA IVA OH IVA Leu

120

100

- 80

> 60 >

0 40

0-4 ______Cys Ser Giy Trp His Figure 111.8: Results of the ATP-PPi exhange assay for HCtEAO.JVA (A) and HctEAc (B) and represent average of duplicate experiments. Abbreviations: 0-IVA, 2-oxo-isovaleric acid; 2-OH IVA, 2-hydroxy-isovaleric acid; 2-oxo-3-OH IVA, 2-oxo-3-hydroxy isovaleric acid; 2,3-diOH IVA, 2,3- dihydroxy isovaleric acid; Va!, L-valine; 2-oxo-Leu, 2-oxo L-leucine; Leu, L- leucine; Ala, L-alanine; His, L-histidine; Gln, L-glutamine; Trp, L-tryptophan; Cys, L-cysteine; Ser, L-serine; Gly, L-glycine. 115

Cloning and overexpression of HctA (acyl-ACP synthetase) In order to examine the substrate specificity of HctA, the entire ORF was cloned and expressed as 6x His-fusion protein in E. coli. Optimal protein expression was observed at 25°C and induction with 0.4 mM IPTG (Figure 111.9). The protein was purified to near homogeneity using Ni2taffinity chromatography and awaits further characterization through ATP-PPi exchange assays. Methods to synthesize 5,5-dichiorohexanoic acid, an important substrate for this assay are currently being explored at Christine Willis's laboratory at the University of Bristol, UK.

kDa 1 2 3 4 5 6 7

- 68.4 kDa

Figure 111.9: Overexpression and purification of HctA. Protein marker (1), total protein extract (2), insoluble fraction (3), soluble fraction (4), eluted HctA (5,6) and dialyzed HctA (7). HctD HctE HctF Module I Module 2 Module 3 HctA HctB K H APC

AMP

S HO) OH Cl HO)(

CI OH Enzymatic domains: C Condensation ASAcyI-ACP synthetase A Adenylation CI HalPutative halogenase PCP Peptidyl carrier protein ACP Acyl carrier protein CyCyclization KS Ketosyrithase OxOxidase AT Acyltransferase Post-N RPS TE Thioesterase Cl modification? CM C-methyl transferase Oxy P5O monooxygenase KR Ketoreducatse CI

Module 2 Q HctG or HctH ,-

A Module 4 HctGorHctH 02'o 0 (Y >t?O HO). W PI'ø..- W >- OH OH "T'd120 >O >-: HO) Figure 111.10: The proposed biosynthesis of hectochiorin. Insets 1 and 2: In modules 2 and 4, the adenylationdomains are proposed to activate 2-oxo-isovaleric acid, which is reduced in situ to 2-hydroxy isovaleric acid by the embedded KR domain and further oxidized by HctG or HctH to form 2,3-dihydroxy isovaleric acid. 117

Proposed biosynthesis of hectochiorin This report describes the cloning and preliminary characterization of 38 kb of DNA from the marine cyanobacterium Lyngbya majuscula that is putatively involved in the biosynthesis of hectochiorin (1). The overall genetic architecture and domain organization encountered in this cluster is consistent with the arrangement expected for the biosynthesis of hectochiorin, and its identity is further strengthened by biochemical characterization of the adenylation domains. The presence of a hybrid histidine kinase/response regulator protein about 10 kb upstream of the hctgene cluster leads us to speculate that the expression of this cluster is probably regulated by signal transduction mechanism, although there is no evidence to support this. Indeed to date, there is very little understanding of the regulation of natural product biosynthetic pathways in cyanobacteria. HctA, an acyl-ACP synthetase homolog, is proposed to activate free hexanoic acid in an ATP-dependent maimer and thus initiate hectochiorin biosynthesis (Figure 111.10). HctA shares a high similarity to j amaicamide JamA (obtained from the same L. majuscula strain), which has been shown to biochemically activate 5-hexynoic acid through an ATP-PPi exchange assay (Edwards et al., 2004). The acyl group is then likely transferred from the acyl-adenylate to the ACP domain in the C-terminal region of HctB. The N-terminal region of HctB shows similarity to barbamide BarB 1 and B2 (Chang et al., 2002), coronamic acid CmaB (Ullrich and Bender, 1994), and syringomycin SyrB2 (Zhang et al., 1995) and weak similarity to phytanoyl-CoA hydroxylases (Mukherji et al., 2001). Sequence alignments of these proteins with HctB show that they share conserved residues involved in co-factor binding, suggesting that the enzyme mechanism may require iron (II) and 2-oxoglutarateas cofactors (Figure 111.5) (Mukherji et al., 2001; Kershaw et al., 2001). BarBiand B2are believed to mediate the transformation of leucine to trichioro-leucine during the biosynthesis of barbamide (Chang et al., 2002). Similarly, SyrB2 is postulated to be involved in the chlorination of threonine during syringomycin biosynthesis (Zhang et al., 1995) and CmaB in the conversion of isoleucine to coronamic acid in coronatine biosynthesis (Ulirich and Bender, 1994). Hence, we speculate that HctB is involved in 118 the formation of the gem dichioro group in hectochiorin based on its location in the cluster and proposed biochemical properties.

Located betweenhctBandhctDis an IS element that encodes for a putative transposase. IS elements are fairly small segments (1.5 -2.0 kb) with a simple genetic organization which encode functions involved in their mobility (Mahillon and Chandler, 1998). IS elements are believed to play a major role in the plasticity of bacterial genomes and are often involved in the assembly of gene clusters with specialized functions such as pathogenicity, antibacterial activity or catabolic pathways (Mahillon et al., 1999). The occurrence of transposases at the boundaries of several cyanobacterial gene clusters has been previously reported (Christiansen et al., 2003; Edwards et al., 2004; Moffitt and Neilan, 2004; Tillett et al., 2000; Becker et al., 2004). However, it is interesting to note that the HctC transposase is present within the putativehctgene cluster. It is likely that these transposases may play an important role in the acquisition and distribution of secondary metabolite gene clusters between different strains and genera. The chlorinated hexanoate initiator of hectochiorin biosynthesis is next proposed to undergo one round of ketide extension by the PKS module (KS-AT-CM- KR-ACP) found in HctD. The AT- domain in this module appears to be specific for malony!-CoA (Figure III.6B) (Haydock et al., 1995). The C-MT transferase possesses a signature motif (LExGxGxG) that is also present in the C-methyl transferases from the jamaicamide A and curacin A biosynthetic gene clusters (Figure III.6C). This is consistent with precursor feeding experiments to several polyketide pathways in which methyl branching at C-2 positions of several cyanobacterial polyketides all derive from SAM (Gerwick et al., 2001; Chang et al., 2004; Edwards et al., 2004). 119

