Cloning And Prokaryotic Expression Of Genes Involved In The Whole Biosynthetic Pathway Of Cholesterol-lowering Drug Lovastatin

Hyperlipidemia is a major elicitor of cardiovascular diseases,and controlling the level of blood lipid is an effective way to prevent such diseases.Statins have become the most important antilipidemic drugs because of inhibiting the activity of HMG-Co A reductase,which is the key enzyme during the synthesis of cholesterol.At present,statins are mainly produced by the metabolic engineering of their original filamentous fungi(such as Aspergillus terreus and Penicillium),or derived from its main form lovastatin through biological / chemical transformation.Although they have also been de novo synthesized from different sources(e.g.in Saccharomyces cerevisiae and Pichia pastoris),there is still lack of such a complete research in another excellent microbial platform,i.e.E.coli system,only with conversion tests in some aspects of the synthetic pathway.Therefore,this work is aimed at the heterologous biosynthesis of lovastatin in bacteria.Firstly,we cloned the genes of the whole lovastatin synthetic pathway from Aspergillus terreus,and then analyzed their recombinant expression in E.coli.The main results obtained are as follows:1.Gene cloning of all enzymes in lovastatin biosynthetic pathway and construction of their prokaryotic expression vectorsThe essential enzymes of lovastatin biosynthesis pathway include Lov A,lov B,lov C,lov D,lov F,lov G and Lov A redox partner CPR(i.e.Lov PR).All genes of these enzymes were cloned,with specific primers designed according to the genome draft sequence(NT?165939)of Aspergillus terreus strain NIH2624.(1)The gene fragments of Lov A,lov C,lov D and CPR were amplified from the total RNA of Aspergillus terreus by RT-PCR,and then subcloned into plasmid p ET32a(+)to obtain their corresponding prokaryotic expression vectors,by Nde I/Xho I digestions or Exnase II-mediated recombinant cloning.(2)The intronless Lov G gene was amplified directly from the total genomic DNA of Aspergillus terreus,and then subcloned into plasmid p ET32a(+)by Nde I/Xho I digestions to obtain its corresponding prokaryotic expression vector.(3)Because Lov B(?9 KB)and Lov F(?7.4 KB)genes were too large to be obtained by one-step RT-PCR in our preliminary experiments,the strategy of segmentation amplification and cloning was specially conceived.Through RT-PCR combined with genomic PCR and / or exon assembly,the full-length genes of lov B,lov F were successfully obtained from Aspergillus terreus,and their corresponding prokaryotic expression vectors with the backbone of p ET32a(+)were also constructed,through Exnase II-mediated recombinant cloning.(4)All constructed prokaryotic expression vectors were verified by sequencing.The results showed that there were no,or only a few and unimportant differences in DNA and deduced protein sequences for each cloned gene,as compared with its reference.2.Recombinant expression of all enzymes of lovastatin biosynthesis pathway in E.coliAll constructed prokaryotic expression vectors were each transformed into E.coli strain BL21(DE3)and induced by IPTG.The expression level and solubility of the recombinant proteins were analyzed by SDS-PAGE.The results showed that all the enzymes of lovastatin biosynthetic pathway could be efficiently expressed in E.coli,with clear bands of correct size on SDS-PAGE gels.However,their solubility was generally low(10%-40%)under 37 °C induction,especially for Lov B and Lov F.However,by changing the induction conditions,such as using overnight induction at low temperature(25 °C),their soluble expression in E.coli could be significantly enhanced.In summary,according to the current literature,this work might be the first time to conduct a complete recombinant expression analysis in E.coli for the enzymes of lovastatin biosynthesis pathway from the filamentous fungi Aspergillus terreus.The results showed that they could be efficiently expressed but have a low yet applicable solubility.In particular,Lov A(belonging to cytochrome P450 monooxygenase which is usually difficult to express in prokaryotic bacteria without endomembrane system),Lov B(?330 k Da)and Lov F(?270 k Da)with large molecular size,were successfully expressed,which is certainly a breakthrough for E.coli.Conclusively,all of these findings have laid a solid foundation and given great feasibility for the creation of an E.coli platform for heterologous biosynthesis of lovastatin or its derivatives.Our next work will be to further improve the soluble expression of these enzymes,and ultimately reconstitute the complete pathway of lovastatin biosynthesis in E.coli.