Use of a nonhomologous end joining deficient strain (Δku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N -Methyl-Dimethylallyltryptophan to Chanoclavine I

“…As the recipient strain, a ku70-deficient derivative of the producer strain P1 was used, characterized by an enhanced efficiency for homologous recombination (5).…”

“…2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2,5,7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway.…”

Fungal Transformation: From Protoplasts to Targeted Recombination Systems

“…Additional enzymes (DNA ligase IV and the protein Xrcc4) complete the DNA repair, following the integration. Due to the interest in decreasing nonhomologous recombination, the NHEJ system has been investigated in some non-conventional yeasts (e.g., Kluyveromyces lactis (Kooistra et al 2004 )) and several fi lamentous fungi, including Neurospora crassa (Ninomiya et al 2004 ;Ishibashi et al 2006 ), Claviceps purpurea (Haarmann et al 2008 ), several aspergilli [ A. nidulans (Nayak et al 2006 ), A. niger (Meyer et al 2007 ), A. fumigatus (Krappmann et al 2006 ), A. oryzae and A. soyae (Takahashi et al 2006 ), and A. parasiticus (Chang 2008 )], and P. chrysogenum (Snoek et al 2009 ). Mutants defective in the targeting proteins Ku70 and/or Ku80 have been isolated for each of these fungi.…”

Ergot Alkaloids

“…This fl exible biosynthesis scheme and the natural variability of the amino acid-binding domains of the LPS1 enzymes are the basis for the high variability of the ergot peptide alkaloid spectrum in the different natural chemical races of C. purpurea (see Table 14.1 ). This knowledge opens up interesting biotechnological perspectives: to generate C. purpurea strains producing single alkaloids, by knocking out lpsA1/2 and/or lpsC genes (exemplifi ed, e.g., by [ 56 ]) or even strains with new specifi cities by introducing "designer" lps genes.…”