Table 111.2: Substrate specificity of A domains from the first modules in HctE and HctF. The binding pocket amino acids involved in substrate recognition determined using the method of (Stacheihaus et al. 1999 and Challis et a!, 2000) of A-domains from the first module of HctE and HctF (this study) were compared to residues from previously characterized A-domains BarE (Chang et al., 2002) McyG (Tillett et al., 2000)and NdaC (Moffitt and Neilan, 2004) involved in

A- Binding pocket amino acids domain Substrate 235236239278299301322330 HctE-A0. V G V W L A L F 2-oxo- IVA isovaleric acid HctF-A0. V G V W L A L F 2-oxo- IVA isovaleric acid BarE-A V G I I V G G T 2-oxo-leucic acid NdaC-A V G I W V A A S phenylacetate McyG-A V G I W V A A S phenylacetate GrsA-Phe D A W T I A A I phenylalanine

Next, HctE is composed of two NRPS modules that are proposed to incorporate DHiv and cysteine. Module 2, which presumably catalyzes the incorporation of DHiv, is unique in that it possesses a KR domain embedded between the A and PCP domains. Analysis of the A-domain binding pocket residues which confer substrate specificity using the previously described methods (Challis et al., 2000; Stacheihaus et al., 1999) revealed that the conserved Asp235 involved in ionic interaction with the amino group of the substrate amino acid was replaced by Va1235 (Table 111.2). This type of codon specificity suggestsa preference for a non a-amino acid substrate such as a hydroxy- or keto-acid. Furthermore, this A-domain is most closely related to the barbamide BarE A-domain, which also lacks the conserved Asp235 and shows high specificity for both the non-chlorinated and trichiorinated 2- oxo derivatives of L-leucine (P.Flatt and W.H.Gerwick, unpublished) and only modest activation of corresponding a-amino acids (Chang et al., 2002). Other previously characterized cyanobacteria! A-domains with the missing Asp235 residue include McyG and NdaC A-domain, both of which are involved in activatinga phenylacetate 120

starter unit in the biosynthesis of microcystin and nodularin, respectively (Table 111.2) (Moffitt and Neilan, 2004; Tillett et al., 2000). Biochemical support for HCtEAOWA function was obtained through the ATP-PPi exchange assay in which the A-domain was found to activate the 2-oxo derivative of valine over other substrates (Figure III.8A). The exchange assays also showed that this A-domain activates 2-OH WA, 2- oxo-3-hydroxy isovalerate and DHiv, suggesting that the A-domain hasa fairly relaxed substrate specificity. Based on its location within module 2 in HctE, we propose that the embedded KR domain may be involved in the in situ reduction of 2-0 WA to 2-OH WA (Figure 111.10 Inset 1). Recently, the presence of a KR domain in an NRPS module has been reported in the partially characterized cereulide gene cluster(ces)fromBacillus cereus and is believed to be involved in the formation of 2-WA (Ehling-Schulz et al., 2005). In cereulide biosynthesis, this has been validated by labeled precursor feeding studies which concluded that 2-OH WA acid is derived from L-valine and is reduced to 2- WA during the biosynthetic process (Kuse et al., 2000). Although the HctE and Ces KR exhibit only weak similarity to KR domains from PKS modules, they dopossess all the key conserved amino acid residues required for enzymatic catalysis (Figure III.6D) (Reid et al., 2003). Next, 2-oxo-IVA undergoes 3-hydroxylation, a reaction presumably catalyzed by either HctG or HctH. HctG and HctH are heme-dependent cytochrome P450 monooxygenases with conserved C-terminal motifs, GxHxCxGxxLxR, which are involved in heme-binding and show greatest similarity to TaH from the TA antibiotic biosynthetic gene cluster (Varon et al., 1992) and a P450monooxygenase from spirangiene biosynthesis (unpublished, accession #: AJ505006). TaH is responsible for a specific post PKS hydroxylation at C-20 of antibiotic TA (Paitan et al., 1999b; Paitan et al., 1999a). P450 monooxygenases involved in post assembly modification are also seen in other polyketide gene clusters such as those involved in the biosynthesis of erythromycin (Andersen and Hutchinson, 1992) and epothiolone (Molnar et al., 2000). However, P450 monooxygenases that function during preassembly to generate -hydroxyl amino acid substrates are encountered in NRPS 121

gene clusters such as those involved in the biosynthesis of the coumarin antibiotics (Steffensky et al., 2000; Wang et al., 2000). A common theme in these lattergene clusters is the occurrence of a heme oxygenase in conjunction withan A-PCP didomain. The substrate amino acid is activated by the A-domain and is transferred to the holo-PCP domain to yield an aminoacyl-S-enzyme intermediate which isf3- hydroxylated by the heme monooxygenase. In novobiocin biosynthesis, NovH (A- PCP) activates the amino acid tyrosine to L-tyrosyl-AMP, which accumulates on its PCP domain. Its partner enzyme, NovI, is a heme dependent cytochrome P450 monooxygenase that specificially hydroxylates this intermediate to formf3- hydroxytyrosine (Chen and Walsh, 2001). Similarly, during biosynthesis of the nucleoside antibiotic nikkomycin, NikPl activates histidine and the L-histidinyl-S- NikP 1 serves as a substrate for the heme monooxygenase, NikQ, which introduces the 3-hydroxyl group (Chen et al., 2002). Such heme monooxygenases are thought to have evolved in conjunction with corresponding NIRPS's to act on substrates that are presented as L-aminoacyl-S-enzyme complexes, thus ensuring that only a select pool of the substrate is modified (Chen et al., 2001). Although a A-PCP didomain scaffold is not adjacent to the heme oxygenase in this cluster, it is plausible that HctG and/or

HctH may catalyze the 3-hydroxylation of 0-NAin transduring the preassembly process (Figure 111.10 Inset 1).It is interesting to note that there are two heme dependent oxygenases in the hct gene cluster and the extent of their involvement, timing of 3-hydroxylation and biochemical mechanism await further characterization. 122

The second module in HctE shows high similarity to NRPS's specific for the incorporation and heterocyclization of cysteine. Analyses of the amino acids in the binding pocket of the adenylation domain are consistent with conserved amino acid residues for cysteine recognition (Table 111.3). Notably, the condensation domain in this module shows the presence of conserved core motifs required for heterocyclization (Figure 111.11) (Schwarzer et al., 2003). In vitro biochemical characterization of the HctEAc domain demonstrated that it activates L-cysteine preferentially (Figure III.8B). An FMN-dependent oxidase domain is embedded between the A9-A10 core motifs of the adenylation domain and presumably catalyzes the oxidation of the thiazoline to a thiazole. Similar oxidase domains involved in thiazole ring formation from the epothilone and bleomycin gene clusters have been biochemically characterized and show high similarity to this domain from the hctgene cluster (48% identity) (Schneider et al., 2003).

