10.6092/issn.2531-7342/12700
Fungal secondary metabolites are promising grounds of many antibiotic compounds; this happens because of the unique biosynthetic capabilities of the organism in adaptation with various environments. Some of the potential environmental conditions or habitats stimulate fungi to produce bioactive compounds; these include various stress factors like temperature, osmotic changes and pollution. Traditional approaches used to isolate fungal antibiotics are mainly mono-culture-based and it trails behind the ever-expanding needs of the clinical world. A recent progress made in the culture-based approach is the co-culture of microbes, which creates a competing environment for the fungi resulting in the induction of hidden biosynthetic pathways. The revolutionizing impacts of the post-genomic era also aided these search in the form of various omics-based and biosynthetic approaches. These approaches not only facilitate the invention of all-new compounds but contribute in the modification of existing compounds through which the compounds can serve as better drug candidates.
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Zobel S, Boecker S, Kulke D, Heimbach D, Meyer V, Süssmuth RD (2016) Reprogramming the biosynthesis of cyclodepsipeptide synthetases to obtain new enniatins and beauvericins. ChemBiochem 17(4):283-287. https://doi.org/10.1002/cbic.201500649
A review on different approaches to isolate antibiotic compounds from fungi
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                 IJM - Italian Journal of Mycology
                 ISSN 2531-7342 - Vol. 50 (2021): 99-116
                 Journal homepage: https://italianmycology.unibo.it/

Review

                     A review on different approaches to isolate
                          antibiotic compounds from fungi

                                          Jyotsna Baby, Toji Thomas

Department of Botany, St. Thomas College Palai, Arunapuram P.O. Pala, Kerala-686574, India

Corresponding author e-mail: tojidr@yahoo.com


Article info
Received 7/4/2021; accepted 7/7/2021
https://doi.org/10.6092/issn.2531-7342/12700

Abstract
Fungal secondary metabolites are promising grounds of many antibiotic compounds; this happens because of the unique
biosynthetic capabilities of the organism in adaptation with various environments. Some of the potential environmental
conditions or habitats stimulate fungi to produce bioactive compounds; these include various stress factors like tem-
perature, osmotic changes and pollution. Traditional approaches used to isolate fungal antibiotics are mainly mono-cul-
ture-based and it trails behind the ever-expanding needs of the clinical world. A recent progress made in the culture-based
approach is the co-culture of microbes, which creates a competing environment for the fungi resulting in the induction of
hidden biosynthetic pathways. The revolutionizing impacts of the post-genomic era also aided these search in the form
of various omics-based and biosynthetic approaches. These approaches not only facilitate the invention of all-new com-
pounds but contribute in the modification of existing compounds through which the compounds can serve as better drug
candidates.

Keywords
Co-culture, novel antibiotics, omics-based, secondary metabolites, mutasynthesis


1. Introduction
Microbial metabolites have proved as rich reservoirs of antibiotic compounds with potential clinical
applications (Sanglier et al., 1996). Isolation of bioactive compounds from microbial cultures
remains far advantageous than chemical synthesis and the method has provided numerous antibiotic
compounds with promising clinical applications till date (Shlaes, 2010). Fungal metabolites draw
much attention in this regard, and whose history dated back to the discovery of penicillin in 1929
(Fleming, 1929). Filamentous fungi own many unique metabolic pathways and produce a huge share
of bioactive compounds possessing clinical use; hence, filamentous fungi serve as the potential
producers of antibiotic compounds (Kuck et al., 2014).
   As the usage of antibiotics became common, the problem of drug resistance emerged. For
example, once an antibiotic drug is introduced into clinical practice, it has a limited period of success
as selection takes place among the target bacteria with intrinsic or acquired resistance mechanisms
(Chandra and Kumar, 2017). Consequently, it is essential to find out novel compounds that may be
able to suppress drug resistant bacteria. Traditional approaches are mainly focused on finding natural
bioactive molecules from cultured microbes (Berdy, 2012). Even though this culture-based approaches
remained as the backbone of the golden antibiotic era, it stayed behind and usually failed to identify

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
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novel lead compounds that could neutralize drug resistant strains. Moreover, the rate of incidences of
reporting and re-isolation of known compounds has been more and more frequent (Corley and Durley,
1994). Hence, there is a need for a paradigm shift in the approaches of drug discovery processes,
which include identifying unexplored microbial reservoirs, novel culture approaches, omics-based
approaches, etc. (Kealey et al., 2017). Here we review the different methods for antibiotic compound
isolation from filamentous fungi, and focus on emerging trends like searching for fungal strains from
previously unexplored sites, exploring various culture-based methods, omics-based and biosynthetic
approaches.

