Preparation and Regeneration of Protoplasts from the Ethyl Vincamine Producing Fungus CH1 (Geomyces sp.)

Abstract

Vinpocetine, a semi-synthetic compound derived from the alkaloid vincamine, exhibits effective pharmacological activities for the treatment and prevention of cerebrovascular circulation and vascular cognitive disorders. Vinpocetine can be produced through a one-step chemical reaction beginning with ethyl vincamine, and a two-step chemical reaction beginning with vincamine. In our previous study, the endophytic fungus CH1, Geomyces sp., was isolated and identified as a producer of ethyl vincamine, which was first obtained by endophytic fungal fermentation. However, the production was largely limited. Fungal protoplasts are a valuable experimental tool for physiological and genetic research such as protoplast fusion, gene transfer and metabolite production. In this paper, we optimized some key factors for the preparation and regeneration of protoplasts from strain CH1. Using an enzymes mixture consisting of cellulase (2.0%, w/v), glusulase (3.0%, w/v) and driselase (1.0%, w/v) in osmotic stabilizer (0.7 mol/L NaCl), the highest yield of protoplasts (6.78×107/mL) was obtained with mycelia after 72 h at pH 5.0-6.0 by digesting for 1.5 h at 30°C. After purification of the prepared protoplasts, they were regenerated in the regeneration medium using a bilayer plate culture method.