Thinking big about small beings--the (yet) underdeveloped microbial natural products chemistry in Brazil.

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Thinking big about small beings – the (yet) underdeveloped microbial natural products chemistry in Brazil Laura P. Ioca, Pierre-Marie Allard and Roberto G. S. Berlinck ´

Covering: up to 2013 Natural products isolated from microorganisms from Brazil are comprehensively reviewed, including the microbial sources, bioactivities and biosynthesis. Analysis of trends related to the research on microbial natural products in Brazil is discussed in detail. An outlook to future perspectives of research in Brazil is presented considering the development of state-of-the-art strategies and Received 1st November 2013

approaches that have emerged during the last decade, aiming to investigate and better understand the microbial world and its chemical and biochemical capabilities. Finally, the importance of

DOI: 10.1039/c3np70112c www.rsc.org/npr

microbial natural products biodiscovery is discussed in the context of the BIOTA-FAPESP program in Brazil.

1 2

Introduction Microbial secondary metabolites isolated in Brazil: structures, biological activities and biosynthesis 2.1 Alkaloids 2.2 Peptides and peptide derivatives 2.3 Terpenes 2.4 Polyketides 2.5 Metabolites of mixed shikimate and terpene biosynthesis 2.6 Metabolites of mixed terpene and alkaloid biosynthesis 2.7 Metabolites of mixed polyketide and amino acid biosynthesis 2.8 Metabolites of mixed terpene and polyketide biosynthesis 2.9 Metabolites of mixed alkaloid, amino acid and polyketide biosynthesis 2.10 Metabolites of unknown biosynthetic origin 3 Current trends in microbial natural products chemistry in Brazil 4 Thinking big 5 Microbial diversity and biodiscovery, and science policy 6 Acknowledgements 7 References

1

Introduction

For centuries microorganisms have fascinated investigators who soon realized the importance of bacteria, fungi and Instituto de Qu´ımica de S˜ ao Carlos, Universidade de S˜ ao Paulo, CP 780, CEP 13560-970, S˜ ao Carlos, SP, Brasil. E-mail: [email protected]

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viruses for human, animal and plant health, not only detrimentally, but also as producing many useful biochemicals such as vitamins and antibiotics, as well as in promoting processes such as fermentation and organic matter transformation and decomposition, which could directly affect life. Currently there is a largely accepted view that microorganisms constitute, if not the most, certainly one of the most important forms of life to provide biotechnological tools to transform organic matter as well as in the production of useful chemicals and biochemicals. Such a view is not only justied by the incredible diversity and uniqueness of microbial secondary metabolism.1–4 Microorganisms are also versatile in allowing genetic manipulations aimed at changing the expression of secondary metabolism,5–9 in performing an array of large scale processes,10,11 in enabling access to its genome while using mining and cloning techniques,12–16 and in providing several technological and scientic tools consequential of the enhancement and improvement of microbial metabolic capabilities,17,18 leading to both new knowledge and technological innovation. A single most signicant example of microorganisms’ importance was the discovery of the polymerase chain reaction (PCR) enzyme from the bacterium Thermus aquaticus that allows the amplication of large amounts of DNA for genomic analyses for multiple purposes19 that has completely revolutionized biological science research and human society as a whole since the late 1980's. Considering the evident relevance of the research on microbial secondary metabolism during the last 60 years, it is

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rather surprising that the rst microbial natural product discovered by Brazilian researchers in Brazil was reported only in 2000.20 Since then the investigation of microorganism metabolites has experienced a slow, but continuous, growth in Brazil, although still somewhat erratic. Analysis of literature data collected for this review aims to show how the research on microbial natural products has developed during the last decade in Brazil and clarify the reason why it has been so slow to develop. To the best of our knowledge, the research on microbial metabolites in Brazil has been only partially addressed in the reviews “Current Status, Challenges and Trends on Natural Products in Brazil”,21 “Challenges and Rewards of Research in Marine Natural Products Chemistry in Brazil”22 and “Chemical biology: a modern strategy for the natural products research”,23 which mention only selected examples of metabolites isolated from microbial sources in Brazil.

The purpose of the present review is to provide a comprehensive state-of-the-art discussion of the research on microbial metabolites developed by Brazilian researchers, including the isolation, biological activities and biosynthesis of such compounds. Results from the investigation of crude extracts obtained from microbial growth media, or dealing with microbial biotransformations or biocatalytic experiments, are not herein included. The organization of the topics follows the biosynthetic origin of the isolated and identied metabolites, in increasing complexity, and include the mention of the respective biological activities, if any. The biosynthesis of a few microbial metabolites is included. Current trends on the research of microbial metabolites by Brazilian researchers are also discussed and an outlook and perspectives have been included aiming to stimulate the debate on how the research on microbial natural products in Brazil should be envisaged in the next years.

Laura Pavan Ióca graduated in Physical and Biomolecular Sciences at the Instituto de F´ısica de S˜ ao Carlos, Universidade de S˜ ao Paulo, in 2013. During her undergraduate studies she was a six month visiting student at the Scripps Institution of Oceanography under the supervision of Paul R. Jensen. Currently she is an MSc student at the Instituto de Qu´ımica de S˜ ao Carlos, Universidade de S˜ ao Paulo, under the supervision of Roberto Berlinck. Laura's research interest includes the investigation of micro-organisms metabolism and biosynthesis of secondary metabolites.

2 Microbial secondary metabolites isolated in Brazil: structures, biological activities and biosynthesis

Pierre-Marie Allard graduated in Pharmacy at the University of Rennes in 2007. He did his PhD under the supervision of Drs. Françoise Guéritte and Marc Litaudon at the Institut de Chimie des Substances Naturelles, Gifsur-Yvette, France, in 2011. During 2012 and 2013 Dr Allard was a post-doctoral fellow at the Instituto de Qu´ımica de S˜ ao Carlos, Universidade de S˜ ao Paulo, under the supervision of Roberto Berlinck, investigating secondary metabolites from marinederived fungi. Currently he is a postdoctoral researcher at the University of Geneva with Prof Jean-Luc Wolfender. His research interests focus on the metabolomics of fungal natural products.


2.1

Alkaloids

Alkaloids have been seldom isolated from microbial culture media by Brazilian researchers. Certainly this is not a matter of choice, but rather a consequence of strain selection towards the isolation of secondary metabolites. The culture of Eupenicillium sp., an endophytic fungus found within the leaves of Murraya paniculata, yielded four novel alkaloids named as alantrypinene B (1), alantryleunone (2), alantryphenone (3) and alanditrypinone (4). Structures 1–4 have been established by analysis of spectroscopic data, while the

Roberto G. S. Berlinck joined the Instituto de Quimica de S˜ ao Carlos, Universidade de S˜ ao Paulo in 1993, as assistant professor. He was appointed as associate professor in 2001 and full professor in 2010. During 1997 and 1998 he spent a six month sabbatical with Professor Raymond J. Andersen (UBC). Since 1994 his research interests are related to the isolation, identication, biological activities and biosynthesis of natural products from marine invertebrates and microorganisms. Since 2009 he is member of the BIOTA-FAPESP steering committee.

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relative stereochemistry was established by analysis of NOE NMR spectra.24

The UPLC-ESI-MS/MS analysis of the culture medium extract produced by Streptomyces sp., an actinomycete associated with the ascidian Eudistoma vannamei colleted at Taiba Beach located on the northeastern coast of Brazil, indicated the presence of the well-known alkaloid staurosporine (5), previously also isolated from the same ascidian. The cytotoxic activity of a purchased sample of 5 was evaluated on tumor cell lines HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (CNS glioblastoma). The observed IC50 were 0.04, 0.1 and 0.27 mg mL1, respectively.25 Bioassay-guided fractionation of the organic extract from the culture medium of the bacterium Pseudoalteromonas sp. isolated from marine sediments at the northeastern coast of Brazil, led to the isolation of prodigiosin (6). Prodigiosin showed cytotoxic activity against the tumor cells HCT-8 (colon carcinoma), HL-60 (leukemia), MDA-MB-435 (melanoma) and SF-295 (CNS glioblastoma), with IC50 at 0.05, 0.06, 0.19 and 0.06 mg mL1, respectively. Prodigiosin (6) was also tested on ErbB-2 overexpressing cell. The ErbB-2 gene has been shown to regulate the development of breast cancer cells. The cytotoxic activity of prodigiosin against HB4a-C3.6 (moderate expression), HB4a-C5.3 (high) and HB4a parental showed IC50 at 0.04, 0.26 and 4.6 mg mL1, respectively.26

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2.2

Peptides and peptide derivatives

The large majority of peptide and peptide derivatives isolated by Brazilian researchers have been obtained from fungi culture media. The dipeptides leucyl-4-hydroxyproline (7) and 8-amino[1,4]diazonane-2,5-dione (8) were produced by Streptomyces acrimycini along with N-acetyltyramine. This actinomycete was isolated from samples of marine sediments obtained 12 to 15 m deep at the North coastline of S˜ ao Paulo State. Structures of compounds 7 and 8 were identied by analysis of spectroscopic data.27 Fumiquinozoline F (9) was isolated from the CHCl3 extract of the culture medium of Penicillium corylophilum isolated from a soil sample from S˜ ao Carlos City. Compound 9 displayed antibacterial activity against Micrococcus luteus and Staphylococcus aureus at 99 and 137 mg mL1, respectively.28

