Construction Of Genetic Transformation System In Hyperthermophilic Archaea Sulfolobus Tokodaii

Genetic transformation systems,including protein expression and gene knockout systems,play a key role in the functional analysis of genes and gene products.For eukarya and bacteria,genetic systems are very well established and continue to be extremely powerful for the elucidation of the functions of genes.However,the development of genetic system in archaea is lagging behind.The main problem is the availability and development of efficient selectable markers.In the past two decades, although numerous plasmids and viruses have been reported in hyperthermophilic genus Sulfolobus species,the development of these genetic elements into useful genetic tools for Sulfolobus has lagged behind those made with methanogenic and halophilic archaea.Several genetic systems have been published in the past 10 years, but they often turned out to be not transferable to other laboratories or to require extensive expertise.Only very recently the first genetic tools started to emerge that can be used reproducibly in different laboratories.At the present time,complete genome sequences are available for three Sulfolobus species,namely,S.solfataricus,S.tokodaii and S.acidocaldarius.For S. solfataricus and S.acidocaldarius,genetic systems have been established,and have greatly promoted functionanl analysis of genes.However,for S.tokodaii,there has been no report on genetic system.Therefore,the development of genetic tools for S. tokodaii is of great importance.PyrE and pyrF encode orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase,respectively.The enzymes pyrEF catalyse the last two steps of the de novo uridine monophosphate synthesis pathway. pyrEF were used extensively as auxotrophic selectable marker.In this paper,we have firstly constructed a knockout vector p5EF3 for deleting lacS gene coding forβ-galactosidase in S.tokodaii.In genetic experiments,lacS is used extensively as reporter gene for examining protein expressin level of expression vector or for analyzing promoter activity.Therefore,the obtained lacS knockout strain would be used as a new host strain for reporter gene system,and the native lacS interfering with reporter gene system would be invoided.In archaea,3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-CoA reductase) plays a key role in the isoprenoid lipid biosynthesis.Simvastatin is a potatial competitive inhibitor of HMG-CoA reductase,and therefore inhibits the growth of archaea cells.The overexpression of HMG-CoA reductase gene can reduce the inhibitory effects of simvastatin on cell growth.Simvastatin and its analog mevinolin have been used as antibiotics for archaea genetics.However,they have not been used in Sulfolobus.Therefore,in this paper,we examined the growth of wild type S.tokodaii in the presence of various concentrations of simvastatin.At concentration of 15μM simvastatin,growth was not observed for at least 168 hours,indicating this concentration would be suitable for selecting transformants with resistance against simvastatin.We next constructed the HMG-CoA reductase overexpression cassette under the control of the glutamate dehydrogenase promoter and then we have constructed pyrE gene knockout vector p5PH3 for S.tokodaii using HMG-CoA reductase overexpression cassette as selectable marker.By electroporation of vector p5PH3 into wild-type S.tokodaii,we have obtained uracil auxotrophic strains.Further identification at molecular level is in undergoing.Besides,to examine whether the HMG-CoA reductase from S.tokodaii possesses expected activity,we cloned the gene encoding presumed HMG-CoA reductase from S.tokodaii,expressed in E.coli and purified by heat treatment and ion exchange chromatograph,and we have determined the expected HMG-CoA reductase activity.