Post-Genome Research on the Biosynthesis of Ergot Alkaloids

• Abstract
• Introduction
• Molecular Biological Investigation of Ergot Alkaloid Biosynthesis in Claviceps purpurea
• Identification of the Biosynthetic Gene Cluster of Fumigaclavine C in the Genome Sequence of Aspergillus fumigatus
• The Structural Similarities and Differences of the Ergot Alkaloids from C. purpurea and A. fumigatus are Perfectly Reflected by the DNA Sequences
• Outlook
• Acknowledgements
• References

Abstract

Genome sequencing provides new opportunities and challenges for identifying genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of Aspergillus fumigatus by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in Claviceps purpurea. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. fumigatus, which show remarkable similarities to dimethylallyltryptophan synthase from C. purpurea and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i. e., the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. purpurea, the identification of the biosynthetic gene cluster of fumigaclavine C in A. fumigatus opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.

Introduction

The biosynthesis of ergot alkaloids (e. g., ergotamine, Fig. [1]) has been studied for more than 40 years, especially in the Claviceps spp., mainly by feeding experiments [1], [2], [3]. The results of these feeding experiments proved unequivocally that the clavine skeleton is derived from L-tryptophan and dimethylallyl diphosphate [2], [3]. However, little is known about the enzymes involved and the exact reaction sequence of the biosynthesis of ergot alkaloids. The exceptions are the formation of dimethylallyltryptophan (DMAT) and its N-methylation. The responsible enzymes have been investigated in vitro as partially purified or crude enzyme extracts from the producer. Recently, the groups of Tudzynski and Keller have cloned, sequenced and identified the biosynthetic gene cluster of ergot alkaloids in C. purpurea. The function of several genes was proven by gene inactivation experiments [4], [5].

The genera Aspergillus and Penicillium produce a series of ergot alkaloids of the clavine type [6], e. g., fumigaclavines (Fig. [1]). Structurally, fumigaclavines have the same skeleton as lysergic acid (Fig. [1]). Both substance groups are expected to share similar reaction steps at the beginning of their biosynthesis.

A. fumigatus is a filamentous and ubiquitous opportunistic fungal pathogen of human and animals. It is the most frequent cause of invasive aspergillosis in immunocompromised individuals. The pathogenicity of A. fumigatus is poorly understood and there is limited knowledge on the basic biology of the organism. To understand the biology of the organism and to identify biochemical pathways that are critical for the pathogenicity and survival of A. fumigatus, which would act as potential targets of new antifungal compounds, the genome of A. fumigatus isolate AF293 was sequenced by an international consortium [7].

From the genome sequence of A. fumigatus, we could identify a DNA region by a bioinformatic approach, which is probably involved in the biosynthesis of fumigaclavine C. We have proven the function of this gene cluster experimentally by cloning and heterologous expression of the two prenyltransferase genes, fgaPT1 and fgaPT2, and by purification and biochemical characterization of the gene products. FgaPT2 and FgaPT1 catalyze the first and the last steps in the biosynthesis of fumigaclavine C, respectively (Fig. [1]) [8], [9].

Molecular Biological Investigation of Ergot Alkaloid Biosynthesis in Claviceps purpurea

Using the dmaW sequence, which codes a dimethylallyltryptophan synthase, the groups of Tudzynski and Keller have identified a putative biosynthetic gene cluster of ergot alkaloids from C. purpurea [4]. This cluster extends over 68.5 kb and contains a homologue of dmaW, cpd1, 4 different non-ribosomal peptide synthetase (NRPS) and several oxidase genes [4]. The functions of three genes, i. e., two peptide synthetase genes and a cytochrome P450 monooxygenase gene have been proven by knock-out experiments [4], [5].

Identification of the Biosynthetic Gene Cluster of Fumigaclavine C in the Genome Sequence of Aspergillus fumigatus

The genome of A. fumigatus AF293 consists of eight chromosomes with a total length of 29.4 megabases containing 9,926 predicted genes [7]. Since the genes for the biosynthesis of secondary metabolites are usually clustered in bacteria and fungi [10], we tried to find the putative biosynthetic gene cluster of fumigaclavine C by screening the whole genome sequence of A. fumigatus with the cpd1 sequence from C. purpurea [4], coding the enzyme DMATS, which is also expected to be involved in the biosynthesis of fumigaclavines. Seven homologues of cpd1 were found in the genome sequence, i. e., Afu2g17990 and Afu2g18040 on chromosome 2, Afu3g12930 on chromosome 3, Afu7g00170 on chromosome 7, Afu8g00210, Afu8g00250 and Afu8g00620 on chromosome 8, respectively [7]. One of them, Afu2g18040 = fgaPT2, encodes a protein with 54 % identity to DMATS from C. purpurea on the amino acid level. The other six ORFs show lower but significant sequence similarities of about 30 % to DMATS, indicating that FgaPT2 is very likely to catalyze the same or a very similar reaction as DMATS and the other DMATS homologues are probably involved in the prenylation reactions of different substances.

