Study On Genes Related To Sorbitan Metabolism In Common Ketone - Based

The two-step fermentation technique of vitamin C,the only microbial transformation used in large scale production,was first established by our country.High conversion ratio is the conspicuous quality of the two-step fermentation technology which includes two fermentation procedures and needs three kinds of microorganism.It will be of great value to vitamin C production in our country if the engineered bacterial could be constructed,in which the related genes were introduced by using recombinant DNA technology.We have isolated an enzyme,sorbose dehydrogenase(SDH),from ketogulonigenum vulgate, which is able to catalyze the conversion of L-sorbose to 2-KGA directly in vitro.However,when expressed in E.coli,the SDH could not complete the conversion,The reason may be lack of electron acceptor of SDH in E.coli.We find that a kind of CytC may be the electron acceptor.Firstly,Yeast two-hybrid system and bacterial two-hybrid system were used to examine the interaction between SDH and Cytochrome C.But we did not found direct interaction between the two proteins in yeast or bacterial.Then,the cosmid pKC505-sdh was constructed,which was used to construct genomic library of ketogulonigenum vulgate.From the library,three positive clones were screened,two of them could convert L-sorbose to 2-KGA,and the other one could catalyze L-sorbosone dehydrogenation. By constructing sub-clone,the complete gene of sorbosone dehydrogenase was isolated.The work of looking for the electron acceptor of SDH is continued.