nature protocols | VOL.4 NO.12 | 2009 | �

protocol

IntroDuctIonTerrestrial fungi have for long been known as a rich source of biologically active secondary metabolites. Since the discovery of penicillin by Sir Alexander Fleming in 1928, which resulted in a breakthrough in the treatment of bacterial infections, fungi have become an important source of drugs for the treatment of a variety of diseases. Beside other well-known antimicrobial agents of fungal origin like fusidic acid and griseofulvin1, new semi-synthetic antifungal drugs like anidulafungin (Eraxis), caspafun-gin (Cancidas) and retapamulin (Altabax) are likewise derived from fungal metabolites2,3. With the discovery of cyclosporine isolated from Tolypocladium inflatum in 1971 an important step in immunopharmacology was taken and improvements in the field of organ transplantation and treatment of autoimmune diseases are still in progress, like the introduction of substances such as the fungal-derived mycophenolic acid (Myfortic) to the market in 2004 (ref. 2).

Further pharmacologically important fungal metabolites include antilipidemic drugs collectively known as ‘statins’ with their parent compounds mevastatin and lovastatin isolated from Penicillium citrinum and Aspergillus terreus, respectively. Statins reduce blood cholesterol levels and are used for the treatment of coronary diseases2,4.

Fungal metabolites are, however, not only indispensable for medicine but are also important for plant protection, as demon-strated by the discovery of the strobilurines that were first isolated from Strobilurus sp. and served as lead compounds for synthetic fungicidals such as trifloxystrobin (Flint)5.

However, ‘the rediscovery of high numbers of previously described metabolites has to some extent precluded the study of traditional terrestrial sources of fungi’6 and recently, interest of natural product chemists and pharmacologists alike has turned to so far less investigated habitats and ecological niches such as the oceans. The oceans cover nearly three-quarters of the earth’s surface and can be considered a ‘soup’ of essentially all imaginable types of microbes6,7. Ecological niches, e.g. deep-sea hydrothermal vents, mangrove forests, algae and sponges provide distinct habitats for the isolation of specific micro-organisms8. Marine

micro-organisms including bacteria, cyanobacteria, microalgae and fungi have become an important source of new pharma-cologically active metabolites. Recent reviews demonstrate the importance of these organisms as potential sources of pharma-ceutical leads6–25. Especially, marine fungi have shown promis-ing potential as sources of new and bioactive natural products as suggested by the chemical diversity of their secondary meta-bolites6 even though sample collection and preparation as the first step might be more difficult for marine-derived fungi than for terrestrial material because of difficulties inherent to the collection in marine environment such as the need for scuba diving. Although the most famous group of bioactive compounds obtained from marine-derived fungi are still the cephalosporines with cephalosporine C, first isolated by G. Brotzu in 1945 from a marine strain of Acremonium chrysogenum26, there are also more recent promising examples. These include halovir and several naturally occurring analogs, which are potent inhibitors of Herpes simplex viruses 1 and 2. Synthetic studies that were carried out for these substances allowed insight into structure–activity relationships22,27. The new alkaloid sorbicillactone A isolated from the sponge-derived fungus Penicillium chrysoge-num showed promising activity against leukemia cells without exhibiting notable cytotoxicity. Large-scale fermentation of the fungus yielded sufficient amounts of the compound for preclinical investigations22,28. Overall, research on marine- derived fungi has led to the discovery of more than 270 new natural products mainly from the early 1990s to mid 2002 (with more than two-thirds of these compounds isolated from sponge- and plant-derived fungi), whereas more than 330 new metabolites were reported only in the 5 years period between 2002 and 2006 (refs. 6,12,22,29) including numerous compounds with new carbon skeletons. This sharp rise in numbers indicates a growing interest in marine-derived fungi as sources of new bioactive metabolites.

The aim of this paper is to provide a detailed protocol on the purification and cultivation of fungi from various selected marine macro-organisms (sponges, algae and mangrove plants),

Methods for isolation of marine-derived endophytic fungi and their bioactive secondary productsJulia Kjer, Abdessamad Debbab, Amal H Aly & Peter Proksch

Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. Correspondence should be addressed to P.P. (proksch@uni-duesseldorf.de).

Published online XX XX 2009; doi:10.1038/nprot.2009.233

Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some of them featuring new carbon frameworks hitherto unprecedented in nature. these compounds are of interest as new lead structures for medicine as well as for plant protection. the aim of this protocol is to give a detailed description of methods useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on rapid ‘in house’ screening systems. some results exemplifying the positive outcomes of the protocol are given at the end. From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at least 3 months has to be scheduled.

� | VOL.4 NO.12 | 2009 | nature protocols

protocol

as well as on the isolation, characterization and structure eluci-dation of biologically active secondary metabolites produced by these fungi when grown in liquid or on solid media. An overview including an approximate time schedule is shown in Figure 1.

In this protocol we describe the isolation of endophytic marine-derived fungi from inner parts of living tissues of marine inverte-brates, algae and mangrove plants. Endophytic fungi are referred to as fungi that spend, at least, part of their life inside healthy tissues of host organisms regardless of the fact whether the host is a plant or an animal30. For the isolation of marine-derived fungi, especially for the isolation of fungi from sponges, alterna-tive methods compared with those described in this protocol are available. For example, instead of cutting pieces from surface-sterilized samples, it is also possible to drop squeeze water from the sponge on an agar slant or to excise an inner piece of the tis-sue after thoroughly rinsing it with sterilized sea water without previous surface sterilization31–33. Likewise, several other media for successful isolation and fermentation of marine fungi like potato dextrose agar, barley spelt solid substrate or solid malt extract medium17,31 as well as addition of specific nutrients or of sub-lethal doses of fungicides to restrict fast-growing fungi34, have been described in the literature. Numerous fermenta-tion conditions such as shaking versus static cultures, applica-tion of different temperature regimes or employment of larger fermenters that can provide varying aeration17,31,35, have likewise been reported. However, all conditions employed in cultivat-ing marine-derived endophytic fungi suffer from limitations as different cultivation techniques usually select only a small

fraction of the fungal community present, thereby providing a limited and selective window of the actual diversity that is present in a given sample36. Furthermore, it must be remembered that different culture conditions might also result in different chemical patterns of metabolites formed, as already stated, in the OSMAC (‘one strain, many compounds’) concept that makes deliberate use of the chemical plasticity of microorganisms as a response to varying conditions of culture37. However, after conducting numerous fermentation experiments with endophytic fungi using different culture conditions, we discovered the methods described in this protocol as being most suitable for our pur-poses, as they generally gave reproducible results regarding the isolation of endophytic fungi and regarding the patterns of secondary metabolites that are produced by these fungi32,38–40. In our hands we have obtained optimum results when cultur-ing fungi on different solid or in liquid media at room tem-perature (20–25 °C), followed by extraction of mycelia and growth media with organic solvents. Flow charts briefly describ-ing the workup procedures of the respective fungal cultures and the time needed for each step are shown in Figures 2–4. The crude extracts are then separated using various chromatographic techniques and the isolated bioactive compounds are analyzed by HPLC–DAD for their purity followed by structure elucidation by MS and NMR using state of the art techniques. Chiral compounds may be derivatized to determine their absolute configuration. Chromatographic separation of fungal extracts usually proceeds guided by bioassays, thereby directing the isolation strategy towards the discovery of bioactive constituents. After the extraction of cultures, the obtained extracts are treated in a similar manner as described earlier for extracts from marine macro-organisms. As the chromatographic separation, structure elucidation and bioactivity screening for extracts derived from marine macro-organisms have been described in detail in a previous protocol41, only those steps that differ from the described procedure are depicted in this protocol.