Analysis of the flanking regions of fgaPT2 leads to the identification of a putative biosynthetic gene cluster for fumigaclavine C (Fig. [2]). This gene cluster spans a DNA range of 22 kb (from bp 2,907,012 to bp 2,929,163 in AAHF01000001.1) and comprises 11 ORFs. The cluster containing fgaPT2 is the only gene cluster found in the genome sequence of A. fumigatus that contains several homologous genes from the cluster of the ergot alkaloids from C. purpurea (see below), indicating that this gene cluster is probably responsible for the biosynthesis of a similar compound as ergot alkaloids from C. purpurea.

Experimental evidence that the identified gene cluster is involved in the biosynthesis of fumigaclavine C was provided by the heterologous expression of the two prenyltransferase genes, fgaPT2 and fgaPT1 and biochemical characterization of the overproduced proteins. Our results showed that FgaPT2 converts L-tryptophan to DMAT in the presence of DMAPP and therefore catalyzes the first pathway-specific step in the biosynthesis of ergot alkaloids. FgaPT1 prenylates fumigaclavine A at position 2 of the indole nucleus and therefore is involved in the last step of the fumigaclavine C biosynthesis. Involvement of fgaPT2 in the biosynthesis of fumigaclavine C was also demonstrated by an inactivation experiment [11].

The Structural Similarities and Differences of the Ergot Alkaloids from C. purpurea and A. fumigatus are Perfectly Reflected by the DNA Sequences

Fumigaclavines are produced by A. fumigatus [6], but not by the fungal family of the Clavicipitaceae, e. g., C. purpurea [6]. Conversely, the amides and peptides of lysergic acid are produced by C. purpurea, but not by A. fumigatus [6]. In contrast, agroclavine (Fig. [1]) was identified in both fungi [6]. Therefore, we hypothesize that the early stages of the pathway of ergot alkaloids up to agroclavine are shared by A. fumigatus and C. purpurea, whereas later steps in the pathways differ in the two fungi (Fig. [1]). Support for this hypothesis is provided by comparison of both biosynthetic gene clusters. Seven homologous genes are found in the biosynthetic gene cluster of fumigaclavines in A. fumigatus AF293 (Fga) and of ergot alkaloids in C. purpurea (Cp) (Fig. [2]) [8], [11]. The sequence identities between FgaPT2 and Cpd1, FgaOX1 and CpOx1, FgaOX2 and CpOx2, FgaOX3 and Cpox3, FgaOrfA and CpOrfA, FgaOrfB and CpOrfB as well as FgaCat and Cpcat2 were found to be significantly high, namely 54 %, 37 %, 60 %, 52 %, 40 %, 53 % and 55 % on the amino acid level, respectively. These seven genes may be involved in the formation of agroclavine, the common structural feature of fumigaclavines from A. fumigatus and of ergot alkaloids from C. purpurea [4], [9] (Fig. [1]).

In comparison to ergot alkaloids from C. purpurea, fumigaclavine C contains no peptide residue, but an acetoxy group at C-7 and a prenyl moiety at C-2. In accordance with these structural features, the fumigaclavine C cluster, in contrast to the ergot alkaloid cluster from C. purpurea, contains no peptide synthetase genes, but genes which may encode enzymes for the synthesis of fumigaclavine C starting from agroclavine, e. g., a putative hydroxylase (FgaP450 - 2), a putative O-acetyltransferase (FgaAT) and the prenyltransferase FgaPT1 [8], [11]. The putative functions of these genes are given in Fig. [1].

Outlook

Identification of the putative biosynthetic gene cluster of fumigaclavine C from the genome sequence of A. fumigatus by a bioinformatic approach demonstrates the potential for finding genetic information for biosynthesis of secondary metabolites in genome sequences, which have been rapidly increased in number in the past years. By the middle of May 2006, genome sequences of more than 380 organisms became available. It can be expected that the biosynthesis of secondary metabolites will play an important role in the post-genome research.

