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protocol

Methods for isolation of marine-derived endophytic


fungi and their bioactive secondary products
Julia 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 18 February 2010; 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
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

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.

INTRODUCTION
Terrestrial fungi have long been known as a rich source of bio- micro-organisms including bacteria, cyanobacteria, microalgae
logically active secondary metabolites. Since the discovery of and fungi have become an important source of new pharma-
penicillin by Sir Alexander Fleming in 1928, which resulted in cologically active metabolites. Recent reviews demonstrate the
a breakthrough in the treatment of bacterial infections, fungi importance of these organisms as potential sources of pharma-
have become an important source of drugs for the treatment of a ceutical leads6–25. Especially, marine fungi have shown promis-
variety of diseases. Beside other well-known antimicrobial agents ing potential as sources of new and bioactive natural products as
of fungal origin like fusidic acid and griseofulvin1, new semi­ suggested by the chemical diversity of their secondary meta­
synthetic antifungal drugs like anidulafungin (Eraxis), caspafun- bolites6, even though sample collection and preparation as the
gin (Cancidas) and retapamulin (Altabax) are likewise derived first step might be more difficult for marine-derived fungi than
from fungal metabolites2,3. With the discovery of cyclosporine for terrestrial material because of difficulties inherent to the
isolated from Tolypocladium inflatum in 1971 an important step in collection in marine environment, such as the need for scuba
immunopharmacology was taken, and improvements in the field diving. Although the most famous group of bioactive compounds
of organ transplantation and treatment of autoimmune diseases obtained from marine-derived fungi are still the cephalosporines
are still in progress, like the introduction of substances such as with cephalosporine C, first isolated by G. Brotzu in 1945 from
the fungal-derived mycophenolic acid (Myfortic) to the market a marine strain of Acremonium chrysogenum26, there are also
in 2004 (ref. 2). more recent promising examples. These include halovir and
Further pharmacologically important fungal metabolites include several naturally occurring analogs, which are potent inhibitors
antilipidemic drugs collectively known as ‘statins’ with their parent of Herpes simplex viruses 1 and 2. Synthetic studies that were
compounds mevastatin and lovastatin isolated from Penicillium carried out for these substances allowed insight into structure–
citrinum and Aspergillus terreus, respectively. Statins reduce blood activity relationships22,27. The new alkaloid sorbicillactone A
cholesterol levels and are used for the treatment of coronary isolated from the sponge-derived fungus Penicillium chrysoge-
diseases2,4. num showed promising activity against leukemia cells without
Fungal metabolites are, however, not only indispensable for exhibiting notable cytotoxicity. Large-scale fermentation of
medicine but are also important for plant protection, as demon- the fungus yielded sufficient amounts of the compound for
strated by the discovery of the strobilurines that were first isolated preclinical investigations22,28. Overall, research on marine-
from Strobilurus sp. and served as lead compounds for synthetic derived fungi has led to the discovery of more than 270 new
fungicidals such as trifloxystrobin (Flint)5. natural products mainly from the early 1990s to mid 2002
However, ‘the rediscovery of high numbers of previously (with more than two-thirds of these compounds isolated from
described metabolites has to some extent precluded the study of sponge- and plant-derived fungi), whereas more than 330 new
traditional terrestrial sources of fungi’6 and recently, interest of metabolites were reported only in the 5-year period between 2002
natural product chemists and pharmacologists alike has turned and 2006 (refs. 6,12,22,29), including numerous compounds with
to so far less investigated habitats and ecological niches such as new carbon skeletons. This sharp rise in numbers indicates
the oceans. The oceans cover nearly three-quarters of the earth’s a growing interest in marine-derived fungi as sources of new
surface and can be considered a ‘soup’ of essentially all imaginable bioactive metabolites.
types of microbes6,7. Ecological niches, e.g. deep-sea hydro­thermal The aim of this paper is to provide a detailed protocol on
vents, mangrove forests, algae and sponges, provide distinct the purification and cultivation of fungi from various selected
habitats for the isolation of specific micro-organisms 8. Marine marine macro-organisms (sponges, algae and mangrove plants),

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protocol
Host organisms
Small-scale fermentation
in liquid medium 3 Weeks

