Minireviewmbt_179 544..563 Bioactive Compounds from Marine Bacteria and Fungi Abdessamad Debbab,1 Amal H. Aly,1 Wen H. Lin2 and Peter Proksch1* 1Institut f��r Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine Universitaet Duesseldorf, Germany. 2National Research Laboratories of Natural and Biomimetic Drugs, Peking University, Health Science Center, 100083 Beijing, People���s Republic of China. Summary Marine bacteria and fungi are of considerable impor- tance as new promising sources of a huge number of biologically active products. Some of these marine species live in a stressful habitat, under cold, light- less and high pressure conditions. Surprisingly, a large number of species with high diversity survive under such conditions and produce fascinating and structurally complex natural products. Up till now, only a small number of microorganisms have been investigated for bioactive metabolites, yet a huge number of active substances with some of them fea- turing unique structural skeletons have been isolated. This review covers new biologically active natural products published recently (2007���09) and highlights the chemical potential of marine microorganisms, with focus on bioactive products as well as on their mechanisms of action. Introduction Since the beginning of mankind nature has been contrib- uting considerably to drug discovery for human beings by providing remedial treatments. One of nature���s treasures is the marine biotope, which occupies almost three quar- ters of the earth���s surface (Fenical, 1993 Whitehead, 1999). Marine natural products play an increasingly important role in biomedical research and drug develop- ment, either directly as drugs or as lead structures for bioinspired chemical drug synthesis (Molinski et al., 2009). Many marine natural products, especially those isolated from macroorganisms, have already undergone clinical trials (Newman and Cragg, 2004). During the last decades, however, repeated isolation of known metabo- lites and a reduced hit-rate of novel compounds from marine macroorganisms were observed. Hence, natural product chemists are turning their interest to so far less investigated drug sources, such as marine fungi and bac- teria, which turned out to be a vast untapped reservoir of metabolic diversity. Thus, research on chemistry of natural products derived from marine microorganisms has increased tremendously in recent years due to the demand for compounds having potential pharmaceutical applications or economical value as cosmetics, drugs, fine chemicals and functional personal-care products (Andersen and Williams, 2000). In contrast to macroor- ganisms, microorganisms represent promising natural product sources having the advantage of feasible and sustainable production of large quantities of secondary metabolites with reasonable cost, by large-scale cultiva- tion and fermentation of the source organisms1 (Waites et al., 2001). Furthermore, to adapt and survive in the marine ecosystem, characterized by very special condi- tions that differ from those found in other habitats, marine microorganisms sometimes accumulate structurally unique bioactive secondary metabolites not found in ter- restrial organisms (Bhakuni and Rawat, 2005). Natural products research still has an enormous unex- ploited potential, and the significant advantages and dis- advantages of natural product-derived molecules as drug candidates for development have been highlighted in numerous articles (Rogers, 2004). The importance of natural product discovery from microorganisms started only after the large-scale production of penicillin (1) during World War II (numbers 1���101 in bold refer to chemical structures 1���101 at the end of this article). After the end of the war, pharmaceutical companies refocused on the search for new bioactive biomolecules. In the 1970s for example, cholesterol biosynthesis inhibitors, compactin (2) (Larsen et al., 2007) and mevinolin (3) (Araki and Konoike, 1997) were discovered. The discovery of com- pactin and mevinolin enabled the development of the Received 15 January, 2010 accepted 28 March, 2010. *For correspondence. E-mail proksch@uni-duesseldorf.de Tel. (+49) 211���8114163 Fax (+49) 211���8111923. 1For detailed description of different isolation and culturing methods of marine fungi see Kjer and colleagues (2010), and marine bacteria see Stafsnes and colleagues (2010). Microbial Biotechnology (2010) 3(5), 544���563 doi:10.1111/j.1751-7915.2010.00179.x �� 2010 The Authors Journal compilation �� 2010 Society for Applied Microbiology and Blackwell Publishing Ltd

highly successful statin therapeutics (4) (Endo, 1992), which even today are considered as block buster in phar- maceutical sales. The discovery of streptomycin, gentami- cin, omegamycin (5) and other antibiotics pushed the pharmaceutical industry to implement large research and developing programs based on natural product discovery, with a recent emphasis on marine microbial fermentation based technologies. New bioactive natural products from marine bacteria and fungi The marine environment is extremely complex and con- tains a huge diversity of life forms. The water column of the oceans contains approximately 106 bacterial cells per millilitre (Hagstr��m et al., 2002). Marine bacteria and fungi are of great interest as novel and rich sources of biologi- cally active products. They live in close association