Analysis And Modification Of The Synthetic Pathway Of Monascus Lovastatin

Lovastatin is a substance that can inhibit the synthesis of cholesterol and is currently one of the most effective drugs for treating hyperlipidemia and regulating human cholesterol.Monascus can produce a variety of beneficial secondary metabolites,and lovastatin is one of them.In this project,we cloned lovastatin synthesis-related genes from Monascus pilosus GN-01,and constructed overexpression plasmids and knockout plasmids with the cloned genes,and transformed Monascus protoplasts mediated by PEG.Genes were overexpressed or knocked out in Monascus,and then the transformed strains were fermented to screen high-yielding lovastatin strains.The main research results are as follows:1.Using the pBC-hygro plasmid as an overexpression vector,the 7 genes related to lovastatin biosynthesis: mok C,mok D,mok E,mok F,mok H,mok I,and Lae A were respectively connected to the p BC-hygro plasmid,and 7 genes were successfully constructed The overexpression plasmid.Then,using the PEG-mediated protoplast transformation method,the overexpression plasmids of these 7 genes were transformed into Monascus respectively,and the overexpression strains selected were subjected to shake flask fermentation,and the yield of each gene overexpression strain lovastatin was measured in liquid phase.,Thus obtained 7 single gene overexpression high-yield strains.Compared with the lovastatin production of the original strain GN-01,the lovastatin production of the overexpression strains C1,D1,E1,F1,H3,I4,and L3 increased by 30.89%,29.67%,44.24%,58.60%,33.50,%,48.62%,33.27%,respectively.Among them,the overexpression strain F1 had the highest yield,reaching1299.78 mg/L.2.The mok C gene,the glycerol-3-phosphate dehydrogenase promoter(gpd)gene and the mok D gene were concatenated by overlapping PCR,and then the concatenated fragments of these three genes were ligated to the p BC-hygro plasmid to complete the mok C-mok D double Construction of gene overexpression plasmid.Then through the PEG-mediated protoplast transformation method,the double gene overexpression plasmid was introduced into Monascus pilosus GN-01,and the double gene overexpression strains CD5 and C1,D1 and GN-01 with the highest lovastatin yield were selected.At the same time,shake flask fermentation was carried out,and the production of lovastatin was measured in liquid phase.Compared with the lovastatin production of the original strain GN-01,the double gene overexpression strain CD5 lovastatin production increased by 46.62%,reaching 1188.21 mg/L.At the same time,compared with the lovastatin production of single gene overexpression strains C1 and D1,the double gene overexpression strain CD5 also increased by 10.98% and 12.13%,respectively.3.Using the pGKO2 plasmid as the knockout vector,first connect the upstream homology arm of the cloned pig R gene to the p GKO2 empty plasmid multiple cloning site to obtain the p GKO2-pig R-up plasmid;then connect the downstream homology arm to p GKO2-pig R-up.The pig R knockout plasmid p GKO2-pig R-up-dn was successfully constructed at another multiple cloning site of the up plasmid.Then,the pig R knockout plasmid was transformed into Monascus GN-01 by PEG-mediated protoplast transformation.The three selected pigment knockout strains were subjected to shake flask fermentation,and the production of lovastatin was detected in liquid phase and the production of Monascus pigment in the fermentation broth was determined by ultraviolet spectroscopy.Compared with the original strain GN-01,the output of lovastatin of knockout strains △pig R1,△pig R2,and △pig R3 reached1331.51 mg/L,1280.29 mg/L,and 1367.49 mg/L,which increased by 62.27%,56.46%,67.12%,respectively.The production of orange pigment and red pigment of knockout strains dropped sharply,and the yield of yellow pigment also decreased significantly.The total pigment output decreased by 87.78%,88.27%,and 86.28% respectively.