+EtOAc Homogenization and


1–5: 1 week 1–5: Isolation of fungi Several min
UltraTurrax extraction

Cell suspension

6: 2 weeks 6: Purification Filter Several min


on malt agar
7: Taxonomic identification
(by PCR and gene sequencing, Filtrate
7: 2 d Pure culture followed by BLAST search)

Several hours

Water phase EtOAc


© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

8: Small-scale
Based on chemical
fermentation on rice or
screening/bioactivity results in large- Evaporate
in liquid medium for
scale fermentation of promising strains
screening purpose
8: Each 3– Residue ~1 h per l EtOAc
4 weeks
90% MeOH
+ n-Hexane
~1 h
8: Large-scale
9–11: 1–3 d 9–11: Extraction with EtOAc fermentation
90% MeOH n-Hexane
12–14: Fractionation and
isolation of secondary products
Figure 2 | Estimation of the time required for fermentation of fungi and
workup of extracts from liquid medium on a small-scale basis.
16: Bioassays
(cytotoxicity and antimicrobial activity)

12–16: At least
2–4 weeks limited and selective window of the actual diversity that is present
15: Structure elucidation
in a given sample36. Furthermore, it must be remembered that
Figure 1 | Schematic overview on important steps involved in the isolation different culture conditions might also result in different chemi-
of fungi from marine sources and in the isolation and identification of their cal patterns of metabolites formed, as already stated, in the
secondary metabolites.
OSMAC (‘one strain, many compounds’) concept that makes
deliberate use of the chemical plasticity of microorganisms as
a response to varying conditions of culture 37. However, after
as well as on the isolation, characterization and structure eluci­ conducting numerous fermentation experiments with endo-
dation of biologically active secondary metabolites produced phytic fungi using different culture conditions, we discovered
by these fungi when grown in liquid or on solid media. An the methods described in this protocol were most suitable for
overview including an approximate time schedule is shown our purposes, as they generally gave reproducible results regard-
in Figure 1. ing the isolation of endophytic fungi and regarding the patterns
In this protocol we describe the isolation of endophytic marine- of secondary metabolites that are produced by these fungi32,38–40.
derived fungi from inner parts of living tissues of marine inverte- In our hands, we have obtained optimum results when cultur-
brates, algae and mangrove plants. Endophytic fungi are referred to ing fungi on different solid or in liquid media at room tem-
as fungi that spend at least part of their lives inside healthy ­tissues perature (20–25 °C), followed by extraction of mycelia and
of host organisms regardless of whether the host is a plant or an growth media with organic solvents. Flow charts briefly describ-
animal30. For the isolation of marine-derived fungi, especially for ing the workup procedures of the respective fungal cultures
the isolation of fungi from sponges, alternative methods to those and the time needed for each step are shown in Figures 2–4.
described in this protocol are available. For example, instead of The crude extracts are then separated using various chromatographic
cutting pieces from surface-sterilized samples, it is also possible techniques, and the isolated bioactive compounds are analyzed
to drop water from a sponge on an agar slant or to excise an inner by HPLC–DAD for their purity followed by structure elucidation
piece of the tissue after thoroughly rinsing it with sterilized sea by MS and NMR using state of the art techniques. Chiral compounds
water without previous surface sterili­zation31–33. Likewise, several may be derivatized to determine their absolute configuration.
other media for successful isolation and fermentation of marine Chromatographic separation of fungal extracts usually proceeds
fungi, like potato dextrose agar, barley spelt solid substrate or solid guided by bioassays, thereby directing the isolation strategy
malt extract medium17,31 as well as the addition of specific nutri- towards the discovery of bioactive constituents. After the extraction
ents or of sub-lethal doses of fungicides to restrict fast-growing of cultures, the obtained extracts are treated in a similar manner
fungi34, have been described in the literature. Numerous fermen- as described earlier for extracts from marine macro-organisms.
tation conditions, such as shaking versus static cultures, applica- As the chromatographic separation, structure elucidation and
tion of different temperature regimes or employment of larger bioactivity screening for extracts derived from marine macro-
fermenters that can provide varying aeration17,31,35, have likewise organisms have been described in detail in a previous protocol41,
been reported. However, all conditions employed in cultivat- only those steps that differ from the described procedure are
ing marine-derived endophytic fungi suffer from limitations depicted in this protocol.
as different cultivation techniques usually select only a small Two examples taken from our group that focus on polyketides
fraction of the fungal community present, thereby providing a of fungal origin will be used to highlight the structural variability

