甾醇酰基转移酶基因高表达对酵母菌麦角甾醇合成的影响

Effect of Overexpression of Sterol-acyl Transferase on Ergosterol Production in Yeast Strains

通过PCR扩增克隆到含酵母菌甾醇酰基转移酶基因ARE2编码序列和上游调控序列的DNA片段ARE2 1及仅含编码序列的DNA片段ARE2 2。分别以ARE2启动子 ,乙醇脱氢酶基因ADH1启动子和铜抗性基因CUP1启动子及ADH1终止子为调控元件构建了酵母菌表达质粒pHX2 ,pHXA2和pHXC2。表达质粒分别转化酿酒酵母单倍体菌株YS5 8和以前通过细胞杂交构建的麦角甾醇高产菌株YEH5 6。通过营养缺陷互补和铜抗性筛选到转化子 ,质粒上的ARE2基因在YS5 8和YEH5 6中都实现了活性表达 ,使细胞内甾醇酯化水平升高 ,并导致细胞麦角甾醇含量的提高。对转化菌株的培养条件进行了初步研究 ,在优化条件下 ,重组转化菌株YEH5 6 (pHX2 )、YEH5 6 (pHXA2 )和YEH5 6 (pHXC2 )的麦角甾醇含量分别是受体菌YEH5 6的 1 3、1 3和 1 4倍。

DNA fragment ARE2 1 containing the coding sequence and the 5′regulatory sequence of ARE2 gene and fragment ARE2 2 containing only the coding sequence of ARE2 were amplified from the chromosomal DNA of yeast strain YEH56 by PCR. Three plasmids, pHX2, pHXA2 and pHXC2, containing sterol acyltransferase gene ( ARE2 ) under the control of ARE2, ADH1 or CUP1 promoters respectively and the copper resistance gene as the selection marker were constructed, and they were then introduced into yeast strain YS58 and YEH56. ARE2 gene on plasmids expressed functionally in transformants. Ergosterol production in recombinant strains was enhanced, which is mainly due to the increase of sterol esterification. Under the optimal culture condition, ergosterol content in recombinant strains YEH56(pHX2), YEH56(pHXA2) and YEH56(pHXC2) was 1 3, 1 3 and 1 4 fold of that in strain YEH56 as control.