2. Potential habitats for fungal isolation
Antimicrobial compounds are synthesized by fungi as secondary metabolites (Keller et al., 2005) and
are not inevitable for their vital life processes (Madigan et al., 1997). These compounds are normally
generated at the end of their life cycle or in stressful conditions, which necessitate them to fight
against the odds of nature including both abiotic and biotic stresses (Davies, 1985). Hence, unusual
environments are promising storehouses of novel metabolites generating microbes (Park et al., 2009).
Antibiotic compounds isolated from filamentous fungi belonging to some of the stressed habitats are
described below. Recently discovered novel antibiotic compounds (from 2000 onwards) from fungi
along with their chemical structure are provided in Supplementary Table 1.

2.1. Regions with temperature extremes
By the 1950s the exploration of microorganisms from extreme conditions began to hurry up
(Yogabaanu et al., 2017). Cold adapted fungi are actually a huge repository of novel antibiotics. In
these extreme conditions, they are highly prone to many stresses such as high UV, low nutrients,
and extremely low freezing temperatures (Nishiyama, 1977; Montiel, 2000). The occurrence and
survival of fungal communities in the extreme cold temperature in Antarctica favoured their unique
biochemical pathways to synthesize new bioactive metabolites (Santiago et al., 2012). Various species
of Penicillium, Aspergillus, Cadophora, Pseudogymnoascus, Paraconiothyrium, Purpureocillium,
Toxicocladosporium are prominent among these (Santiago et al., 2012; Vieira et al., 2018). Recent
studies reported a vast array of antibacterial and antifungal compounds from fungi inhabiting Arctic
and Antarctic zones (Lo Giudice and Fani, 2016) such as geomycin (Li et al., 2008) chanoclavine I,
griseofulvin, roquefortine C and D, elymoclavine, mycelianamide, fulvic acid (Frisvad et al., 2004),
amphotericin (Svahn et al., 2015) etc. from polar soils. Godinho et al. (2015) investigated the diversity
and bioactive capabilities of fungal communities in the oligotrophic soil sample collected from
Ellsworth Mountains, Antarctica. Two species of Penicillium viz. Penicillium allii-sativi Frisvad,
Houbraken & Samson and P. brevicompactum Dierckx exhibited production of antibiotics penicillin
and mycophenolic acid respectively. Li et al. (2008) obtained a pure culture of Antarctic ascomycete,
Pseudogymnoascus sp. from the soil samples collected from Field Peninsula of King George
Island, Antarctica and they isolated geomycins B and C. These compounds demonstrated antifungal
properties towards Aspergillus fumigatus Fresen. and antibacterial action towards Staphylococcus
aureus, Escherichia coli and Streptococcus pneumoniae. Yogabaanu et al. (2017) conducted a study
on the antimicrobial activity by 40 cold friendly soil fungi from both poles (Arctic and Antarctic).
About 45% of fungal cultures produced antimicrobial compounds in culture and they exhibited
antimicrobial action towards at least one of the five bacterial pathogens investigated. The fungal
strains which exhibited prominent bioactivity were again subjected to temperature manipulation
studies and the studies revealed the roles of culture conditions on bioactivity. Likewise, studies were
also reported from high temperature regions like deserts, for example, Awaad et al. (2012) isolated
two new antibiotic compounds, which were butyrolactone I derivatives, produced by Aspergillus
terreus Thom from desert soil.

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2.2 Mangroves
Mangrove ecosystem is another stressed one, found at land-sea interface having a variety of stresses
such as temperature, salinity, anoxia, UV, etc. (Kathiresan and Bingham, 2001). They are unique
ecosystem composed of diverse microbes, which act as potential agents for the isolation of novel
pharmaceutically active compounds. This is because, the microbiota in mangrove ecosystem are
constantly adapting to the changing environmental conditions through the biosynthesis of various
secondary metabolites (Thatoi et al., 2013). The microbial communities of the mangrove ecosystem
remain largely unexplored (more than 99%) and those which are chemically characterized accounted
only 5% of the known ones (Xu, 2015). Liu et al. (2018) conducted a study at South China Sea,
and isolated two novel compounds ergosterdiacids A and B from Aspergillus sp. DM29 from the
rhizospheric soil of Aegiceras corniculatum, a mangrove plant. These compounds demonstrated
antibacterial activity towards Mycobacterium tuberculosis. Likewise, He et al. (2019) cultured
Penicillium pinophilum Thom SCAU037 from the mangrove sediment collected from the roots of
Rhizophora stylosa at Techeng Isle, China and isolated novel compounds pinophilone A-E, funicone
derivatives. The compounds exhibited growth inhibition of Mycobacterium smegmatis and S. aureus.