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protocol

Large-scale Fermentation
fermentation 3 Weeks on rice medium 4– 6 Weeks

+EtOAc Overnight + 30 min


Filter
Mycelium Medium
EtOAc extract
+MeOH
Several hours EtOAc Several hours
UltraTurrax Evaporate ~1 h per l EtOAc

Cell suspension Residue

~1 h 90% MeOH small scale, ~ 1 h


Filter
+ n-Hexane large scale, several hours
Water phase EtOAc
MeOH extract

~1 h Evaporate Evaporate ~1 h
90% MeOH n-Hexane
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

per l MeOH per l EtOAc


Residue Residue
Figure 4 | Estimation of the time required for fermentation and workup of
90% MeOH 90% MeOH
Several hours Several hours extracts from rice medium.
+ n-Hexane + n-Hexane

90% MeOH n-Hexane 90% MeOH n-Hexane


during workup. Taxonomic identification of fungal strains is
Figure 3 | Estimation of the time required for fermentation of fungi and achieved by DNA amplification and sequencing of the fungal ITS
workup of extracts from liquid medium on a large-scale basis. region39 as described in Box 1. However, fungi can also be identi-
fied by morphological characteristics using e.g., a commercially
of natural products obtained from marine-derived fungi. Other available service such as Centraalbureau voor Schimmelcultures
results can be found in refs. 33,38,39,42–44. (CBS), Utrecht, The Netherlands.

Experimental design Biological assays.  Biological activities of extracts, fractions


Sampling and general consideration before isolation of fungi.  and pure compounds are tested in a number of rapid ‘in house’
Transport the collected material in suitable containers (for bioassays that can be used for bioactivity-guided isolations.
sponges, algae or the like containing sea water). For animal and Those used in our laboratory are briefly described in Box 2.
algal tissues with high water contents, isolation of fungi must be
carried out within the next hours to avoid growth of ambient Controls.  Negative controls during the isolation of endophytic
bacteria. Isolation of fungi from plant material like mangrove fungi from marine organisms are necessary to detect contami-
leaves can take place within 1–2 d as long as the samples are nation from external parts of the slices, from phylloplane or
kept at low temperature (5–10 °C) and excess condensation from airborne micro-organisms. Therefore, surface-steri-
is prohibited. Otherwise there is the risk that phylloplane fungi lized marine samples are pressed onto a petri dish containing
will colonize the plant material leading to false results during the isolation medium before aseptic cutting and inoculation on
isolation of endophytes. a second agar plate. If fungal growth can be observed on the
negative control dishes, the positive plates are not used for
Identification of fungi.  As we restrict ourselves to cultivation further purification procedures that aim at the isolation of
of non-pathogenic fungi, any fungal cultures with documented true endophytic fungi.
pathogenicity45,46 towards humans are discarded immediately after For all bioassays, negative and positive controls to compare the
taxonomic identification and excluded from further investigation. effectiveness of the tested fractions and pure compounds have to
Thus, identification of the isolates is carried out at an early stage be used as described (Box 2).

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 C, http://www.ncbi.nlm.
nih.gov/Genbank/, National Institute of Biotechnology Information, Bethesda, Maryland, USA) with the help of Blast-Algorithmus.

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Box
  2 | BIOACTIVITY SCREENING TESTS
Agar diffusion assay
According 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 TESTS
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

Brine shrimp assay


Eggs 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.