6: Purificationon malt agar

Pure culture

7: Taxonomic identification(by PCR and gene sequencing,followed by BLAST search)

8: Small-scalefermentation on rice orin liquid medium forscreening purpose

8: Large-scalefermentation

16: Bioassays(cytotoxicity and antimicrobial activity)

9–11: Extraction with EtOAc

12–14: Fractionation andisolation of secondary products

Host organisms

15: Structure elucidation

1–5: Isolation of fungi

Based on chemicalscreening/bioactivity results in large-scale fermentation of promising strains

1–5: 1 week

6: 2 weeks

7: 2 d

8: Each 3–4 weeks

9–11: 1–3 d

12–16: At least2–4 weeks

Figure � | Schematic overview on important steps involved in the isolation of fungi from marine sources and in the isolation and identification of their secondary metabolites.

Homogenization andextraction

Cell suspension

Filtrate

Water phase EtOAc

Residue

90% MeOH n-Hexane

Small-scale fermentationin liquid medium

+EtOAcUltraTurrax

Filter

Evaporate

90% MeOH+ n-Hexane

3 Weeks

Several min

Several hours

~1 h per 1 EtOAc

~1 h

Several min

Figure � | Estimation of the time required for fermentation of fungi and workup of extracts from liquid medium on small-scale basis.

nature protocols | VOL.4 NO.12 | 2009 | 3

protocol

Two examples taken from our group that focus on polyketides of fungal origin will be used to highlight the structural variability of natural products obtained from marine-derived fungi. Other results can be found in ref.33,38,39,42–44.

Experimental designSampling and general consideration before isolation of fungi. Transport the collected material in suitable containers (for sponges, algae or the like containing sea water). For animal and algal tissues with high water contents, isolation of fungi must be carried out within the next hours to avoid growth of ambient bacteria. Isolation of fungi from plant material like mangrove leaves can take place within 1–2 d as long as the samples are kept at low temperature (5–10 °C) and excess condensation is prohibited. Otherwise there is the risk that phylloplane fungi will colonize the plant material leading to false results during the isolation of endophytes.

Identification of fungi. As we restrict ourselves to cultivation of non-pathogenic fungi, any fungal cultures with documented pathogenicity45,46 towards humans are discarded immediately after taxonomic identification and excluded from further investigation.

Thus, identification of the isolates is carried out at an early stage during workup. Taxonomic identification of fungal strains is achieved by DNA amplification and sequencing of the fungal ITS region39 as described in Box 1. However, fungi can also be identi-fied by morphological characteristics using e.g., a commercially available service such as Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands.

Biological assays. Biological activities of extracts, fractions and pure compounds are tested in a number of rapid ‘in house’ bioassays that can be used for bioactivity-guided isolations. Those used in our laboratory are briefly described in Box 2.

Controls. Negative controls during the isolation of endophytic fungi from marine organisms are necessary to detect contami-nation from external parts of the slices, from phylloplane or from airborne micro-organisms. Therefore, surface-steri-lized marine samples are pressed onto a petri dish containing isolation medium before aseptic cutting and inoculation on a second agar plate. If fungal growth can be observed on the negative control dishes, the positive plates are not used for further purification procedures that aim at the isolation of true endophytic fungi.

For all bioassays, negative and positive controls to compare the effectiveness of the tested fractions and pure compounds have to be used as described

. Q1Q1

Medium

Water phase EtOAc

Residue

90% MeOH n-Hexane

Mycelium

Cell suspension

MeOH extract

Residue

90% MeOH n-Hexane

Large-scalefermentation

+MeOHUltraTurrax

EtOAc

Filter

Evaporate Evaporate

90% MeOH+ n-Hexane

90% MeOH+ n-Hexane

3 Weeks

Several hours

~1 hper 1 EtOAc

Several hoursSeveral hours

Several hours

~1 h

~1 hper 1 MeOH

Figure 3 | Estimation of the time required for fermentation of fungi and workup of extracts from liquid medium on large-scale basis.

Fermentationon rice medium

EtOAc extract

+EtOAc Filter

Residue

90% MeOH n-Hexane

Evaporate

90% MeOH+ n-Hexane

4–6 Weeks

Overnight + 30 min

~1 h per 1 EtOAc

small scale, ~ 1 hlarge scale, several hours

Figure 4 | Estimation of the time required for fermentation and workup of extracts from rice medium.

Box 1 | IDENTIFICATIoN oF FUNGI Taxonomic identification of the fungal strains is achieved by DNA amplification and sequencing of the fungal ITS region39.

For this purpose, a piece of fungal mycelium (0.5 cm2) is sampled from an agar plate, lyophilized in a freeze dryer and powdered in a mixer mill after adding a tungsten carbide bead.

DNA isolation is carried out using the DNeasy Plant Mini Kit according to the manufacturer’s protocol. The procedure includes cell lysis, digestion of RNA by RNAse A, removing of precipitates and cell debris, DNA shearing, DNA precipitation and purification. This is followed by DNA amplification using Hot StarTaq Master Mix Kit and the primer pair ITS1 and ITS4 in an iCycler thermocycler. The PCR product is loaded onto agarose gel (2% agarose in 100 ml TBE buffer, 10 µl SybrSafe). After electrophoresis at 70 V for 60 min, the band corresponding to the desired PCR product (~size 550 bp) is removed from the gel slice using the PerfectPrep Gel Cleanup Kit following the manufacturer’s protocol.

Pure PCR products are submitted for sequencing together with the primer ITS1 to a commercial service (e.g., SeqLab GmbH, Goettingen and BMBF, Düsseldorf, Germany) and the base sequence is compared with publicly available databases (GenBank

) with

the help of Blast-Algorithmus.Q10Q10

4 | VOL.4 NO.12 | 2009 | nature protocols

protocol

MaterIalsREAGENTS

Bacto agar (BD, cat. no. 214010)Malt extract (Merck, cat. no. 105391)Artificial sea salt (Sera, cat. no. 05420)Chloramphenicol (Sigma, cat. no. C0378)Sodium hydroxide (‘NaOH’, Sigma, cat. no. S5881)Hydrochloric acid (‘HCl’, Sigma, cat. no. 258148)Yeast extract (Fluka, cat. no. 70161)Peptone (Merck, cat. no. 111931)Glucose (Caelo, cat. no. 5247)Rice (parboiled, any supermarket brand)Glycerin (Roth, cat. no. 4043)Penicillin G (Sigma, cat. no. P3032)Streptomycin (Sigma, cat. no. S6501)Gentamycin (Serva, cat. no. 22185)Nystatin (Sigma, cat. no. N4014)Antimicrobial detergent Melsept SF (B. Braun, cat. no. 18907)Tungsten carbide bead (Qiagen, cat. no. 69997)Agarose (Sigma, cat. no. A9414)TBE buffer (Merck, cat. no. 106177)DNeasy Plant Mini Kit (Qiagen, cat. no. 69104)Hot StarTaq Master Mix Kit (Qiagen, cat. no. 203443)RNAse A (Invitrogen, cat. no. 12091-039)Primers: ITS1 and ITS4 (Invitrogen; individual order)SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102)DNA loading buffer (Eppendorf, cat. no. 0032 006.850)PerfectPrep Gel Cleanup Kit (Eppendorf, cat. no. 0032 007.740)

