Heterogenous Overexpression Of MenA In Bacillus Subtilis Natto

Vitamin K2(VK2,MK-n),menaquinone,is an indispensable fat-soluble vitamin in human metabolism,it has many important physiological functions.Bacillus subtilis natto which is considered as a probiotic bacteria by FDA is used to produce VK2 for its high content.Studies have shown that overexpression of 1,4-dihydroxy-2-naphthalic acid prenyltransferase(EcMenA)which is one of the key enzymes during producing VK2 in Escherichia coli can significantly improve the yield of VK2.However,1,4-dihydroxy-2-naphthoate prenyltransferase(BsnMenA)which has a low abundance in Bacillus subtilis natto has been rarely reported so far.Therefore,the crystal structure and specific catalytic process of BsnMenA are still unclear.In this report,BsnMenA was not only predicted and analyzed by bioinformatics,but also studied by subcellular localization experiments,cloning and overexpressed in Escherichia coli,which laid a theoretical foundation for further study of BsnMenA in the future.The main research contents and results are as follows:(1)Bioinformatics software was used to predict the phylogenetic relationship,basic property,subcellular localization,structural domain and motifs searching and space structure of BsnMenA.From the perspective of phylogenetic tree,MenAs in Bacillus subtilis were highly similar to each other,but far from other families,such as Escherichia coli.According to prediction of the physical,chemical properties and subcellular localization of the enzyme,it was found that BsnMenA was a kind of intermembrane protein.Structural domain and motifs searching showed that there was a highly conserved UbiA structural domain in BsnMenA from 34 to 278.Moreover,there were two Asp-rich motifs,NxxxDxxxxxD and NNxxDxxxD,from 74 to 84 and from 204 to 212,respectively.Homologous sequence alignment showed that 74,204,205 Aspa ragine and 208,84,78,212 Aspartic acid were the key amino acid sites in BsnMenA Through the method of folding identification modeling,three-dimensional space structure of BsnMenA was constructed,and there were 9 transmembrane helixes forming an active central cavity which was embedded in the phospholipid bilayer,which were very similar with 4-hydroxybenzoate prenyltransferase(ApUbiA)in Aeropyrum pernix.Since the space structures of BsnMenA and ApUbiA are highly similar,the conserved domain sites which are related to the catalytic function of BsnMenA and its catalytic mechanism were deduced.(2)To further investigate the subcellular localization of BsnMenA in E.coli.In this chapter,the recombinant bacteria,BL21/pET28a-menA-egfp(MEP28)and BL21/pET28a-egfp-menA(EMP28)were successfully constructed by overlapping PCR amplification.Subsequently,the two recombinant strains as well as the positive control BL21/pET28a-egpp(EP28)and the negative control BL21/pET28a(P28)were fermented and induced under the same culture conditions where the concentration of IPTG was 0.2 mM,induction temperature was 28 ?,and the shaking speed was 150 rpm.After the induced fermentation,the samples were observed by laser confocal fluorescence microscopy,and the results were as follows:When the induction time was 20 min,there were obvious fluorescence in the cell membranes of MEP28 and EMP28,while the whole cell of EP28 was fulfilled with fluorescence and P28 had no obvious fluorescence.This experiment not only verified that BsnMenA was a membrane protein again,but also fully demonstrated that menA and egfp could be expressed and correctly folded in Escherichia coli.(3)In order to further research cloning and overexpression of BsnMenA in Escherichia coli.Firstly,menA which were amplified by PCR were connected to pET22b(+)and pET28a(+),respectively.And then were transformed into JM109,then JM109/pET28a-menA and JM109/pET22b-menA were constructed.After fermentation,the recombinant plasmids were extracted and transformed into BL21(DE3)again.BL21/pET28a-menA(MP28)and BL21/pET22b-menA(MP22)were obtained.Secondly,When LB mediums were used as fermentation mediums,MP22,MP28,BL21/pET22b(P22)and BL21/pET28a(P28)were induced under the condition of low temperature and the final concentration of IPTG,0.2 mM and 0.8 mM,respectively.The crude samples were prepared for SDS-PAGE detection.No obvious protein bands were observed in MP22 and MP28.Lastly,when LB mediums with 0.5%glucose were used as fermentation mediums,MP22,MP28,P22 and P28 were induced under the condition of low temperature and the final concentration of IPTG,0.5 mM.Through SDS-PAGE detection,it was found that BsnMenA was obviously expressed in the crude sample of MP22,and the band was equal with 38 KD.While no obvious protein bands were detected in SDS-PAGE of MP28.Through the experiments,it was found that MP22 could overexpress BsnMenA in LB medium with 0.5%glucose,and a suitable method for heterogenous overexpression of BsnMenA was established,which laid a certain theoretical foundation for systematic fermentation condition optimization of MP22 later and the study on the enzymatic properties of BsnMenA.