MATERIALS
REAGENTS • Chromatographic stationary phases: Different materials from different
• Bacto agar (BD, cat. no. 214010) suppliers are possible, in our laboratory material is used as described in
• Malt extract (Merck, cat. no. 105391) a previous protocol41
• Artificial sea salt (Sera, cat. no. 05420) • Laminar air-flow (Herasafe HS15, Heraeus)
• Chloramphenicol (Sigma, cat. no. C0378) • pH meter (420 Aplus, Orion)
• Sodium hydroxide (‘NaOH’, Sigma, cat. no. S5881) • Autoclave (Varioklav, H&P)
• Hydrochloric acid (‘HCl’, Sigma, cat. no. 258148) • Microcentrifuge (Biofuge pico, Heraeus)
• Yeast extract (Fluka, cat. no. 70161) • Freeze dryer (Lyovac GT2, pump Trivac D10E, Savant)
• Peptone (Merck, cat. no. 111931) • PCR machine (iCycler, Bio-Rad)
• Glucose (Caelo, cat. no. 5247) • UV transluminator (GVM 20, Syngene)
• Rice (parboiled, any supermarket brand) • Mixer mill (MixerMill MM30 Retsch, Haan, Germany)
• Glycerin (Roth, cat. no. 4043) • Power supply for electrophoresis (PowerPac 300, Bio-Rad)
• Penicillin G (Sigma, cat. no. P3032) • Ultra Turrax (T18 basic, IKA)
• Streptomycin (Sigma, cat. no. S6501) • Vacuum pump (type 4EKF 56CX-4, Greiffenberger Antriebstechnik)
• Gentamycin (Serva, cat. no. 22185) • Nitrogen generator (UHPN 3001, Nitrox)
• Nystatin (Sigma, cat. no. N4014) • Deep freezer ( − 80 °C, 86-Freezer, Forma Scientific)
• Antimicrobial detergent Melsept SF (B. Braun, cat. no. 18907) • Shaker SM 30 A (Edmund Bühler, cat. no. 6101 000)
• Tungsten carbide bead (Qiagen, cat. no. 69997) • Balance (BL1500, Sartorius)
• Agarose (Sigma, cat. no. A9414) • HPLC, MS and NMR: the equipment as described in a previous protocol is used41
• TBE buffer (Merck, cat. no. 106177) REAGENT SETUP
• DNeasy Plant Mini Kit (Qiagen, cat. no. 69104) Organic solvents for separation and purification  Many organic solvents
• Hot StarTaq Master Mix Kit (Qiagen, cat. no. 203443) of varying polarities may be used for chromatographic separation and
• RNAse A (Invitrogen, cat. no. 12091-039) purification procedures, including acetone (! CAUTION highly flammable
• Primers: ITS1 and ITS4 (Invitrogen; individual order) and irritant), acetonitrile (ACN) (! CAUTION highly flammable and toxic),
• SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102) dichloromethane (DCM) (! CAUTION harmful), ethanol (EtOH) (! CAUTION
• DNA loading buffer (Eppendorf, cat. no. 0032 006.850)
highly flammable), ethyl acetate (EtOAc) (! CAUTION highly flammable
• PerfectPrep Gel Cleanup Kit (Eppendorf, cat. no. 0032 007.740)
and irritant), n-hexane (! CAUTION highly flammable, irritant, harmful,
Solvents:
dangerous for the environment and toxic for reproduction), n-butanol
• Solvents for extraction and chromatographic separation (see Reagent
Setup) (n-BuOH) (! CAUTION flammable, harmful and irritant) and methanol
• Solvents for high performance liquid chromatography, HPLC, for (MeOH) (! CAUTION highly flammable and toxic). They all are of analyti-
measuring optical rotation, for NMR spectroscopy are used as described cal grade. ! CAUTION All organic solvents must be handled carefully. Wear
in a previous protocol41 protective clothing, safety glasses and gloves. They should be handled under a
• Spray reagents are used as described in a previous protocol41 fume hood and stored in a ventilated solvent cabinet.
• Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (‘Marfey’s reagent’, Fluka, Medium A for isolation of fungal strains from marine macro-organisms
cat. no. 42095)
and mangrove plants 
• Standard amino acids (ICN, Sigma)
EQUIPMENT Bacto agar 15 g
• Sterile plastic petri dishes, diameter 9.4 cm (Greiner, cat. no. 633161) Malt extract 15 g
• Parafilm M (Marienfeld, cat. no. 74 038 10)
Artificial sea salt 24.4 g (for isolation from mangroves 10 g)
• Sterile tubes, 15 ml, 120 mm × 17 mm (Sarstedt, cat. no. 62553)
• Susceptibility paper disks (5 mm diameter, Oxoid) Chloramphenicol 0.2 g
• All micro-organisms are acquired from DSMZ–Deutsche Sammlung von pH 7.4–7.8 (adjusted with NaOH/HCl)
Mikroorganismen und Zellkulturen GmbH
Dem. water fill up to 1,000 g
• Artemia salina (Dohse Aquaristik, cat. no. 21350)

