Analyses of ergot alkaloid production in Aspergillus leporis and an easD knockout and sequences for phylogenetic analyses of rugulovasine-associated genes

Ergot alkaloids are fungal specialized metabolites that are important in
agriculture and serve as sources of several pharmaceuticals. Aspergillus
leporis is a soil saprotroph that possesses two ergot alkaloid
biosynthetic gene clusters encoding lysergic acid amide production. We
identified two additional, partial biosynthetic gene clusters within the
A. leporis genome containing some of the ergot alkaloid synthesis (eas)
genes required to make two groups of clavine ergot alkaloids,
fumigaclavines and rugulovasines. Clavines possess unique biological
properties compared to lysergic acid derivatives. Bioinformatic analyses
indicated the fumigaclavine cluster contained functional copies of easA,
easG, easD, easM, and easN. Genes resembling easQ and easH, which are
required for rugulovasine production, were identified in a separate gene
cluster. The pathways encoded by these partial, or satellite, clusters
would require intermediates from the previously described lysergic acid
amide pathway to synthesize a product. Chemical analyses of A. leporis
cultures revealed the presence of fumigaclavine A. Rugulovasine was only
detected in a single sample, however, prompting a heterologous expression
approach to confirm functionality of easQ and easH. An easA knockout
strain of Metarhizium brunneum, which accumulates the rugulovasine
precursor chanoclavine-I aldehyde, was chosen as expression host. Strains
of M. brunneum expressing easQ and easH from A. leporis accumulated
rugulovasine as demonstrated through mass spectrometry analysis. These
data indicate that A. leporis is exceptional among fungi in having the
capacity to synthesize products from three branches of the ergot alkaloid
pathway and for utilizing an unusual satellite cluster approach to achieve
that outcome.