Increased Accumulations Of Ganoderic Acids In Ganoderma Lucidum By Integrating Molecular And Hybrid Breeding Techniques

Ganoderm lucidum,a traditional Chinese medicinal mushroom,can generate numerous bioactive chemicals,including ganoderic acids(GAs)and polysaccharides.GAs,a type of secondary metabolite produced by this medicinal fungus,exhibit a variety of essential pharmacological properties,including anti-HIV,antimetastasis,and antitumor properties.Unfortunately,the accumulation of GAs is currently insufficient for clinical trials and commercial uses.It was shown that integrating molecular and hybrid breeding techniques in rice and maize can not only increase the quantity of secondary metabolites,but also reduce breeding blindness.Yet,there are no reports of enhanced accumulations of secondary metabolites in mushrooms through the integration of molecular and hybrid breeding techniques.Two monokaryons,G.260125 and G.260124,of the dikaryon CGMCC5.0026 were isolated using the protoplast monokaryogenesis procedure,and the monokaryons strains were confirmed by clamp connection and single nucleotide polymorphism(SNP)sequencing.Thereafter,the PEGmediated transformation system was utilized to transform the G.260124 strain with the Vitreoscilla hemoglobin gene(24+vgb)and the G.260125 strain with the squalene synthase gene(25+sqs).Subsequently,the mutant strain v+s which is a hybrid of strain 24+vgb and 25+sqs was created and confirmed by PCR,clamp connection,and SNP sequencing.The accumulations of GAs of CGMCC5.0026 strain and v+s strain was analyzed in liquid static culture conditions.The cell growth of v+s strain was not obviously different from that of the CGMCC5.0026 strain.However,the highest contents of individual ganoderic acid(GA)-T,GA-S and GA-Me in the v+s strain were 124.876±14.322,27.945±2.672 and 68.154±1.775μg/100 mg dry weight(DW),which were 1.24-,1.52-,and 1.28-fold greater than in the CGMCC5.0026 strain,respectively.Additionally,we analyzed the accumulations of intermediate metabolites(squalene and lanosterol)and the transcript levels of two important genes,squalene synthase gene(sqs)and lanosterol synthase gene(ls),in GA biosynthesis under liquid static culture conditions in order to investigate the possible mechanism.In the v+s strain,the highest levels of squalene and lanosterol were 0.866±0.097 and10.638±0.50 μg/100 mg DW,which were 2.04-and 1.89-fold higher than in the CGMCC5.0026 strain,respectively.The highest expression levels of sqs and ls in the v+s strain were 3.07 and 2.42 times higher than in the CGMCC5.0026 strain,respectively.The increased GAs biosynthesis precursor supply and the up-regulated expression levels of GA biosynthetic genes in the v+s strain may account for the increased GA accumulation.The accumulations of GAs in the mycelia mat,the primordium,and the mature fruiting body of strains CGMCC5.0026 and v+s were also investigated.Results showed that the GA-T,GA-S,and GA-Me contents of the mycelia mat in the v+s strain were 21.456±5.659,2.045±0.929 and7.124±0.491 μg/100 mg DW,which were 3.38-,8.78-,and 1.73-fold higher than those in the CGMCC5.0026 strain,respectively.The GA-N,GEA-E,GA-A,GEA-D and GA-F contents of the primordium were 66.481±6.2,6.864±0.883,126.993±33.241,57.972±10.554 and 34.96±3.236 μg/100 mg DW in the v+s strain,representing 1.52-,1.66-,1.43-,1.53-and 1.57-fold greater than those in the CGMCC5.0026 strain,respectively.The GA-I,GAB,GA-K,GA-A,GA-H and GA-F contents of the mature fruiting body were31.147±2.167,20.719±0.687,22.446±2.94,43.654±1.924,47.677±5.369 and60.568±3.281 μg/100 mg DW in the v+s strain,representing 1.8-,2.5-,2.12-,1.8-,1.99-and 1.61-fold higher than those in the CGMCC5.0026 strain,respectively.Our results suggested that the integrating molecular and hybrid breeding techniques increased the accumulation of GAs during fruiting body development in G.lucidum.In summary,we used the molecular and hybrid breeding techniques for the first time to increase the accumulations of GAs in the mycelium and fruiting body of G.lucidum.It was discovered that the increased GA content in the engineered strain was relevant to the accumulation of more intermediate metabolites and the upregulation of GA biosynthetic gene transcription.This work will be helpful for efficient production of GAs and the breeding of G.lucidum and other mushroom.