Seven diketopiperazines, cyclo-(L)-Pro-Gly (10), cyclo-(L)-Pro(L)-Val (11), cyclo-(L)-Pro-(L)-Leu (12), cyclo-(L)-4-OH-Pro-(L)-Leu (13), cyclo-(L)-Pro-(L)-Pro (14), cyclo-(L)-Pro-(L)-Phe (15) and cyclo(L)-4-OH-Pro-(L)-Phe (16), were isolated from the fermentation broth of Aspergillus fumigatus, a fungus obtained from a soil sample collected in Pantanal, Mato Grosso do Sul State. Compounds 10–16 were inactive in antimicrobial assays against S. aureus and M. luteus. The absolute conguration of diketopiperazines 10–16 was determined using a modied version of Marfey's method.29 Compound 11 was also produced in the culture medium of Penicillium sp. isolated from leaves of Alibertia macrophylla. Its antifungal activity was evaluated against Cladosporium cladosporioides and C. sphaerospermum and in acetylcholinesterase (AChE) inhibitory activity. Compound 11 showed a minimum amount for inhibition of fungi growth at 50 mg for both Cladosporium strains, and presented a minimal inhibitory activity at 10 mg, while the positive control galanthamine was active at 1 mg, as inhibitors of acetylcholinesterase (AChE).30 The fungus Papulaspora immersa was isolated from the roots and leaves of Smallanthus sonchifolius. Aer culture in rice, it produced tyrosol (17).31 Compound 17 was also obtained from a culture of Diaporthe helianthi, an endophytic fungus of Luehea duvaricata,32 as well as from Glomerella cigulata associated with Vigueira arenaria.33


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with the experimentally measured ECD curves also allowed the determination of the absolute conguration of 24. Compounds 18, 19 and 21–24 were unable to promote the survival of the nematode Caenorhabditis elegans infected with the human opportunistic pathogen Enterococcus faecalis.34 The known brasiliamides A (25) and B (26) and the new brasiliamide F (27) have been isolated from the growth medium of Penicillium brasilianum, an endophytic fungus found in the root bark of Melia azedarach. The bacteriostatic activity of brasilamide (28) was evaluated against Bacillus subtilis and showed MIC at 250 mg mL1.35

Three new diketopiperazines 18, 20 and 24 were isolated from two endophytic fungi obtained from distinct Viguiera species. From V. robusta, Colletotrichum gloeosporioides yielded colletopiperazine (18) and bisdethiobis(methylthio)gliotoxin (19), while Penicillium crustosum produced the diketopiperazines 21 and 22 as well as fusaperazine E (24). Fusaperazine C (20) and the diketopiperazine 23 were isolated from the culture of Fusarium sp. isolated from V. arenaria. While the relative conguration of 21–24 was assigned by interpretation of NOE data, the absolute conguration of 19 was determined by comparison of its ECD data with those reported in the literature. Theoretical calculations of the ECD data and comparison

Two new destruxins, [b-Me-Pro] destruxin E chlorohydrin (28) and pseudodestruxin C (29), along with the known destruxin E chlorohydrin (30), [Phe,3 N-Me-Val5] destruxin B (31), roseotoxin B (32), roseocardin (33), isariin B (34)36,37 and isariin (35),37 were produced by a marine-derived fungus Beauveria felina obtained from the seaweed Caulerpa sp. The major compounds 29, 31 and 32 were evaluated against Mycobacterium tuberculosis H37 Rv and in cytotoxic assays. Compound 32 showed good cytotoxic activity against SF-295 (human CNS), MDA-MB435 (human breast), HCT8 (colon carcinoma) and HL60 (leukemia) cancer cells lines, with IC50 at 1.09, 1.30, 0.90 and 0.14 mg mL1 respectively.36 The absolute stereochemistry of the 5-chloro-2,4-dihydroxypentanoic acid in [b-Me-Pro] destruxin E chlorohydrin (28) has been assigned as 2(R),4(S) using the J-based conguration method in conjunction with a variable temperature NMR analysis of the corresponding R-MPA ester derivative. The absolute conguration of the common aminoacid residues in 28 and 29 was assigned using Marfey's method. Beauvericin (36) has been isolated from the extract of liquid culture of Fusarium oxysporum, an endophytic fungus of Smallanthus sonchifolius. Beauvericin showed cytotoxic activity at 3.02, 3.17 and 2.39 mg mL1 against the tumor cell lines HCT8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (CNS glioblastoma) respectively.38


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(44), 3,12-dihydroxycadalene (45) and 3,11,12-trihydroxycadalene (46). The relative conguration of 42–46 was assigned by analysis of NOESY experiments. Compounds 42–46 were evaluated as antifungals against C. cladosporioides and C. sphaerospermum. Compounds 43 and 46 showed potent activity, with a detection limit of the antifungal activity at 1 mg, almost the same of the positive control nystatin. The same compounds 42–46 were also tested for cytotoxic activity against HeLa (human cervical cancer) cell line. Compound 45 was the most active, with an IC50 at 20 mmol L1, while 44 and 46 displayed cytotoxicity at 100 and 110 mmol L1, respectively.40 Pumilacidins A (37), B (38), C (39), D (40) and E (41) were identied in the Bacillus pumilus culture medium organic extract, an endophytic bacterium of cassava collected at the Amazon forest.39

2.3

Terpenes

Terpenes constitute the second largest class of metabolites isolated by Brazilian researchers from cultures of microbial strains, particularly fungi. The culture medium extract of Phomopis cassiae, an endophytic fungus associated with Cassia spectabilis, showed moderate antifungal activity. Five new cadinane sesquiterpenoids were subsequently isolated from this extract: ()-(7S*,9S*,10S*)-3,9,12-trihydroxycalamenene (42), and its diastereomer (+)-(7S*,9R*,10S*)-3,9,12-trihydroxycalamenene (43), (+)-(7S*,10S*)-3,12-dihydroxycalamenene

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Two new diastereoisomeric compounds, ()-(7R*,9S*,10R*)3,9-dihydroxycalamenene (47) and ()-(7R*,9R*,10R*)-3,9-dihydroxycalamenene (48) were isolated along with the known 3hydroxycalamen-8-one (49) and aristelegone A (50) from a culture of Phomopsis cassia obtained from leaves of Cassia spectabilis. Compounds 47–50 were equally active as antifungal agents against C. cladosporioides and C. sphaerospermum and as inhibitors of AChE, with minimum inhibitory concentration at 5 mg for the fungi and 3 mg for the enzyme.66

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displayed potent cytotoxic activity with IC50 at 0.05, 0.20, 0.16 and 0.09 mg mL1 against each of the cell lines, respectively. Compound 56 was active at 6.24, 14.11, 17.03 and 5.29 mg mL1 and 59 was active at 6.26, 1.44, 2.15 and 0.70 mg mL1, respectively.31,42 The biosynthesis of aphidicolin (54) was investigated using feeding of [1-13C]-D-glucose into the culture medium of Nigrospora sphaerica. The optimal production of 54 was observed at the 8th day of growth, when the medium was harvested aer feeding with the labeled precursor at day one. 13C NMR analysis of puried 54 indicated that its biosynthesis proceed via the transformation of glucose into mevalonate, which was shown to be the precursor of the whole aphidicolin carbon skeleton.44

The fungus Lentinus strigosus obtained from a culture collection was selected aer a screening of fungal extracts as inhibitors of trypanothione reductase (TryR). Fractionation of the L. strigosus culture medium extract led to the isolation of two triquinane sesquiterpenoids, hypnophilin (51) and dihydrohypnophilin (52). Compound 51 was active, with an IC50 at 0.8 mM. The same metabolite 51 displayed trypanomicidal and cytotoxic activities against Trypanosoma cruzi amastigotes and inhibited human lymphocyte proliferation at 2.5 and 8.9 mM, respectively. Dihydrohypnophilin (52) was devoid of any bioactivity.41

Nigrospora sphaerica, Phoma betae42 and Papulospora immersa31 were found in association with Smallanthus sonchifolius. When cultivated in solid media, N. sphaerica produced pimara-7,15-dien-3b-ol (53), aphidicolin (54) and ergosterol peroxide (55), the last one being also produced by Penicillium janthinellum43 and Phoma betae.42 The growth in liquid culture of P. betae yielded (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (56). Fractionation of the extract from P. immersa liquid culture led to the isolation of 56, (24R)-stigmast-4-en-3-one (57), 24-methylenecycloartan-3b-ol (58) and of the new (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (59). The absolute conguration of 56–58 was assigned by comparison with data reported in the literature. The 24R conguration within 59 was assumed as identical to the conguration of the same stereogenic center in 56.31 All compounds were evaluated on cytotoxic assays against HCT-8 (colon carcinoma), MDA-MB435 (human breast), SF-295 (CNS glioblastoma) and HL-60 (leukemia) cancer cell lines. Compounds 53, 55, 57 and 58 were inactive. Compound 54


