Clonging And Expression Of Desaturase Genes From Mucor Circinelloides EIM-10

As one of Mucorales, Mucor circinelloides shows important economic value, which can produce a variety of unsaturated fatty acids. To future explore the metabolic pathways and molecular mechanism of Mucor circinelloides, we cloned and analyzed△12-desaturase and△9-desaturas gene’s cDNA sequence from Mucor circinelloides EIM-10. The results show that,△12-desaturase and△9-desaturas gene’s cDNA sequence from Mucor circinelloides EIM-10has length of about1191bp and1359bp respectively, encodeing the protein397and453amino acids respectively. Homology analysis suggest the△9-desaturase sequence was94%identities with the△9-desaturase of Amylomyces rouxii (GenBank:AY995173.1) and the△12-desaturase sequence was also94%identities with the△12-desaturase of Mucor circinelloides (GenBank: AF533361.1) respectively. Its desaturase activity was confirmed by expression of it under the inducible promoter of galactose in Saccharamyces Cerevisiae in the present of Linoleic acid as substrate, total lipids were extrated from them, a new fatty acid was detected in the lipid fraction by GC-MS (gas chromatography-mass spectrum), the results suggested the recombinant cell pYMCD12converted the Oleic acid to Linoleic acid with a conversion efficiency of36.401%.To improve enzymatic activity and understand the effect of promoter MD6p and GAP to△12-desaturase, we clone GAP promoter sequencr from pichia expression vector pGAPZa, and then connect the GAP to pYMD12vector so as to successfully built pYGAPMD12expression vector; Besides we build pYMD6pMD12expression vector by replacing△6-desaturase cDNA with△12-desaturase gene in pYMD6pMD6. Then pYGAPMD12and pYMD6pMD12expression vectors were introduced into the S. cerevisiae INVScl by the lithium-acetate method. The transformants were selected by plating on synthetic agar plates lacking uracil (SC-ura). S. cerevisiae harbouring pYGAPMD12and pYMD6pMD12were cultured in uracil-lacking synthetic complete (SC) medium without containing2%glucose. After extracting the total lipids, we conducted the Corresponding GC-MS. The results suggested that compared to the pYMD12, conversion efficiency of the recombinant cell pYMD6pMD12and pYMD6pMD12increase18.432%and32.771%respectively. Addition, pYMD6pMD12 could not convert C16:1to C16:2, but pYGAPMD12can convert C16:1to C16:2with a conversion efficiency of32.943%.