ABSTRACT
A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5-7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.
ABSTRACT
The biosynthetic gene cluster of γ-aminobutyric acid (GABA)-containing fungal cyclic heptapeptides unguisins A (1) and B (2) was identified in the fungus Aspergillus violaceofuscus CBS 115571. In vitro enzymatic reactions and gene deletion experiments revealed that the unguisin pathway involves the alanine racemase UngC to provide d-alanine, which is then accepted by the first adenylation domain of the nonribosomal peptide synthetase (NRPS) UngA. Intriguingly, the hydrolase UngD was found to transform unguisins into previously undescribed linear peptides. Subsequently, heterologous production of these peptides in Aspergillus oryzae was achieved, in which we established a methodology to readily introduce a large NRPS gene into the fungal host. Finally, genome mining revealed new unguisin congeners, each containing a (2R,3R)-ß-methylphenylalanine residue.