A strain of Penicillium brasilianum, obtained from a soil sample collected at the Cerra do Sip´ o National Park, Minas Gerais State, produced austin (60) and dehydroaustin (61). Both 60 and 61 were inactive as antibacterial agents as well as inhibitors of AChE. Structures of both 60 and 61 were established by analysis of spectroscopic data.45

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Two new sesquiterpenes, 9,15-dihydroxypresilphiperfolan-4oic acid (62) and 15-acetoxy-9-hydroxypresilphiperfolan-4-oic acid (63), were isolated together with the known phaseolinone (64) and phomenone (65) from the extract of Xylaria sp. culture medium, obtained from the leaves of Piper aduncum. The relative stereochemistry of 63 and 64 was assigned by analysis of NMR NOE data. Diazomethane esterication of 63 allowed the observation of additional NOE signals necessary for the full relative stereochemical assignment. Relative conguration of 65 was determined by comparison with literature data. The new compounds 62 and 63 were inactive as antifungal agents against C. cladosporioides and C. sphaerospermun. The same compounds were tested against CHO cell line (Chinese hamster ovary). Phaseolinone (64) displayed cytotoxicity with IC50 at 200 mM, while phomenone (65) displayed antifungal activity at 10 mg.46

Two new sesquiterpenes, cupressolide A (66) and B (67), along with the known 68 and 69, were produced by Xylariaceous sp., an endophytic fungus of Cupressus lusitanica leaves. The relative conguration of 66 and 67 was established by analysis of NOE data.47 Deoxycholic acid (70) and cholic acid (71) were produced by Pseudoalteromonas sp., a bacterium strain isolated from marine sediments collected at the Northeastern Brazilian coastline.26

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ovary) cancer cell lines. Compound 72 also exhibited weak antifungal activity with detection limit concentration at 25 mg.48 A new guaiane mannoside, (1R,4S,5S,7R,10R)-10-hydroxyguaianol 10-O-b-mannopyranoside (73), was puried from the culture medium crude extract of an Eutypa-like fungus obtained from the stems of Murraya paniculata. The relative stereochemistry of 73 was determined by analysis of NOE data. The absolute conguration of 73 was inferred from the repeated natural occurrence of 7R congured guaiane-type sesquiterpenoids.49

A species of Penicillium strain was obtained from the root bark of Melia azedarach, and produced the new meroterpenes preaustinoids A (74), B (75),50 A1 (76), A2 (77), B1 (78)51 as well as austinoneol A (79), neoaustin (80), 7-b-acetoxy-dehydroaustin (81) and dehydroaustin (61).52 The relative conguration of 74– 78 was established by analysis of NOESY experiments. Compound 74 exhibited bactericidal effect at 250 mg mL1

Periconicin B (72) was isolated from a culture of the endophytic fungus Periconia atropurpurea obtained from Xylopia aromatica and showed cytotoxic activity with IC50 at 8 mM against HeLa (human cervix carcinoma) and CHO (Chinese hamster

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against Escherichia coli, Pseudomonas aeruginosa and Bacillus sp., preaustinoid B (75) displayed bacteriostatic effect against E. coli, Staphylococcus aureus, P. aeruginosa and Bacillus sp. at 250 mg mL1, compounds 80 and 81 showed the same minimum inhibitory concentration against E. coli, while 61 was inactive and 76–79 were not tested.50,52 Pestalotiopis sp. was isolated as an endophytic fungus of the trunk bark of Pinus taeda, and yielded three new highly oxygenated caryophyllene sesquiterpenes, taedolidol (82), 6epitaedolidol (83), and pestalotiopsolide A (84). Geometry optimization studies and NOE experiments allowed the assignment of the relative stereochemistry of 82–84.53 Ergosterol 5,8-endo-peroxide (55), ergosterol (85) and the new neocyclocitrinol (86) have been isolated from Penicillium janthinellum associated with Melia azedarach fruits. The structures of 55, 85 and 86 were established by analysis of NMR and MS data. Neocyclocitrinol (86) was isolated as a mixture of diastereoisomers. The relative conguration of the modied steroid skeleton was assigned by analysis of NOE experiments.43

The liquid culture of Penicillium sp., an endophyte of Mauritia exuosa, yielded ergosterol (85), ergosterol peroxide (55), brassicasterol (87) and cerevisterol (88). Compounds 55, 85, 87 and 88 were inactive against S. aureus, M. luteus and E. coli.54 A new triterpene, pisosteral (89), and four known ones, pisosterol (90), 3-epi-pisosterol (91), an isomer of pisosterol (22hydroxy, 23-acetyl) (92) and 93, have been isolated from the fruiting bodies of Pisolithus tinctorius, associated with Eucalyptus sp. The same fungus was obtained from Pinus taeda, and yielded four known triterpenes aer cultivation, lanosterol (94), agnosterol (95), 96 and 97. The relative stereochemistry of 89 was established by examination of NOE data.55


2.4

Polyketides

Polyketides constitute the largest group of secondary metabolites isolated by Brazilian researchers from both fungi and bacteria cultures. Penicillium simplicissimum isolated from a soil sample of the Brazilian Cerrado produced penicillic acid (98).56 The same compound was isolated from P. brasilianum obtained from a soil sample collected at the Serra do Cip´ o National Park, Minas Gerais State,45 as well as from Aspergillus sp. isolated from the ascidian Eudistoma vannamei.57 Penicillic acid (98) showed MIC at 512 mg mL1 against Staphylococcus aureus and S. typhimurium, and at 256 mg mL1 against Listeria

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monocytogenes, Bacillus cereus, Escherichia coli and Citrobacter freudii. It was also active in the acetylcholinesterase (AChE) assay and exhibited cytotoxic activity with IC50 at 4.46 and 8.76 mg mL1 against MDA-MB-435 (melanoma) and HCT-8 (colon cancer) tumor cell lines, respectively.45,57 Agrocybin (99) was obtained from a culture of Agrocybe perfecta, obtained from the basidiomycete culture collection of the Instituto de Botˆ anica, S˜ ao Paulo State. This compound showed IC50 at 2 mM in the recombinant trypanothione reductase enzyme (TryR) assay and killed 60% of the trypomastigote form of Trypanosoma cruzi at 680 mM. Compound 99 showed 50% inhibitory activity of lymphocyte peripheral mononuclear cells (PBMC) at 3.4 mM. The same compound displayed weak cytotoxic activity against the tumor cells lines UACC-62 (melanoma), MCF-7 (mammary cancer) and TK-10 (kidney), with IC50 at 28.5, 21.0 and 47.0 mM, respectively.58

Bioassay-guided fractionation of the extract obtained from the culture medium of an Aspergillus fumigatus soil strain in the presence of autoclaved growth media of S. aureus, S. epidermidis, Micrococcus luteus, P. aeruginosa, E. coli and B. subtilis led to the isolation of two aromatic compounds that inhibited the growth of S. aureus, M. luteus and Candida albicans. The antibiotic compounds were identied as 3,4-dimethoxyphenol (100) and 1,3,5-trimethoxybenzene (101). Although remarkable changes were observed in the chromatographic prole of the crude extract produced by A. fumigatus aer addition of sterilized media where pathogenic strains have been grown, only compounds 100 and 101 were isolated and identied.59

1-O-a-D-Glucopyranoside (102) was obtained from the liquid culture of Penicillium sp. an endophytic fungus of the roots of Mauritia exuosa and was inactive on antibacterial assay against S. auerus, M. luteus and E. coli.54 Two new aromatic polyketides, ethyl 2,4-dihydroxy-5,6dimethylbenzoate (103) and phomopsilactone (104) were produced along with the known compound 2-hydroxyphenylacetic acid (105) by Phomopsis cassiae, an endophyte of Cassia spectabilis. Compounds 103–105 were tested against C. cladosporioides and C. sphaerospermum. Both 103 and 104 were active at 1 mg. In a cytotoxic assay against the tumor cell line HeLa (human cervical cancer), 104 showed weak cytotoxic activity at 200 mmol L1 and 105 was active at 10 mmol L1.60

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The strain Glomerella cingulata, found in association with Viguiera arenaria, produced nectriapyrone (106). This compound showed very weak cytotoxic activity against tumor and normal cell lines JURKAT (leukemia) and PMBC (peripheral blood mononuclear cells), with IC50 at 476.4, 1463 and 2667 mg mL1, respectively.33 The fungus Penicillium paxilli was isolated from the marine sponge Mycale angulosa. The known pyrenocines B (107) and A (108) and the new pyrenocine J (109) have been isolated from P. paxilli culture medium extract, which displayed cytotoxic activity against the tumor cell lines MDA-MB435, CNS295 and HL60, and identied by analysis of spectroscopic data.61