Solvents:Solvents for extraction and chromatographic separation (see REAGENT SETUP)Solvents for high performance liquid chromatography, HPLC, for measuring optical rotation, for NMR spectroscopy are used as described in a previous protocol41

Spray reagents are used as described in a previous protocol41

Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (‘Marfey’s reagent’, Fluka, cat. no. 42095)Standard amino acids (ICN, Sigma)

EQUIPMENTSterile plastic petri dishes, diameter 9.4 cm (Greiner, cat. no. 633161)Parafilm M (Marienfeld, cat. no. 74 038 10)Sterile tubes, 15 ml, 120 mm × 17 mm (Sarstedt, cat. no. 62553)Susceptibility paper disks (5 mm diameter, Oxoid)All micro-organisms are acquired from DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHArtemia salina (Dohse Aquaristik, cat. no. 21350)

••••••••••••••••••••••••••

•

•

••

•

•••••

•

Chromatographic stationary phases: Different materials from different suppliers are possible, in our laboratory material is used as described in a previous protocol41

Laminar air-flow (Herasafe HS15, Heraeus)pH meter (420 Aplus, Orion)Autoclave (Varioklav, H&P)Microcentrifuge (Biofuge pico, Heraeus)Freeze dryer (Lyovac GT2, pump Trivac D10E, Savant)PCR machine (iCycler, Bio-Rad)UV transluminator (GVM 20, Syngene)Mixer mill (MixerMill MM30

)

Power supply for electrophoresis (PowerPac 300, Bio-Rad)Ultra Turrax (T18 basic, IKA)Vacuum pump (type 4EKF 56CX-4, Greiffenberger Antriebstechnik)Nitrogen generator (UHPN 3001, Nitrox)Deep freezer ( − 80 °C, 86-Freezer, Forma Scientific)Shaker SM 30 A (Edmund Bühler, cat. no. 6101 000)Balance (BL1500, Sartorius)HPLC, MS and NMR: the equipment as described in a previous protocol is used41

REAGENT SETUPOrganic solvents for separation and purification Many organic solvents of varying polarities may be used for chromatographic separation and purification procedures, including acetone (! cautIon highly flammable and irritant), acetonitrile (ACN) (! cautIon highly flammable and toxic), dichloromethane (DCM) (! cautIon harmful), ethanol (EtOH) (! cautIon highly flammable), ethyl acetate (EtOAc) (! cautIon highly flammable and irritant), n-hexane (! cautIon highly flammable, irritant, harmful, dangerous for the environment and toxic for reproduction), n-butanol (n-BuOH) (! cautIon flammable, harmful and irritant) and methanol (MeOH) (! cautIon highly flammable and toxic). They all are of analytical grade. ! cautIon All organic solvents must be handled carefully. Wear protective clothing, safety glasses and gloves. They should be handled under a fume hood and stored in a ventilated solvent cabinet.Medium A for isolation of fungal strains from marine macro-organisms and mangrove plants

Bacto agar 15 g

Malt extract 15 g

Artificial sea salt 24.4 g (for isolation from mangroves 10 g)

Chloramphenicol 0.2 g

pH 7.4–7.8 (adjusted with NaOH/HCl)

Dem. water ad 1,000 g.

m

•

••••••••••••••••

Q2Q2

Q3Q3

Box 2 | BIoACTIVITY SCREENING TESTS agar diffusion assayAccording to the Bauer–Kirby test (DIN 59040), susceptibility disks are impregnated with 250 or 500 µg of crude extracts and 50 or 100 µg of pure compounds, respectively, and placed on agar plates that have been inoculated with either the standard bacterial strains Bacillus subtilis DSM 22109 ( = ATCC 11774), Escherichia coli DSM 10290 ( = ATCC 15766), the yeast Saccharomyces cerevisiae DSM 1333 ( = ATCC 9763) or the fungi Cladosporium herbarum DSM 63422 or Cladosporium curcumerinum DSM 62122.

The plates are checked for inhibition zones after 24 h of incubation (37 °C for bacteria, 27 °C for yeast) or after 7 d (22 °C for fungi), respectively.

Negative controls are only treated with the respective solvents, positive controls are treated with penicillin G, streptomycin and gentamycin (for bacteria) or nystatin (for fungi)33,47.cYtotoXIcItY testsBrine shrimp assayEggs of Artemia salina are hatched in artificial seawater at room temperature and under daylight conditions. After 48 h, 20 nauplii are transferred into each test vial using a binocular and seawater is added to 5 ml. Pure compounds are dissolved in 40 µl of DMSO to give final concentrations of compounds of 10 or 100 p.p.m. The experiment is kept under illumination and mortality is determined after 24 h by counting the survivors with the aid of a ×3 magnifying glass. Vials containing 40 µl of DMSO (negative controls) are also prepared48.The microculture tetrazolium (Mtt) assay is carried out as previously described for marine macro-organisms41.

nature protocols | VOL.4 NO.12 | 2009 | �

protocolThe contents are combined in a flask big enough so that it is filled not

more than two-thirds, covered and autoclaved at 121 °C for 20 min. After cooling down to ~60 °C, the medium is poured in sterile plastic petri dishes, ~25 ml medium per plate, under aseptic conditions. The prepared agar plates can be kept under sterile conditions for upto 1 week before inocula-tion. ! cautIon Flasks must not be closed tightly when being autoclaved. The temperature of the autoclave has to be below 80 °C before it can be opened to prevent hot steam from emerging. Heat protective gloves and safety glasses should be worn.Medium B for purification and short term storage of fungal strains

Bacto agar 15 g

Malt extract 15 g

Artificial sea salt 24.4 g (for mangrove-derived fungi 10 g)

pH 7.4–7.8 (adjusted with NaOH/HCl)

Dem. water ad 1,000 g

The contents are combined in a flask big enough so that it is filled not more than two-thirds, covered and autoclaved at 121 °C for 20 min. After cooling down to ~60 °C, the medium is poured in sterile plastic petri dishes, ~25 ml medium per plate, under aseptic conditions. The prepared agar plates can be kept under sterile conditions for upto 1 week before inoculation. ! cautIon Flasks must not be closed tightly when being autoclaved. The temperature of the autoclave has to be below 80 °C before it can be opened to prevent hot steam from emerging. Heat protective gloves and safety glasses have to be worn.Liquid Wickerham’s medium

Yeast extract 3 g

Malt extract 3 g

Peptone 5 g

Glucose 10 g

Artificial sea salt 24.4 g (for mangrove-derived fungi 10 g)

pH 7.2–7.4 (adjusted with NaOH/HCl)