Table 111.3: Substrate specificity of the adenylation domains in the second modules present in HctE and HctF. Amino acid residues involved in substrate recognition from adenylation domains of the second modules of HctE and HctF were identified using the method of Stachelhaus et al. 1999 and Challis et a!, 2000 and compared to other cyanobacterial A-domains involved in activating cysteineBarGA2and CurF. The numbering of binding pocket amino acid residues correspond to GrsA-Phe A-domain numbering.

A-domain Binding pocket amino acids Predicted substrate 235236239278299301322330 HctE-Ac D L Y N L S L I cysteine

HctF-Ac, D L Y N A L L I cysteine

BarG-Ac D L Y N L S L I cysteine

CurF-Ac, D L Y N V D L I cysteine 123

Zi Z2 C3 Z3

HetEcyc FPLTDIQQAYWLGR---RAVVLPDGQQ---flALLADSWS---LPPAPELP--- HctFcyc FPLTDIQQAYWLGR- - -RAVVLPDGQQ- - -DALThDGWS- - -LPPAPELP- - BarGcyc FPLTEIQQAYWLGR---RMVILPNGEQ---DALIP.DAWS---LPPAPELP--- CurFcyc FPLNDIQQAYWIGR- - -RI4VVLADGNQ- - -DMLXFDAWS- - -LPPAPEIP - - Cons. FPLTxxQxAYxxGR- --RAx.xxGxQ- - -DxxxxDxxS- - -LPxxPxxP- - -

HctEcyc TNSTVLLWADILTCW- - -GDFT- - -GVVFTSVL- - -QVWLDHQVLEEQGELFQWD HctFcyc TNSTVLLAADVLTP.W- - -GDT-- -GFTSTL---QVWLDHQVEQGLFQWD BarGcyc TPSGLLAMiDFIAYW- - -GDFT- - -GVVFTSTL- - -QVWLDNSVAEQNGALLLIW CurFcyc TPSGVgSAFASVLNYW- - -GT- - -GVSTL- - -QVLLDHrEEKGALAPSW Cons. TPXXXLXXXXXXVLXXW- - -GDFT- - -GVVFTSxL- - -QVxLDxQxicxxxxxxxxxW

Figure 111.11: Alignment of heterocyclization domains from barbamide BarG and curacin A CurF with heterocyclization domains from the hct gene cluster show presence of consensus core motifs.

Next, modules 4 and 5 in HctF are believed to catalyze the incorporation of the second DHiv and cysteine units, respectively. The modular organization of HctF is identical to that present in HctE. The A-domain in module 4 exhibits a remarkably high similarity to the A-domain in module 2 (95% identical) with both adenylation domains sharing identical binding pocket amino acid residues (Table 111.2). Thus, the A-domain in module 4 is expected to catalyze the activation and incorporation of the second 0-IVA that is reduced in situ to 2-OH IVA by the embedded KR domain and then further oxidized by HctG or HctH to form DHiv (Figure 111.10 Inset 2). Although the incorporation of the two DHiv units is catalyzed by modules that have the same domain organizations, it is intriguing that their regiochemical orientation is different, with the secondary alcohol of DHiv-1 forming the ester with the preceding polyketide unit and the tertiary alcohol of DHiv-2 forming the ester with the preceding cysteine- derived residue. It is unknown how the alternate orientations of DHiv are achieved by these very similar modules. Module 5 in HctF appears to catalyze the incorporation of a second L-cysteine unit into hectochlorin (1). The A-domain binding pocket amino acids are in consensus 124

for the recognition of cysteine (Table 111.3) (Challis et aL, 2000). Conserved regions were again identified for the heterocyclization domain, which catalyzes the cyclization of cysteine (Figure 111.10). An oxidase domain is present between the A9 and Al 0 conserved motifs and is again likely involved in the oxidation of thiazoline to thiazole. Finally, an embedded type I thioesterase which possesses conserved GxSxG motif is present at the C-terminal end of HctF, and may be involved in catalyzing the release and cyclization of hectochiorin. A candidate gene product to introduce the acetate group at C-14 in hectochlorin (1) is lacking and leads us to speculate that this may be catalyzed by an acetyl transferase not present in thehctgene cluster and likely as a post-NRPS modification. The natural products chemistry of marine cyanobacteria is amazingly diverse and is a direct reflection of the unique biosynthetic capabilities of these organisms. L. majuscula is clearly the most prominent producer of novel secondary metabolites among these organisms. Thehctgene cluster adds to the growing repertoire of cyanobacterial hybrid PKS-NRPS pathways and contains several novel elements. An interesting feature of this cluster is the juxtaposition of two multimodule ORF 's with identical organization suggesting that it may have arisen through a gene duplication event. Sequence alignment of HctE and HctF indicate >90% identity in certain regions. Analysis of the putativehctgene cluster has revealed novel motifs such as the presence of an embedded KR domain in an NRPS module, which is likely involved in reducing an 0-IVA precursor. This is the first report of thepresence of a KR domain in an NRPS module from a cyanobacterial gene cluster, to the best ofour knowledge. Other notable features include an acyl-ACP synthetase,a putative halogenase, two P450 monooxygenases and the presence ofan IS element within the cluster. Another striking feature of this gene cluster is the presence of homologs of recognizable genetic elements such as JamA from the j amaicamide A gene cluster and BarB 1 from the barbamide gene cluster that are found in new combinations (HctA and HctB). The isolation of each new cluster has illustrated the remarkable versatility of cyanobacterial biosynthetic machinery and provides the foundation for the harnessing the activity of unique tailoring enzymes. The continued discovery of new molecules 125

from these metabolically talented organisms will providea bountiful source of novel bioactive natural products that may be developed as potential therapeutics. Additionally, efforts focused at dissecting the biosynthesis of these metabolites at the molecular genetic level along with whole-genome sequencing of several cyanobacteria will enable us to fully exploit the biochemical diversity of these organisms. 126

EXPERIMENTAL

Bacterial strains and culture conditions Escherichia coli strain DH1 OB was routinely used in this study as a host for DNA cloning and sequencing while E. coli strain EPI 300 was used as a host for construction of the fosmid library and amplification of fosmid clones. E. coli DH1OB containing pGEM-T Easy, pBluescript or pGEM3ZF(+) was grown at 37°C overnight in Luria-Bertani medium (LB) with a final concentration of ampicillin at 100 pg!ml. E. coli EPI 300 harboring fosmid clones were grown at 37°C in LB medium with chloramphenicol at a final concentration of 12 tg/ml. Lyngbya majuscula JHB was collected from Hector Bay in 1996 and is maintained as a unigal culture in our laboratory (Marquez et al., 2002).