Panepoxydone (110) was produced by a fungal strain obtained from a culture collection Lentinus strigosus in liquid culture medium. Neopanepoxydol (111) also was isolated and showed inhibitory activity of the trypanothione reductase enzyme, of Trypanosoma cruzi intracellular amastigotes and of human lymphocyte proliferation, with IC50 at 38.9, 8.7 and 1.3 mM, respectively.41 The Eutypa-like fungus isolated from the stems of Murraya paniculata produced 3-hydroxy-5-phenylmethyl-(3S,5R)-tetrahydrofuran-2-one (112). The absolute conguration of 112 was assigned as (3S,5R) aer comparison of specic optical rotation with the same compound prepared by synthesis.49 The fungus Epicoccum nigrum, found in association with Saccharum offinarum (sugarcane), yielded 4,5-dimethylresorcinol (113), mellein (114), 5-hydroxymellein (115) and the novel carbon skeleton-bearing epicolactone (116). Epicolactone (116) was isolated as a racemic mixture. Its structure and relative conguration was assigned by X-ray crystallography analysis.62


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Dimethyl terephthalate (123) and 8-hydroxy-6-methoxy-3methylisocoumarin (124) were isolated from cultures of Phoma betae, an endophyte of Smallanthus sonchifolius. Compounds 123 and 124 were inactive in cytotoxic assays against the tumor cell lines HCT-8 (colon carcinoma), MDA-MB435 (human breast), SF-295 (CNS glioblastoma) and HL-60 (leukemia).42 The endophytic fungus Colletotrichum gloeosporioides was found in association with the leaves of Cryptocarya mandioccana. Fractionation of its culture medium extract led to the isolation of ()-cis-4-hydroxy-6-deoxyscytalone (125) and (4R)-4,8-dihydroxy-a-tetralone (126). Both compounds were tested as antifungal agents, and showed a minimum amount to inhibit the growth of C. cladosporioides and C. sphaerospermum of 5 mg.64 A strain of Aspergillus sp. obtained from the ascidian Eudistoma vannamei produced (R)-mellein (117), cis-4-hydroxymellein (118) and trans-4-hydroxymellein (119). Compounds 117–119 were devoid of cytotoxic activity against MDA-MD-435 (melanoma) and HCT-8 (colon cancer).57 No evidence has been provided whether (R)-mellein (117) isolated from cultures of Aspergillus sp.57 is identical or not to mellein (114) isolated from cultures of Epicoccum nigrum.62

Compounds 115 and 119 have also been isolated from a culture of Penicillium sp. together with 8-methoxymellein (120) and orcinol (121). This fungus was isolated from Alibertia macrophylla. Each compound was tested as an antifungal agent against C. cladosporioides and C. sphaerospermum. The minimum amount to inhibit the growth of both Cladosporium strains was 50 mg for 115, while 119 and 121 showed the same results with 5 and 10 mg each. The amount of 114 to inhibit both Cladosporium strains was 10 and 25 mg, respectively. Neither 115 or 119–121 were active as inhibitors of AChE.30 The fungus Mycoleptodiscus indicus, obtained as an endophyte of Borreria verticillata, yielded eugenitin (122). Compound 122 was tested in an Aspergillus niveus glycomylase activation assay and it doubled the enzyme hydrolytic activity at 5 mM.63


The endophytic fungus Curvularia sp., obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado, produced two new compounds 2,3-dihydro-2-methylbenzopyran-4,5-diol (127) and (20 S)-2-(propan-20 -ol)-5-hydroxybenzopyran-4-one (128), reported together with the known (2R)2,3-dihydro-2-methyl-5-methoxy-benzopyran-4-one (129) and 2methyl-5-methoxy-benzopyran-4-one (130). The S absolute conguration at the stereogenic center of 128 was assigned by optical rotation comparison with literature data of an analogous compound. Compounds 127–130 were tested against C. cladosporioides and C. sphaerospermum, as well as in cytotoxicity tests against HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cancer cell lines. Compounds 128 and 130 displayed weak antifungal activity, with detection inhibitory limit at 10 mg. Compound 128 increased the proliferation of HeLa and CHO cell lines at 70 and 25%, respectively.65

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Papulaspora immersa was obtained from roots and leaves of Samallanthus sonchifolius, and yielded two new compounds, 2,3epoxy-1,2,3,4-tetrahydronaphthalene-c1-c-4,8-triol (131) and papulasporin (132), along with the known 5-hydroxymellein (115), (3R,4R)-4-hydroxymellein (133), 6,8-dihydroxy-3-methylisocoumarin (134), 4,8-dihydroxy-a-tetralone (135), naphthalene-1,8-diol (136), 6,7,8-trihydroxy-3-methylisocoumarin (137) and 7-hydroxy-2,5-dimethylchromone (138). The relative conguration of 131 was established by analysis of NOE experiments.31

Norliquexanthone (145) was isolated from a culture of Penicillium raistrikii, isolated from the marine sponge Axinella cf. corrugata, while (S)-8-methoxy-3,5-dimethylisochroman-6-ol (146) was isolated from the culture medium of P. steckii obtained from a marine alga of the genus Sargassum. The structure of 146 could be distinguished from that of its isomer 6-methoxy-3,7dimethylisochroman-8-ol (147) by detailed analysis of spectroscopic data. Penicillium sp. isolated from the ascidian Didemnum granulatum yielded 13-desoxy-phomenone (148).61 The fungus P. citrinum was obtained from another seaweed, Caulerpa sp., and produced citrinin (149) in liquid culture media.69

A new metabolite named terreinol (139) and the known terrein (140) and terreic acid (141), were isolated from the EtOAc extract of Aspergillus terreus culture medium, a strain isolated from the Brazilian Atlantic Rain Forest. The biosynthetic origin of terreinol from acetate was established using feeding experiments with [1-13C]-D-glucose.67 This was the rst biosynthesis investigation of a microbial metabolite performed in Brazil.

The organic extract of Penicillium janthinellum culture medium, an endophyte of Melia azedarach fruits, yielded the well-known mycotoxin citrinin (149) along with emodin (150),

The polyketides 2-hexyl-3-methyl-butanodioic acid (142), griseofulvin (143) and 7-deschloro-griseofulvin (144) were produced by Xylaria sp., an endophytic fungus associated with Palicourea marcgravii. All compounds were evaluated as antifungal agents. Compound 142 was the only active, with MIC at 10 mg mL1 against C. cladosporioides and C. sphaerospermum.68

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citreorosein (151) and the new modied anthraquinone, janthinone (152). Compounds 149–152 were evaluated as antibacterial agents against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. None of 149–152 were active against E. coli. Compound 149 displayed bactericidal effect at 125 mg mL1 against P. aeruginosa and bacteriostatic effect at 31.25 mg mL1 against B. subtilis. Emodin (150) showed bactericidal effect at 62.5 and 250 mg mL1 against P. aeruginosa and B. subtilis, respectively.70 A strain of Penicillium was isolated from the same plant, and produced 138 and fusarindin (153), while P. herquei produced 150–152 together with dihydrocitrinone (154) and citrinin H1 (155). Citrinin H1 (155) was more active than 149 against E. coli and B. subtilis, with bactericidal effect at 15.63 and 125 mg mL1, respectively.71 Penicillium janthinellum from Melia azedarach fruits produced citrinin (149) and a citrinin dimer, dicitrinol (156), which showed less effective bactericidal activity than citrinin against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis.72 The known cytosporone J (157) and the new cytosporones O (158), P (159) and Q (160) were obtained from the solid medium culture of Cytospora sp. found in association with the mistletoe of Phoradendron perrottetii.73

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A strain of a Penicillium fungus was isolated from a Brazilian Cerrado soil sample and produced three new compounds: methyl 6-acetyl-4-methoxy-5,7,8-trihydroxyphthalene-2-carboxylate (163), methyl 6-acetyl-4-methoxy-7,8-dihydroxyphthalene2-carboxylate (164) and methyl 6-acetyl-4-methoxy-5,8-dihydroxyphthalene-2-carboxylate (165). Compound 163 displayed antifungal activity against Candida albicans with MIC at 32 mg mL1, Bacillus cereus and Listeria monocitogenes at 64 mg mL1 and against Staphylococcus pyogenes, Listeria sp. and S. typhimurium at 128 mg mL1.74 Alternaria sp. isolated from Trixis vauthieri produced altenusin (166), alternariol (167) and alternariol methyl ether (168). Altenusin (166) inhibited trypanothione reductase enzyme with IC50 at 4.3 mM.75

The fungus Phoma sorghina isolated from the leaves of the medicinal plant Tithonia diversifolia, produced the new dendryol E (169) and F (170), as well as 1,7-dihydroxy-3-hydroxymethyl-9,10-anthraquinone (171), along with the known 1,7dihydroxy-3-methyl-9,10-anthraquinone (172), phomarin (173) and pachybasin (174). The relative stereochemistry of dendryols E (169) and F (170) was established by analysis of NOE data.76

The new 2,4-dihydroxy-6-[(10 E,30 E)-penta-10 ,30 -dienyl]-benzaldehyde (161) and 6,8-dimethoxy-3-(20 -oxo-propyl)-coumarin (162) have been isolated from Periconia atropurpurea, an endophytic fungus obtained from leaves of Xylopia aromatic. Polyketides 161 and 162 were tested against HeLa (cervix carcinoma) and CHO (Chinese hamster ovary) cell lines. Compound 161 increased the proliferation of HeLa cells to 37% at 2 mM and CHO cells to 38% at 20 mM. Compound 162 was not active.48