Dem. water ad 1,000 g

All contents are mixed thoroughly. In all, 300 ml of the media is poured in 1,000 ml Erlenmeyer flasks. The flasks are covered with a tissue lid and autoclaved at 121 °C for 20 min. ! cautIon Tissue lids must be covered with aluminum foil to prevent soaking during autoclaving. The temperature of the autoclave has to be below 80 °C before it can be opened to prevent hot steam from emerging. Heat protective gloves and safety glasses have to be worn. The medium can be stored for 1–2 d before inoculation.Solid rice medium

Rice 100 g

Dem. water 110 g

The 1,000 ml Erlenmeyer flasks with the contents are covered with a tissue lid and kept standing overnight to allow swelling of the rice kernels before autoclaving at 121 °C for 20 min. ! cautIon Tissue lids must be covered with aluminium foil to prevent soaking during autoclaving. The temperature of the autoclave has to be below 80 °C before it can be opened to prevent hot steam from emerging. Heat protective gloves and safety glasses have to be worn. The medium can be stored for 1–2 d before inoculation.MexA medium for long-term storage

Malt extract 20 g

Yeast extract 0.1 g

Glycerin 50 g

Artificial sea salt 24.4 g (for mangrove derived fungi 10.0 g)

Bacto Agar 13 g

Dem. water ad 1,000 g

The contents are combined in a flask big enough so that it is filled not more than two-thirds, covered and autoclaved at 121 °C for 20 min. ! cautIon During autoclaving the flasks must not be tightly closed. The temperature of the autoclave has to be below 80 °C before it can be opened to prevent hot steam from emerging. Heat protective gloves and safety glasses have to be worn.

After cooling down to ~60 °C the medium is poured in sterile tubes, each with ~6 ml under aseptic conditions. The prepared flasks can be kept under sterile conditions for upto 1 week before inoculation. crItIcal All the autoclaved media have to be stored under aseptic conditions to prevent contamination with ubiquitous microbes from the environment.

proceDureIsolation and cultivation of fungi from marine organisms ● tIMInG 6–9 weeks�| Cut the samples (pieces of sponge tissue, algal biomass or mangrove leaves or other organs) into small segments of approximately 1 cm × 1 cm and rinse three times with sterile sea water to eliminate adherent surface debris. crItIcal step It is crucial to maintain aseptic conditions by working under a laminar air-flow for the isolation, purification, transfer and growth of the fungal cultures to prevent contamination by ubiquitous micro-organisms.

�| Immerse a piece of the sample in EtOH 70% (vol/vol) for 60–120 s for surface sterilization. crItIcal step If the treatment with EtOH is too short, the sterilization of the outer part is not complete; if the sterilization time is too long, EtOH kills fungi in the inner parts of the tissue.

3| Dry the piece of tissue with sterile cotton cloth (or rinse with sterile artificial sea water) to stop the sterilization with EtOH.

4| Streak the piece carefully over the surface of a first petri dish containing isolation medium A with sterilized tweezers, then put it back onto sterile cotton cloth and cut it into smaller segments with a sterile razor blade (negative control).

�| Place the small pieces on a second petri dish containing isolation medium A so that the freshly cut edges are in direct contact with the agar surface. Seal the agar dish with parafilm, label and store it at 20–25 °C.Cultures are kept between 20 °C and 25 °C under daylight. Fungal growth from the cut segments usually begins after 2–3 d until 14 d after the onset of experiment.? trouBlesHootInG

6 | VOL.4 NO.12 | 2009 | nature protocols

protocol

6| Usually different fungal strains will develop from one sample. Isolate the individual strains by transferring hyphal tips growing out of the cut tissue pieces with a sterile loop onto a fresh petri dish containing medium B. crItIcal step For purification of the fungal strains this step might be repeated several times until the colony is deemed pure following macroscopic and microscopic analysis.

Depending on the culture condition and different fungal strains, growth can be observed after 2–3 d. Pure strains must grow for ~1–2 weeks before further workup.

7| Submit pure fungal strains for taxonomic identification as described in Box �.

8| For small-scale and large-scale fermentation, inoculate a pure fungal strain in a 1,000 ml Erlenmeyer flask containing either 300 ml of liquid Wickerham’s medium or 210 g of solid rice medium. For this purpose, cut a strain that covers the surface of the inoculated petri dish into small pieces of ~1.5 cm × 1.5 cm and transfer these pieces with a sterile loop into an Erlenmeyer flask containing the sterilized medium.

Cultivation is carried out at room temperature under static conditions and daylight. Depending on the fungal growth, cultures on liquid medium are incubated for 3–4 weeks, while on rice for 4–6 weeks.

9| Bring the fermentation to an end by adding 250 ml EtOAc to the culture flask and leave the flask closed for at least 24 h. EtOAc will increase the wettability of the spores and decrease the number of spores, which will get airborne once the flask is opened.! cautIon To prevent the distribution of fungal fragments or spores, flasks should only be opened under a laminar air-flow.

�0| For long-term storage, transfer the pure fungal strains to 10 ml BD Falcon tubes containing ~5 ml MexA medium. When growth can be observed (after ~3 d, depending on the fungal strain), freeze the fungal strain stepwise: first place it in a fridge at 4 °C for 2 h, then in a freezer at − 20 °C for 2 h and finally place it in a deep freezer at − 80 °C. pause poInt Frozen pure fungal strains can be stored at − 80 °C.For recovery from − 80 °C, thaw the frozen culture quickly in a water bath at 37 °C and transfer a small piece with a sterile loop to a petri dish containing medium B.! cautIon To prevent contamination of the environment with viable material, equipment that has been in contact with fungal material must be autoclaved at 134 °C for 15 min before reuse or disposal.

extraction of small scale cultures ● tIMInG ~� d��| Extract the cultured material by following the steps in options (A) small-scale liquid medium (Fig. �); (B) small-scale rice medium (Fig. 4); (C) large-scale liquid medium (Fig. 3); or (D) large-scale rice medium (Fig. 4).(a) small-scale liquid medium (Fig. �) (i) Mix the content of the culture flask including the added 250 ml of EtOAc thoroughly in an Ultraturrax at 4,000 µ min − 1

for cell destruction and extraction for 10 min. (ii) Filter the mixture under vacuum using a Buchner funnel and discard the mycelium residue. (iii) Transfer the culture filtrate into a separation funnel. Separate the EtOAc and H2O phases and extract the aqueous phase

twice with 300 ml EtOAc each. Wash the combined EtOAc phases with 100 ml dem

. water to eliminate any remaining

sea salt. crItIcal step Each step of the extraction should be carried out with care under a fume hood. Complete separation of the two immiscible liquid phases should be achieved before continuing. For optimal extraction of the fungal biomass, usually three cycles are sufficient. However, if the EtOAc phase is still colored after the third extraction, further exhaustive extraction with EtOAc should follow until the color fades. ! cautIon To prevent contamination of the environment with viable material, an antimicrobial detergent (Melsept SF) has to be added to the medium and to the disrupted fungal cells and kept in it for at least 1 h before disposal. Auto-claving is not possible at this stage because of the remaining traces of EtOAc that may cause explosion in the autoclave.

(B) small-scale rice medium (Fig. 4) (i) Cut the culture medium containing the mycelium into small pieces to allow exhaustive extraction with EtOAc.