DNA isolation and construction of fosmid library DNA from Lyngbya majuscula JHB for PCR amplification was obtained using DNeasy Plant Mini Kit (Qiagen, Valencia, CA). High molecular weight DNA for construction of the library from L. majuscula JHB was extracted using phenol followed by purification with Genomic Tip Kit (Qiagen, Valencia, CA) (Chapter.II). The high molecular weight DNA was size selected (-' 40 kb), end repaired, and ligated into the copy control fosmid vector pCC1FOS according to instructions provided with the CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI). Plasmid DNA isolation was carried out using commercially available kits (Qiagen, Valencia, CA). Other standard DNA manipulations such as restriction digests, ligations and transformations were carried out using standard methods (Sambrook et al., 1989). Library screening, labeling of probes, detection and Southern analysiswere performed according to the protocols provided with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Molecular Biochemicals, Manheim, Germany). Prehybridization and hybridization of probes were carried out at 42°C and stringency washes were carried out at 55°C. 127

PCR cloning of heterocyclization-adenylation domain probe PCR amplification of probe fragments used to screen the genomic library in this study was carried out using Taq DNA-polymerase (Invitrogen, Carlsbad, CA) using manufacturer's recommendation regarding template and primer concentrations in an EppendorfMastercycler. The following thermocycler conditions were used for amplification: denaturation at 94°C for 30 s, annealing at 48°C for 30s, extension at 72°C for 1 mm for 30 cycles. Degenerate primers were designed basedon conserved domains found in the heterocyclization-adenylation domain regions (HetForward-5'- CCG GAT AAG CGA CAT GAA CCN TTY CCN YTN A-3' and AdenReverse-5'- CAT CAG GGC GGG CAC NSW RTT CCA-3'). These primers were used to amplify an approximately 1.7 kb fragment. The PCR product was purified usinga commercially available kit (Qiagen, Valencia, CA), cloned into pGEM-T Easy (Promega, Madison, WI) and unique clones were sequenced. Amplification of an approximately 700 bp region of the ketosynthase domain was carried out using previously designed primers KS1Up 5'-MGI GAR GCI HWI SMI ATG GAY CCI CAR CAl MG-3' and KSD1 5'- GGR TCI CCI ART SWI GTI CCR TG-3'(Beyer et al., 1999). All primers were obtained from Tnvitrogen (Carlsbad, CA).

Sequence analysis of fosmids Sequencing of fosmids pHctl and pHctII was carried out using shotgun cloning. Fosmid DNA was subjected by partial digest using AluI or Sau3AI (0.10- 0.25 units/.xg of fosmid DNA for 1 hour). The partially digested fosmid DNAwas then separated on a 1% agarose gel and fragments of size 1 kb-2. 5 kb were selected. The AluI fragments were ligated into pGEM3Zf-(+) treated with SmaTJCIAP (calf intestinal alkaline phosphatase) and Sau3AI fragments were cloned into pBluescript that was digested with BamHT and ClAP-treated and random subcloneswere selected for sequencing reactions. Sequencing was carried out using Big Dye ITT terminator cycle sequencing (PE biosystems) at the Central Services Lab, Center for Gene Research and Biotechnology, Oregon State University. Resulting gaps in the sequences were filled using primer walking with sequence specific primers. The 128

sequences were edited and assembled using VectorNTl's ContigAssembly software program (InforMaxmc,Frederick, MD). Analyses of DNA and protein sequences were carried out using the NCBI (National Center for Biotechnology Information) BLAST server. All alignments were conducted using ClustalW (Thompson et aL, 1994).

Cloning, expression and purification of adenylation domains from HCtEO..1VA and HctEc adenylation domains The adenylation domains of HctE were amplified by PCR using Vent® DNA polymerase (New England Biolabs) according to manufacturer's recommendations. The following primers (5 'tail in italics; restriction digest sites in bold) HCtEAOIVA- Forward3-5'GGA ATT CCA TAT GGA AAC TGA CAA CTT OAT CGA C 3' and HctEAOWA-Reverse3-5' CCG CTC GAG GGA AGA CAG ATT AAA AAC AGG 3' were used to amplify the adenylation domain from module 2 HctE (amino acids 787- 1391) and the primers HctEAc-Forward3-5'GGA ATTCCA TAT GTT CCA GAG TTA CAC AGA TTA T 3' and HctEAc-Reverse3-5' CCG CTC GAG ATC AAA TTC TGA CAG TGG TAA were used to amplify the adenylation domain of HctE3 (amino acid 2450-2956). The PCR products were cloned in-frame witha carboxy- terminal 6xHis fusion at theNdeIand XhoI sites of the pET2Ob(+) expression vector (Novagen). Both constructs were verified by sequencing. Overexpression of proteins was carried out in E. coli BL21 (DE3) and both proteins were purified in an identical manner. Overnight cultures expressing HctE2 A-domain or HctE3 A-domain were diluted 1:100 in 1 liter of LB-broth containing 100 g/m1 ampicillin andgrown at 18°C for about 12-18 hours without IPTG induction. Cellswere harvested by centrifugation, resuspended in lysis buffer (20 mM Tris-HC1 {pH8], 300 mM NaC1 and 20 mM imidazole) and lysed by sonication for six 10s bursts at 75 mAmps using a UPC 2000U sonicator (Ultrasonic Power Corporation, Freeport, IL). Recombinant proteins were purified using nickel chelate chromatography (Qiagen, Valencia, CA). Protein lysates were incubated with Ni-agarose beads for 2 hours at 4°C. The protein- 129

Ni-agarose slurry was then washed three times with wash buffer (20 mM Tris-HC1 [p11=8], 300 mM NaC1 and 40 mM imidazole) and proteins were eluted with elution buffer (20 mM Tri-HC1 [pH=8], 300 mM NaC1 and 250 mM imidazole). Purified proteins were dialyzed overnight at 4°C against storage buffer (50 mM Tris-HC1 [p11=8], 1 mlvi EDTA [p11=8], 100 mlvi NaC1, 10 mlvi MgC12, 1 mlvi DTT and 10% glycerol) using Spectra/P or dialysis membrane with a molecular weight cutoff of 12,000-14,000 Da (Spectrum Labsmc,Rancho Dominguez, CA). The dialyzed proteins were aliquoted, flash frozen in liquid nitrogen and stored at -80°C. Protein concentration was determined by Bradford protein assay and protein purity was assessed by analysis of protein lysates on a 12% SDS-PAGE followed by staining with Coomassie blue staining.

