Effect Of UDP-galactose-4-epimerase Gene(GalE) Overexpression On Ganoderma Lucidum Polysaccharides Biosynthesis

Known as “superior herb" in the Far East,Ganoderma lucidum(Reishi)has been used as functional food and preventive medicines for more than 2000 years for its numerous clinical efficacies,including strengthening the body,calming the nerves,soothing the liver,improving eyesight and even extending life expectancy.Polysaccharides,one of the most important pharmacologically active ingredients of G.lucidum,have been reported to possess anti-tumor,antioxidant,immunomodulatory,hypoglycemic effect,etc.The biological activities of G.lucidum polysaccharides are closely related to their structure.Overexpression of key biosynthetic genes like UDP-glucose pyrophosphorylase gene(UGP)and Phosphoglucomutase gene(PGM)has been proved to have promoted G.lucidum polysaccharides formation,yet the impact on their structures and bioactivities remained unclear.Galactose is an important monosaccharide of G.lucidum polysaccharides.Many studies have found that the anti-tumor activity of polysaccharides can be improved by increasing the proportion of galactose in polysaccharides.As a key enzyme gene of the galactose metabolic pathway(Leloir pathway),udp-glactose-4-epimerase gene(Gal E)has been proved to be involved in many important physiological processes in vivo,and be crucial for cell growth and polysaccharide generation of bacteria and plants.However,its function in fungi is obscure.Therefore,we undertook the research below to find out the influence on the biosynthesis of G.lucidum polysaccharides through Gal E gene overexpression in a submerged culture of Reishi.Firstly,the GalE gene was amplified from genome DNA of G.lucidum.The full length of Gal E gene was 1530 bp,encoding a total of 373 amino acids.The phylogenetic analysis showed that Gal E gene was highly homologous with other fungi,and the protein similarity reached more than 87%.The vector pJW-EXP-GalE was made by homologous recombination and transferred into G.lucidum protoplast to establish Gal E gene overexpression strain.The effects of Gal E gene overexpression on the growth of G.lucidum cells,yield of polysaccharides and transcription levels of relavant genes by liquid fermentation were investigated.It was found that Gal E gene could promote the growth of G.lucidum cells,increase its biomass by 10.63%,reaching 10.620±0.295 g/L.And the yields of extracellular polysaccharide(EPS)and intracellular(IPS)production were1.815±0.004 g/L and 27.599±1.323 mg/100 mg,which were enhanced by 10.46% and12.8% comparing to wild G.lucidum(WT),respectively.The expression levels of galactose-1-phosphate uridine transferase gene(Gal T)in galactose metabolic pathway were 2.43 times higher than in WT strain.Upon extraction and purification of Lingzhi polysaccharides,we found that the molecular weights of Gal E strain were higher than that of WT strain,and the molecular weight of main polysaccharide increased from 8723.2 k Da to 11913.9 k Da.At the same time,overexpression of Gal E gene had no effect on monosaccharide constituents of G.lucidum polysaccharides,but the proportion of each component was changed-galactose and mannose increased significantly(increased from 18.97%,12.04% to23.15%and 20.12% respectively)while glucose decreased significantly(decreased by19.86%,from 66.00% to 52.90%).The research of EPS on antioxidant activities indicated that the recombinant strain owned far more higher antioxidative effects than WT strain.Specifically,DPPH radical scavenging activity was 35.449±0.535%,which increased by 73.34%;ferrous metal ions chelating activity was 26.614±3.995%,which increased by 54.01%;and scavenging activity against superoxide radical increased by36.2%,the average value of which was above 80%.Overexpression of GalE gene has not only enhanced accumulations of IPS and EPS,but also increased the molecular weights,the component ratio of galactose and mannose,and the antioxidative activities of EPS.This study laid a foundation for further study of the effect of this regulatory mechanism on other biological activities and structures of G.lucidum polysaccharides,also suggested that regulation of genes in biosynthetic pathways other than glucose may be a new direction for further study of oriented regulation of polysaccharide components to improve pharmacological activities.