with soft-bodied marine organisms, which lack obvious struc- tural defence mechanisms, and thus rely on chemical defence by production of bioactive secondary metabo- lites, either by themselves or by associated microflora, to survive in their extreme habitat (Jensen and Fenical, 1994). In the last decades, the number of reported sec- ondary metabolites from marine bacteria and fungi has steadily increased (Fenical, 1993 Kobayashi and Ishiba- shi, 1993 Bernan et al., 1997 Faulkner, 1997 1998 1999 2000 2001 Blunt et al., 2003 2004 2005 2006 2007 2008 2009 Hill, 2003), thus reflecting the growing attention by groups from academia and industry. In the year 2007 alone 961 new compounds were described from marine microorganisms reflecting an increase of 24% compared with the number of compounds reported for 2006 (Blunt et al., 2009). In the following, selected examples of new secondary metabolites from bacteria and fungi, published in the period 2007���09, that live in association with marine macroorganisms, such as sponges, algae and mangrove plants, are presented, with special emphasis on bioactive products and their modes of their action, as well as source organisms and place of origin. Chemical structures are only shown for new com- pounds, or for previously reported compounds with newly reported biological activities. Antimicrobial secondary metabolites Since the discovery of penicillin in 1928 (Fleming, 1929), intensive studies, mainly on soil-derived bacteria and fungi, demonstrated that microorganisms are a rich source of structurally unique bioactive substances (Fenical, 1993). The increasing need for new antimicrobial agents able to control emerging diseases or resistant strains of microorganisms inspired a growing number of research groups to explore the oceans for new bioactive compounds. Throughout the years, extensive screening programs were developed worldwide and great efforts have been devoted aiming of the isolation of new metabo- lites from marine microorganisms. Cultures of the marine bacterial isolate Brevibacillus laterosporus PNG276 obtained from Papua New Guinea yielded a new lipopeptide named tauramamide (6), together with its methyl (7) and ethyl esters (8). Structures were elucidated by analysis of NMR and MS data and by chemical degradation as well as by total synthesis of tauramamide (6) and tauramamide ethyl ester (8). Com- pounds 6 and 8 showed potent [minimum inhibitory con- centration (MIC) values of 0.11 mM] and relatively selective activity against the important Gram-positive human pathogen Enterococcus sp. The ethyl ester (8) showed weaker activity against multidrug-resistant Sta- phylococcus aureus, but neither compound was apprecia- bly active against the yeast C. albicans. Tauramamide (6) is a new lipopeptide antibiotic that contains two D amino acids and is acylated at the N-terminus. Both structural features are hallmarks of non-ribosomal peptide synthase biosynthetic origin (Desjardine et al., 2007). A member of the new bacterial genus Marinispora (strain NPS008920) was isolated from a sediment sample collected in Cocos Lagoon, Guam. Chemical investigation of this strain afforded a series of novel 2-alkylidene-5- alkyl-4-oxazolidinones, lipoxazolidinone A (9), B (10) and C (11). Compounds 9���11 showed broad spectrum antimi- crobial activities similar to those of the commercial antibi- otic linezolid (Zyvox) (Barbachyn and Ford, 2003). Lipoxazolidinones A (9), B (10) and C (11) and the hydrolysis product 12 were screened against a panel of various Gram-positive and Gram-negative bacteria. Com- pound 9 showed broad spectrum activity, with MIC values ranging from 1.56 to 15.57 mM against Gram-positive bac- teria and 37.38 mM against two strains of Haemophilus influenzae. Compounds 10 and 11 showed also broad spectrum antibacterial activity, albeit with lesser overall potency than 9. In contrast, the hydrolysis product of 12 showed only weak activity against MSSA (methicillin- sensitive S. aureus), indicating the importance of an intact oxazolidinone ring system. While the oxazolidinone het- erocycle is a common structural motif shared by the lipoxazolidinones and linezolid, the compounds are clearly distinguished as 4- and 2-oxazolidinones, respec- tively, and each class is uniquely substituted. Thus, the 4-oxazolidinones offer a unique scaffold of compounds with antibiotic therapeutic potential (Macherla et al., 2007). The new marine actinomycete, NPS12745, was recently isolated and described from a marine sediment collected off the coast of San Diego, California. The analy- sis of the full-length 16S rRNA sequence indicated that NPS12745 is a novel strain of the recently described Marine Bioactive Compounds 545 �� 2010 The Authors Journal compilation �� 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 544���563