Penicillium sclerotiorum obtained from a Brazilian Cerrado soil sample56,77 produced sclerotiorin (175) and isochromophilone IV (176). Sclerotiorin (175) displayed bacteriostatic and fungistatic activity against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Streptococcus pyogenes and Candida albicans at the same amount against all strains, 128 mg. Compound 176 displayed bacteriostatic and fungistatic effect at 64 mg against the same strains, except S. aureus, for which the bacteriostatic activity was weaker (128 mg).77

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Koninginin A (177), E (178) and F (179) were produced by Trichoderma koningii, an endophyte of Strychnos cogens. Compounds 177–179 were tested as inhibitors of the edemainducing, myotoxic and enzymatic activities of the venom of the snake Bothrops jararacussu and as inhibitors in an enzymatic assay against a homolog form of phospholipases A2 (bjPLA2 group IIB) and human secreted PLA2 protein fusion (hsPLA2 – group IIA). Compounds 178 and 179 were more active as inhibitors of group IIB than of IIA, while 177 was completely inactive.78

The fungus Curvularia senegalensis was obtained from a soil sample of the Brazilian Cerrado. This strain produced an oil rich in phthalates, seven of which (180–186) have been identied by infrared and mass spectrometry analyses.79 Roridin A (187) was isolated from cultures of Trichoderma sp. obtained

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from the sponge Mycale angulosa and identied by analysis of spectroscopic data.61 The major compound of the lichen Cladina kalbii, collected at Ibabaina county, Sergipe State, was isolated and identied as atronorin (188). It inhibited dose-dependently inammatory processes in vivo on mice at 200 and 400 mg kg1 and signicantly reduced abdominal writhing by 52.6 and 61.3% at the same dosages when compared with acetic acid-induced effects.80 Different species of lichens collected at Mato Grosso do Sul State have been chemically investigated. Parmotrema tinctorum yielded atranorin (188), ethyl orsellinate (189)81 and lecanoric acid (190).81,82 Atranorin (190) and protocetraric acid (191) have been isolated from P. dilatatum.82 Salazinic acid (192) and lichexanthone (193) were obtained from P. lichexanthonicum.81 Salazinic acid (192) was also obtained from Rimelia cetrata.82 The organic extract of the lichen Pseudoparmelia sphaerospora yielded hypostictic acid (194). Norstitic acid (195) and usnic acid (196) were obtained from the organic extract of Ramalina sp.81 Usnea meridionalis gave (+)-(12R)-usnic acid (197).82 Usnic acid (196) was also isolated from Usnea subcavata together with diffrataic acid (198).81,82 Perlatolic acid (199) was obtained from the organic extract of Cladina confusa found in a decoration shop.83,84 Fumarprotocetraric acid (200) was isolated from Cladonia verticillaris.82 Compounds 188–200 were tested in different assays. Compounds 192, 19782 and 19984 showed potential immunological modulating activities on the release of NO by 197 and of H2O2 by 192 and 199, in culture of mice peritoneal macrophages. Compounds 188 and 190 were tested as antioxidants and showed IC50 at 167.25 and 42.87 mM, respectively.85 These same compounds were also evaluated against the tumor cell lines HEp2 (human cervical carcinoma, derivative of HeLa), MCF7 (human breast cancer), 786-0 (renal carcinoma), B16–F10 (melanoma) and Vero (normal cell). Compound 191 showed IC50 > 50 mg mL1 for all cell lines while 190 was active at 31.2, 70.3, 47.3, 64.8 and 28.1 mg mL1, respectively.86 The lethal activity of 188 and 199 was evaluated against the brine shrimp Artemia salina, and showed LD50 at 24.1 mM112 and 495 mM, respectively.87 Compound 199 showed antifungal activity with MIC at 10 mg against Cladosporium sphaerospermum and antibacterial activity against E. coli and S. aureus with a 10 mm zone inhibition.83 Atranorin (188), ethyl orsellinate (189), lecanoric acid (191), salazinic acid (192), lichexanthone (193), norstitic acid (195), usnic acid (196) and diffrataic acid (198) were also evaluated against Mycobacterium tuberculosis.81 Diffrataic acid (198) was the most active one, with MIC at 41.7 mg. Compounds 188, 191, 193, 197, 198 and 199, along with divaricatic acid (201) and psoromic acid (202), were tested against UACC-62 and B16–F10 melanoma cells.88 The acids 191, 199 and 201 showed the most potent cytotoxic activity against UACC-62 melanoma cells, with growth inhibitory concentrations (GI50) at 0.52, 2.7 and 3.3 mg mL1, respectively, while the acids 199 and 201 were the most cytotoxic against B16–F10 melanoma cells, with GI50 at 4.4 and 18.0 mg mL1, respectively.88 Five polyketides were isolated from the lichen Ramalina peruciana using HPLC-UV detection. The lichen was collected at Paran´ a State. Atranorin (188), 40 -O-demethylsekikaic acid (203), ramalinolic acid (204), sekikaic acid (205) and homosekikaic acid (206) were identied by analysis of spectroscopic data.89


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The solid culture of Cladosporium uredinicola isolated from Psidium guajava fruits, yielded four new depsides, 3-hydroxy-2,5dimethylphenyl 2,4-dihydroxy-3,6-dimethylbenzoate (207), 3-hydroxy-2,4,5-trimethylphenyl 2,4-dihydroxy-3,6-dimethylbenzoate (208), 3-hydroxy-2,5-dimethylphenyl 3-[(2,4-dihydroxy3,6-dimethylbenzoyl)oxy]-6-hydroxy-2,4-dimethylbenzoate (209) and 3-hydroxy-2,4,5-trimethylphenyl 3-[(2,4-dihydroxy-3,6dimethylbenzoy)oxy]-6-hydroxy-2,4-dimethylbenzoate (210). Compounds 207, 209 and 210 were evaluated against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis. Compound 207 showed bactericidal activity


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against B. subtilis and P. aeruginosa at 25 mg mL1, and against E. coli and S. aureus at 250 mg mL1. Compound 210 showed bacteriostatic effect at 250 mg mL1 against E. coli and S. aureus, and at 25 mg mL1 against B. subtilis and P. aeruginosa, while 209 displayed bacteriostatic activity against all strains at 250 mg mL1.90 Compounds 207 and 209 were also tested as inhibitors of photosynthesis. Both 207 and 209 inhibited partially the reaction of photosynthesis II, showing potential as new herbicidal leads.91 The known T2-toxin (211), as well as a mixture of 8-n-butyrylneosolaniol (212) and 8-isobutyrylsolaniol (213), were obtained from a culture of Fusarium sp. and showed fungicidal activity against diverse strains of the fungus Paracoccidioides brasiliensis. Compound 211 displayed antifungal activity in the range of 75–640 mmol L1, while the mixture of 212 and 213 was active in the range between 160–960 mmol L1.92 The extract of the lichen Pseudoparmelia hypomiltha yielded secalonic acid A (214), which displayed potential immunological modulating activities by the release of H2O2.82 Secalonic acid A was also isolated from the lichen P. sphaerospora, although its structure was incorrectly drawn.81 Phomopsis longicolla was obtained from the thallus of the red seaweed Bostrychia radicans. Aer cultivation in solid media, mycophenolic acid (215) and dicerandrol C (216) have been isolated and identied. Compounds 215 and 216 were devoid of

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antibacterial activity against S. aureus and S. saprophyticus, while 216 displayed bacteriostatic effect at 1 mg mL1 against S. aureus and bactericidal effect at 2 mg mL1 against S. saprophyticus.93 A strain of Fusarium oxysporum was isolated from the medicinal plant Smallanthus sonchifolius. In solid culture F. oxysporum produced anhydrofusarubin (217) which was evaluated against the tumor cell lines HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (CNS glioblastoma) and it showed IC50 at 9.85, 6.23 and 6.32 mg mL1, respectively.38 The fungus Chaetomium globosum was isolated from the leaves of Viguiera robusta. This endophyte produced six novel polyketides, 50 -epichaetoviridin A (218), 40 -epichaetoviridin F (219), 12b-hydroxychaetoviridin C (220), chaetoviridins G (221), H (222) and I (223), along with the known chaetoviridins A (224), B (225), C (226), D (227), E (228) and 40 -epichaetoviridin A (229). Application of the Mosher method allowed the assignment of correct the structure of 229 as 40 -epichaetoviridin A instead to that of the 50 epimer of chaetoviridin A. Additionally, analysis of NOE data enabled the assignments of the absolute conguration of compounds 218–223 as depicted. All compounds were inactive in an in vivo pathogenicity assay with Caenorhabditis elegans infected with Enterococcus faecalis.94 Cosmomycin (230) isolated from a strain of Streptomyces sp., was shown to effectively bind 16-mer double stranded DNA as effectively as daunorubicin, measured by gel mobility shi assays and ESI-MS analysis.95

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Streptomyces sp. AMC 23 cultures. The mixture of 231 and 232 showed phytotoxic activity against the microalga Chlorella vulgaris at 10 mg mL1.96 The novel polyketides 240 and 241 were produced in a co-culture of two marine-derived fungal strains, Penicillium sp. Ma(M3)V and Trichoderma sp. (Gc(M2)1, however due to the scarcity of material the compounds could not be tested in bioassays.97 This was the rst published report of microbial co-culture experiments leading to the production of identied metabolites performed in Brazil, although the results of other co-culture experiments that have been previously performed in Brazil have been presented in scientic meetings.

