Production of Vincristine and Vinblastine by the Endophytic Fungus Botryosphaeria laricina Strain (CRS1) is Dependent on Stimulating Factors Present in Catharanthus roseus

Abstract

Aims:To isolate vinca alkaloid producing endophytic fungi from Catharanthus roseus and the evaluation of the factors which enhance the vincristine production.Background:An endophytic fungus Botryosphaeria laricina (CRS1) isolated from Catharanthus roseus demonstrated vinca alkaloid production under certain conditions.Objective:o Understand the conditions under which the fungus was able to produce vincristine and vinblastine.Methods:Fungal isolates from C. roseus were grown in liquid culture and screened for alkaloid production. The strain (CRS1) producing catharanthine was sequenced and matched with GenBank. This isolated strain was studied for production of vinca alkaloids and the conditions required for vincristine and vinblastine production.Results:Eight endophytic fungi were isolated from the fresh aerial parts of C. roseus. Only CRS1, demonstrated catharanthine production. DNA sequencing of CRS1 gave a 100% match with the GenBank accession number, KC509580.1, which is related to the Botryosphaeria laricina strain JAS6. CRS1 produced only catharanthine when cultured in Czapek’s peptone liquid medium (CZ). Addition of C. roseus fresh plant extract (8.0 mL) to the culture medium (4.0 L) stimulated the production of catharanthine (3.2 mg), catharanthinic acid (0.3 mg), N-demethyl-vinblastine (0.3 mg), vinblastine (2.8 mg) and vincristine (2.4 mg). However, if the added plant extract was preheated (80 ˚C, for 15 min), no vinca alkaloids appeared other than catharanthine. To identify the active fractions of the plant extract stimulating vinca alkaloid production, the extract was dialyzed in buffer at 4 ˚C through 20 kDa MW cutoff membrane to separate into two fractions of molecules above and below 20 kDa MW. Only the fraction containing molecules above 20 kDa was able to transform catharanthine to vincristine and vinblastine. When the dialysis was performed in water instead of buffer, the larger fraction could only produce catharanthine and vinblastine. Other conditions such as the presence of light:dark (12:12 h), fructose (30.0 g L-1), glucose (30.0 g L-1), Cu2+(0.1 mM) ions, L-tryptophan (0.1%) and succinic acid (1%) did not induce alkaloid production.Conclusions:The catharanthine producing fungal strain B. laricina (CRS1) could only produce the two vinca alkaloids, vinblastine and vincristine from catharanthine in the presence of active components larger than 20 kDa MW present in the plant extract of C. roseus.