A combined strategy for the overproduction of complex ergot alkaloid agroclavine

Microbial cell factories(MCFs)and cell-free systems(CFSs)are generally considered as two unrelated approaches for the biosynthesis of biomolecules.In the current study,two systems were combined together for the overproduction of agroclavine(AC),a structurally complex ergot alkaloid.The whole biosynthetic pathway for AC was split into the early pathway and the late pathway at the point of the FAD-linked oxidoreductase EasE,which was reconstituted in an MCF(Aspergillus nidulans)and a four-enzyme CFS,respectively.The final titer of AC of this combined system is 1209 mg/L,which is the highest one that has been reported so far,to the best of our knowledge.The development of such a combined route could potentially avoid the limitations of both MCF and CFS systems,and boost the production of complex ergot alkaloids with polycyclic ring systems.

Production of Ergot Alkaloids by the Japanese Isolate Claviceps purpurea var. agropyri on Rice Medium

Ergot alkaloids (EAs) are secondary metabolites produced by ergot fungi (e.g., Claviceps purpurea), which are parasites of Gramineae grasses. EAs and their analogs are used to treat migraine, postpartum uterine bleeding, and Parkinson's syndrome. Recent studies have reported additional new bioactive activities of EAs and their analogs, making them essential compounds for drug development, drug repositioning, and clinical applications. EAs are produced industrially by field cultivation of ergot or liquid fermentation in the mycelial phase, but there are few published studies of the production of EAs by cereal culture and thus this approach is poorly understood. This study searched for Claviceps strains that produce EAs cultured artificially in the mycelial phase, then the selected strains were cultured on cereal media (white rice, brown rice, and rye) to examine their ability to produce EAs on each medium. C. purpurea var. agropyri produced the Clavine-type EAs pyroclavine (1), festuclavine (2), and agroclavine (3) in the mycelial phase. When cultured with white rice, brown rice, or rye, C. purpurea var. agropyri produced 1 - 3 on all cereal media. The total amount of 1 - 3 in each cereal medium (150 g of cereal per Roux flask) was 2220.5 ± 564.1 μg for white rice, 920.0 ± 463.6 μg for brown rice, and 595.4 ± 52.1 μg for rye. The white rice medium supported the highest production of 1 - 3, with the total amount of EAs (150 g of white rice per Roux flask) being about 34 times higher than that in the T25 liquid medium (190 mL per 1 L Erlenmeyer flask) (equivalent amount per flask).