marine actinomycete genus Marinispora. Chemical inves- tigation of this novel strain yielded a series of new chlori- nated bisindole pyrroles, lynamicins A-E (13���17). The bisindole pyrrole derivatives are small molecules, and include chromopyrrolic acid which was previously isolated from Chromobacterium violaceum (Hoshino et al., 1993) as well as lycogarubins A-C known from the myxomycete Lycogala epidendrum (Fr��de et al., 1994 Hashimoto et al., 1994). Lynamicins A���E represent the first haloge- nated bisindole derivatives. The antibacterial activity of the compounds against a panel of Gram-positive and Gram-negative bacteria was studied and substances 14 and 15 were found to be active against S. aureus (MSSA, MRSA: methicillin resistant), Staphylococcus epidermidis and Enterococcus faecalis, suggesting potential for treat- ment of nosocomial infections (McArthur et al., 2008). Screening of 100 bacteria, which were isolated from intestinal tract of fish that landed on the Baluchistan coast that borders the Gulf of Karachi, Pakistan, afforded an isolate of Pseudomonas stutzeri (CMG 1030) that showed pronounced inhibitory activity against several pathogenic bacteria, including MRSA strains. Chemical investigation of the ethyl acetate extract yielded a new antibacterial metabolite named zafrin (4b-methyl-5,6,7,8���tetrahydro-1 (4b-H)���phenanthrenone) (18). Screening of zafrin indi- cated activity against a panel of important clinical and environmental microorganisms. The MIC of zafrin (235.85���589.62 mM) compared favourably with other novel antimicrobials such as 2,4-diacetylphloroglucinol (2.38���4.76 mM) (Isnansetyo et al., 2003). Interestingly, the killing rate of zafrin against Bacillus subtilis was faster than for ampicillin, vancomycin or tetracycline. Zafrin does not target the bacterial cell wall and its pattern of lysis resembles that of compounds such as nisin (14.91 mM) and Triton X-100, which disrupt the cell membrane. It was suggested that the mode of action of zafrin is via the disruption of the cytoplasmic membranes, since the mol- ecule is amphiphilic (Uzair et al., 2008). The extract of an Egyptian strain of Nocardia sp. ALAA 2000, which was isolated from the marine red alga Lau- renica spectabilis collected from Ras-Gharib coast of the Red Sea, Egypt, was found to be active against patho- genic microorganisms with MIC values ranging from 0.1 to 10 mg ml-1. Chemical and biological screening of the crude extract of this strain afforded the new bioac- tive compound ayamycin [1,1-dichloro-4-ethyl-5-(4-nitro- phenyl)-hexan-2-one] (19), which is structurally unique since it contains both chlorine and rarely observed nitro groups, beside being structurally related to compounds such as chrysophanol 8-methyl ether (20), asphodelin (21) and justicidin B (22). The isolated compounds were tested for their antimicrobial activities against Gram- positive and Gram-negative bacteria as well as against pathogenic fungi such as Candida albicans, Aspergillus niger and Botrytis fabae. The most active compound was the new ayamycin (19) with MIC values ranging from 0.31 to 1.57 mM (El-Gendy et al., 2008). From a marine sediment sample (collected near La Jolla, CA) at a depth of 51 m, the actinomycete strain CNQ-418 was isolated. This strain shared 89.1% 16S rNRA gene sequence similarity with its nearest neighbour Streptomyces sannurensis. The crude extract exhibited strong antibiotic activity. Two prominent products named marinopyrroles A (23) and B (24), were isolated and iden- tified as new secondary metabolites. X-ray analysis of marinopyrrole B showed that the natural product exists as an atropoenantiomer with the M-configuration. The newly isolated substances 23 and 24 displayed noteworthy activity against methicillin-resistant S. aureus, with an MIC90 of less than 2 mM. For each of the compounds, cytotoxicity against a human cancer cell line (HCT-116: colon carcinoma) was less pronounced (8.8 and 9.0 mM for compounds 23 and 24 respectively) (Hughes et al., 2008). The marine-derived fungus Nigrospora sp. was isolated from a sea fan that was collected near Similan Island, Thailand. It represented the first example of marine- derived Nigrospora sp., as Nigrospora species were up to now known only as plant endophytes. When grown in 500 ml Erlenmeyer flasks containing potato dextrose broth for 4 weeks, this strain produced four new metabo- lites named nigrospoxydons A���C (25���27) and nigrospo- rapyrone (28), together with nine known compounds. The crude ethyl acetate extract obtained from the culture broth showed antibacterial activity against standard S. aureus ATCC 25923 (SA) and methicillin-resistant S. aureus (MRSA), with MIC values of 64 and 128 mg ml-1 respec- tively. Only compound 25 and the known compound (+)- epoxydon (29) showed activity against both strains, while the remaining compounds were inactive. Compound 25 was more active than 29 against SA (MIC 152.38 mM), but was less active against MRSA (MIC 304.76 mM). Com- pound 29 gave an MIC value of 820.51 mM against both strains (Trisuwan et al., 2008). A marine Aspergillus species (family Trichocomaceae) was isolated from the surface of the marine brown alga Sargassum horneri collected at Gadeok Island, Busan, Korea. The fungal broth yielded a new polyoxygenated decalin derivative, dehydroxychlorofusarielin B (30), which was found to exhibit mild antibacterial activity against S. aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus with MIC values of 142.36 mM for all strains (Nguyen et al., 2007). A marine-derived Penicillium sp. PSU-F44 was isolated from the same sea fan species (Annella sp.). Fungi of the genus Penicillium are known to produce a large variety of compounds with a wide range of biological and pharmacological activities. The fungal broth yielded two 546 A. Debbab, A. H. Aly, W. H. Lin and P. Proksch �� 2010 The Authors Journal compilation �� 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 3, 544���563