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The contents are combined in a flask big enough so that it is filled not All contents are mixed thoroughly. In all, 300 ml of the media is poured
more than two-thirds, covered and autoclaved at 121 °C for 20 min. After in 1,000 ml Erlenmeyer flasks. The flasks are covered with a tissue lid and
cooling down to ~60 °C, the medium is poured in sterile plastic petri dishes, autoclaved at 121 °C for 20 min. ! CAUTION Tissue lids must be covered
~25 ml medium per plate, under aseptic conditions. The prepared agar with aluminum foil to prevent soaking during autoclaving. The temperature
plates can be kept under sterile conditions for upto 1 week before inocula- of the autoclave has to be below 80 °C before it can be opened to prevent hot
tion. ! CAUTION Flasks must not be closed tightly when being autoclaved. steam from emerging. Heat protective gloves and safety glasses have to be
The temperature of the autoclave has to be below 80 °C before it can be worn. The medium can be stored for 1–2 d before inoculation.
opened to prevent hot steam from emerging. Heat protective gloves and Solid rice medium 
safety glasses should be worn.
Rice 100 g
Medium B for purification and short term storage of fungal strains 
Dem. water 110 g
Bacto agar 15 g
The 1,000 ml Erlenmeyer flasks with the contents are covered with a tissue
Malt extract 15 g 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
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

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


pH 7.4–7.8 (adjusted with NaOH/HCl) 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
Dem. water ad 1,000 g steam from emerging. Heat protective gloves and safety glasses have to be
worn. The medium can be stored for 1–2 d before inoculation.
The contents are combined in a flask big enough so that it is filled not
MexA medium for long-term storage 
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, Malt extract 20 g
~25 ml medium per plate, under aseptic conditions. The prepared agar plates
Yeast extract 0.1 g
can be kept under sterile conditions for up to 1 week before inoculation.
! CAUTION Flasks must not be closed tightly when being autoclaved. The Glycerin 50 g
temperature of the autoclave has to be below 80 °C before it can be opened Artificial sea salt 24.4 g (for mangrove derived fungi 10.0 g)
to prevent hot steam from emerging. Heat protective gloves and safety glasses
Bacto Agar 13 g
have to be worn.
Liquid Wickerham’s medium  Dem. water ad 1,000 g

Yeast extract 3g 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
Malt extract 3g During autoclaving the flasks must not be tightly closed. The temperature of the
Peptone 5g 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.
Glucose 10 g
After cooling down to ~60 °C the medium is poured in sterile tubes, each
Artificial sea salt 24.4 g (for mangrove-derived fungi 10 g) with ~6 ml under aseptic conditions. The prepared flasks can be kept under
pH 7.2–7.4 (adjusted with NaOH/HCl) sterile conditions for up to 1 week before inoculation.  CRITICAL All the
autoclaved media have to be stored under aseptic conditions to prevent
Dem. water ad 1,000 g
contamination with ubiquitous microbes from the environment.

PROCEDURE
Isolation and cultivation of fungi from marine organisms ● TIMING 6–9 weeks
1| 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.

2| 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).

5| 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

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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 1.

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
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

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 become 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.

10| 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 ~2 d


11| Extract the cultured material by following the steps in options (A) small-scale liquid medium (Fig. 2); (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. 2)
(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 deminearalized 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.

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! 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.
(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
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

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


12| 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 2 or the MTT assay described in ref. 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.

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

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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 analysis
Marfey’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.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

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 Steps 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.

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

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

15| 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 Box 3) 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).

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

17| 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 strain (growth ~1–2 weeks)
Step 7: see Box 1
Step 8: ~5 min per strain (growth ~3–5 weeks)
Steps 9 and 10: each ~5 min (plus 24 h extraction)
Step 11 A(i–iii): 45–60 min
Step 11 B/D(i): 5/30 min (plus 1 d for extraction)

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Step 11 B/D(ii): ~10 min ( plus 1 d for extraction) (twice)


Step 11 B/D(iii): ~10 min
Step 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 h
Steps 12–17: The timing of these steps is dependent on chosen columns, complexity of the extract and the kind of isolated
compounds.
See also Figures 1–4.

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

Table 1 | Troubleshooting table.

