Biosynthetic Characterization, Heterologous Production, and Genomics-Guided Discovery of GABA-Containing Fungal Heptapeptides

The biosynthetic gene cluster of gamma-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)-beta-methylphenylalanine residue.

Automated summary

The biosynthetic gene cluster for the cyclic heptapeptides unguisins A and B, which contain the neurotransmitter gamma-aminobutyric acid (GABA), was discovered in the fungus Aspergillus violaceofuscus CBS 115571. Enzymatic reactions and gene deletion experiments showed that the unguisin pathway involves the alanine racemase UngC to produce D-alanine, which is then utilized by the first adenylation domain of the nonribosomal peptide synthetase UngA. Surprisingly, the hydrolase UngD was found to convert unguisins into previously unknown linear peptides. Heterologous production of these peptides was achieved in Aspergillus oryzae, and genome mining revealed new unguisin variants, each containing a (2R,3R)-beta-methylphenylalanine residue.