Cloning, Expression And Functional Analysis Of Mevastatin Biosynthetic Gene MlcH From Penicillium Citrinum And Its Site-directed Mutagenesis

Currenly, cholesterol-lowering compound that significantly reduces the risk of heart diseases associated with hypercholesterolemia is increasing rapidly. Hypercholesterolemia is known to be one of the prime risk factors for cardiovascular disease such as arteriosclerosis and coronary heart disease. Statins,as the inhibitor of3-hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase, effectively lower the Synthesis of endogenous cholesterol and add compensatively the Synthesis of Low Density Lipoprotein in organs cells, improve the uptake of Low Density Lipoprotein in blood and reduce the contents of Total Cholesterol. It is tabulated, as the first-line drugs for curing hypercholesterolemia.with remarkable cholesterol-lowering effect, less side effect and high security,statin have been at the top of the list of global best-selling drugs in the therapeutic treatment of hypercholesterolemia. Mevastatin is first found produced by Penicillium citrinum and used as a substrate for microbial conversion to pravastatin sodium. Lovastatin is the first approved HMG-CoA reductase inhibitor by FDA as the structural analogs of mevastatin.The research on mevastatin biosynthesis gene cluster and putative biosynthesis pathway reveals that mevastatin is a typical polyketide compound.Studying on keypoint enzyme in biosynthesis pathway of mevastatin provided new bases for developing statin compound. Nine predicted genes for mevastatin biosynthesis were located within a 38-kb region and were transcribed when mevastatin was produced. mlcH contains 1257bp and encodes a putative transesterase of 418 amino acids (46.7 kDa).MlcH displays 76% identity with LovD,a transesterase required for lovastatin biosynthesis. LovD,the first acyltransferase reported in statin biosynthesis which catalyze the transacylation reaction that regioselectively transfers the α-methylbutyryl group from the diketide synthase to the C8 hydroxyl group of monacolin J to yield lovastatin. An one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J by using Escherichia coli strain overexpressing the acyltransferase LovD was developed for its broad substrate specificities. Whether MlcH has the same broad substrate specificities which could convert 6-hydroxyl-6-desmethylmonacolin J to huvastatin, a excellent cholesterol-lowering compound, could be an attractive route.Here we focus on cloning and functional analysis of an mevastatin biosynthetic gene mlcH from mevastatin-producing Fungus Penicillium citrinum. A 1257bp mlcH cDNA was amplified from P. citrinum by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. There was one point mutation at the site of 1064 which changed glutamic acid to glycine. Then cDNA was subcloned into prokaryotic expression vector pET28a (+), and the recombinant plasmid was transformed into E.coli BL21 (DE3). The recombinant protein MlcH was purified by Ni2+ affinity chromatograph.The insoluble proteins was renatured in dialysis buffer consist of L-Arg,Triton X-100 and glycerine. In 4 mM mevastatin,10 mM MlcH 50μl HEPES assay buffer,A single new compound matching the expected retention time as a result of hydrolysis of mevastatin was detected. The optimal pH conditions and reaction temperature for MlcH hydrolysis was pH 8.0 and 45℃after gradient experiment. Homology model structure of MlcH was generated by the Swiss-Model Repository based on the crystal structure of EstB from B. gladioli and analyzed by pymol. Cys65 was predicted to be highly exposed which in charge of spurious aggregation. Then C65A Site-directed Mutagenesis was completed by the mean of megaprimer PCR.MlcH hydrolysis activity was assayed successful in vitro.Protein engineering was used for rational improving solubility of the recombinant protein which provided condition for the whole-cell biocatalysis of huvastatin.MlcH developed as a statin analogs biocatalysis is undergoing.