Enhancement Of Ganoderic Acids And Polysaccharide Production In Ganoderma Lucidum By The Heterologous Expression Of Vitreoscilla Hemoglobin Gene

Ganoderma luicidum (G. lucidum), 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 study mainly focus on optimizing fermentation conditions, adding inducers and overexpression of homologous genes, such as 3-hydroxy-3-methylglutaryl coenzyme A reductase gene(hmgr), squalene synthase gene (sqs), phosphoglucomutase gene (pgm) and uridine diphosphate glucose pyrophosphorylase gene (ugp). Expression of heterologous gene to improve GAs accumulation and polysaccharide production in G. lucidum has no report yet. Vitreoscilla hemoglobin (VHb), a homodimeric oxygen binding protein, can reinforce cellular respiration intensity. Thereby, VHb has been used to increase cell density, metabolite synthesis in industrial applications. Expression of VHb in heterologous hosts can improve oxidative metabolism, cell growth, protein synthesis, bioremediation and metabolite production.Expression of Vitreoscilla hemoglobin (VHb) gene, vgb, was under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter in G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419　nm for the transfornant. In this study, we heteroexpressed vgb in G. lucidum to increase GAs and polysaccharide production.The effects of VHb expression on the GAs accumulation, lanosterol and the transcription of GA biosynthesis genes were investigated. In the vgb-bearing G. lucidum, the maximum contents of GA-S, GA-Mk, GA-Me, and GA-T were 19.1±1.8,191.5± 13.1,45.2±2.8, and 34.6±2.1μg/100 mg dry cell weight, respectively, which were 1.4,1.9,2.0, and 2.2 fold higher than those obtained in the wild-type (WT) strain. Moreover, the maximum lanosterol content in the vgb-bearing G. lucidum was 1.28 fold lower than that in the WT strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), squalene synthase (SQS), and lanosterol synthase (LS) genes were up-regulated by 1.6,1.5 and 1.6 fold, respectively, in the vgb-bearing G. lucidum.The effects of VHb expression on the polysaccharide production and the transcription of polysaccharide biosynthesis genes were also investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100 mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the WT strain. The transcription levels of phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and (3-1,3-glucan synthase (GLS) gene were up-regulated by 1.51,1.55 and 3.83 fold, respectively, in the vgb-bearing G. lucidum.The vgb-bearing G. lucidum has increased GAs accumulation and polysaccharide production in submerged condition. This work highlights the potential of VHb to enhance G. lucidum GAs accumulation and polysaccharide production by large scale fermentation.