2.3. Polluted sites
Many of the past studies were focused on natural extreme environments, but a recent trend is to
focus on sites that have become stressed due to unchecked human interventions. Acid mine drainage
is one among these characterized by heavy metal contamination, highly acidic soil and many other
pollutants, which make an arena of competition between endemic microbes, resulting in the generation
of novel antimicrobial compounds (Johnson and Hallberg, 2003). Park et al. (2009) isolated a novel
metabolite glionitrin A, as they co-cultured bacterial strain Sphingomonas KMK-001 and fungal strain
A. fumigatus, KMC-901, derived from abandoned coal mine of Young-dong, Korea. This compound
exhibited notable antibacterial activity towards a series of bacteria including methicillin resistant S.
aureus and weak antifungal activity towards some pathogenic strains. Noticeably, the monoculture
of both these microbes failed to produce this bioactive compound revealing the potential of stressful
interactions in the production of novel compounds.
    Heavily antibiotic contaminated sites have also recently become a field of interest (Kristiansson
et al., 2011). The bacteria thriving in such an environment developed antibiotic resistance and thus it
became inevitable for the endemic fungi to resort to novel compounds. Svahn et al. (2012) assessed
the antimicrobial potential of fungal isolates sampled from river sediment from Isakavagu stream in
Andhrapradesh, India, located downstream to the outlet of a waste water treatment plant receiving
waste waters from drug manufacturers, with high antibiotic contamination including fluoroquinolone
antibiotics. They identified 61 strains many of which displayed activity against multidrug resistant
bacteria. They also identified gliotoxin, a toxin produced from A. fumigatus. Even though this
compound exhibited significant antimicrobial activity, its toxicity towards mammalian cells hinders
its development as a potential drug candidate. Bharadwaj et al. (2017) characterized six fungal isolates
belonging to four genera viz. Aspergillus, Fusarium, Penicillium and Rhizopus having antibacterial
activity from soil sample collected from a pharmaceutical site and provided optimum conditions for
fermentation. The bioactivities of the fungal isolates were compared with established antibiotics and
much better antibacterial activities were exhibited by crude extracts from the fungal isolates towards
the test pathogens.

3. Different approaches in antibiotic compound isolation
Various approaches employed in antibiotic compounds isolation are discussed below and briefly
outlined (Fig. 1).


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3.1. Culture-based approaches
This method involves the culture of desired fungal strains in laboratory conditions. Culture-based
approaches include monoculture and co-culture depending on whether the fermentation is maintained
as the culture of a single strain or the culture of two interacting microbes (Gomez-Flores et al., 2017).

3.1.1. Monoculture approach
This is the traditional culture-based approach followed in the fermentation of many isolated fungal
strains for the extraction of desired product. Most of the antibiotic isolation protocols from microbes
during the past century followed this approach. The pure culture of fungus is inoculated into the
fermentation medium, providing optimum conditions for the production of secondary metabolites
(Hauser, 2006).




 Fig. 1 – Different approaches in antibiotic discovery from filamentous fungi. The drug discovery pipeline starts with
 the traditional culture-based approaches and advances towards the recent omics-based and biosynthetic approaches.
 Each approach and its subsections are indicated with the same coloured boxes. The pipeline ends with the discovery
 of novel antibiotic compounds with further modifications for increased bioactivity.

3.1.2. Co-culture approach
Microbial monocultures suffer a limitation of lacking interaction between microbial communities
as in the natural environment. As a result, many biosynthetic gene clusters (BGCs) in fungi stand
cryptic, thus transcriptionally inactive under the standard conditions in laboratories (Scherlach and
Hertweck, 2009). A solution to this is the co-cultivation of microbes, which has already attained
success in isolating many novel secondary metabolites (Netzker et al., 2015; Molloy and Hertweck,
2017; Netzker et al., 2018). One of the first initiatives in this regard was reported by Fenical laboratory
at the Scripps Institute of Oceanography. They co-cultured the marine fungus Pestalotia sp. CNL 365
(an epiphyte on the brown alga Rosenvingea sp. from Bahamas Islands) with the marine bacterium
CNJ 328. The fungal strain is cultured in Fernbach flasks having sea water based fermentation medium
with shaking. Ten millilitres of the culture of marine bacterium in the same medium is poured into
the Fernbach flasks after 24 hours of initiation of fungal culture, followed by a mixed fermentation
period of 6 days. The purified culture extracts showed the occurrence of pestalone, a novel antibiotic

