The Study On High Production Of Lovastatin By Agrobacterium-mediated LovD Gene

Lovastatin is one of the most effective drugs in the treatment of cholesterol in the hyperlipidemia in the world.At present,lovastatin is mainly chemically synthesized.Monascus has a variety of secondary metabolites that are effective for humans,including lovastatin.The 3-hydroxy-3-methyl-glutaric acid monoterpene CoA reductase in humans plays a key role in regulating the synthesis of cholesterol.The physiological function of lovastatin is to inhibit the activity of this enzyme,thereby achieving cholesterol The reduction of the amount,in which the acyltransferase LovD is very important in the biosynthetic pathway of lovastatin,will increase the production of lovastatin by the study of the LovD gene.In this study,through the UV mutagenesis screening Monsus strain M120-1 as the basic material,mediated by Agrobacterium tumefaciens LovD gene transformation research.The total RNA was extracted from the strain of Monascus rubra.The LovD gene was cloned by reverse transcription-polymerase chain reaction(RT-PCR),then ligated and transformed into E.coli.The single factor experiments were used to optimize the transformation conditions.The transformants were identified by high performance liquid chromatography(HPLC)analysis of high-yield lovastatin strains.The use of Agrobacterium tumefaciens-mediated method to construct high quality and efficient Monascus genetic transformation system is expected to lay the foundation for the gene function of Monascus lovastatin.Research result:(1)Total RNA was extracted from M120-1 and cDNA sequence was synthesized.After purification and recovery,it was successfully ligated with the expression vector and finally transformed into Escherichia coli successfully.(2)The optimal genetic transformation system obtained after optimization was as follows:the protoplast concentration was 1×105 cells/mL;connect Monascus to PDA medium and culture at 30 ℃ for 13 days;Agrobacterium tumefaciens concentrations were incubated until the OD600 value was 0.6;acetosyringone(AS)concentration was 140 μmol/L.(3)The optimized system was used to transform Monascus strains.A total of 1000 transformants were obtained.Twelve randomly selected 12 strains were identified by PCR,10 strains LovD genes were successfully integrated into Monascus genome.(4)The successfully transformed strain was cultured and analyzed by HPLC for lovastatin.The yield of MD-5 lovastatin was as high as 0.55 mg/mL,which was 63%higher than that of the original strain.Subculture 9 generations showed good genetic stability.