res of the ergocorine/ergokryptine producing Claviceps purpurea strain, Fb299.
The results showed that the radioactivity from l-valyl-(1- 14 C)-l-valyl-l-proline
was incorporated only after breakdown of the precursor into its component
amino acids. The results provided a basis for further investigations in this field.
Incorporation of natural amino acids by variation of amino acid I, II, and III is
reviewed [2]. Table 2 contains a summary of the research work done on the
directed biosynthesis of Claviceps using different synthons and incorporation
strategies.
3
Molecular Biology
The application of molecular biology to ergot alkaloid biosynthesis in Claviceps
purpurea has been reviewed [3, 6, 8]. In the first review, the authors have discussed genetic recombination, gene amplification, transposition, and fungal
cloning vectors, specifying that mitochondrial DNA or mitochondrial plasmids
may serve as a basis for development of a eukaryotic cloning system for the
fungus. In the second review, mutation, selection, and genetic recombination
have been highlighted, especially the development of a transformation system
and the widespread homology between mitochondrial plasmids and mitochondrial DNA in Claviceps purpurea. The third review focuses on transformation
systems and application of newer techniques such as restriction enzyme
mediated integration (REMI) for mutagenesis, pulse-field-gel electrophoresis
(PFGE) for karyotype analyses, and PCR methods such as random amplified
polymorphic DNA (RAPD) for identification/differentiation of C. purpurea. The
work done by Tudzynski and Arntz to identify the genes which are expressed
during alkaloid biosynthesis by differential cDNA screening led to the identification of gene coding for DMAT-synthase as an alkaloid pathway specific
gene, (for details see [73]), thus confirming earlier work [74] wherein partial
sequence information for the purified enzyme DMAT-synthase was obtained
and a degenerate oligonucleotide mixture was used to identify and amplify
segments of the gene. The complete gene and near full length cDNA were
cloned in a yeast expression vector and sequenced. The reviews of 1990 [3] and
1996 [6] say that the application of modern molecular biology has been limited
in this system due to the complex life cycle and long generation periods of the
fungus. However, the review from 1997 [8] is very optimistic and the authors
feel that application of modern molecular biology will open up interesting new
perspectives for the analysis of ergot alkaloid biosynthesis.
In this part of our review we mention further interesting work on the
molecular biology of the fungus not covered in the earlier reviews. In addition,
very recent work on the enzymology of Claviceps purpurea is presented.
The peptide synthetase gene families of Acremonium coenophialum and
Claviceps purpurea were investigated [75]. Hybridization analyses indicated
that the four fragments cloned from Acremonium coenophialum represented
three different peptide synthetase genes, most of which were present in multiple
copies in the genome of the fungus. Each of the three clones from Claviceps
purpurea appeared to be from a different peptide synthetase gene, only one of
12
J. Mukherjee · M. Menge
The results showed that the radioactivity from l-valyl-(1- 14 C)-l-valyl-l-proline
was incorporated only after breakdown of the precursor into its component
amino acids. The results provided a basis for further investigations in this field.
Incorporation of natural amino acids by variation of amino acid I, II, and III is
reviewed [2]. Table 2 contains a summary of the research work done on the
directed biosynthesis of Claviceps using different synthons and incorporation
strategies.
3
Molecular Biology
The application of molecular biology to ergot alkaloid biosynthesis in Claviceps
purpurea has been reviewed [3, 6, 8]. In the first review, the authors have discussed genetic recombination, gene amplification, transposition, and fungal
cloning vectors, specifying that mitochondrial DNA or mitochondrial plasmids
may serve as a basis for development of a eukaryotic cloning system for the
fungus. In the second review, mutation, selection, and genetic recombination
have been highlighted, especially the development of a transformation system
and the widespread homology between mitochondrial plasmids and mitochondrial DNA in Claviceps purpurea. The third review focuses on transformation
systems and application of newer techniques such as restriction enzyme
mediated integration (REMI) for mutagenesis, pulse-field-gel electrophoresis
(PFGE) for karyotype analyses, and PCR methods such as random amplified
polymorphic DNA (RAPD) for identification/differentiation of C. purpurea. The
work done by Tudzynski and Arntz to identify the genes which are expressed
during alkaloid biosynthesis by differential cDNA screening led to the identification of gene coding for DMAT-synthase as an alkaloid pathway specific
gene, (for details see [73]), thus confirming earlier work [74] wherein partial
sequence information for the purified enzyme DMAT-synthase was obtained
and a degenerate oligonucleotide mixture was used to identify and amplify
segments of the gene. The complete gene and near full length cDNA were
cloned in a yeast expression vector and sequenced. The reviews of 1990 [3] and
1996 [6] say that the application of modern molecular biology has been limited
in this system due to the complex life cycle and long generation periods of the
fungus. However, the review from 1997 [8] is very optimistic and the authors
feel that application of modern molecular biology will open up interesting new
perspectives for the analysis of ergot alkaloid biosynthesis.
In this part of our review we mention further interesting work on the
molecular biology of the fungus not covered in the earlier reviews. In addition,
very recent work on the enzymology of Claviceps purpurea is presented.
The peptide synthetase gene families of Acremonium coenophialum and
Claviceps purpurea were investigated [75]. Hybridization analyses indicated
that the four fragments cloned from Acremonium coenophialum represented
three different peptide synthetase genes, most of which were present in multiple
copies in the genome of the fungus. Each of the three clones from Claviceps
purpurea appeared to be from a different peptide synthetase gene, only one of
12
J. Mukherjee · M. Menge