离子注入介导甘草基因组DNA转化酵母菌

Transformation of Glycyrrhiza uralensis genomic DNA into yeast mediated by ion implantation

目的以甘草酸为目标产物,探讨氮离子(N+)和氩离子(Ar+)注入介导甘草基因组DNA在酵母菌中的转化。方法通过N+和Ar+注入介导乌拉尔甘草基因组DNA在异常汉逊酵母Hansenula anomala中随机转化,转化后的酵母菌经斜面传代和液体培养后,用醋酐-浓硫酸定性检识和RP-HPLC方法,检测重组酵母菌培养液中甘草酸和甘草次酸的量。结果获得了生物合成甘草酸和/或甘草次酸的重组酵母菌5株。液体培养96h,RP-HPLC测试其培养液中甘草酸最高量114.49mg/L,18α-甘草次酸和18β-甘草次酸最高量分别为0.56和0.81mg/L。TLC检测发现其中1株重组酵母菌的培养液中含一种未知的红色组分。结论采用离子注入介导甘草基因级DNA大分子转化技术,可获得易于人工培养的产生甘草酸等次生代谢产物的微生物工程菌株。

Objective With the sole object of glycyrrhizic acid products, the methods were investigated for Glycyrrhiza uralensis genomic DNA transformation into Hansenula anomala by nitrogen and argon ion implantation. Methods The genomic DNA from G. uralensis was randomly transferred into H. anomala by nitrogen and argon ion bombardment. The recombined yeasts were cultured by the slant and liquid cultivation, in which the contents of glycyrrhizic acid and glycyrrhetic acid were determined by acetic anhydried-H2SO4 qualitative test and RP HPLC determination. Results Five recombined yeast strains that produced glycyrrhizic acid and glycyrrhetic acid were obtained. After cultured in liquid medium for 96 h, the highest content of glycyrrhizic acid in the cultivation liquid was 114.49 mg/L and that of 18α-glycyrrhetic acid and 18β-glycyrrhetic acid were respectively 0.56 and 0.81 mg/L by RP-HPLC. A kind of unknown red component was found in the cultivation liquid of one reeombined strain by TLC. Conclusion The reeombined yeast strains of producing glycyrrhizic acid could be obtained G. uralensis genomic DNA transformation into yeast mediated by the ion implantation.