Cloning, Expression And Functional Analysis Of Mevastatin Biosynthetic Gene MlcF

Cardiovascular diseases have been the gravest diseases with their morbidity and mortality both ranking the first overtaking that of tumors,posing severe threats to mankind.The increase of total cholesterol level,especially low density lipoprotein-cholesterol（LDL-C） level,is crucial for the nosogenesis and development of diseases.3-hydroxy- methylglutaryl coenzyme A（HMG-CoA） reductase inhibitor（Statin） is a group of very active antihypercholesterolemic agents that function by limiting cholesterol biosynthesis by competitively inhibiting the HMG-CoA reductase,the rate limiting enzyme in cholesterol biosynthesis.At present,Statins,with remarkable cholesterol-lowering effect,less side effect and high security,have been at the top of the list of global best-selling drugs in the therapeutic treatment of hypercholesterolemia,the total sale of the statin drugs in global market reached US$ 39 billion in 2008, with a market share of 5.5%worldwide Mevastatin（ML-236B,compactin） is the earliest found statin compound,also the main substrate in the course of pravastatin biotransformation.Therefore,mevastatin has played a significant role in the development of statins.Research on mevastatin biosynthesis gene cluster and putative biosynthesis pathway reveals that mevastatin is a typical polyketide compound,which provides new ideas and insights to improve yields by molecular method.The biosynthesis of mevastatin starts from Acetyl-CoA,the initiation element,and the elongation element is malonyl CoA.The biosynthesis is accomplished with the joint action of all the genes in the biosynthetic gene cluster.Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced.The predicted amino acid sequences are encoded by these nine genes,mlcA-mlcH and mlcR. mlcF encodes a putative oxidoreductase of 251 amino acids.MlcF bears some similarity to dihydrofolate reductases and also shares 57%identity with a putative polypeptide.The function of mlcF remains unknown,but MlcF is presumed to have oxidoreductase activity,which might be required an as yet unknown step in ML-236B biosynthesis.This thesis mainly focuses on cloning and functional analysis of a mevastatin biosynthetic gene mlcF from mevastatin-producing fungus Penicillium citrinum.A 756 bp mlcF eDNA was amplified from citrinum by reverse transcription-polymerase chain reaction（RT-PCR）,and sequence alignment shows that it is the same with the reported sequence by NCIB,which contains 756 bp and（G+C） represents 50.93%of total number of bases,encodes 251 amino acids（27.9 kDa）,and the isoelectric point is 8.43, shares 57%homology with orf5 in lovastatin biosynthesis gene cluster from lovastatin-producing fungus Aspergillus terreus.The gene was subcloned into prokaryotic expression vector pET28a（+）,and the recombinant plasmid was transformed into E.coli BL21（DE3）,and induced by IPTG.The recombinant protein MlcF performed as inclusion body,denaturized by 8 M urea,and purified by Ni²⁺ affinity chromatograph,then renaturized by urea concentration gradient.Bioinformatics analysis established the foundation of MlcF of P.citrinum in the structural and functional aspect.MleF sequence was delivered to ExPAsy servers to recognize the protein Motif.It contains a N-glycosylation site,2 Casein kinaseⅡphosphorylation sites,2 N-myristoylation sites,a Protein kinase C phosphorylation site,and 2 Tyrosine kinase phosphorylation sites.It was predicted that there was no signal peptide,by neural networks and hidden Markov models,and no transmembrane domain by TMpred.Online protein prediction software PSIPRED predicted that MlcF was composed of 6α-helixes,7β-sheets and many coils.Circular Dichroism was used to determine the secondary structure feature of this protein.The modeling of MlcF of P.citrinum was based on the structure of FSH 1/YHR049W in Saccharomyces cerevisiae.With the increasing demand of statin drugs and the development of molecular biotechnology, strain-improvement began to use molecular biotechnological methods,more and more functional genes were discovered and cloned.People are eager to know the crystal structure of proteins which are encoded by these genes,to pursue a better functional research.But due to limitation of technology,it is difficult to gain the crystallized protein.Therefore,3D-structure prediction of unknown protein by the existing structure became an important method.By the means of protein-protein interaction networks,gene dynamic expression research,analysis of protein expression level,protein location and gene functional analysis,it is hoped that this can facilitate better understandings of the creation and regulation mechanisms of intra-cellular physiological reactions,reveal the function of MlcF in the pathway for ML-236B biosynthesis.