Overexpression Of The Homologous Squalene Epoxidase Gene Alone And In Combination With The N-terminally Truncated 3-hydroxy-3-methylglutaryl Coenzyme A Gene Leads To Increased Ganoderic Acid Production In Ganoderma Lingzhi

Ganoderma lingzhi,as a famous traditional Chinese medicinal mushroom,has been used as a traditional medication for the prevention and treatment of various human diseases for several thousand years.Ganoderic acids(GAs)and polysaccharide produced by this fungi possess a variety of bioactivities such as anticancer,anti-HIV virus and improving immunity.Due to the important pharmacological functions of GAs and polysaccharide,the interest in G.lucidum has increased around the world in recent years,and many researchers have focused on studying fermentation conditions to accelerate mycelial growth and optimize the total GAs and polysaccharide production.However,commercial application of mycelia fermentation is still limited due to the low production of GAs and polysaccharide.Previous reports on the yield of Ganoderma lingzhi were mainly focused on the optimization of culture conditions and the addition of inducers.With the key genes of Ganoderma lingzhi metabolic pathway cloned,overexpression of Ganoderma lingzhi metabolic pathway gene was used in the production of GAs by overexpression of HMGR gene,SQS gene and LS gene increased the yield of GAs,but overexpression of others genes in Ganoderma lingzhi synthesis pathway have not been reported.Squalene epoxidase(SE),catalyzes the first oxidation reaction in the synthesis pathway of G.lingzhi,and catalyzes the production of oxidosqualene by squalene.Manipulation of SE genes has been applied to the production of secondary metabolites in other species.However,overexpression of SE gene to increase GAs was not reported.Here,a putative squalene epoxidase gene,a key gene in the biosynthesis of GAs,was cloned and overexpressed in G.lingzhi.Overexpression of the homologous SE gene produced approximately 1.4-times higher production of individual GAs than the wild-type(WT)strain.In the SE gene overexpression the maximum contents of GA-T,GA-S,GA-Mk,and GA-Me were 57.7±4.2,19.1±2.1.4.3±0.2 and 33.6±2.4μg/100 mg dry cell weight,respectively,which were 3.2,2.4,1.8,and 2.9 fold higher than those obtained in the wild-type(WT)strain.In addition,the SE gene overexpression resulted in up-regulation of SE and lanosterol synthase genes in ganoderic acid(GA)biosynthetic pathway.The results indicated that SE plays an important role in stimulating GAs accumulation in G.lingzhi.In the metabolic pathway of G.lingzhi,the previous study was mainly based on overexpression of individual gene.Because of the complexity of the synthetic pathway of G.lingzhi,this strategy only improves the moderated yield of GAs.Co-overexpression of multiple genes has increased the yield of terpenoids in other species,whereas co-overexpression of multiple genes has not been reported in G.lingzhi.3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)is a rate-limiting enzyme in the MVA metabolic pathway of G.lingzhi.The overexpression of HMGR gene in G.lingzhi enhances the accumulation of precursor material,and SE is the first regulatory enzyme in the steroid or saponin synthesis pathway.Therefore,high expression of HMGR gene and SE gene may be higher in GAs production.Furthermore,the homologous SE and N-terminally truncated 3-hydroxy-3-methylglutaryl coenzyme A(HMGR)genes were simultaneously overexpressed in G.lingzhi.The combinational overexpression of the SE with HMGR genes further enhanced production of individual GAs compared to SE or HMGR gene alone.The maximum contents of GA-T,GA-S,GA-Mk and GA-Me in the transgenic strain were 54.3±3.8,157.5±4.9,25.5±1.2 and 73.2±8.7μg/100mg,respectively,which were 4.6-,4.87-,2.4-.6.9-times higher than those of the WT strain.Our results demonstrate that manipulation of multiple biosynthetic genes is a powerful approach to enhance GA production in G.lingzhi.