Recombinant Expression Of Azoreductase And Gene-knockout Of Mtra On Shewanella Oneidensis MR-1

Due to the widely distribution of Shewanella and its outstanding performance of ability in dissimilatory metal reduction and dye decoloration,Shewanella has been one of the popular research nowadays.However,the genetic research of genue Shewanella is still a mystery.The lack of genetic tool,such as vector and high efficient knock-out system is the limitation on molecular studies of Shewanella.Thereforce,the suitable plasmids ang genetic manipulating tool on Shewanella are urgent and requird.Two kind of E.coli-Shewanella shuttle plasmids,i.e.,pETSXM2 and pSB1C3-repB were investigated in this study.We found that both plasmids were able to express in E.coli BL21(DE3)and Shewanella oneidensis MR-1 under driven by promoter placI.The recombined azoRI(encoded as AzrS)could be over-expressed in plasmid pETSXM2 and pSB1C3-repB.The maximum yield of recombined AzrS from pSB1C3-repB-azoRI/BL21 reached up to 18366.5U/L,against on methyl red as substrate.What's more,recombined AzrS appeared enzyme activity in MR-1 for the first time.Besides,recombined AzrS also showed activities of several substrates,with a rank of methyl red>methyl orange>congo red.The mtr proteins on outer membrane of Shewanella are strictly important to itself,especially on dissimilatory metal reduction and dye decoloration.In this study,the gene of mtrA in MR-1 was knockout by the suicide plasmid of pDS3.0.After knockout of mtrA gene,the activity of ferric reductase using FeCl3 as substrate drop from 6.5U/L to 0.9U/L,while the activity using ferric citrate as substrate drop from 60.5U/L to 5.0U/L,respectively.According to the of ferric reductase results,we suggested that the mtrA is a key protein of electron transport chain in Shewanella.