For optimum extraction the mycelium with added extraction solvent should be kept on a shaker for 1 d. (ii) Filter the contents under vacuum using a Buchner funnel and repeat the extraction with EtOAc until exhaustion. (iii) Wash the combined EtOAc phases with 300 ml water to eliminate remaining sugar and starch.

crItIcal step The aqueous and EtOAc phases should be left in a separation funnel until complete separation of the two immiscible liquid phases is achieved. For optimal extraction of the fungal biomass, usually three cycles are sufficient. However, if the EtOAc phase is still colored after the third extraction, further exhaustive extraction with EtOAc should follow until the color fades.

Q4Q4

nature protocols | VOL.4 NO.12 | 2009 | 7

protocol

! cautIon To prevent contamination of the environment with viable material, an antimicrobial detergent (Melsept SF) has to be added to the medium and to the disrupted fungal cells and kept in it for at least 1 h before disposal. Autoclaving is not possible at this stage because of the remaining traces of EtOAc that may cause explosion in the autoclave.(c) large-scale liquid medium (Fig. 3) (i) Separate fungal mycelia from the culture media and cover the mycelia with ~5 liters of MeOH.

! cautIon To prevent the distribution of fungal fragments or spores, flasks are only opened under a laminar air-flow. The mycelia are left in MeOH overnight.

(ii) Disrupt and extract the cells for 10 min at 4000 µ min − 1 using an Ultraturrax. (iii) Filter the mixture under vacuum using a Buchner funnel. (iv) Repeat extraction in the same manner until exhaustion (2–3 times). (v) Extract the culture media in the same manner as described for the extraction of small-scale cultures to obtain the

EtOAc extract. ! cautIon To prevent contamination of the environment with viable material, antimicrobial detergent has to be added to the extracted mycelia and growth medium and kept in it for at least 1 h before disposal. Autoclaving of the residues is not possible because of the remaining solvent.

(D) large-scale rice medium (Fig. 4) (i) The extraction of the large-scale rice media is carried out in the same manner as described for the small-scale cultures

using ~10 liters of EtOAc. ? trouBlesHootInG

Workup of obtained crude extracts ● tIMInG several days–weeks��| Workup of the crude extracts following the steps in option (A) for small–scale extracts and option (B) for large-scale extracts, respectively.(a) small-scale extracts (i) Dry the EtOAc extract (either from rice or liquid culture) under vacuum (~200 mbar) using a rotary evaporator at

40 °C to give a solid or oily residue. Depending on the amount of EtOAc used and on the size of the round-bottom flask this step takes about 40 min liter − 1 of EtOAc. pause poInt The dried extract can be stored in the deep freezer until further workup.

(ii) Partition the residue between n-hexane and 90% (vol/vol) aqueous MeOH in a ratio of 1:1 (vol/vol) (~150 ml each). crItIcal step The solvent fractionation should be carried out with care under a fume hood until complete separation of the two immiscible liquid phases is achieved.

(iii) Dry each fraction under vacuum (~200 mbar) using a rotary evaporator at 40 °C to give an oily or solid residue. (iv) Submit all fractions to TLC, analytical HPLC, LC–MS and also to bioactivity assays (see Box 3, ref. 41). (v) Further investigation depends on the results of the chemical and biological screening and on the taxonomic identity

of the fungus. If the fungus is non-pathogenic and if the extract displays promising activity in bioactivity screening, cultivate the fungal strain on a large-scale basis either in 20 liters of liquid Wickerham’s medium (67 Erlenmeyer flasks) or on 4.2 kg solid rice medium (20 flasks). From these large-scale fermentations, usually an amount of crude extract sufficient for the subsequent isolation and structural identification of pure secondary metabolites can be obtained.

(B) large-scale extracts (i) Dry the EtOAc or the MeOH extracts (either from rice or liquid culture) under vacuum (~200 mbar) using a rotary

evaporator at 40 °C to give solid or oily residues. Depending on the volume of EtOAc used and on the size of the round flask this step takes about 40 min liter − 1 of the solvent. pause poInt The dried extract can be stored in the deep freezer until further workup.

(ii) Partition each residue between n-hexane and 90% (vol/vol) MeOH in a ratio of 1:1 (vol/vol), using as little solvent as possible. crItIcal step The solvent fractionations should be carried out with care under a fume hood. Complete separation of the two immiscible liquid phases should be achieved before continuing.

(iii) Submit all fractions to TLC, analytical HPLC, LC–MS and also bioactivity assays such as the agar-diffusion assay or brine shrimp assay described in Box � or the MTT assay described in reference 41. crItIcal step Based on the obtained results, the success of the solvent fractionation can be monitored easily by differences in both bioactivities and HPLC/TLC profiles of the different fractions.

�3| In accordance with the diverse properties of the components of the fractions, two different procedures for purification can be applied:

8 | VOL.4 NO.12 | 2009 | nature protocols

protocol

For low- or medium-polar compounds refer to option (A), for polar compounds refer to option (B).(a) low- or medium-polarity fractions (i) Fractions containing low- or medium-polar compounds are further fractionated and purified using MPLC methods like

VLC. Then, further purification proceeds by column chromatography (CC) using either normal or reversed stationary phase and a suitable mobile phase to elute the components. Refer to reference 41 for advice on the choice of stationary phase and how to setup the experiment.

(B) polar fractions (i) Highly polar fractions contain water-soluble organic compounds. In our experience, a good procedure is to use

reversed phase CC (e.g., RP-18) or ion-exchange resin beds such as HP-20 eluted gradually from water to MeOH, to eliminate the remaining mineral salts and sugars present in these fractions. Refer to REAGENT SETUP for further advice

.

Chromatographic methods are carried out as described in a previous protocol dealing with workup of extracts derived from marine macro-organisms41

.

? trouBlesHootInG

�4| Continue the purification procedures until compounds of sufficient purity is obtained to allow structural elucidation.

��| Structure elucidation is carried out using various spectroscopic methods, mainly MS and NMR (1 d and 2 d). For the elucidation of the absolute configuration of new chiral natural products, derivatization methods such as the preparation of Mosher esters (see reference C

) or by using Marfey’s method (see Box 3) are sometimes necessary (alternatively, the

absolute configuration may be elucidated by X-ray crystallography or by CD spectroscopy followed by quantum chemical calculations32).

�6| Once the individual components are isolated in pure form and structurally identified, they should be subjected to bioactivity testing.

�7| Study the structure–activity relationships to identify optimized compounds which may serve as drug leads from natural sources.