Western blot analysis Western blot analysis was carried out according to previously described methods (Sambrook et al., 1989). Proteins were transferred onto nitrocellulose membrane (Immobilon P, Millipore, Massachusetts) using Trans-Blot Electrophoretic Transfer Cell (Biorad, California) according to manufacturer's instruction accompanying the transfer unit. The membrane was first blocked in 5% nonfat dry milk in TTBS (20 mM Tris, 137 mM NaC1, 0.1% Tween, pH 7.6) for one hour. The membrane was the incubated with a-His antibody, the primary antibody (1:1000) in 1% non-fat dry milk /TTBS for 1 hour. After three washes in TTBS (20 minutes each), the membrane was incubated with horseradish peroxidase (HRP) conjugate secondary antibody (1:7500) for one hour. The membrane was washed four times in TTBS (20 minutes each) and chemiluminiscent detection using standard ECL kit was used to visualize protein bands. 130

Substrate-dependent ATP-pyrophosphate [32PPiI exchange assay All amino acid substrates used in this assay were obtained from Sigma (St Louis, MO) or Lancaster (Windham, NH). 2-oxo-3-hydroxy isovaleric acid and DHiv were synthesized using previously described methods (Hanson et al., 1990; Luesch et al., 2000b). The substrate-dependent ATP-PPi exchange assay was performed by incubating 2 M of HCtEOWA or HctEcin reaction buffer (50 mM Tris-HC1 [pH=8], 100 mM NaC1, 10 mM MgC12, 1 mM DTT) containing 0.5 tCi tetrasodium [32P] pyrophosphate (NEN-Perkin Elmer, Boston, MA) and 2 mM of the respective amino acid or oxo-derivative for 1 hour at 28°C. The reaction was quenched by the addition of stop-mix (500 il of 1.2% (w/v) activated charcoal, 0.1 M tetrasodium pyrophosphate and 0.35 M perchioric acid). The charcoal pellet was pelleted and washed three times with wash buffer (0.1 M tetrasodium pyrophosphate and 0.35 M perchioric acid) and resuspended in 500 d of water. The bound radioactivity was determined using a Packard TriCarb 2900 liquid scintillation counter.

Cloning, expression and purification of HctA The adenylation domains of HctE were amplified by PCR using Vent® DNA polymerase (New England Biolabs) according to manufacturer's recommendations. The following primers (5 'tail in italics; restriction digest sites in bold) were used for the amplification of HctA. HctAForward: 5' GGA ATT CCA TAT GAA TAC TAA GAA AAA ATT TAC CTC T 3' and HctAReverse: 5' CCG CTC GAG CCC CAG ATG CGC CCC ATT TTT 3'. The PCR products were cloned in-frame witha carboxy-terminal 6xHis fusion at theNdeIand XhoI sites of the pET2Ob(+) expression vector (Novagen) and verified by sequencing. Overexpression of proteins was carried out in E. coli BL21 (DE3) and overnight cultures expressing HctA were diluted 1:100 in 1 liter of LB-broth containing 100 p.g/ml ampicillin and grown at 25°C until0D600 = 0.4 before induction with 0.4 mM IPTG. Protein purification was carried out as described above. 131

Accession number The nucleotide sequence of the hectochiorin biosynthetic gene described in this work has been submitted to GenBank under the accession number AY974560. 132

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CHAPTER IV

EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF ADENYLATION DOMAINS FROM THE CARMABIN BIOSYNTHETIC GENE CLUSTER

INTRODUCTION

A Caribbean collection of the cyanobacterium,Lyngbyamajuscula (designated as strain 1 9L) was an extraordinarily rich source of bioactive metabolites and yielded several novel metabolites including barbamide (53) (Orjala and Gerwick, 1996), the curacins (curacin A 14) (Gerwick et al., 1994) and antillatoxin (25) (Orj ala et al., 1995). The more polar lipid extract yielded minute quantities ('1 mg) of a lipopeptide, carmabin A (55) (Hooper et al., 1998). The cyanobacteriumwas subsequently recollected and carmabin A was re-isolated along with the closely related carmabin B (56) (Hooper et al., 1998). Carmabin A was the first L. majuscula-derived metabolite to possess a terminal acetylene funcationality (Gerwick et al., 2001). The producing organism has been maintained in stable culture in our laboratory for 12 years. Although carmabin A was initially isolated as an antiproliferative agent(IC50 6.58 tg/mL against MRC-5 embryonic lung cell), the re-isolatedpure compound proved to be inactive in the NC1-60 cell lineassay (Hooper et al., 1998). A related metabolite, dragonamide (57) has also been isolated from a Panamanian strain of L. majuscula (Jiménez and Scheuer, 2001). Interesting structural features of carmabin A, such as its terminal functionalization, stimulated efforts to clone and identify thegene cluster involved in its biosynthesis. A genomic DNA library ofLyngbyamajuscula 19L was screened with KS-domain probes amplified using previously described primers (Beyer et al., 1999). This led to the identification of two overlapping cosmids, pLM32 and pLM23 as putative candidates for the carmabin biosynthetic gene cluster (car) (Z. Chang and D.H. Sherman, unpublished). Sequence analyses of these two cosmids led to the 139 mapping of an approximately 42 kb region consisting of ten ORFs that may be involved in the biosynthesis of carmabin A (Figure IV.1) (Z. Chang, J. Junyong and D.H. Sherman, unpublished).