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compound. The monoculture of both could not produce the novel compound suggesting the role of
bacterial competition in the induction of the antibiotic biosynthetic pathway (Cueto et al., 2001).
In the same manner, Degenkolb et al. (2002) communicated the isolation of antibiotic compounds
acremostatins A, B and C from the co-culture of Acremonium sp. Tbp-5 (an endophytic fungus of
Taxus baccata) and Mycogone rosea Link DSM 12973. The co-culturing of microbes creates a
tension of competition between microbes which induce them to produce compounds, which are not
usually produced when left undisturbed (Park et al., 2009). In the same way, Emericella sp. produced
emericellamides A and B in competing co-culture with Salinispora arenicola (a marine actinomycete).
Both these compounds exhibited modest inhibition towards methicillin resistant S. aureus (Oh et al.,
2007). Schroeckh et al. (2009) described the synthesis of orsellinic acid from Aspergillus nidulans
(Eidam) Winter as the fungus was co-cultured with Streptomyces rapamycinicus. The mixed culture
conditions were able to activate a silent gene cluster of fungi. Same kind of finding was also conveyed
by Konig et al. (2013) in which S. rapamycinicus activated a silent gene cluster of A. fumigatus
producing fumicyclines. This compound is proposed to be involved in fungal defence mechanism as
they exhibited moderate inhibition of S. rapamycinicus. Meng et al. (2015) also successfully isolated
novel compounds, citrifelin A and B, which are citrinin adducts having tetracyclic ring structure from
the co-culture of two fungi, Penicillium citrinum Thom and Beauveria felina (DC.) J.W. Carmich.
Monocultures of both fungi failed to generate the novel compound and it exhibited inhibition of
several pathogenic strains. Another latest innovation of co-culture materialized the production of
novel compounds berkeleylactones A-H, 16-membered ring macrolide antibiotic obtained from the
co-cultivation of two fungi Penicillium fuscum (Sopp) Biourge and Penicillium camemberti Thom/
clavigerum Demelius by Stierle et al. (2017). The culture also yielded known antibiotic compounds
A26771B, citrinin and patulin. Recently, Liu et al. (2020) reported a new antifungal compound from
Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson against the
plant pathogen Botrytis cinerea Pers., as they co-cultured two fungi. The culture plates showed clear
inhibition towards the pathogen and the new bioactive compound was identified using Matrix-assisted
laser desorption/ionization (MALDI) time-of flight (TOF) - imaging mass spectroscopy (IMS) as
lipopeptaibol (leucinostatin Z).
   Co-culture not only induces novel bioactive compound production, but enhances the productivity
of metabolites. The metabolic productivity of P. pinophilum was reported to be enhanced during
the co-culture with Trichoderma harzianum Rifai (Nonaka et al., 2011). Along with the increased
production of known compounds like stromemycin, penicillide, pestalasin A, a novel compound
secopenicillide C was also isolated.

3.2 Omics-based approach
Culture-based approaches are inefficient in the sense having only limited access to the vast biosynthetic
capabilities of the genome for the reason that they are not usually displayed under the undisturbed
lab environment (Gross, 2009). The development of omics-based techniques has contributed much
to the revitalization of novel antibiotic discoveries by providing an insight to the cryptic biosynthetic
gene clusters (BGCs) encoding secondary metabolites (Palazzotto and Weber, 2018). Here we discuss
some relevant omics-based approaches employed in the antibiotic discovery from fungi.
3.2.1. Genomic mining
In the post-genomic era, genomic mining has proved to perform a crucial position in the identification
of untold lead compounds for clinical use. Thousands of microbial genome sequences were made
available, and provided large grounds for this exploration. Along with this, various computational
tools and methods have been devised to guide this resource hunting (Ziemert et al., 2016).
    The core of genome mining lies in its ability to predict genes that can code for novel bioactive
compounds by in silico approaches (Machado et al., 2017). Among the huge genome data, BGCs, which