● tIMInG

Steps 1–5: 15–30 min per isolate (growth ~1–2 weeks)Step 6: 5 min per strainStep 7: see Box �Step 8: ~5 min per strain (growth ~3–5 weeks)Steps 9 and 10: each ~5 minStep 11 A(i–iii): 45–60 minStep 11 B/D(i): 5/30 min (plus 1 d for extraction)

Q5Q5

Q6Q6

Q7Q7

Q8Q8

Box 3 | DERIVATIZATIoN METHoDS Determination of absolute stereochemistry by Mosher reaction has been described in detail in a protocol on isolation and structural elucidation of metabolites from marine invertebrates38,41.Determination of the absolute configuration of amino acids by Marfey’s analysisMarfey’s reagent (FDAA = Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) is used as a reagent for derivatization of D- and L-amino acids that are obtained after hydrolysis of cyclic or linear peptides to determine their absolute configuration. The obtained diastereoisomers can be easily differentiated and identified by their retention times following HPLC analysis on RP columns and comparison with commercially available D- and L-amino acids that have been treated in the same way47,49.1. Mix 50 µl of 50 mM of each commercially available standard amino acid (D- or L-form) that is of interest in H2O, 20 µl 1 M NaHCO3 and 100 µl of 1% Marfey’s reagent in acetone and heat at 40 °C for 1 h.2. Stop the reaction by addition of 10 µl of 2 M HCl.3. Freeze the derivatized product to − 80 °C, dry it in a freeze dryer, redissolve it in MeOH and analyze it by HPLC or by LC–MS.4. Hydrolyze your isolated peptide (0.5–1 mg) in 1–2 ml 6 N-HCl at 110 °C for 24 h under N2 atmosphere until complete hydrolysis and liberation of the amino acids.5. Cool the hydrolysate containing the mixture of free amino acids, dry it and redissolve it in water to achieve a final concentration of ~50 µM. Proceed in the same way as applied to standard amino acids (see 1–3

).

6. Compare the retention times (HPLC or LC–MS) of the derivatized standard amino acids and of the derivatized amino acids obtained following hydrolysis of the peptide to distinguish D- and L-amino acids.

Q11Q11

nature protocols | VOL.4 NO.12 | 2009 | 9

protocol

Step 11 B/D(ii): ~10 min ( plus 1 d for extraction) (twice)Step 11 B/D(iii): ~10 minStep 11 C(i): ~5 min (plus extraction overnight)Step 11 C(i–iv): 20–30 min (2–3 times)Step 11 C(v): 2–3 hSteps 13–17: The timing of these steps is dependent on chosen columns, complexity of the extract and the kind of isolated compounds.See also Figures �–4.

? trouBlesHootInGTroubleshooting advice can be found in table �.

antIcIpateD resultsExamples taken from our group that illustrate the applica-tion of the described procedures for the isolation of sponge- and mangrove-derived fungi and the subsequent isolation and structure determination of bioactive compounds will be described.

anthraquinones and betaenones (Fig. �)Two new betaenone derivatives (�,�) and three new 1,3,6,8-tetrahydroxy anthraquinone congeners (3–�) were obtained from the fungus Microsphaeropsis sp. isolated from the inner parts of the marine sponge Aplysina aerophoba32.

The EtOAc extract of the fungus that had been grown in liquid medium (including 2.4% (wt/vol) of artificial sea salt) under static conditions at room temperature for 41 d, was evaporated under reduced pressure. The residue was partitioned between EtOAc (4 × 400 ml) and H2O (400 ml) to eliminate the remaining salt. The organic phase was taken to dryness and subjected to VLC on silica gel using step gradient employing CH2Cl2 and MeOH (100:0, 98:2, 95:5, 90:10, 80:20 and 30:70 vol/vol) as solvent systems.

taBle � | Troubleshooting table.

step problem possible reason solution

4 Fungal growth on negative controls

Insufficient surface sterilization If you are not sure how delicate the surface of your source organism is, then try different times for surface sterilization, e.g. 30, 60 and 120 s. The plates with growth on the respec-tive negative controls or those without any growth can be discarded

6 No growth at all Time of surface sterilization too

long,

fungi residing in the tissue are killed

11 Insufficient separation of the two phases

Formation of an emulsion because of extracted compounds

Leave standing overnight in the separation funnel; centrifu-gation also helps

13 No or insufficient separation

Elution of the column is too fast Inappropriate stationary or mobile phases Column too small

Reduce the speed of the mobile phase (for Sephadex material ~10 drops min − 1, silica < 20 drops min − 1, depending on the size of the column) Always test on TLC to check stationary and mobile phase before column chromatography Combine all fractions again and find a better separation system

13, 14 Loss of substance Adsorption on stationary phases Always keep some of your fraction to be able to repeat separation steps

Q20Q20

O

O

H

HO OHOH

H

O

N

O

H

HO OHOH

H

O

O

O

OH

OH

OH

HOO

O

OH

OH

OH

HO

O

O

OH

OH

OH

HO

HO

OH

O

O

Figure � | New betaenones and anthaquinones from the sponge-derived fungus Microsphaeropsis sp.

�0 | VOL.4 NO.12 | 2009 | nature protocols

protocol

Fractions 3 and 4 from the obtained ten fractions were pooled and chromatographed over a Sephadex LH-20 column with MeOH as eluent. From the obtained 17 fractions, fractions 6 and 7 contained the betaenone congeners, whereas the anthraquinone derivatives were obtained from the yellow colored fractions 11, 12 and 13.

Purification of the betaenones was accomplished by chromatography on an RP-18 column. 10-Hydroxy-18-methoxybetaenone (�) was obtained from fraction 7 with MeOH:H2O:TFA (95:5:0.1 vol/vol) as eluent and 10-hydroxy-18-N-2-naphthyl-N-phenyl-aminobetaenone (�) from fraction 6 with MeOH:H2O:TFA (90:10:0.1 vol/vol) as eluent.

The anthraquinone congeners were purified by semi-preparative HPLC on a C18 column using a gradient of H2O and MeOH as follows: 0 min, 80% MeOH (vol/vol); 20 min, 90% MeOH (vol/vol); 25 min, 90% MeOH (vol/vol); and 30 min, 100% MeOH (vol/vol).

All compounds were readily identified by their spectroscopic data. Through-bond homonuclear and heteronuclear correla-tions were used to establish assignments and atom connectivities. The relative stereochemistry of 10-hydroxy-18-methoxy-betaenone (�) was determined from a ROESY experiment.

As similar metabolites had previously been described as inhibitors of protein kinases, which are of particular interest as targets for the development of novel anticancer drugs, the isolated compounds were tested for inhibition of protein kinases in vitro.

For a first assessment of the kinase inhibitory potential, an isoenzyme of the protein kinase family C, the cyclin-dependent kinase 4 in complex with its activator cyclin D1 and the tyrosine kinase domain of the epidermal growth factor receptor were selected.

Of the two obtained betaenone derivatives only 10-hydroxy-18-methoxybetaenone (�) inhibited all three kinases (with ED50 values of 36.0, 11.5 and 10.5 µM, respectively), whereas 10-hydroxy-18-N-2-naphthyl-N-phenylaminobetaenone (�) showed no inhibitory effect, thus suggesting the enone side chain as a promising position for further optimization of such compounds towards higher potency and better selectivity.

alternaria metabolites (Fig. 6)From the endophyte Alternaria sp. isolated from leaves of the Chinese mangrove plant Sonneratia alba, several natural products, some of them structurally related to alternariol (6–��), perylene quinones and new compounds (��–�7) were isolated40.