OCH3 CH3 H ccI NS OCH3 HNCH3 H 'H Barbamide (53) Curacin A (14)

Ant illatoxin (25)

0 0

0 Carmabin A (55) -0CH3

Carmabin B (56) OCH3

I 0

Dragonamide (57) 140

ORF PKS PKS NRPS NRPS NRPS NRPS car AB D E - F . G H I

KS-AT-DH-MT-ER-KR-ACP KS-AT-DH-MT-ER-KR-ACP C-AN-PCP C-ATY,-N-Mt-O.Mt-PCP-AmT Hydrolase!TE I C-AphNMt.PCP CAN4.lt-PCP 2.8 kb pLM32

pLM23

Figure IV. 1: Genetic architecture of the carmabin A biosyntheticgene cluster (Z. Chang, J. Junyong, D. H. Sherman and W.H. Gerwick, unpublished).

Table IV.!: Deduced functions of open reading frames in thecar gene cluster

Protein Size Proposed function Sequence % Reference (amino similarity Identity, acids) (protein, Similarity organism) CarA --402 Acyl-ACP synthetase JamA, 96%,98% (Edwards et L. ma] uscula al., 2004) CarB 321 Desaturase JamB, 95%,98% (Edwards et L.majuscula al., 2004) CarC 100 ACP JamC, 95%, 98%(Edwards et L. majuscula al., 2004) CarD 2549 PKS JamJ, 68%,80% (Edwards et L.majuscula al., 2004) CarE 2575 PKS JamJ, 69%,81% (Edwards et L. majuscula al., 2004) CarF 1545 NRPS BarG, 67%,81% (Chang et al., L. ma] uscula 2002) CarG 1135 NRPS JamO, 49%,68% (Edwards et L. majuscula al., 2004) CarH 1546 NRPS BarG, 52%,67% (Chang et al., L. majuscula 2002) Carl 2303 NIRPS NcpB, 55%,71% (Beckeretal., Nostoc ATCC 2004) 53789 CarJ 293 Predicted Nostoc 67%,78% hydrolase/thioesterasepunctforme PCC 73102 141

Complete sequence information for CarA is currently lacking and sequencing of cosmid pLM32 is in progress to obtain this information. Additionally, sequencing will also provide insights into the region directly upstream of CarA and thus enable proper delineation of thecarcluster. Overall,carappears to be generally co-linear and fully consistent with the structure of carmabin A (55). Based on their similarity to JamA-C proteins (Table IV. 1), it is proposed that the CarA-C proteins comprisea system for the generation of a hexynoic acid starter unit (Edwards et al., 2004). The starter unit is then expected to undergo two rounds of ketide extension catalyzed by CarD and CarE, both of which possess KS-AT-DH-MT-ER-KR-ACP domain organization. Next, the incorporation of the four amino acids (N-methyl phenylalanine, alanine, N-methyl alanine and N- methyl 0-methyl tyrosine) is most likely catalyzed by the proteins CarF-I (Table IV.2). CarF and CarH contain embedded N-methyl transferase domains, while Carl contains an N-methyl transferase domain along withan 0-methyl transferase domain (Schwarzer et al., 2003). Finally, release of carmabin A (55) is likely catalyzed by the TE encoded by CarJ. 142

Table IV.2: Substrate specificity of A-domains in car. Amino acid residues involved in substrate recognition from adenylation domains of CarF, CarG, CarH and Carl were analyzed using the method of Challis et al. and Stacheihaus et al. The predicted amino acid specificity is completely consistent with the observed amino acids of carmabin A (55). A-domain Binding pocket amino acids Predicted substrate 235236239278299301322330 CarF D A W T V A A V phenylalanine

CarG D L F N N A L T alanme

CarH D L F N N A L T alanine

Carl D A S T V A A V tyrosine

In order to confirm the activities of the NRPS adenylation domains and to provide further biochemical evidence for the involvement of thisgene cluster in the biosynthesis of carmabin A (55), we sought to overexpress the adenylation domains and analyze them using the ATP-PPi exchange assay (Stachelhaus and Marahiel, 1995). Herein, the cloning, overexpression and biochemical characterization of two adenylation domains from the carmabin gene cluster is reported. 143

RESULTS AND DISCUSSION

Cloning and overexpression of adenylation domains

Since methods for targeted gene disruption are not yet available inL. majuscula, overexpression and biochemical characterization of the adenylation domains is important for establishing the role ofcar in the biosynthesis of carmabin A

(4). A similar method has been used for otherL.majuscula derived biosynthetic gene clusters (Chang et al., 2002; Edwards et al., 2004), and other cyanobacterialgene clusters (Becker et al., 2004; Hoffmann et al., 2003). Primers for the amplification of the A-domains from the car cluster were based on the start site of the construct designed for the expression ofj amaicamide JamO which has been successfully expressed (Edwards et al., 2004). The stop site of CarG A domain was based on that of JamO construct, however the CarF, CarH and Carl A- domains had to be truncated because of embedded tailoring domains and consequently, stop sites were designed downstream of the QVKRIG motif (A8 motif). The adenylation domains were amplified, cloned into the pET2Ob(+) expression vector with a C-terminal 6x His-tag and transformed into E. coli BL2 1 (DE3) cells for expression. Optimal protein expression was obtained for CarG and Carl A-domains at 25°C and induction with 0.4 mM IPTG. Western blot analysis using a-His antibodies was performed to confirm heterologous protein expression. However, protein expression was not observed for the CarF and CarH proteins although expression was examined under several temperature conditions (18°C, 22°C, 25°C and 30°C) and with different IPTG concentrations (0.2 mM, 0.4mM) to optimize expression and yield. Our initial goal was to characterize all four A-domains of the car cluster, but we succeeded in overexpressing and biochemically characterizing only two, CarG and Carl A-domains. In general, functional expression of adenylation domains hasproven to be challenging. Complete absence of protein expression, very low expression levels or inactivity of expressed proteins is frequently encountered. In the case of chloroeremomycin biosynthetic gene cluster, although low levels of protein expression for CepA adenylation domain was observed, the proteinwas inactive in subsequent 144

