Enhanced Production Of Ganoderic Acids In Static Liquid Culture Of Ganoderma Lucidum By Addition Of Small Molecules And A Preliminary Study On Their Acting Mechanism

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. Despite this, the low content of ganoderic acids is always the bottle neck for the application. We had reported a two-stage cultivation strategy that significantly increased ganoderic acids'production, but it still far beyond the needs for application. In this work, the addition of two little molecular elicitors, phenobarbital and miconazole, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation of G. lucidum.The addition of 100μM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4±0.6mg/g cell dry weight (DW) and increases in the levels of ganoderic acids-Mk, -T, -S and -Me in the treated cells by 30-60% (compared to those with DMSO). Meanwhile, transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), squalene synthase (SQS) and lanosterol synthase (LS) in the triterpene biosynthetic pathway were up-regulated, and the accumulation of lanosterol, a key intermediate, was found to decrease under phenobarbital induction. These results indicate phenobarbital induced biosynthesis of ganoderic acids with more lanosterol was being converted to ganoderic acids.The optimum treatment for miconazole was 10μM on day 7. The maximum content of total ganoderic acids was 30.4±0.4mg/g DW, and the levels of ganoderic acids-Mk, -T, -S and -Me in the treated cells were increased by 20-35% (compared to those with DMSO). In the miconazole-grown cells there was ergosterol depletion leading to accumulation of lanosterol, an important biosynthetic intermediate, resulting in ganoderic acid increasing. We also detected the transcriptions of four key genes (hmgr, sqs, ls,and 14α-demethylase (14α-ldm)), and all of them showed depletion compared to control (those with DMSO), indicating miconazole may acted through other ways, which need further research.This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum. These important informations provide a good reference to manipulating production of ganoderic acids and other triterpenes in high potency.