Vitreoscilla Hemoglobin Gene Expression In S.cerevisiae As2.346

In order to increase the transformation efficiency of the intermediate of levonorgestrel, which is a over-the-counter drug currently often used in contraception, Vitreoscilla hemoglobin gene (vgb) was transformed into S.cerevisiaeAs2.346. S.cerevisiaeAs2.346 is a industrial strain of the intermediate of levonorgestrel. Vitreoscilla hemoglobin gene (vgb) encodes Vitreoscilla Hemoglibin (VHb) which has the function of trapping extra cellular oxygen to inside. Which type of the plasmid and the resistance labelling fit to S.cerevisiaeAs2.346 is the problem. Through the experiments, we found S.cerevisiaeAs2.346 was sensitive to the formaldehyde. That the YCp plasmid can steadily replicate in S.cerevisiaeAs2.346 was also proved. Recombinant plasmid pSPUV129 carrying vgb gene and formaldehyde resistance was constructed and transformed into S.cerevisiaeAs2.346, using a efficient transformation "LiAc transformation". PCR and Dot bolt hybridization technologies were used in the experiment, in which the S.cerevisiaeAs2.346 transformant carrying vgb gene had been proved.In the experiment of shake flask, furthermore, vgb was successfully expressed in transformed cells under different conditions. For example, with sufficient oxygen supply, the density of transformant is much higher than that of the contrast strain. With rare oxygen supply, the density of transformant is also much higher than that of the contrast strain. Generally, the transformation efficiency of the intermediate of levonorgestrel in transformant was increased remarkably compared with the reference strain. For instance, under oxygen-rich condition, the transformation efficiency of the intermediate of levonorgestrel in transformant has been increased by 1.53 times to 73.67% compared with the contrast strain's 48.31%. Under oxygen-limited condition, the transformation efficiency of the intermediate of levonorgestrel in transformant is 60.60%, that of the contrast strain is almost 0%. The result demonstrated that Vitreoscilla hemoglobin gene (vgb) contributed to both the growth of S. cerevisiae As2.346 and the transformation efficiency of the intermediate of levonorgestrel.