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encode secondary metabolites, are potent resources of innovative antibiotic molecules (Cimermancic
et al., 2014; Tracanna et al., 2017). BGCs also represent elements that can enhance the production of
both already known natural compounds and engineered compounds (Olano et al., 2014). The genome
mining can effectively predict the putative structure of the metabolites from BGCs. This approach is
helpful for screening and targeting potential compounds from a huge data; thus avoiding the tedious
task of screening every cryptic BGC (Zerikly and Challis, 2009).
    In dealing with fungi, many of the enzymes involved in their secondary metabolite biosynthesis
are large and complex with multiple modules and domains (Walsh et al., 2001). Furthermore, the
number of BGCs is much greater than anticipated; this is because of the cryptic cluster of genes that
remain hidden under the laboratory conditions. Bok et al. (2006) performed LaeA (Loss of aflR-
expression A - a regulatory protein) based genome mining in A. nidulans and thus deciphered its
secondary metabolome. This work involved a unique way to evaluate the gene regulation of secondary
metabolism. Cain et al. (2020) sequenced the genome of the Ascomycete sp. F53 and performed
mining for the purpose of rigorously exploring the biosynthetic capabilities of this endophytic fungus.
35 putative BGCs were revealed in the study, along with a distinctive azaphilone BGC which paved
the discovery of lijiquinone, an azaphilone polyketide. The compound showed antifungal activity
towards Cryptococcus albidus (Saito) Skinner and Candida albicans (C.-P.Robin) Berkhout. The
various ways to improve the manufacture of already known compounds also came under the targets of
genome mining. Van den Berg et al. (2008) sequenced the genome of Penicillium chrysogenum Thom
and recognized various genes relevant to penicillin formation, consequently provided the potential
of genomics-driven manipulation of metabolites. Wang et al. (2016) sequenced the genome of P.
lilacinum and through comparative genomics strategy using bioactive characters; they identified 20
genes concerned with leucinostatin biosynthesis. Through various bioassays, leucinostatins revealed
bioactivity against Phyotophthora capsici and P. infestans, by inhibiting their growth. Thus, the
potential of P. lilacinum as a biocontrol agent was revealed and it was effectively used for the same.
    Different techniques employed in the genome mining included bioinformatic methods, gene
inactivation method, heterologous expression, use of transcription activators and inactivation of
inhibitors (Scheffler et al., 2013). In the bioinformatics approach, entire genome or gene of interest
is sequenced, after that gene prediction is done by various tools such as BLAST, GOLD, HMMER,
SBSPKS, NORINE, antiSMASH, etc. (Nikolouli and Mossialos, 2012).
    The gene inactivation method deals with the manipulation of the organism using a gene knock out
approach. Here the product of a knocked out gene is unknown. This could efficiently overcome the
difficulty arising from inaccurate gene predictions using bioinformatics tools (Gross et al., 2007). An
example of this method involves the study conducted by Chiang et al. (2008) for the elucidation of
emericellamide biosynthetic pathway. They studied six genes of A. nidulans using six strains with
one of the genes knocked out in each strain. Only one knocked out exhibited a change from wild type
indicating the silent nature of other genes. Furthermore, the gene of the concerned strain was found to
be implicated in the biosynthesis of bioactive compounds emericellamides A and C-F.
    As opposed to gene inactivation, heterologous expression involves the transfer of a cryptic gene
from an organism to a host that can express the gene (Gross, 2007). The product of the cryptic gene
(transgene) is expressed in the heterologous host. LC/MS analysis is performed and is used to compare
the fermentation broth of transgenic host, with the broth of the host lacking this transgene. Metabolites
present in the fermentation broth of the transgenic host with target BGC; but it is absent in the broth of
the host lacking this BGC. It is assumed to be the products of the cryptic BGC (Challis, 2008). Bailey
et al. (2016) heterologously expressed pleuromutilin gene cluster from Clitopilus passeckerianus
(Pilát) Singer in the host Aspergillus oryzae (Ahlburg) E. Cohn. This was the initially reported in
heterologous expression involving cross-phylum transfer of a gene cluster from basidiomycete to
ascomycete host. The study was conducted for strain improvement and increased antibiotic production.

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    Another method involves transcription activators, particularly used when the gene of interest is
unable to be transferred to a host cell. In order to turn on these cryptic genes, activator genes already
present in the cell are employed and thus the product is induced to form (Challis, 2008). Using this
approach, Bergmann et al. (2007) successfully induced a cryptic BGC in A. nidulans, which generated
in the formation of new polyketide synthase–nonribosomal peptide synthetase (PKS-NRPS), a hybrid
metabolite. As a means to activate the cryptic BGC, an inducible activator gene, apdR (Aspyridones
cluster regulator) in this cryptic gene cluster was identified and overexpressed. They amplified the
activator and cloned into pAL4 (phenylalanine Ammonia-lyase 4), an expression vector with inducible
promoter, alcAp (alcohol dehydrogenase promoter) of A. nidulans. The inducing conditions resulted
in PKS-NRPS hybrid synthetase activation and the induced strains produced aspyridones A and B.
    Another technique of the genome mining employs the expression of a cryptic gene by removing
the inhibitor, which repress the product formation from the target gene. The product formed can be
isolated and identified. This method suffers from a limitation; it is useful only in instances where an
inhibitor is present in the organism, which repress the gene and the target gene can be activated when
the inhibition is removed. In most cases, an inhibitor is not the reason, but the lack of activators.
Therefore, this method is least employed in the discovery of bioactive compounds (Scheffler et al.,
2013).

