Exploitation of fungal secondary metabolites old and new Geoffrey by dfsiopmhy6

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									                               Exploitation of fungal secondary
                               metabolites old and new
                               Geoffrey Turner


                               q
Fungal secondary                         The fungal kingdom offers enormous bio-
                                         diversity, with around 70,000 known species,        Table 1. Some fungal peptides
metabolites have
been exploited by                        and an estimated 1.5 million species in total.      Non-ribosomal peptide synthetase genes have been
                               Most of these are filamentous fungi, which differ from        characterized for those shown in bold type.
scientists for many            the yeasts not only in their more complex morphology
years. Geoffrey                and development (e.g. asexual and sexual structures), but     ACV                        Aspergillus nidulans
Turner describes               also in their greater metabolic complexity. In particular,                               Penicillium chrysogenum
some current                   they are known for production of secreted enzymes                                        Acremonium chrysogenum
applications                   and secondary metabolites, many of which have been
and shows how                  exploited by Man. Genetic analysis of secondary meta-         Ergotpeptides              Claviceps purpurea
increasing                     bolic pathways over the past 10 years has revealed some       Alamethicin                Trichoderma viride
knowledge of                   common themes and offered new approaches to the               Cyclopeptin                Penicillium cyclopium
fungal gene                    exploitation of natural products.                             HC-toxin                   Cochliobolus carbonum
structure and                     The best known fungal secondary metabolites in
                               commercial production are the β-lactam antibiotics            Tentoxin                   Alternaria alternata
metabolic pathways                                                                           Ferrichrome                Aspergillus quadricinctus
                               penicillins G and V and cephalosporin C, produced for
is paving the way for          over 50 years, with continuous strain and fermentation
the development of                                                                           Echinocandin               Aspergillus nidulans
                               improvement programmes. During the past 15 years,             Cyclosporin                Tolypocladium inflatum
new drugs.                     most of the genes encoding the biosynthetic steps have
                               been characterized, leading to a detailed understanding       Destruxin                  Metarhizium anisopliae
                               of the biochemistry and regulation of these pathways.         Enniatin                   Fusarium oxysporum
                               Nevertheless, the long history of traditional strain          Beauvericin                Beauveria bassiana
                               improvement by mutagenesis and screening had already
                               put into place many of the changes that an applied      peptide synthetases in fungi and bacteria. Although the
                               molecular biologist might have considered after iso-    peptide products show a wide range of biological
                               lating the genes. These include increased gene copy     activity, from antibiotics to pathogenicity factors (Table
                               number and enhanced transcription, and limit the scope  1), the biosynthetic mechanism is conserved, and the
                               for further yield improvement. A more sophisticated     genes responsible are instantly recognizable from their
                               approach, the engineering of a hybrid cephalosporin     modular organization (Fig. 1). A module of some 600
                               pathway in the penicillin producer Penicillium chrysogenum,
                                                                                       amino acids is required for each amino acid incorporated
                               was achieved as an alternative route to semi-synthetic  into the peptide. Amino acids are recognized, adenyl-
                               cephalosporins, but its commercial advantage has yet to ated, and covalently bound to a module via a 4′-
                               be established.                                         phosphopantetheine cofactor, and less well conserved
                                                                                       regions are probably involved in peptide bond
                             q Non-ribosomal peptide synthesis                         formation. The final peptide is released as a linear or
                             One of the fascinating results of genetic analysis of cyclic structure, depending on the system.
                             β-lactam biosynthesis was the discovery that the first      Cyclosporin A, a product of Tolypocladium inflatum
                             step, synthesis of the tripeptide δ-(L-α-aminoadipyl)- (Fig. 2), was identified by screening in the 1970s as
                             L-cysteinyl-D-valine (ACV) from precursor amino an antifungal and anti-lymphocytic compound, and
                             acids, was catalysed by a multifunctional enzyme exploited as an immunosuppressant, revolutionizing
                             closely related to those responsible for synthesis of the organ transplant surgery. Subsequent studies on its
                             antibiotics gramicidin and tyrocidin by Bacillus species. mode of action as an inhibitor of cyclophilin, a peptidyl
                             While non-ribosomal peptide biosynthesis and its prolyl isomerase involved in calcium signalling
                             evolutionary significance had already been described by following antigen recognition by T-cells, opened up
                             Fritz Lipmann ‘before cloning’, gene isolation and DNA avenues for discovery of new immunosuppressants.
                             sequencing have revealed a large and growing family of Interestingly, the compound also has anti-Plasmodium
                                                                                                                      activity. Cyclosporin is an
      Fig. 1. Modular arrangement in peptide synthetases                                                              undecapeptide, assembled
                                                                                                                      by a synthetase consisting
      ACV synthetase                                                                                                  of a single polypeptide
                                                        Adenylation        N-Methylation    Thioester formation       with a molecular mass
                                                                                                                      of some 1.7 million Da.
      Cyclosporin synthetase                                                                                          Some of its 11 modules
                                                                                                                      contain inserted domains
                                                                                                                      responsible for N-methyl-
                                                                                                                      ation of the respective