The crude EtOAc extract obtained after fermentation of the fungus on solid rice medium was partitioned between n-hexane and 90% (vol/vol) aqueous MeOH. After VLC of the MeOH extract using silica gel as stationary phase and a step gradient employing CH2Cl2 and MeOH as solvent systems, fraction 3 (80% (vol/vol) CH2Cl2) was chromatographed over silica gel using CH2Cl2 and MeOH (90:10 vol/vol) as eluent mixture. Fractions 3 and 4 were combined and re-chromato-graphed by normal-phase VLC using a step gradient of CH2Cl2 and MeOH as eluent. Fraction 5 was further purified using a RP-18 column and H2O:MeOH (7:3 vol/vol) as eluent. Fraction 3 yielded altenusin (6), fraction 5 gave altenuene (7), 4′-epialtenuene (8) and 2,5-dimethyl-7-hydroxychromone (9) after semi-preparative HPLC using an RP-18 column with MeOH:H2O (25:75 vol/vol) as eluent. Alternariol (�0) and altertoxin I (�3) were obtained from fraction 7 and final purification was carried out by semi-preparative HPLC using a RP-18 column with MeOH:H2O (35:65 vol/vol) as eluent.

The new metabolite alternarienonic acid (��) was obtained from the com-bined VLC fractions 5 and 6 following purification by Sephadex LH-20 CC with MeOH as eluent.

OO

OH

HO

OH

OO

OH

HO

O

OO

HO

HO

OH OO

HO

HO

O

OH

O

O

HO

OH

OOH

O

OHOH

OH O

OHO

HO

OHHH

HOOC OHHO

HO

O

COOHOH

OO

HO

OH O

OOH

OHOH

OOH

O

OHCOOH

OOH

O

OH

COOH

OH

OH

Figure 6 | Natural products from the mangrove-derived fungus Alternaria sp.

nature protocols | VOL.4 NO.12 | 2009 | ��

protocol

Fraction 5 of the VLC separation yielded the new natural products xanalteric acid I (�4) and II (��), which were purified over silica gel using CH2Cl2 and MeOH as solvent system and subsequent semi-preparative HPLC with MeOH and H2O as eluent.

Upon fermentation for 3 weeks in liquid Wickerham’s medium, compounds 6–8, �0, �3, �4 and �� were likewise detected as constituents of the crude extract using HPLC, whereas 9 and �� were missing. In addition, the known compounds alternariol-5-O-methylether (��) and the perylene derivatives stemphyperylenol (�6) and alterperylenol (�7) were also obtained after fractionation of the extract by VLC using silica gel as stationary phase followed by semi-preparative HPLC, thus confirming the ‘OSMAC’ concept that a given microbial strain can yield different metabolites upon varying fermentation conditions.

Both crude EtOAc extracts (obtained following fermentation on solid medium or in liquid medium) exhibited promising antiproliferative activities as detected by the MTT assay. Alternariol (�0) and altenusin (6) exhibited strong antiprolifera-tive activities against the murine L5178Y cell line with EC50 values of 1.7 µg ml − 1 and 6.8 µg ml − 1, respectively, whereas the new metabolites epialtenuene (8), alternarienonic acid (��), xanalteric acid I (�4) and II (��), as well as altenuene (7), altertoxin I (�3) and 2,5-dimethyl-7-hydroxychromone (9) were inactive in this bioassay38.

acknoWleDGMents We are indebted to numerous previous coworkers and to several colleagues who were indispensable for our studies on marine-derived fungi. Continued support by BMBF to P.P. is gratefully acknowledged.

autHor contrIButIons

Published online at http://www.natureprotocols.com/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/.

1. Grove, J.F., MacMillan, J., Mulholland, T.P.C. & Rogers, M.A.T. Griseofulvin part I. J. Chem. Soc. 3949–3958 (1952).

2. Butler, M.S. The role of natural product chemistry in drug discovery. J. Nat. Prod. 67, 2141–2153 (2004).

3. Abramovits, W., Gupta, A. & Gover, M. Altabax (retapamulin ointment), 1%. Skinmed 6, 239–240 (2007).

4. Dewick, P.M. Medicinal Natural Products. A Biosynthetic Approach. (John Wiley & Sons Ltd., Baffins Lane, Chichester, UK, 2006

).

5. Balba, H. Review of strobilurine fungicide chemicals. J. Environ. Sci. Health B. 4�, 441–445 (2007).

6. Bugni, T.S. & Ireland, C.M. Marine-derived fungi: a chemically and biologically diverse group of micro-organisms review. Nat. Prod. Rep. ��, 143–163 (2004).

7. König, G.M. et al. Natural products from marine organisms and their associated microbes. Chembiochem 7, 229–238 (2006).

8. Kohlmeyer, J. & Kohlmeyer, E. in Marine Mycology, The higher fungi (Academic

Press; New York, San Francisco, London, ) 54–69 (1979).

9. Schulz, B. in Bioactive Fungal Metabolites–Impact and Exploitation, 20 (British Mycological Society,

International Symposium Proceedings:

University of Wales, Swansea, UK, 2001).10. Baker, D. & Alvi, K. Small-molecule natural products: new structures, new

activities. Curr. Opin. Biotechnol. ��, 576–583 (2004).11. Pietra, F. Secondary metabolites from marine microorganisms: bacteria,

protozoa, algae and fungi. Achievements and prospects. Nat. Prod. Rep. �4, 453–464 (1997).

12. Blunt, J.W. et al. Marine natural products. Nat. Prod. Rep. ��, 35–94 (2008).

13. Faulkner, D.J. Highlights of marine natural products chemistry (1972–1999). Nat. Prod. Rep. �7, 1–6 (2000a).

14. Zhang, H.W., Song, Y.C. & Tan, R.X. Biology and chemistry of endophytes. Nat. Prod. Rep. �3, 753–771, Erratum 828–830 (2006).

15. Mayer, A. & Gustafson, K. Marine pharmacology in 2005–2006: antitumor and cytotoxic compounds. Eur. J. Cancer (2008

).

16. Bernan, V.S., Greenstein, M. & Maiese, W.M. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43, 57 (1997).

17. Höller, U. et al. Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol. Res. �04, 1354–1365 (2000).

18. Jensen, P.R. & Fenical, W. Marine microorganisms and drug discovery: current status and future potential. In Drugs from the Sea (Karger Publishers, Basel) 6–29 (2000).

19. Jensen, P.R. & Fenical, W. Secondary metabolites from marine fungi. In Fungi in Marine Environments (ed. Hyde, K.D.) (Fungal Diversity Press, Hong Kong) (2002

).

20. Koenig, G.M. & Wright, A. D. trends in marine biotechnology. In Drug Discovery from Nature (eds. Grabley, S & Thiericke, R), (Springer Verlag. Berlin) 180–187 (1999).

21. Verbist, J.F., Sallenave, C. & Pouchus, Y.F. Stud. Nat. Prod. Chem. �4, 979 (2000).

22. Ebel, R. Secondary metabolites from marine derived fungi. In Frontiers in Marine Biotechnology (eds. Proksch, P. & Müller, W.E.G.) (Horizon Scientific Press, Norwich,) 73–143 (2006).

23. Laatsch, H. Marine bacterial metabolites. In Frontiers in Marine Biotechnology (eds. Proksch, P. & Müller, W.E.G.) (Horizon Scientific Press, Norwich, 2006

).

24. Gerwick, W. The secondary metabolites and biosynthetic gene clusters of marine cyanobacteria. In Frontiers in Marine Biotechnology (eds. Proksch, P. & Müller, W. E. G.), (Horizon Scientific

Press, Norwich) 179–224

(2006).25. Shimizu, Y. & Li, B. Microalgae: special problems and methology.

