Unveiling nonribosomal peptide synthetases from the ergot fungus involved in the formation of diverse ergopeptines.

Ergopeptines or their derivatives are widely used for treating neurodegenerative and cerebrovascular diseases. The nonribosomal peptide synthetase-d-lysergyl peptide synthetase A (LPSA) determines ergopeptine formation but the detailed mechanism remains to be elucidated. Here, we characterized two LPSAs from Cp-1 strain through heterologous expression in feeding with d-lysergic acid. We proved that Cp-LPSA1 catalyzed the formation of ergocornine, -ergocryptine, and -ergocryptine, precisely controlled by the substrate specificity of its three modules. Cp-LPSA2 was initially inactive but could be restored to catalyze -ergosine formation. Using this platform, we validated that P1-LPSA1 and P1-LPSA2 from the reported P1 strain catalyzed ergotamine and -ergocryptine formation, respectively. Typically, the non-ribosomal peptide codes implicated in every module of the LPSAs were defined and elucidated, in which certain key residues could play a switched role for substrate specificity and product interconversion. By constructing chimeric LPSAs through module assembly, the production of the desired ergopeptines was achieved. Notably, 1.46 mg/L of -ergocryptine and 1.09 mg/L of ergotamine were produced respectively by mixed-culture of No. 24 (fermentation supernatant) and the recombinants of . Our findings provide insights into the biosynthetic mechanism of ergopeptines and lay a foundation for directed ergopeptine biosynthesis.