Molecular analysis of ergot alkaloid biosynthesis in Claviceps spec.

PAUL TUDZYNSKI
Institut für Botanik
Westfälische Wilhelms-Universität Münster
Schlossgarten 3
D-48149 Münster
tudzyns@uni-muenster.de
http://www.uni-muenster.de/Biologie.Botanik/agtudzynski/Tudzynsk.htm

References
Haarmann T, Machado C, Lübbe Y, Correia T, Schardl CL, Panaccione DG, Tudzynski P (2005) The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution. Phytochemistry 66: 1312-1320.
The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster (Tudzynski et al., 1999) was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5 kb) contains 4 different non-ribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.

Haarmann T, Ortel I, Tudzynski P, Keller U (2006) Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathway. ChemBioChem. 7: 645-652.
The clavines and the D-lysergic acid-derived alkaloid-amides and alkaloid-peptides represent two different families of compounds having in common the indole-derived tetracyclic metergoline ring system. Previous work has shown that D-lysergic acid is biosynthetically derived from the clavine alkaloids. The recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from D-lysergic acid peptides (ergopeptines)-producing Claviceps purpurea shows that it most probably contains all genes necessary for D-lysergic acid synthesis and in addition those encoding the assembly of D-lysergic acid peptides such as ergotamine. To address the role of oxygenase genes of the alkaloid gene cluster, inactivation by gene disruption of the only cytochrome P450 monooxygenase gene of this gene cluster was performed. The resultant mutant accumulated agroclavine, elymoclavine and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis suggesting a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (encoding a clavine oxydase, CLOA). Retransformation of the mutant with the intact cloA gene restored ergopeptine synthesis also. These data show that CLOA catalyses the step of conversion of clavines to D-lysergic acid; it acts as a critical enzyme in the ergot alkaloid gene cluster, bridging the biosynthesis of the two different families of alkaloids.

Schardl CL, Panaccione DG, Tudzynski P (2006) Ergot alkaloids - biology and molecular biology. Alkaloids Chem. Biol. 63: 45-86.

Haarmann T, Lorenz N, Tudzynski P (2007) Use of a nonhomologous end joining deficient strain (Dku70) of the ergot fungus Claviceps purpurea for identification of the nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genetics Biol. (in press)
The ergot fungus Claviceps purpurea uses mainly the non-homologous-end-joining (NHEJ) system for integration of exogenous DNA, leading to a low frequency of homologous integration (1-2 %). To improve gene targeting efficiency we deleted the C. purpurea ku70 gene in two different strains: the pathogenic strain 20.1 and the apathogenic, ergot alkaloid producing strain P1. The mutants were not impaired in vegetative and pathogenic development nor alkaloid production. Gene targeting efficiency was significantly increased (50-60 %) in the Dku70 mutants. The P1 Dku70 strain (producing ergotamine and ergocryptine) was used for targeted deletion of lpsA1, one of the two trimodular NRPS genes present in the alkaloid gene cluster, encoding D-lysergyl peptide synthetases involved in formation of the tripeptide moiety of ergopeptines. Mutants lacking the lpsA1 gene were shown to be incapable of producing ergotamine but were still able to produce ergocryptine, proving that LpsA1 is involved in ergotamine biosynthesis.

[back]