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 (6.78×10 7 /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.

Enhancement of pigment and proximate values of intergeneric hybrid of protoplast fusion of Monascus ruber and Pleurotus ostreatus

“…A recent study optimized several critical variables for the synthesis and regeneration of protoplasts from strain CH1. The highest yield of protoplasts was produced using an enzyme mixture consisting of cellulase, glusulase, and driselase in an osmotic stabilizer [37]. Similarly, another study indicated that in an osmotic stabilizer (0.6 M KCl), Trichothecium roseum protoplasts were extracted using the combining the lytic enzyme.…”

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932

“…Different fungal strains possess cell walls with unique properties requiring the use of specific protoplast isolation conditions including the types and concentrations of cell wall-digesting enzymes ( Li et al, 2017 ; Roth and Chilvers, 2019 ). Moreover, selection of an appropriate type and concentration of an osmotic stabilizer in the protoplasting medium is critical in preventing lysis or shriveling of protoplast due to osmotic stress ( Ren et al, 2018 ; De Wu and Chou, 2019 ). Other than the enzymes and osmotic stabilizers, other factors such as enzymatic hydrolysis temperature, duration of incubation, fungal age and mycelium status need to be optimized for each strain ( Jin et al, 2020 ; Ning et al, 2022 ).…”

Ursodeoxycholic acid production by Gibberella zeae mutants

“…A sharp (more than fivefold) decrease in the yield of G. zeae protoplasts when using 48-h mycelium (Fig. 2 a) may be associated with increased differentiation of the population, a change in the frequency of septation of hyphae, and the appearance of spore cells with a denser cell wall (Ren et al 2018 ). As shown earlier, C. lunatus protoplasts obtained from actively growing mycelium were presumably more resistant to external influences compared to protoplasts obtained from cells of older fungi (Długoński et al 1998 ).…”

“…The use of protoplasts,—single mononuclear cells devoid of a cell wall and surrounded only by a cytoplasmic membrane, can facilitate genetic manipulation and mutagenesis of filamentous fungi and often plays a significant role in strain improvement (Roth and Chilvers 2019 ; Zhou et al 2008 ). In turn, effective protoplast production, release and mutagenesis depend on many factors including fungal species, mycelium state and protoplasting conditions (osmotic stabilizer, temperature, pH, digestion time, lytic enzyme concentration and others) whether a protoplast is mononucleate, binucleate or multinucleate (Kumari and Panda, 1992 ; Coelho et al 2010 ; Ren et al 2018 ).…”