3.2.2. Antisense-guided isolation
Revolutionizing improvements in the area of microbial genomics have paved the way for detection
of novel antibiotics based on antisense-based screening strategies. In this approach, essential genes
are identified in microbes as target for antisense-based inhibition (Bai et al., 2010). The major target
genes include those involved in bacterial growth, multiplication, metabolite synthesis and virulence
(Woodford and Wareham, 2009).
   The antisense RNA is made to express selectively, and it binds to the mRNA of the target gene
resulting in its degradation, thus a reduction in the corresponding gene product. These weakened
strains are exposed to an inhibitor against the targeted gene product usually by screening a natural
compound library (Young et al., 2006). Ondeyka et al. (2006) employed antisense-based strategy to
search for cell-permeable inhibitors of type II fatty acid synthesis pathway (FASII). They conducted
a two plate assay screening over 25,000 fermentation broths of natural compounds, and discovered
phomallenic acid A-C from Phoma sp. as potent inhibitors of β-ketoacyl-[acyl carrier protein (ACP)]
synthase II(FabF)/and III)(FabH) enzymes of FASII pathway (Ondeyka et al., 2006; Young et al.,
2006). Zhang et al. (2009) also employed this approach, targeting ribosomal protein S4 in the search
for novel antibiotics. This protein is synthesized by the gene encoding ribosomal protein S4 (rpsD)
in bacteria and screening is done by two-plate assay using S. aureus, engineered with rpsD antisense
RNA construct. Natural compound extract from an endophytic fungus of Pleosporales MF7028
showed promising results and the compound was identified as pleosporone. Likewise, Parish et al.
(2009) also employed the strategy of antisense-mediated isolation in identifying the inhibitors of
SecA (Type II secretary pathway – a system responsible for the secretion of proteins through cell
membrane – it is a membrane associated ATPase motor), an integral part of secretary machinery
concerned with protein translocation. Secretary pathways are previously unexplored for antibacterial
compounds, thus providing opportunity for the development of drugs with novel structural scaffolds
thus offering some advantage from drug resistance. A manipulated strain of S. aureus with an inducible
secA antisense RNA construct is used to screen over 115,000 natural compounds in a two plate assay.
An extract from the fungus Pseudogymnoascus pannorum (Link) Minnis & D.L. Lindner showed
promising results and an active compound is isolated and is named as pannomycin.



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3.2.3. Metabolomics
Metabolomics, a relatively new domain of omics-based method, includes the systematic characterization
and quantification of the entire metabolites in a given biological sample (Idle and Gonzalez, 2007)
and it has established as a successful method in the discovery of antibiotic compounds from microbes
(Wu et al., 2015). This approach can be divided into targeted and untargeted metabolomics (Schrimpe-
Rutledge et al., 2016).

   Targeted metabolomics - It is generally involved in the identification and quantification of known
compounds and the data it handles are limited (tens to hundreds of compounds) (Schrimpe-Rutledge
et al., 2016). These compounds are previously defined, for example, biochemically annotated and
chemically characterized (Roberts et al., 2012) and thus less employed in the discovery of novel
compounds.

   Untargeted metabolomics - As opposed to the targeted metabolomics, untargeted metabolomics
aims at inspecting as much metabolites as possible (Patti, 2011). Untargeted metabolomics look for
a fingerprint of the metabolome of the target organism with nuclear magnetic resonance (NMR)
spectroscopy or mass spectrometry (MS) data (Smedsgaard and Nielsen, 2004). Its advantage lies
in its ability to produce high resolution data with the aid of MS and NMR techniques (Kind and
Fiehn, 2010; Neumann and Bocker, 2010). Analytical strategies employed in this approach along with
dereplication tools and software are briefly discussed (Hautbergue et al., 2018).
   Analytical techniques employed in labelling of metabolites with a stable isotope called stable isotope
labelling (SIL), helped the marking of fungal secondary metabolites on a complex metabolome. A
labelled substrate is used to label fungal metabolites, thus effectively marking it from the background
and contaminants. This technique was successfully utilized by Klitgaard et al. (2015) while studying
the metabolite synthesized from phenylalanine by labelling phenylalanine in the feed of A. nidulans.
The compounds of relevance were analysed using MS by comparing with the non-labelled control;
they isolated fungisporin and nidulanin A analogues.
   Another key step in metabolomics approach for novel bioactive compound detection is the
screening out of already known compounds from the metabolome called dereplication. It employs
the computer aided strategy in marking known compounds and directs the discovery process towards
unknown compounds by submission of NMR or MS data to databases (Gaudencio and Pereira, 2015).
Different databases are available based on chemical formulae, mass, liquid chromatography (LC)
values, etc. for the exact characterization of molecules. Fungal specialized databases, for example,
AntiBase (Laatsch, 2014), and Dictionary of natural products (http://dnp.chemnetbase.com) are
based on exact mass and chemical formula. Another widely used tool based on MS/MS spectral
data is Global Natural Product Social Molecular Networking (GNPS). It has about 220,000 MS/MS
spectra, which represent more than 18,000 metabolites from the NIST (National Institute of Standards
and Technology), ReSpect -RIKEN tandem mass spectral database (Sawada et al., 2012), MassBank
(Horai et al., 2010) and is based on DEREPLICATOR algorithm (Mohimani et al., 2017).
   Data mining tools: Several tools help in the process of data mining from complex metabolome
such as XCMS (Smith et al., 2006), GNPS, etc. GNPS allows the grouping of compounds on the
assumption that compounds with similar MS/MS spectral properties also share similar structures
(Mohimani and Pevzner, 2016). On the other hand MS2LDA tool helps in describing compounds with
similar MS/MS spectra (Van der Hooft et al., 2016). In dealing with structure elucidation tools of these
natural metabolites, various aids such as NRPquest, which compares multiple peptide compounds on
the basis of MS/MS spectra, are created (Mohimani et al., 2014).
   Oppong-Danquah et al. (2020) performed untargeted metabolomics study in six sea foam-derived
fungal strains included in the genera Penicillium, Plectosphaerella, Emericellopsis and Cladosporium.

