一步重组法构建产龙胆苦苷酵母重组菌的研究  

The construction of Gentiopicroside-producing recombinant yeast using one-step recombinant method

作　　者：钱卫东[1] 赵德志[1] 付云芳[1] 陈雪峰[1] 毛培宏[2] 周颖欣[1] 常凯[1] 谢海艳[1] 

机构地区：[1]陕西科技大学生命科学与工程学院,陕西西安710021 [2]新疆大学物理科学与技术学院离子束生物技术中心,新疆乌鲁木齐830046

出　　处：《陕西科技大学学报（自然科学版）》2014年第5期123-128,共6页Journal of Shaanxi University of Science & Technology

基　　金：国家自然科学基金项目(31100040);陕西省教育厅科研计划项目(2013JK0727);西安市未央区科技计划项目(201210)

摘　　要：利用低能N+注入介导秦艽基因组DNA转化多形汉逊酵母技术,结合颜色反应、薄层层析和高效液相色谱等方法筛选重组菌株,获得1株遗传稳定的能生物合成龙胆苦苷的重组酵母菌,被命名为DL-49.经液体培养90h后,利用HPLC分析其发酵液中的龙胆苦苷,其产量为2.22mg/L.将重组菌株传代培养8代后,分析显示重组酵母菌生物合成龙胆苦苷的产量基本稳定.试验表明,采用低能离子注入介导秦艽基因组转化酵母技术,可以在龙胆苦苷生物合成相关基因信息未知的情况下,直接获得能生物合成龙胆苦苷的酵母重组菌.To obtain an alternative source for the production of Gentiopicroside,a very simple method for achieving one-step whole-pathway assembly of Gentiopicroside biosynthesis in yeast is presented.Genomic DNA segments of medicinal plant Gentiana macrophylla（G.macrophylla）were randomly transferred into Hansenula polymorpha（H.polymorpha）by25KeV nitrogen ions（N^＋）at a dose of 2.0×10^16 ions/cm^2 under the vacuum pressure of 1×10^-3 Pa.One potential stable recombinant yeast strain capable of producing Gentiopicroside was obtained using a combination screening of Fehling′s test and TLC,and designated as DL-49.The potential Gentiopicroside-producing strain DL-49 was further analyzed by HPLC,and the result showed that the sample from strain DL-49 possessed of a retention time and ion peaks consistent with those of the Gentiopicroside standard.The corresponding Gentiopicroside yield was 2.22 mg/L as determined by HPLC.Additionally,the recombinant strain was stable for at least 8generations.One Gentiopicroside-producing recombinant strain was obtained by low energy N＋ion implantation.This would have a valuable application for low-cost production of natural compounds.

关 键 词：低能离子注入 多形汉逊酵母 秦艽 龙胆苦苷 

分 类 号：R282.2[医药卫生—中药学]