Balomycins B1 (231) and B2 (232) have been isolated as a mixture, while balomycins D (233), A1 (234), G (235), H (236), C1-amide (237), C2 (238) and antibiotic NK (239) could be identied by UPLC-ESI-multistage MS from the extract of


Metabolic proling of endophytic Phomopsis and Cytospora strains obtained from diverse trees, coupled with fungi phylogenetic analysis, showed that the occurrence of different classes of polyketides are associated with specic chemotypeproducing strains. Fungal phyletic groups could be assigned using the genetic and chemotaxonomic analyses, showing that the production of secondary metabolites is species-specic and providing further support to the concept that fungal chemotaxonomic analysis is a valuable tool for dereplication and fungi taxonomy.98

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Scheme 1

2.5 Metabolites of mixed shikimate and terpene biosynthesis ()-(S)-Guignardic acid (242) was produced by Guignardia sp., an endophytic fungus isolated from leaves of Spondias mombin, a tropical tree with antimicrobial properties used in traditional medicine. The structure of 242 was established by analysis of spectroscopic data, while its absolute conguration was determined by total synthesis (Scheme 1).99 2.6

Metabolites of mixed terpene and alkaloid biosynthesis

Pencolide (243) was produced by Penicillium sclerotiorum, isolated from Brazilian Cerrado soil samples.56,77 It showed bacteriostatic effect against E. coli, S. aureus, S. typhimurium and S.

pyogenes with MIC at 128 mg, and fungistatic activity against C. albicans at 64 mg.77 Penicillium sp. isolated from the root bark of Melia azedarach produced verruculogen (244), which showed bacteriostatic effect against Escherichia coli at 250 mg mL1.50 The novel alkaloids citrinalin A (245) and B (246) were isolated from the growth medium of fungus Penicillium citrinum, obtained from the seaweed Caulerpa sp. The production of both 244 and 246 was enhanced using experimental design and chemometric analysis. Both alkaloids were inactive against the bacteria S. aureus, E. coli, and P. aeruginosa, and the fungus Candida albicans and didn't show cytotoxic activity against the tumor cell line HTB-129 (breast cancer). X-ray crystallography analysis of citrinalin A (245) allowed to assign its absolute conguration as 1S, 14R, 16S, 22S. For citrinalin B (246), its absolute conguration was determined by analysis of NOESY experiments and by comparison with spectroscopic data obtained for 245. A strain of Penicillium oxalicum obtained from marine sediments produced meleagrine (247) and oxaline (248).69 A strain of Penicillium sp. was isolated from the roots of Mauritia exuosa. This endophyte produced glandicoline B (249) which was active against Staphylococcus aureus, Micrococcus luteus and Escherichia coli at 100 mg mL1. The absolute conguration of 249 was established aer comparison with crystallographic data of its O-methylated analog, meleagrine (247).54 2.7 Metabolites of mixed polyketide and amino acid biosynthesis The unusual amino acid derivative N-acetyl-g-hydroxyvaline lactone (250) was isolated from a species of Streptomyces obtained from marine sediments collected at the North coastline of S˜ ao Paulo State. Differential NOE experiments as well as molecular-modeling calculations allowed to assign the relative stereochemistry of 250 as shown.20 From the conidia of Metarhizium anisopliae var. anisopliae ESALQ-1037, obtained from a culture collection, 2-{[1-carboxy-2(4-hydroxyphenyl)ethyl]amino}-N,N,N-trimethyl-2-oxoethanammonium (251) was isolated and identied. This compound was also detected in the extract of six more strains: M. anisopliae var. anisopliae ARSEF 1095, 5626 and 5749 and M. anisopliae var. acridumisolates ARSEF 324, 3391 and 7486.100

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Methylobacterium mesophilicum, isolated from orange trees infected with the citrus variegated chlorosis (CVC) disease, produced in liquid medium the known (S)-N-dodecanoyl-HSL (252), N-tridecanoyl-HSL (253), (S)-N-(7Z)-tetradecenoyl-HSL (254), (S)-N-(2E,7Z)-tetradecadienyl-HSL (255), (S)-N-tetradecanoyl-HSL (256), together with the new (S)-N-(2E)-dodecenoyl-HSL (257). The S absolute conguration at the lactone moiety of all the acyl homoserine lactones 252–257 was established by chiral gas chromatography analysis using authentic synthetic standards. Antimicrobial assays with the corresponding synthetic compounds against bacterial endophytes isolated from CVC-infected trees (Bacillus sp., Curtobacterium accumfaciens and Nocardiopsis sp.) did not show any activity.101 The fungus Pencillium citrinum isolated from the marine alga Caulerpa sp. produced (E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-

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mobilization, which is directly related to the mast cell degranulation. The results obtained indicated that pyridovericin (260) can be considered as a hit for the development of anti-allergic drugs.103 Phomopsis longicolla was obtained from the thallus of the red seaweed Bostrychia radicans. Aer cultivation in solid media, 18-deoxycytochalasin H (261) was isolated and identied and did not display antibacterial activity against Staphylococcus aureus and S. saprophyticus.93 One new (262) and ve known cytochalasins, 19,20-epoxycytochalasins C (263), D (264), N (265), Q (266) and R (267), were produced by Xylaria sp., an endophytic fungus isolated from leaves of Piper aduncum. Analysis of NOE experiments the relative conguration of

methyldec-8-ene-1,3-dione (258) and 1-(2,3-dihydro-1H-pyrrol-1yl)-2-methyldecane-1,3-dione (259).69 The biosynthesis of both 258 and 259 proceeds via the incorporation of acetate, methionine and ornithine, as established by experiments using 13Clabeled precursors.102 Pyridovericin (260) isolated from the culture medium of Beauveria bassiana obtained from a soil sample, was evaluated as an inhibitor of mast cell degranulation and cytokine secretion.103 Compound 260 was tested as a degranulating agent of RBL-2H3 cells, immortal cell line derived from rat leukemia basophil-like cells. Pyridovericin (260) affected the release of bhexoaminidase in a dose-dependent manner, with an IC50 at 7.4 mM, and mildly inhibited b-hexoaminidase activity. Additionally, compound 260 also inhibited dose-dependently the thapsigargin-induced degranulation of RBL cells, with IC50 at 6.3 mM. Pyridovericin (260) also promoted reduction of Ca2+


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compound 262 to be established.104 Two additional cytochalasins B (268) and D (269), were isolated from the culture medium of the same fungus species, an endophyte of Palicourea marcgravii. Compound 269 was active at 10 and 25 mg mL1 against C. cladosporioides and C. sphaerospermun, respectively,68 while 266 showed a weak fungicidal activity against the same fungi strains, at 100 mg mL1. Compounds 262 and 265 were inactive in cytotoxic assays against CHO cells (Chinese hamster ovarian). Compounds 263, 264, 266 and 267 showed IC50 at 120.0, 90.0, 125.0 and 4.0 mmol L1, respectively. On HeLa cancer cell (human cervical cancer), compounds 262–267 displayed cytotoxic activity with IC50 at 43.0, 1.0, 2.0, 1.0, 3.0 and 3.0 mmol L1, respectively.104

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(leukemia) and B16F10 (melanoma) tumor cells and showed 89.55% and 57.10% of inhibition at 100 mg mL1. Weak antibacterial activity was observed against Staphylococcus aureus by 270 (MIC at 120 mg mL1) and Escherichia coli (MIC at 189 mg mL1).105

2.8 Metabolites of mixed terpene and polyketide biosynthesis The new tricycloalternarene F (273) and two new guignardones, D (274) and E (275), as well as the known guignardone A (276), were isolated from cultures of the endophytic fungus Guignardia mangiferae found in the leaves of Viguiera arenaria.106 The fungus Cochiliobulus sp. isolated from Piptadenia adiantoides produced cochlioquinone A (277) and isocochlioquinone A (278). Both compounds showed leishmanicidal activity against Leishmania amazonensis, with EC50 (half maximum effective concentration) at 1.7 and 4.1 mM, respectively. The same compounds were inactive in cytotoxic assays against the tumor cells MCF-7 (breast cancer), TK-10 (renal carcinoma) and UACC-62 (melanoma).107

Isolated as an endophytic fungus of Viguiera robusta, Chaetomium globosum produced three chaetoglobosins, B (270), D (271) and E (272). Compound 270 was tested against Jurkat

2.9 Metabolites of mixed alkaloid, amino acid and polyketide biosynthesis A compound named GKK1032 (279), known by its antibiotic and cytotoxic activities, was produced by Penicillium sp. isolated from Murraya paniculata. Its structure was assigned by analysis of spectroscopic data.71

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2.10

Metabolites of unknown biosynthetic origin


A low molecular weight polyhydroxybutyrate (280) has been isolated from the growth medium of Burkholderia sp. and identied by analysis of spectroscopic data. Although 280 has been previously synthesized, this was its rst occurrence as a natural product.108