Step Problem Possible reason Solution

4 Fungal growth on Insufficient surface sterilization If you are not sure how delicate the surface of your source
negative controls 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, As described above
fungi residing in the tissue are killed
11 Insufficient separation Formation of an emulsion because of Leave standing overnight in the separation funnel; centrifu-
of the two phases extracted compounds gation also helps
13 No or insufficient Elution of the column is too fast Reduce the speed of the mobile phase (for Sephadex material
separation ~10 drops min − 1, silica  < 20 drops min − 1, depending on the
size of the column)
Inappropriate stationary or mobile Always test on TLC to check stationary and mobile phase
phases before column chromatography
Column too small 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

ANTICIPATED RESULTS 1

Examples taken from our group that illustrate the applica- O N


tion of the described procedures for the isolation of sponge- O O
and mangrove-derived fungi and the subsequent isolation H H
H H
and structure determination of bioactive compounds will be
described (Figs. 5 and 6).
HO OH HO OH
OH OH
O O
Anthraquinones and betaenones
Two new betaenone derivatives (1,2) and three new 3 OH O OH OH
1,3,6,8-tetrahydroxy anthraquinone congeners (3–5) were 4
obtained from the fungus Microsphaeropsis sp. isolated from
OH O OH O
the inner parts of the marine sponge Aplysina aerophoba32. HO OH

The EtOAc extract of the fungus that had been grown in O

liquid medium (including 2.4% (wt/vol) of artificial sea salt) HO OH


under static conditions at room temperature for 41 d, 5 O
OH O OH O
was evaporated under reduced pressure. The residue was
HO
partitioned between EtOAc (4 × 400 ml) and H2O (400 ml)
to eliminate the remaining salt. The organic phase was HO OH
taken to dryness and subjected to VLC on silica gel using O
step gradient employing CH2Cl2 and MeOH (100:0, 98:2, Figure 5 | New betaenones and anthaquinones from the sponge-derived
95:5, 90:10, 80:20 and 30:70 vol/vol) as solvent systems. fungus Microsphaeropsis sp.

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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
(1) 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 (2) 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
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

betaenone (1) 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 (1) 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
(2) 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
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 (6–11), perylene quinones and new compounds (12–17), 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 6 7 8
systems, fraction 3 (80% (vol/vol) O O
CH2Cl2) was chromatographed over HO HOOC OH O OH O OH

silica gel using CH2Cl2 and MeOH (90:10 HO HO HO


vol/vol) as eluent mixture. Fractions 3 O HO HO O
and 4 were combined and re-chromato­
graphed by normal-phase VLC using a 9 10 11
step gradient of CH2Cl2 and MeOH as O
O O
O OH O OH
eluent. Fraction 5 was further purified
using a RP-18 column and H2O:MeOH HO HO
HO O
(7:3 vol/vol) as eluent. Fraction 3 OH O
yielded altenusin (6), fraction 5 gave OH O
altenuene (7), 4′-epialtenuene (8) 12
and 2,5-dimethyl-7-hydroxychromone COOH
OH
(9) after semi-preparative HPLC using 13
OH
OH
an RP-18 column with MeOH:H2O (25:75 HO
O
vol/vol) as eluent. Alternariol (10) and O
OH O
altertoxin I (13) were obtained from
fraction 7 and final purification was 14 15 16 17
carried out by semi-preparative HPLC OH OH OH O OH O
using a RP-18 column with MeOH:H2O O COOH O
COOH
OH
(35:65 vol/vol) as eluent.
OH OH
The new metabolite alternarienonic H H OH
OH
HO
acid (12) was obtained from the com-
bined VLC fractions 5 and 6 following
OH O
purification by Sephadex LH-20 CC OH O O OH OH O

with MeOH as eluent. |


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

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Fraction 5 of the VLC separation yielded the new natural products xanalteric acid I (14) and II (15), 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, 10, 13, 14 and 15 were likewise detected
as constituents of the crude extract using HPLC, whereas 9 and 12 were missing. In addition, the known compounds
alternariol-5-O-methylether (11) and the perylene derivatives stemphyperylenol (16) and alterperylenol (17) 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 (10) 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 (12), xanalteric acid I (14) and II (15), as well as altenuene (7),
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols

altertoxin I (13) and 2,5-dimethyl-7-hydroxychromone (9) were inactive in this bioassay38.

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