氩离子注入介导麻黄基因组DNA转化获得产麻黄碱重组酵母菌

Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation

摘要:

通过氩离子(Ar⁺)注入介导蓝麻黄基因组DNA在异常汉逊酵母(Hansenula anomala)中随机转化,转化后的酵母菌经BTB指示性辅助筛选、斜面传代、液体培养、铜铬盐定性检识和RP-HPLC定量检测,获得了遗传稳定的以葡萄糖为碳源、NaNO₃为氮源生物合成麻黄碱和(或)伪麻黄碱的重组酵母菌3株。液体培养72h,RP-HPLC测试胞外麻黄碱和伪麻黄碱的最高产量分别为11.87mg/L和4.11mg/L;胞内伪麻黄碱最高含量为294.86mg/g干细胞,胞内麻黄碱未检出。分析了Ar⁺注入介导蓝麻黄基因组DNA在酵母菌中的遗传转化效率,探讨了麻黄基因组DNA大分子的完整性对其在酵母菌中遗传转化的影响。

Abstract:

The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, copper chromic salt qualitative test and RP-HPLC determination, 3 strains, l-ephedrine and d-pseudoephedrine producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO₃ as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87mg/L and d-pseudoephedrine 4.11mg/L excellular, d-pseudoephedrine 294.86mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.