3 Current trends in microbial natural products chemistry in Brazil

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have not been much investigated, possibly because of the difficulty in the collection of single lichen species and of the very limited availability of lichen taxonomists in Brazil. However, lichenicolous fungi are poorly known110 and should be better considered as potential sources of novel bioactive secondary metabolites. As for lichens, a reliable microorganism strain identication is essential in microbial biodiscovery programs, aiming not only to nd new and useful products, but also for the description of new and unusual microbial species. Fig. 2 shows that secondary metabolites from microbial strains isolated from plants have been much more intensely investigated by Brazilian researchers than strains isolated from the marine environment or from soil samples. Endophytic fungi and bacteria are, indeed, a quite unique source of structurally diverse and bioactive secondary metabolites, which very oen display potent biological activities in drug-discovery screens, such as antibacterial, antifungal, antiparasitic, neuroprotective, antioxidant and cytotoxic.111–118 Endophytes live in a very particular environment, under a stressing competition for nutrients and space, and disputing the internal host

While collecting literature data to prepare this review, it seemed to us that Kelecom's approach to understand the development of natural products chemistry from marine microorganisms was particularly informative, since it provided information using quantiable data.109 We therefore decided to analyze different data sets in order to have a clearer landscape on the development of microbial natural products chemistry in Brazil. Fig. 1 shows the distribution of microbial strain groups from which secondary metabolites 1–280 have been isolated. Metabolites from fungi are largely predominant. A probable reason for such a trend is that in general fungi provide higher yields of secondary metabolites in culture media than bacteria, and, therefore, have been preferred for studies on bioactive microbial natural products isolation and identication. Lichens

Fig. 1 Percentage of secondary metabolites isolated from bacteria, fungi and lichen in Brazil.


Fig. 2 Sources from which microbial strains that yielded compounds 1–280 have been isolated in Brazil. The total number of compounds/ microbial source in the bar graph include the re-isolation of a same compound from different microorganisms obtained from different sources.

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environment with other strains. Such conditions are likely to inuence the microbial metabolism in the production of compounds that may enhance tness, survival and species maintenance.116,119 Another reason why endophytes have been of much interest is that several potently bioactive secondary metabolites which have been rst isolated from higher plants have been subsequently isolated from plant-associated microbial strains. These include paclitaxel,120,121 camptothecin,122 podophyllotoxin,123–125 hypericin126 as well as vincristine.127 The discovery of the actual biosynthetic origin of plant endophyte metabolites is currently a subject of much interest. Considering endophytes metabolic uniqueness and the array of bioactivities presented by such compounds, the emphasis of Brazilian researchers towards the isolation of bioactive natural products from endophytic microorganisms is largely justiable. Fig. 2 also shows that microbial strains isolated from samples obtained from the marine environment have been more oen investigated by Brazilian natural product chemists than strains isolated from soil samples. This trend can be explained by the recent development of the research in microbial natural products chemistry in Brazil, when the discovery of marine microorganisms' secondary metabolites was in evidence. Microbiological and secondary metabolism investigation of marine microorganisms have been subject of increasing investigation during the last 20 years.109,128–140 Brazilian microbial natural product chemists have not engaged in similar efforts towards the isolation of bioactive secondary metabolites from soil microorganisms. However, the diversity of soil fungi strains, for example, seems to be by far higher than in any other natural environment.141,142 Considering that the ca. 80 000–100 000 described species of fungi are likely to correspond to only 5% of all fungi species,142,143 there is an unimaginable diversity of fungi strains to be uncovered, described and bioprospected. It seems to be of consensus that 95% of fungi have not yet been cultivated, i.e., are essentially unknown.144–146 Estimates of 1.5 million of fungi species proposed in 1991,147 updated to 2.3 million species in 2001145 and reviewed to up to 3.0 million of species in 2012148 point to a very impressive level of fungi biodiversity. Data indicate that the fungal diversity in the tropics is larger than in temperate regions, although the dimension of such difference is complex and difficult to establish.110,148–150 Soil bacteria are also extremely abundant, corresponding to 109 bacterial cells per gram of soil, the largest portion of which is nevertheless difficult to growth in articial media.151 Particularly actinomycetes constitute outstanding producers of exquisite bioactive chemicals. These include both bacterial and fungal antibiotics, anticancer agents, enzyme inhibitors, immunosuppressants, hypocholesterolemic drugs, insecticides, antiparasitics, among many other activities in different bioassays.144,152,153 Considering that Brazil is one of the world's megadiverse countries, there is a very considerable effort remaining to describe microorganisms from distinct organisms and environments. A major description of fungi species in Brazil reported in 1985 was critically assessed in 1997 by Hawksworth and Rossman,110 who showed that such evaluation had to be

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better designed in order to provide reliable data. Description of marine fungal diversity in Brazil is very recent, and was directed to strains associated to marine invertebrates and marine algae.154,155 The marine bacterial diversity from specic marine ecosystems and organisms has been very widely investigated in Brazil, including diversity surveys at estuarine sediments, of hydrocarbonoclastic bacterial communities from mangrove sediments, from deep-water subseaoor rock samples, of a very large hypersaline lagoon, from the marine sponges, ascidians, corals and algae.154,156–167 Studies on the diversity of endophytic microorganisms performed in Brazil is comparable to the studies on marine bacteria diversity, and include surveys on the microbial diversity of Theobroma cacao.168–182 New species of fungi and bacteria described in Brazil during the last ve years have been reviewed, and include species found in different biomes, plants, insects, soil, corals, shes, mangroves, the Amazon forest, the Caatinga biome and several aquatic biomes as well.183 Considering such an immense microbial diversity, a major effort can be envisaged towards the discovery of microbial bioactive secondary metabolites from the Brazilian microorganism biodiversity.

Fig. 3 Biogenetic origin of metabolites 1–280 isolated from microbial sources in Brazil. ALK: alkaloid; PEP: peptide; TER: terpene; PKS: polyketides; MIX: mixed biogenetic pathways.


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Fig. 3 illustrates that half of the metabolites isolated from microbial strains investigated in Brazil are polyketides, followed by terpenes (19%), metabolites of peptidic origin (13%), of mixed biogenetic origin (13%) and alkaloids (2%). It is not known if such trends correspond to the actual panorama of the whole microbial secondary metabolite diversity. A recent analysis by Ebel184 showed that, as for marine fungi, most of the isolated and identied secondary metabolites correspond to polyketides (41%), followed by alkaloids (20%), then terpenes and peptides (both 14%), while prenylated polyketides (8%) and shikimic acid derivatives (2%) correspond to the minority of secondary metabolites isolated from marine fungi. However, such a prole may be directly dependent on the growth conditions of the isolated strains which produce secondary metabolites, since it is well-known that signicant changes in microbial production of natural products correlates directly to the growth media composition as well as to several parameters of growth conditions.61 Fig. 4 shows that polyketides are the predominant group of metabolites isolated from fungi and lichens from Brazil, while peptides and metabolites of mixed biogenetic origin have been predominantly isolated from bacterial strains. Lichens have exclusively yielded polyketides. It remains to be investigated if such trends reect particular evolutionary metabolic

Biogenetic origin of microbial secondary metabolites isolated from fungi, bacteria and lichens.

Fig. 4

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adaptations, since polyketides are by far the largest group of secondary metabolites biosynthezised by fungi185 and lichenicolous fungi.186 Fig. 5 illustrates the biogenetic origin of secondary metabolites isolated from the culture media of microorganisms obtained from distinct sources. Endophytes investigated by Brazilian researchers have been shown to produce predominantly more polyketides, followed by terpenes, metabolites of mixed biogenetic origin and peptides. Soil strains have yielded mainly polyketides and peptides, while strains isolated from marine sediments and algae produce mainly metabolites of peptidic origin. Marine invertebrate strains have yielded largely more polyketides than any other class of natural products. “Pure alkaloids” have been very rarely isolated from microbial strains obtained from any of sources herein considered. It would be worth verifying if such distribution follows the same distribution as microbial metabolites worldwide, although such a survey may be envisaged as a major analysis effort. Additionally, the present survey shows that, while the number of compounds produced by microbial strains isolated from samples other than plants are not large enough in number to allow a consistent analysis of metabolic prole, fungal endophytes clearly show that polyketides and terpenes are predominant over peptides and alkaloids. It would be interesting to compare the metabolic prole of endophytes with plant secondary metabolite distribution in order to verify any possible parallel of co-evolutionary metabolism adaptation between hosts and guests. Such an analysis would raise a number of interesting questions to be investigated. While the output of microbial natural products isolation and identication in Brazil has increased over the years, both in number of compounds isolated and in number of articles published (Fig. 6 and 7), the large majority of the isolated and identied compounds from microorganisms in Brazil is already known. Although this may be a reection of the current difficulty in the isolation of new secondary metabolites,187 it may be also be due to the limited use of dereplication tools before the isolation of compounds produced in growth media. Currently, different dereplication strategies are considered essential to avoid redundancy, waste of time and resources in the isolation

Fig. 5 Biogenetic origin of secondary metabolites isolated from cultures of microorganisms obtained from distinct biological sources (plants, marine sediments, soil, seaweeds, marine invertebrates and other).


