Overexpression Of The Blue Light Recptor Wc-2 Gene Increases Production Of Ganoderic Acids In Ganoderma Lucidum

Ganoderma luciduma,a well-known medicinal fungus,has been employed to cure and prevent human diseases for two thousand years.Ganoderic acids(GAs),are one of the most important active constituents of G.lucidum,exhibit numerous pharmacological activities,such as cytotoxicity to hepatoma cells,antitumor,antimetastasis,and anti-HIV.Nevertheless,low GA production severely limits clinical research and applicability.Thus,research aimed at enhancing the production of GAs has significant practical value.Previous reports showed that blue light stimulates the growth of mycelium of G.lucidum and the accumulation of GAs in Ganoderma.However,whether the blue light receptor WC-2 mediates blue light signal to regulate GA biosynthesis and whether overexpression of the wc-2 gene can improve the production of GAs in Ganoderma lucidum is not yet clear.The blue light receptor wc-2 gene homolog gl24708 was cloned from the G.lucidum genome.By PCR amplification and sequencing analysis,a full sequence of1302 bp encoding 320 amino acids and a predicted molecular weight of 35.2 k Da was determined.Blast protein sequence analysis revealed that the amino acid sequence encoded by the gl24708 was highly similar to the amino acid sequences of wc-2 in Grifola frondosa,Suillus lakei,and Pleurotus ostreatus,by 61.68%,46.87%,and47.44%,respectively.Our results showed that the gl24708 gene may therefore encode the WC-2 protein in the G.lucidum.We next utilized dual sg RNA-mediated CRISPR/Cas9 gene editing technology to delete the wc-2 gene in G.lucidum.Genomic PCR amplification and sequencing analysis showed that the wc-2 gene in the mutant had 788 bp less bases than that in the control strain.Then,the contents of GAs were investigated in the control strain and the mutant under blue light induction condition.Results showed that the total GAs and GA-Mk,GA-T,GA-S,GA-Me contents of theΔwc-2 strain were significantly lower than those of WT control strain.The highest content of total GAs and individual GA-Mk,GA-T,GA-S,and GA-Me were 4.9 mg/100 mg dry weight(DW),53.54,63.42,56.15,and 38.65μg/100 mg DW,which were 32.56%,17.54%、14.95%、9.03%and 20.21%of the control.The findings suggested that WC-2 may regulate the production of GAs in response to a blue light signal.Moreover,we analyzed the effect of wc-2 gene deletion on the production of asexual spores,intermediate metabolites,and expression levels of key GA biosynthetic genes hydroxymethylglutaryl reductase gene(hmgr),lanosterol synthase gene(ls),and squalene synthase gene(sqs).The highest number of asexual spores was 0.63×10~7/cm~2,which was 33.51%of the control,and the expression level of asexual sporulation-related gene gl25098 was also significantly reduced in the mutant.In addition,the accumulation of intermediate metabolites in theΔwc-2 strain was significantly reduced.The highest contents of squalene and lanosterol were 0.276μg/100 DW and 5.13μg/100 DW,which was 15.08%and 49.04%of the control strain,respectively.The expression levels of hmgr,sqs,and ls in theΔwc-2 strain were was30%,21.67%,and 33.31%of the control strain,respectively.Our results suggested that the decreased content of GAs in theΔwc-2 strain may be related to decreased numbers of asexual spores and metabolic flow of the GA biosynthesis.Furthermore,the wc-2 gene was overexpressed in G.lucidum.q RT-PCR results showed that the expression level of the wc-2 gene in the overexpression(OEwc-2)strain was 4.56 times that of the wild-type(WT)strain.A novel approach by combining wc-2gene overexpression and bule light induction was developed to enhance GA production in liquid static cultures of G.lucidum.The maximum contents of total GAs and individual GA-Mk,GA-T,GA-S,and GA-Me were 8.29 mg/100 mg DW,650.66,695.01,635.07 and 354.71μg/100 mg DW respectively under the integrated approach,which were 2.1-,1.75-,-2.55-and 1.74-fold of the WT strain.The contents of squalene and lanosterol were increased by 2.17-and 1.67-fold in this case compared with those in the control.The expression levels of hmgr,sqs,and ls in the OEwc-2 strain were upregulated by 3.54-,6.58-and 6.60-fold respectively.In summary,we presented a fermentation approach to boost GA production in G.lucidum by combining overexpression of the wc-2 gene and blue light induction.These results help improve our understanding of the regulatory mechanisms of GA biosynthesis in Ganoderma and will be valuable to efficient production of GAs on a large scale.