Lipid accumulation and membrane fluidity influence mycelial stability and riboflavin production by the riboflavinogenic fungus Eremothecium ashbyii

摘要

The plant pathogenic filamentous hemiascomycete fungus Eremothecium ashbyii is a natural overproducer of riboflavin (vitamin B2). Preceding riboflavin overproduction, microdroplets of lipid were observed in the hyphae of E.ashbyii as yellow fluorescent bodies on staining with the lipid specific dye Nile blue. Following this the fungus was grown on different substrates-olive oil, sunflower oil and glucose. Lipid accumulation was followed as a time course by gravimetry. The mycelial lipid was fractionated into triglycerides and phospholipids and relative proportions of constituent fatty acids in them was estimated by GC MS during growth and riboflavin overproduction on each substrate. Changes in mycelial morphology were followed as a time course. In parallel, mycelial growth on each medium was converted to protoplasts whose membrane fluidity was monitored by measuring the fluorescence anisotropy using 1,6 - diphenyl - 1,3,5 - hexatriene (DPH). Lipid accumulation and extracellular lipase activity was maximum at 48 h of growth on all growth substrates. Maximum lipase production coincided with maximum lipid accumulation preceding riboflavin overproduction. A GC-MS analysis of fatty acids during growth (48 h) and riboflavin overproduction (96 h) showed the presence of a large percentage of unsaturated fatty acids in triglycerides and phospholipids. Decrease in Octadecadienoic acid (C 18:2) in the triglycerides during the production phase correlated with riboflavin production while mycelial stability correlated to the Octadecenoic acid (C 18:1) content of the phospholipids. Olive oil-grown mycelia showed maximum lipid accumulation, riboflavin production, lipase activity, membrane fluidity, stability and least morphological changes. Maximum riboflavin was obtained on olive oil medium due to a greater decrease in Octadecadienoic acid content in the triglycerides and the stability of olive oil grown mycelia was attributed to the high content of Octadecenoic acid in its phospholipids. Introduction Riboflavin (vitamin B2) is the precursor of the coenzymes Flavin mononucleotide (FMN) and Flavin adenine dinucleotide (FAD) which function as electron acceptors in various oxidation-reduction reactions in the cell. Riboflavin forms an important ingredient of animal feed supplements for monogastric animals such as poultry (Coopermann and Lopez, 1991) as well as multivitamin formulations. Though competitive chemical processes are used for the industrial production of riboflavin, much of the riboflavin required for animal feed purposes is derived by the biotechnical route on account of its cost effectiveness (Vandamme, 1992). Among the microorganisms used in the past and present for the commercial production of riboflavin, the hemiascomycete fungi Eremothecium ashbyii and Ashbya gossypii are important natural overproducers of the vitamin. Commercial fermentations for riboflavin production using E. ashbyii and A. gossypii were first established in 1940 and 1946 respectively (Perkins, et al., 1999; Wickerham, et al.,1946). Much of the current knowledge about riboflavin production has been acquired with mutants of E. ashbyii and A. gossypii. One previously reported interesting feature of riboflavin over production by E.ashbyii during growth on glucose medium is that it is preceded by an accumulation of lipids (Starka, 1957; Pujari and Chandra, 2001). More recently a large number of lipid bodies were reported in an overproducing mutant of E.ashbyii UV-18-57 while absence of lipid bodies was reported in a non flavinogenic mutant of E.ashbyii UV-85 (Pujari and Chandra, 2001). In the closely related A. gossypii, accumulation and degradation of lipid bodies has been shown to accompany riboflavin overproduction (Stahmann et al., 1994). This points to a probable role for lipids in riboflavin overproduction. Another interesting observation regarding riboflavin overproduction by E.ashbyii is that the production and excretion of riboflavin is accompanied by ch