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Secondary metabolites isolated from microbial culture media in Brazil. New compounds correspond to 28.4% of all compounds.

Fig. 7 Number of articles published per year dealing with the isolation, identification, biological activities and biosynthesis of pure natural products isolated from microbial culture media in Brazil.

of known natural products. Nevertheless, known natural compounds can be explored as yet unknown bioactives in several new bioassays, since several of such microbial metabolites were isolated decades ago, when many bioassays still had very limited availability, particularly targeted-based assays.188,189 There is an increasing interest towards the investigation of microbial metabolites by Brazilian researchers, as shown by the number of publications per year dealing with microbial natural products isolation, identication, evaluation of biological activities and investigation of biosynthetic pathways. However, the number of natural products chemistry and pharmacognosy research groups devoted to the investigation of plant secondary metabolism is still large in Brazil. A possible reason for such a trend is that historically the Brazilian natural products chemistry research has well established roots in the search for plant secondary metabolites.21,190,191 Since the 1990's there has been a change towards the search for marine metabolites and in the 2000's towards the investigation of microbial metabolites. The

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diversication of Brazilian natural product research results mainly from the efforts of research groups with strong national and international collaborations. Since the research on microbial natural products needs a strong collaborative effort with microbiologists, there is a clear need to promote the science of microbiology biodiversity and bioprospecting in Brazil.

4 Thinking big As an extraordinary coincidence, the very rst report on the isolation of a microbial natural product in Brazil20 was contemporary to the proposal of a major paradigm shi in microbiology sciences.192 Such change also corresponded to a paradigm shi of natural products sciences in Brazil, then incorporating a new vision to investigate and understand how to explore unusual secondary metabolites, nally looking for its three major sources: plants, animals and microorganisms. Knowledge on microorganisms diversity and metabolism


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gained a completely new momentum when biochemical, genetic and molecular biology tools became available in the early 1990's. As a single example of such impact, in 1985 only eleven bacterial phyla were recognized,193 a number that multiplied by almost ve in less than 20 years aer.194 While it took more than one decade to start the investigation of microbial natural products in Brazil aer the advent of biochemical, genetic and molecular biology tools, Brazilian scientists are now currently aware of the advances in microbial diversity, physiology and metabolism investigations. In order to reinforce the importance of microbial biodiscovery, it is worth mentioning that it has been recently estimated that the discovery rate of new antibiotic producing bacteria will require more than 107 isolates in order to nd the next class of antibiotics. New cultivation approaches and high throughput cultivation apparatuses are needed in order to provide adequate conditions for the discovery of yet uncultured strains in a reasonable time frame.195 On the other hand, it has been stated that less culturable strains could not be used in biodiscovery programs.1 However, if true this assumption may be only temporary, dependent on the development of new technologies that translate the particularities of yet uncultured microorganisms from the microbiology research laboratory into biotechnological and biodiscovery programs, in order to give access to a yet underestimated diversity of new microbial strains and their biosynthetic products.196,197 Such new microbiology and molecular biology technologies will certainly enhance the capabilities to explore the microbial diversity and biotechnology.195 Currently biodiscovery research requires truly innovative approaches in order not only to achieve the isolation of yet not cultured strains, but also to develop reliable and robust conditions for culturing such microorganisms under articial conditions,198 as well as the use of technological advances that allow the deep and wide analysis of microbial secondary metabolism. A recent example of how technological advances may strongly impact the advances of microbiology sciences is the rst report of in situ real-time analysis of bacterial cultures using fast and very sensitive mass spectrometry analysis.199,200 Stewart optimistically looks forward to an outstanding development of microorganism cultivation science during the next 30 years, suggesting that its impact on health, ecology and science will surmount its previous development during the last 300 years.195

5 Microbial diversity and biodiscovery, and science policy Since the Rio 1992 meeting led to the Convention of Biological Diversity, there has been much hope and expectation that its fundamental proposals would be achieved to facilitate agreements between megadiverse and non-megadiverse partners in order to promote collaborative research and development programs on biodiversity conservation and biodiscovery. Unfortunately such hope and expectations were not met.201 This is mainly because the benet sharing paradigm raised too high expectations on the value of biodiversity products, making agreements very difficult or even impossible to be


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accomplished.202–205 Moreover, many megadiverse countries established very strict legislation to regulate the access to biodiversity and its genetic resources that, in combination with lengthy bureaucratic negotiations, terminated bilateral biodiscovery and biotechnological initiatives even before they had started. Aside to elusive nancial returns which are so difficult to address a priori, the benet sharing principle seems to have undervalued the human resources capacity building, knowledge and technology transfer, as well as infrastructure building that are absolutely essential for the success of international collaborative biodiscovery and biodiversity conservation programs. If viewed under a wider perspective, it would be clear that such positive aspects of international collaborative programs provide long-term consequences as, for example, improving education and enhancing scientic knowledge,206 currently considered as key capabilities to be acquired by students looking for highly specialized and high-prot jobs. Restricting and making more difficult the research on biodiversity is a lose-lose damaging weltanschauung that may empoverish scientic development and lead to biological species extinction, loss of knowledge about biological diversity and of the value of biodiversity as well as the discovery of invaluable products to human society and to the environment. Unfortunately the establishment of such a culture was detrimental to society and the environment as a whole while impeding cultural exchange, scientic development as well as valuing human and natural resources. By the end of the 1990's, the national Brazilian legislation and regulations to access its biodiversity and genetic resources started to be settled and was nally formatted by the 2001 provisional act 2,186-16 (http://www.mma.gov.br/estruturas/sbf_dpg/ _arquivos/mp2186i.pdf). The so-called MP 2,186-16 certainly aimed to protect the Brazilian biodiversity. However, as conceived it rather resulted in such a restrictive legislation that practically inhibited any formal international cooperative biodiscovery program between Brazilian and international researchers during the last twelve years. The settling of formal collaborative research programs between Brazilian and international biodiscovery groups would be particularly interesting and important to achieve during that period, not only because Brazil has a long tradition of research on biodiversity and natural products sciences with a considerable number of internationally well recognized research groups devoting efforts towards biodiscovery projects. But also because during the last 20 years Brazil has experienced one of its most stable economic periods with substantial and continuous investment in science and education, resulting in a signicant increase of knowledge output.207 The rst organized research program aimed at promoting knowledge on biodiversity conservation and sustainable use in Brazil is the BIOTA program launched by the S˜ ao Paulo state funding agency FAPESP in 1999, a science-based Brazilian program aiming to investigate all aspects of the S˜ ao Paulo state biodiversity, which is currently expanded to other Brazilian regions.208 The BIOTA-FAPESP program missions initially included cataloguing and characterizing the biodiversity of S˜ ao Paulo state, aiming to improve the research on biodiversity sciences and to provide a sound scientic background for

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policymakers to propose and establish mechanisms for biodiversity conservation and sustainable use. Currently the BIOTAFAPESP program promotes research on virtually all scientic branches related to biodiversity, including research on several of the major biology specialities, sustainable land use, longterm processes, as well as wild biodiversity based biodiscovery and biotechnology under the auspices of the BIOprospecTA subprogram of BIOTA-FAPESP. The BIOTA-FAPESP program established new roadmaps for biodiversity conservation and management in Brazil, resulting in a number of government acts and resolutions aimed at dening policies for biodiversity conservation in S˜ ao Paulo state,209 constituting a ne example of how sounding science may result in establishing effective public policies to the benet of society and environment.210 Consequentially, the national Brazilian SISBIOTA program launched in 2010 was conceived largely based on the BIOTA-FAPESP premises. Even though the BIOTA-FAPESP program has signicantly promoted effective advances to the biodiversity and biodiscovery research initiatives in Brazil, the number of projects dealing with microbial diversity and biodiscovery within the S˜ ao Paulo state BIOTA-FAPESP program and the Brazilian Federal Government SISBIOTA program are still modest, but increasing. Much remains to be done, not only in exploring microbial diversity and biochemical capabilities, but also in developing innovative and groundbreaking scientic and technological approaches to assess the Brazilian microorganism diversity, considering Brazil's megabiodiversity spread among six major distinct regions with completely distinct environmental features. Multidisciplinary collaborative initiatives between Brazilian and international scientists working on distinct specialities is therefore essential to promote the development of the research on microbiology and microbial natural products chemistry in Brazil.

6

Acknowledgements

RGSB thanks David Newman (NIH) for sending reprints of several articles. LPI thanks to the S˜ ao Paulo State Funding Agency FAPESP and the Federal Government Funding Agency CAPES for nancial support as an Undergraduate Investigator Scholarship both at IQSC-USP (FAPESP 2011/08064-2) and at the Scripps Institution of Oceanography (CAPES). PMA thanks FAPESP for a post-doctoral scholarship (2012/04100-7), while RGSB thanks FAPESP for a BIOTA/BIOprospecTA grant (2010/ 50190-2) and the Brazilian Research Counsil of Scientic and Technological Development CNPq for a Researcher Award (301289/2009-3). This review is dedicated to Dr Carlos A. Joly (Instituto de Biologia, Universidade Estadual de Campinas) for his outstanding contributions to the development of biodiversity sciences.

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