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They employed ultra-high performance liquid chromatography with quadrupole time-of-flight mass
spectrometry based molecular networking (UPLC-QToF-MS/MS based MN) in their investigation.
Furthermore, they also tested the antimicrobial activity of organic extracts of these fungal isolates.
The study identified a Penicillium strain as the most productive among them and also identified
novel antimicrobial derivatives such as analogues of fungisporin. Albright et al. (2015) used the
technique of untargeted metabolomics in a study conducted on A. nidulans to track the changes in
small molecule metabolites secreted from the fungus after creating reduction in histone deacetylase
activity (HDACi). Along with metabolites from known and unknown pathways, the study reported
the presence of fellutamide, whose production is enhanced up to ~ 100 fold by HDACi.

3.3. Biosynthetic approaches
Fungi provide a large repository of secondary metabolites, many of which have potential clinical
applications. Some of them cannot be utilized directly due to toxic activities or suboptimal bioactivity.
To circumvent these limitations, non-natural derivatives from these natural compounds via biosynthetic
routes are proposed (Boecker et al., 2016). The different strategies in this regard are outlined here.

3.3.1. Precursor –directed biosynthesis (PDB)
This approach makes use of the processing of precursor analogues created through chemical synthesis
into novel bioactive compounds, since the natural biosynthetic machinery is flexible enough to
accommodate synthetic substrate analogues and process them to modified intermediates. The
application of PDB in creating derivatives of existing molecules has started since 1950s. Brandl
and Margreiter (1954) supplemented the cultures of P. chrysogenum with phenoxyacetic acid and
successfully isolated acid stable, orally applicable penicillin V. Nilanonta et al. (2002) reported
the biosynthesis of beauvericin analogues from Paecilomyces tenuipes (Peck) Samson using PDB
approach. Enhanced production of beauvericin A, beauvericin B, beauvericin C along with beauvericin
was resulted on feeding L-isoleucine or D-alloisoleucine while the feeding of D-isoleucine or
L-alloisoleucine produced allobeauvericin A, B and C. One of the limitations of this method is the
competition between natural substrates and supplemented analogues (Boecker et al., 2016).

3.3.2. Mutasynthesis
This is considered as an advanced approach of combinatorial biosynthesis, and it involves the
creation of mutants lacking some responsible genes concerned with the production of the natural
precursor in the biosynthesis (Weist and Süssmuth, 2005; Kirschning and Hahn, 2012). Such lacking
precursors are complemented by precursor analogues called mutasynthons, which are chemically
synthesized and supplied. One of the limitations of PDB has been rectified, as it avoids competition
between mutasynthons and natural precursor. Moreover, the isolation of the engineered product
also becomes easy as the natural product formation is hindered (Sussmuth et al., 2011). Xu et al.
(2009) identified the gene coding for the enzyme ketoisovalerate reductase (KIVR). This enzyme
is accountable for the production of D-hydroxyisovalerate (D-Hiv), which acts as the precursor for
cyclooligomer depsipeptides (COD) including bassianolide, beauvericin and enniatins which has
antibiotic, antifungal and many other bioactivities. They mutated KIVR genes, thus facilitating the
biosynthesis of beauvericin from mutasynthons. From the results, they found that the precursor
analogues D-2-hydroxybutyrate (D-Hbu) efficiently produced beauvericin G3 while DL-2- hydroxyl-
3-methylvalerate (DL-Hmv) produced beauvericin C and also provided a strategy for a library of
unnatural CODs of beauvericin type.