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      MICROBIOLOGYTODA VOL27/AUG00
amino acids (Fig. 1). This is one of the largest known       applications, such as hastening labour or preventing
enzyme polypeptides, incorporating 40 catalytic              post-partum bleeding, were recognized from the Middle
functions. While these enzymes appear to use a rather        Ages, and semi-synthetic derivatives such as dihydro-
cumbersome way of assembling small peptides, their           ergotamine were developed in the 1940s for treatment
speciality is their ability to escape the bounds set by      of blood pressure and migraine. Ergotamine and semi-
ribosomal peptide synthesis. In addition to incorp-          synthetic derivatives are structural analogues of
orating valine and alanine, cyclosporin synthetase can       serotonin and interact with its receptors. Recent studies
N-methylate leucine, and incorporate 2-butenyl-4-            on the biochemistry and genetics of ergotamine
methyl-L-threonine and α-aminobutyrate.                      biosynthesis have shown that lysergic acid, synthesized
   As medical conditions, including AIDS, resulting in a     by the fungus, is converted to ergotamine via a three-
compromised immune system, have increased in recent          module peptide synthetase which adds alanine, proline
years, systemic fungal infections have increased,            and phenylalanine (Fig. 3).
stimulating a search for better antifungal drugs. The
cyclic lipopeptide echinocandin, probably elaborated         q Polyketide synthetases
by a peptide synthetase, is produced by a number of          Another major family of multifunctional enzymes
fungi, including a sub-species of Aspergillus nidulans.      responsible for biosynthesis of secondary metabolites
Investigated for some time as an antifungal antibiotic, it   are the polyketide synthetases, which are relatives of
interferes with fungal cell wall assembly by inhibiting      fatty acid synthetases. While these have been studied
the synthesis of β-1,3 glucan. Improved semi-synthetic       most intensively in the prokaryotic actinomycetes, they
derivatives produced by Eli Lilly and Merck are currently    are also responsible for assembly of potent carcinogens,
undergoing clinical trials as anti-Candida agents.           the aflatoxins of Aspergillus parasiticus, and the chole-
   Ergot alkaloids produced by the plant pathogen            sterol biosynthesis inhibitor lovastatin, produced
Claviceps purpurea during infection of rye were              commercially by Aspergillus terreus. Lovastatin is an
responsible for outbreaks of St Antony’s Fire, described     inhibitor of hydroxymethylglutaryl (HMG) CoA
as long ago as the 9th century. Victims suffered             reductase, an early step in cholesterol biosynthesis, and
gangrene, convulsions and hallucinations after con-          was developed as a treatment for familial hyper-
suming contaminated rye bread. However, medical              cholesterolaemia. Subsequently, it has been found to
                                                                                           be effective at reducing
                                                                                                                       LEFT:
                                                                                           cholesterol levels in Fig. 2. Tolypocladium inflatum .
                                                                                           individuals with dietary COURTESY BIOCHEMIE GMBH
                                                                                           problems.
                                                                                              In the case of both
                                                                                           polyketide (PKS) and non-
                                                                                           ribosomal peptide synthe-
                                                                                           tases (NRPS), in fungi and
                                                                                           bacteria, these enzymes
                                                                                           often form only part of a
                                                                                           complex pathway involv-
                                                                                           ing many other genes for
                                                                                           synthesis of precursors, or
                                                                                           modification of products.
                                                                                           Indeed, there are now some
                                                                                           prokaryotic examples of
                                                                                           PKS and NRPS modules
                                                                                           co-operating to produce
                                                                                           secondary metabolites,
                                                                                           exemplified by rapamycin,
                                                                                           a new immunosuppressant,
                                                                                           and yersiniabactin, an
                                                                                           iron-chelating siderophore
                                                                                           and pathogenicity factor in
                                                                                           plague. Elucidation of
                                                                                           other pathway components
                                                                                           is aided by the common
                                                                                           observation that the PKS
                                                                                           and NRPS genes are