ATP-PPi exchange assays and lack of activity was attributed to misfoldingor partial unfolding during purification (Trauger and Walsh, 2000). The expression of NRPS domains with chaperone proteins such as GroES/EL has been shown to facilitate functional expression of these domains (Symmank et al., 1999). However, expression in the presence of such chaperones does not always result in the production of active protein and this has been observed for chioroeremomycin A-domains (Trauger and Walsh, 2000) and curacin CurF A domain (P. Flatt, personal communication). Another important factor impacting the functional expression of these proteins is the host organism. For instance, expression of adenylation domains from the baihimycin biosynthetic gene cluster could not be detected in E. coli whereas expression of these proteins was readily achieved in a Streptomyces host (Recktenwald et al., 2002). Similarly, several other proteins have been successfully expressed in a functional form in a Streptomyces sp. heterologous host (Malpartida and Hopwood, 1984; Tang et al., 2000). However, preliminary investigations conducted in our laboratory by P. Flatt and G. Zeller revealed that cyanobacterial genes demonstrateda high level of instability in Streptomyces host systems and often undergo rearrangements. Thus, this organism was not feasible for the expression of cyanobacterial genes. Studies have also shown that protein expression is more efficient in heterologous hosts that are more closely related to the parental strain. For example, in the case of epothilone biosynthesis, heterologous expression in Myxococcus xanthus resulted in higher yields of epothilone when compared to a Streptomyces host (Julien and Shah, 2002; Tang et al., 2000). At present, Nostoc punctiforme ATCC 29133 is being considered an as alternate host for heterologous expression. The genome of this strain has been sequenced and several molecular tools have been developed that allow for the design and development of genetic manipulation experiments (Meeks et al., 2001; Wong and Meeks, 2001) The heterologous expression of functional cyanobacterial proteins posesa major challenge and the development of new methods and strategies is an important goal of current research. Additionally, dissection of these modular systems at the 145 biochemical and genetic levels will enable us to accomplish the heterologous expression of functional proteins.

Biochemical characterization of the adenylation domains CarG and Carl A-domains were purified by using Ni2taffinity chromatography (Figure IV.2A-B). Substrate specificity of the two adenylation domains was demonstrated through the ATP-PPi exchangeassay (Stacheihaus and Marahiel, 1995).

A Marker B Marker (kDa) CarG-AIa (kDa) Carl-Tyr

60 kDa 60 - kDa 55 50

Figure IV.2: Overexpression of CarG (A) (60 kDa) and Carl (B) (49 kDa) adenylation domains. 146

The relative activities of the various amino acid substrates in the ATP-PPi exchange assay are displayed in Figures IV.3A and IV.3B. CarG A-domain activated L-alanine to the highest level (=100%) and L-valinewas activated to about 60%. Other amino acids tested were activated toa much lower level (5%)(Figure IV.3A). Analysis of the binding pocket amino acids in CarG A-domain (Table IV.2) revealed that the residues were identical to those present in JamO A-domain, which has also been shown to activate L-alanine to the highest level (Edwards et al., 2004). Other L- alanine activating A-domains with identical binding pocket amino acids include bleomycin Bim VII (Du et al., 2000), microcystin Mcy A-2 (Tillett et al., 2000) and myxalamid Mxa A (Silakowski et al., 2001). In the case of the Carl A-domain, L-tyro sine was activated to the highest level (=100%) and L-phenyalanine was activated up to 30%, while other amino acids tested, were activated to a much lesser extent (Figure IV.3B). The binding pocket residues in Carl (Table IV.2) are identical to those present in nostocyclopeptide NcpAl tyrosine activating domain (Becker et al., 2004). Biochemical characterization of these two adenylation domains provides further evidence to support the involvement ofcarin the biosynthesis of carmabin A. 147 A 120

100

C) w >60

0 L-AIa L-VaI L-His L-Iso L-Trp L.Ser L-GIn

120

100

80 >

>60

0 L.Tyr L-GIy L-Phe L-Iso L-GIn L-Ser L-His

Figure IV.3: Results for the ATP-PPi exhange assay for CarG A-domain (A) and Carl A-domain (B). Abbreviations: L-Ala, alanine; L-Val, valine; L-His, hisitidine, L-Ile, isoleucine; L-Trp, tryptophan; L-Ser, serine, L-Gln, glutamine; L-Tyr, tyrosine, L-Phe, phenylalanine. EXPERIMENTAL

Cloning, expression and purification of adenylation domains fromcar The adenylation domains of car were amplified by PCR using Vent® DNA polymerase (New England Biolabs) according to manufacturer's recommendations. The following primers (5 'tail in italics; restriction digest sites in bold)were used for PCR amplification of individual A domains. CarF-Forward 5' GGA ATTCCA TAT GGT AAC AGC ACT ACC ATT AAT G 3'; CarF-Reverse 5' CCG CTC GAG TGG TAC TAT ATA TGC TAC TAG ACG 3'; CarG- Forward-5'GGA ATTCCA TAT GTT GCA GTT GCC ACT AAT GAA A 3'; CarG-Reverse 5' CCG CTC GAG TTG TGT CTC CGT CTC AGG 3'; CarH-Forward 5' GGA ATTCCA TAT GAG CCA GTT GCC ATT AAT GAC 3'; CarH-Reverse 5' CCG CTC GAG TCC AGG CTT ATC CTC TCT G 3'; Carl-Forward 5' GGA ATTCCA TAT GCC GAT TAG CCA GTT GCC A 3'; Carl-Reverse 5' CCG CTC GAG CCC TGG AGT ATC TTC CTT GT 3'. The PCR products were cloned in-frame with a carboxy-terminal 6xHis fusion at theNdeIand XhoI sites of the pET2Ob(+) expression vector (Novagen). Intially, 50 mL ofEcoli BL2 1 (DE3) containing each construct were tested for protein expression under various temperature conditions (18°C, 22°C, 25°C and 3 7°C) and concentrations of IPTG (0.2mM and 0.4mM). Protein expression was optimized only for CarG and Carl A-domains. Both CarG and Carl A-domain constructswere verified by sequencing. Overexpression of proteins was carried out inE. coliBL21 (DE3) and both proteins were purified in an identicalmanner. Overnight cultures expressing CarG A-domain or Carl A-domain were diluted 1:100 in 1 liter of LB- broth containing 100 jig/ml ampicillin and grown at 25°C till0D600= 0.4. Cultures were then induced with 0.4 mM IPTG and allowed to grow for an additional eight hours. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris-HC1 {pH=8], 300 mM NaC1 and 20 mM imidazole) and lysed by sonication for six 10 s bursts at 75 mAmps using a UPC 2000U sonicator (Ultrasonic Power Corporation, Freeport, IL). Recombinant proteins were purified using nickel chelate chromatography (Qiagen, Valencia, CA). Protein lysateswere incubated with Ni- agarose beads for 2 hours at 4°C. The protein-Ni-agarose slurry was then washed three times with wash buffer (20 mM Tris-HC1 [pH=8], 300 mM NaC1 and 40 mM imidazole) and proteins were eluted with elution buffer (20 mM Tri-HC1 [pH=8], 300 mM NaC1 and 250 mM imidazole). Purified proteins were dialyzed overnight at 4°C against storage buffer (50 mM Tris-HC1 [pH=8], 1 mM EDTA [pH8], 100 mM NaC1, 10 mM MgC12, 1 mM DTT and 10% glycerol) using Spectra/Por dialysis membrane with a molecular weight cutoff of 12,000-14,000 Da (Spectrum Labsmc,Rancho Dominguez, CA). The dialyzed proteins were aliquoted, flash frozen in liquid nitrogen and stored at -80°C. Protein concentrationwas determined by Bradford protein assay and protein purity was assessed by analysis of protein lysateson a 12% SDS-PAGE followed by staining with Coomassie blue staining.