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3.3.3. Combinatorial biosynthesis
The genetic manipulation of biosynthetic machineries naturally present in the cell for the production
of altered, novel compounds with novel bioactivities is combinatorial biosynthesis (Hopwood et
al., 1985). The manipulations range from combining of enzymes from different pathways to the
modifications in the enzymes themselves (Floss, 2006). Majority of fungal secondary metabolites
are formed mainly by three enzymes viz. Non-Ribosomal Peptide Synthetases (NRPS), Polyketide
Synthases (PKS), Terpene Synthases (TPS) with accompanying tailoring enzymes. The natural
architecture of these key enzymes are modified through insertion, deletion, exchange of domains,
etc. all aiming at the generation of some modified compounds with increased bioactivity (Boecker et
al., 2016). In the context of rush in search for novel antibiotics, Non-Ribosomal Peptide Synthetases
are drawing attention because they are engaged in the synthesis of many clinically important peptide
antibiotics. A study done by Zobel et al. (2016) aimed at one of its subgroups, cyclodepsipeptide
(CDP) synthetases which are a highly similar group of enzymes implicated in the synthesis of cyclo-
hexadepsipeptide and cyclo-octadepsipeptide. For this, they selected depsipeptide synthetases,
beauvericin synthetase (BSYN) for synthesizing beauvericin from Beauveria bassiana (Bals.-Criv.)
Vuill., enniatin synthetase (ESYN) for producing enniatin from Fusarium oxysporum Schlecht.
Emend. Snyder & Hansen and PF1022 synthetase (PSYN) for synthesizing PF1022 from Rosellinia
abscondita Rehm. Engineering of hybrid synthetases were carried out by fusing module 1 of PSYN
with the module 2 of BSYN and ESYN. These hybrid synthetases were heterologously expressed in
Aspergillus niger van Tieghem and E. coli resulting in the formation of hybrid hexadepsipeptides.

3.3.4. Combinatorial libraries
In the search for novel bioactive compounds, combinatorial libraries are gaining ground by allowing
a wide array of compounds to be screened for activity. Depending on the central structure around
which the library is built, it can be divided into biased and generic libraries. In biased library, the
lead compound is either from a natural compound, previously characterized by structure activity
relationship (SAR) studies or a proven compound from the generic library. It has the advantage of
generating a large number of analogues for a given compound in a suitable time frame. In contrast,
generic libraries aim on a much different strategy of building the bioactive candidate around a
pharmacophore model, which is totally unrelated to any proven compound. Thus, it is also less prone
to be prevailing over by the multiple drug resistant strains (Houghten et al., 1991; Pinilla et al.,
1992). Blondelle & Lohner (2000) built a biased synthetic combinatorial library (SCL) based on
an 18-mer lytic peptide formed of lysine and leucine residues (YKLLKKLLKKLKKLLKKL-NH2)
named YLK (Blondelle and Houghten, 1992; Blondelle et al., 1995; Perez-Paya et al., 1996). YLK
exhibited antibacterial action towards gram-positive and gram-negative bacteria as well as limited
antifungal activity towards C. albicans (Blondelle et al., 1996). A huge set of YLK analogues (130,321
analogues) were created in the main SCL, many of which exhibited improved activity against a given
organism than the parent peptide. It is generally involved in the identification and quantification of
known compounds and the data it handles are limited (tens to hundreds of compounds) (Blondelle
and Lohner, 2000).

4. Conclusion
The search for the novel antibiotic drug candidates is never-ending, as the resistance to almost all
known antibiotics is evolving in the target groups. Screening performed among natural products
served much better than that in chemical compound library as nature created more than that of human
imagination. Thus, a wide array of fungal metabolites is potential targets of drug discovery methods.
However, this search is very tedious like finding a needle in a haystack. Traditional monoculture
based culture methods are trailing behind the expanding needs of the clinical world. For this purpose,

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potential habitats have to be explored and novel methods have to be devised. Isolation of fungal
strains from various stressed environments has reported success due to their unique secondary
metabolites through which they get through these various pressures. Also the emerging trends of co-
culture, omics-based and biosynthetic approaches are rejuvenating this search from rapid induction
or detection of potential biosynthetic pathways by combining or modifying existing pathways to
generate novel lead compounds.

Acknowledgements
Authors are thankful to the Council for Scientific and Industrial Research for bestowing CSIR JRF
to conduct the research work of Jyotsna Baby, and it helped to prepare and submit the review paper.
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