In Frontiers in Marine Biotechnology (eds. Proksch, P. & Müller, W.E.G.) (Horizon Scientific Press, Norwich) (2006

).

26. Newton, G.G.F. & Abraham, E.P. Cephalosporine C, a new antibiotic containing sulphur and D-α-aminoadipic acid. Nature �7�, 548 (1955).

27. Dalla Bona, A. et al. Synthesis, conformation, and bioactivity of novel analogues of the antiviral lipopeptide halovir A. J. Pept. Sci. ��, 748–757 (2006).

28. Bringmann, G. et al. Large-scale biotechnological production of the anti-leukemic marine natural product sorbicillactone A. Mar. Drugs �, 23–30 (2007).

29. Blunt, J.W. et al. Marine natural products. Nat. Prod. Rep. �4, 31–86 (2007).

30. Jones, G.E.B., Stanley, S.J. & Pinruan, U. Marine endophyte sources of new chemical natural products: a review. Botanica Marina ��, 163–170 (2008).

31. Schulz, B. et al. Screening strategies for obtained novel, biologically active, fungal secondary metabolites from marine habitats. Botanica Marina ��, 219–234 (2008).

32. Brauers, G. et al. Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis sp.: novel inhibitors of protein kinases. J. Nat. Prod. 63, 739–745 (2000).

33. Lin, W. et al. Novel chromone derivatives from the fungus Aspergillus versicolor isolated from the marine sponge Xestospongia exigua. J. Nat. Prod. 66, 57–61 (2003).

34. Hallermann, J., Berg, G. & Schulz, B. Isolation procedures for endophytic microorganisms. In Microbial Root Endophytes (Soil Biology) (eds. Schulz, B., Boyle, C. and Sieber, T.N.) (Springer

Verlag Berlin Heidelberg)

299–328 (2006

).

35. Klemke, C., Kehraus, S., Wright, A.D. & Koenig, G.M. New secondary metabolites from the marine endophytic fungus Apiospora montagnei. J. Nat. Prod. 67, 1058–1063 (2004).

36. Gao, Z., Li, B., Zheng, C. & Wang, G. Molecular detection of fungal communities in the Hawaiian marine sponges Suberites zeteki and Mycale armata. Appl. Environ. Microbiol. 74, 6091–6101 (2008).

37. Bode, H.B., Bethe, B., Höfs, R. & Zeeck, A. Big effects from small changes: possible ways to explore nature′s chemical diversity. Chembiochem 3, 619–627 (2002).

Q9Q9

Q12Q12

Q21Q21

Q22Q22

Q13Q13

Q14Q14

Q15Q15

Q23Q23

Q16Q16

Q24Q24Q17Q17

Q23

Q23

�� | VOL.4 NO.12 | 2009 | nature protocols

protocol38. Aly, A.H. et al. Cytotoxic metabolites from the fungal endophyte Alternaria

sp. and their subsequent detection in its host plant Polygonum senegalense. J. Nat. Prod. 7�, 972–980 (2008).

39. Wang, S. et al. Chaetopyranin, a benzaldehyde derivative, and other related metabolites from Chaetomium globosum, an endophytic fungus derived from the marine red alga Polysiphonia urceolata. J. Nat. Prod. 69, 1622–1625 (2006).

40. Kjer, J. Xanalteric acids I and II and related phenolic compounds from an endophytic Alternaria sp. isolated from the mangrove plant Sonneratia alba. in press

.

41. Ebada, S.S., Edrada, R.A., Lin, W. & Proksch, P. Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates. Nat. Protoc. 3, 1820–1831 (2008).

42. Xu, J. et al. Cytosporones, coumarins, and an alkaloid from the endophytic fungus Pestalotiopsis sp. isolated from the Chinese mangrove plant Rhizophora mucronata. Bioorg. Med. Chem. �7, 7362–7367 (2009).

Q18Q18

43. Jadulco, R. et al. New macrolides and furan carboxylic acid derivative from the sponge-derived fungus Cladosporium herbarum. J. Nat. Prod. 64, 527–530 (2001).

44. Proksch, P. et al. Sponge-associated fungi and their bioactive compounds: the Suberites case. Botanica Marina ��, 209–218 (2008).

45. Technische Regeln für biologische Arbeitsstoffe. TRBA 460: Einstufung von Pilzen in Risikogruppen. BArbBl. Heft �0, 78–84 (2002).

46. Sichere Biotechnologie. Classification of biological agents: fungi. Merkblatt B007e (2003

).

47. Ashour, M. et al. Kahalalide derivatives from the Indian sacoglossan mollusk Elysia grandifolia. J. Nat. Prod. 69, 1547–1553 (2006).

48. Meyer, B.N. et al. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 4�, 31 (1982).

49. Marfey, P. Determination of D-amino acids. Part II: use of a bifunctional reagent 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591–596 (1984).

Q19Q19

nature protocols | VOL.4 NO.12 | 2009 | �3

Nature Protocol

Manuscript ID

Author

Editor

Publisher

QUERY FORM

Query No. Nature of Query

AUTHOR:

The following queries have arisen during the editing of your manuscript. Please answer queries by making the requisite cor-rections directly on the galley proof. It is also imperative that you include a typewritten list of all corrections and comments, as handwritten corrections sometimes cannot be read or are easily missed. Please verify receipt of proofs via e-mail

Nature Publishing Group

AQ1 Please specify where is it described?

AQ2 Please provide the name of the company for MixerMill MM30.

AQ3 Please check as to what does ‘ad 1000 g’ mean in tables 2, 3, 4 and 6.

AQ4 Please confirm whether dem. water stands for demineralized water in step iii of option A.

AQ5 Please check that this is still correct?

AQ6 Okay to insert here??

AQ7 Please check whether it is option C or is it a reference to box 3. Please check and confirm.

AQ8 Please check the timing provided in the TIMING section is not in accordance with that provided in the PROCEDURE section along with the subheadings for the step numbers indicated.

AQ9 Please supply the author contributions.

AQ10 Please provide the name of the city, state (if applicable) and country for Genbank.

AQ11 Please check Box 3 ‘see 1–3’ of what?

AQ12 Please provide the title and page range in reference 4.

AQ13 Please provide the updates in reference 15.

AQ14 Please provide the page range in reference 19.

AQ15 Please provide the page range in reference 23.

�4 | VOL.4 NO.12 | 2009 | nature protocols

Nature Protocol

Manuscript ID

Author

Editor

Publisher

QUERY FORM

Query No. Nature of Query

AUTHOR:

The following queries have arisen during the editing of your manuscript. Please answer queries by making the requisite cor-rections directly on the galley proof. It is also imperative that you include a typewritten list of all corrections and comments, as handwritten corrections sometimes cannot be read or are easily missed. Please verify receipt of proofs via e-mail

Nature Publishing Group

AQ16 Please provide the page range in reference 25.

AQ17 Please provide the place of the publisher in reference 34.

AQ18 Please provide the updates in reference 40.

AQ19 Please provide the updates in reference 46.

AQ20 Solution for the problem?

AQ21 Please provide the title of chapter/article, location as a single city.

AQ22 Please provide title of the chapter/article and page range.

AQ23 Please provide the country details for Refs. 22, 23 and 24.

AQ24 Please provide the single city, country of publisher.