The identification of the fumigaclavine C cluster in A. fumigatus provides a convenient way to identify the candidate genes for the common steps at the early stage of the biosynthesis of ergot alkaloids by comparison of this cluster with that of C. purpurea. It also provides additional opportunities to investigate the formation of the clavine skeleton with genes from both clusters. Gene inactivation experiments could be carried out in either of the organisms. All of these experiments will expand our understanding of the biosynthesis of the pharmaceutically important ergot alkaloids from C. purpurea.

The next challenge for investigation on the biosynthesis of the ergot alkaloids is to elucidate the function of genes found in both clusters, the functional genomics. This can be done by heterologous expression of the genes in E. coli, Saccharomyces cerevisiae or other hosts and by subsequent biochemical investigation of the enzymatic reaction with the overproduced proteins. Alternatively, the genes can be deleted from the genome by genetic manipulation and the function of the genes will be elucidated by subsequent analysis of the secondary metabolites in the defective mutants.

Using information obtained from functional genomics, new ”designed” compounds could be obtained by genetic engineering. It could also be possible to increase the yield of a desired compound by manipulation of the regulatory genes.

Acknowledgements

The work in our group was supported by a grant from the Deutsche Forschungsgemeinschaft (SPP 1152: Evolution of metabolic diversity, to S.-M. Li).

References

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Shu-Ming Li

Institut für Pharmazeutische Biologie und Biotechnologie

Heinrich-Heine-Universität Düsseldorf

Universitätsstr. 1

40225 Düsseldorf

Germany

Phone: +49-211-81-14180

Fax: +49-211-81-11923

Email: shuming.li@uni-duesseldorf.de

References

• 1 Floss H G. Biosynthesis of ergot alkaloids and related compounds.  Tetrahedron. 1976;  32 873-912
  CrossrefPubMedSearch in Google Scholar
• 2 Williams R M, Stocking E M, Sanz-Cervera J F. Biosynthesis of prenylated alkaloids derived from tryptophan.  Top Curr Chem. 2000;  209 97-173
  PubMedSearch in Google Scholar
• 3 Gröger D, Floss H G. Biochemistry of ergot alkaloids - Achievements and challenges. In: Cordell GA The Alkaloids. Vol. 50 San Diego; Academic Press 1998: p 171-218
  Search in Google Scholar
• 4 Haarmann T, Machado C, Lübbe Y, Correia T, Schardl C L, Panaccione D G. et al . The ergot alkaloid gene cluster in Claviceps purpurea: Extension of the cluster sequence and intra species evolution.  Phytochemistry. 2005;  66 1312-20
  CrossrefPubMedSearch in Google Scholar
• 5 Haarmann T, Ortel I, Tudzynski P, Keller U. Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways.  ChemBioChem. 2006;  7 645-52
  CrossrefPubMedSearch in Google Scholar
• 6 Flieger M, Wurst M, Shelby R. Ergot alkaloids - sources, structures and analytical methods.  Folia Microbiol (Praha). 1997;  42 3-30
  PubMedSearch in Google Scholar
• 7 Nierman W C, Pain A, Anderson M J, Wortman J R, Kim H S, Arroyo J. et al . Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus .  Nature. 2005;  438 1151-6
  CrossrefPubMedSearch in Google Scholar
• 8 Unsöld I A, Li S -M. Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus .  Microbiology. 2005;  151 1499-1505
  CrossrefPubMedSearch in Google Scholar
• 9 Unsöld I A, Li S -M. Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification and characterization of fumigaclavine C synthase FgaPT1.  Chembiochem. 2006;  7 158-64
  CrossrefPubMedSearch in Google Scholar
• 10 Keller N P, Turner G, Bennett J W. Fungal secondary metabolism - from biochemistry to genomics.  Nat Rev Microbiol. 2005;  3 937-47
  CrossrefPubMedSearch in Google Scholar
• 11 Coyle C M, Panaccione D G. An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus .  Appl Environ Microbiol. 2005;  71 3112-8
  CrossrefPubMedSearch in Google Scholar

Shu-Ming Li

Institut für Pharmazeutische Biologie und Biotechnologie

Heinrich-Heine-Universität Düsseldorf

Universitätsstr. 1

40225 Düsseldorf

Germany

Phone: +49-211-81-14180

Fax: +49-211-81-11923

Email: shuming.li@uni-duesseldorf.de