Substrate-dependent ATP-pyrophosphate [32PPiI exchange assay All amino acid substrates used in this assay were obtained from Sigma (St Louis, MO) or Lancaster (Windham, NH). The substrate-dependent ATP-PPi exchange assay was performed by incubating 2 mM of CarG or Carl A domains in reaction buffer (50 mM Tris-HC1 [pH=8], 100 mM NaC1, 10 mM MgC12, 1 mM DTT) containing 0.5 tCi tetrasodium[32P] pyrophosphate (NEN-Perkin Elmer, Boston, MA) and 2 mM of each amino acid for 1 hour at 28°C. The reaction was quenched by the addition of stop-mix (500 tl of 1.2% (w/v) activated charcoal, 0.1 M tetrasodium pyrophosphate and 0.35 M perchloric acid). The charcoal pelletwas pelleted and washed twice with wash buffer (0.1 M tetrasodium pyrophosphate and 0.35 M perchioric acid) and resuspended in 500 tl of water. The bound radioactivitywas determined using a Packard TriCarb 2900 liquid scintillation counter. 150

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Tillett, D., Dittmann, E., Erhard, M.,von Dohren, H., Borner, T., and Neilan, B. A. (2000). Structural organization of micro cystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology 7, 753-764.

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Wong, F. C. and Meeks, J. C. (2001). The hetF gene product is essential to heterocyst differentiation and affects HetR function in the cyanobacterium Nostoc punctiforme. Journal of Bacteriology 183, 2654-2661. 153

CHAPTER V

CONCLUSIONS

The marine environment is a rich source of biologically active natural products and continues to be a major source of molecules for drug discovery and development. Among marine organisms, marine cyanobacteria are proving to be an outstanding source of biologically active natural products. Moreover, there is growing recognition that much of the unique chemistry isolated from marine invertebrates is actually of cyanobacterial origin. The continued discovery of new compounds from these metabolically talented organisms will provide a range of new chemicals with potential applications in biotechnology, pharmaceuticals and agriculture. The astounding structural diversity encountered in cyanobacterial secondary metabolites can be attributed to their complex biosynthetic systems, consisting of polyketide synthases and nonribosomal peptide synthetases This thesis has focused on two secondary metabolite biosynthetic gene clusters present in a Jamaican isolate of the cyanobacteriumLyngbya majuscula.This strain has been a source of several interesting molecules including hectochiorin, a metabolite with remarkable antifungal and cytotoxic properties. Initial attempts to clone and characterize the hectochiorin biosynthetic gene cluster from this organism led to the identification of a cryptic gene cluster,LMciyl.This allowed us to develop a novel interdisciplinary approach aimed at identifying the metabolite produced byLMcryl. The first step in this process was demonstration of its expression by RT-PCR which was successful. Feeding experiments using labeled amino acid precursors is currently in progress and identification of the metabolite will be realized using select 2D-NMR techniques. Genomics guided approaches, such as the one discussed here, will become a vital part of novel methodologies designed to identify and characterize new chemical entities. Such methods also allow for the identification of new metabolites 154 present in very low concentrations that may otherwise be missed during routine chemical isolation procedures. Using a more specific cloning strategy, the hectochlorin biosynthetic gene cluster (hct) was subsequently isolated and sequenced. hct adds to the growing repertoire of marine cyanobacterial gene clusters. The genetic architecture and domain organization appear to be colinear with respect to its biosynthesis and consists of eight ORF's spanning 38 kb with several novel features. A striking feature of this cluster is the contiguity of two ORF's with identical modular organization suggesting that the cluster may have arisen through a gene duplication event. Another unusual feature of the cluster is the presence of KR domains in two peptide synthetase modules which are predicted to be involved in the formation of the two DHiv units and appears to be the first report of such an arrangement in a cyanobacterial secondary metabolite gene cluster. Also present are an acyl-ACP synthetase that may be involved in the activation of a hexanoic acid starter unit, and two cytochrome P450 monooxygenases which are likely involved in the formation of the DHiv units. A putative halogenase, at the beginning of the gene cluster, is predicted to form 5,5-dichloro hexanoic acid. Two adenylation domains from HctE along with the HctA acyl-ACP synthetase were expressed heterologously in E. coli. While the two adenylation domains were successfully characterized through biochemical experiments, the acyl-ACP synthetase awaits further biochemical characterization. The characterization of each new gene cluster from a marine cyanobacterium has illustrated the extraordinary versatility of their biosynthetic machinery. Efforts focused at understanding and dissecting the biosynthesis of these metabolites at the molecular genetic level will provide the foundation for harnessing these systems through combinatorial engineering. Additionally, detailed biochemical characterization of the unique tailoring enzymes associated with cyanobacterial gene clusters will also facilitate the engineered biosynthesis of novel metabolites. The development of alternate hosts such a Nostoc punctiforme for the heterologous expression of individual proteins or entire gene clusters is also of paramount importance, as such systems can generate an unlimited supply of metabolites for 155 further biomedical or biotechnological use. At present, only a few marine cyanobacteria are amenable to laboratory cultivation and advances in this area will enable further investigations of the natural product diversity of these organisms. A better understanding of the complex metabolic processes governing symbiosis will be vital in order to exploit the biosynthetic potential of cyanobacterial symbionts and enhance their application in biotechnology. Finally, whole-genome sequencing of several cyanobacteria will be crucial for gaining a better understanding of the regulation of these gene clusters, their arrangement within the genome, their distribution among different strains and their probable role in the marine environment. Functional genomic studies which provide a well-defined blueprint of the genome will be indispensable for the design of rational approaches to search for and harness secondary metabolite gene clusters involved in the biosynthesis of valuable natural products. 156

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