                                                                                                  MICROBIOLOGYTODAYVOL27/AUG00                  119
                                                                                           Webwatch
 Fig. 3. Ergotamine biosynthesis in Claviceps purpurea


               D-Lysergic acid




                       Alanine        Phenylalanine       Proline


                               3-module peptide synthetase                                 Free online access until Sept 1 2000,
                                                                                           hosted by BioMedNet
                                                                                           Go to bmn.com
                      Ergotamine                         LA-Ala-Phe-Pro                    Download current issues and back issues
                                                                                           to Jan 1998.
Further reading                  located within large gene clusters, which include genes
                                 for the other steps. Recent examples include the
Lipmann, F. (1971).
Attempts to map a process        lovastatin and ergotamine gene clusters.
evolution of peptide
biosynthesis. Science 173,     q Future drug discovery
875–884.                       While the examples of useful fungal secondary meta-
Kleinkauf, H. &                bolites described above have been discovered via
von Döhren, H. (1996). A       traditional natural product screening methods, the
nonribosomal system of         subsequent genetic analyses suggest alternative             Seward Ltd manufacture the Stomacher® range
peptide biosynthesis. Eur J    approaches based on the highly conserved and easily         of lab blenders, accessories and consumable
Biochem 236, 335–351.          recognizable module, domain and motif structures of         Stomacher bags and Simplettes. Seward also
Penalva, M.A., Rowlands,       the biosynthetic enzymes. Either genomic data mining        manufactures the Clear-spin® microcentrifuge
R.T. & Turner, G. (1998).      or PCR-based approaches could be used to discover new       and Cryosect® cryo-microtome.
The optimization of penicillin enzymes and secondary metabolic pathways, providing
biosynthesis in fungi. Trends  opportunities for product discovery where natural           Website www.seward.co.uk
Biotechnol 16, 483–489.        expression is low and the product cannot be easily          email info@seward.co.uk
Tudzynski, P. & others         detected in conventional screens. For example, the
(1999). Evidence for an ergot  Canadian company TerraGen aims to use a gene-based
alkaloid gene cluster in       approach to screen fungi which are difficult to culture,
Claviceps purpurea. Mol Gen    such as those found in lichens.
Genet 261, 133–141.               A common feature of commercially exploited fungal
Kennedy, J. & others (1999). secondary metabolites is that natural products have been
Modulation of polyketide       chemically modified to yield semi-synthetic derivatives.
synthase activity by accessory An attractive additional approach would be to redesign
proteins during lovastatin     the biosynthetic pathways, including the multi-
biosynthesis. Science 284,     functional enzymes. This approach, using detailed
1368–1372.                     knowledge of gene structure and pathway biochemistry,       Free online access until Sept 1 2000,
McDaniel, R. & others          and taking advantage of the modular structure of the        hosted by BioMedNet
(1995). Rational design of     enzymes, has already led to promising progress in the
aromatic polyketide natural    case of polyketide synthetases of the prokaryotic           Go to bmn.com
products by recombinant        actinomycetes, though success has been more limited so
assembly of enzymatic                                                                      Download current issues and back issues
                               far for peptide synthetases, where a better understanding   to Jan 1998.
subunits. Nature 375,
                               of enzyme structure and function is needed.
549–554.
Stachelhaus, T., Schneider,      q Geoffrey Turner is Professor of Genetics
A. & Marahiel, M.A. (1995).      in the Department of Molecular Biology and
Rational design of peptide
                                 Biotechnology at the University of Sheffield,
antibiotics by targeted
replacement of bacterial and
                                 Firth Court, Sheffield S10 2TN
fungal domains. Science 269,     Tel. 0114 222 6211; Fax 0114 272 8697;
69–72.                           email g.turner@shef.ac.uk

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