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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 To investigate the transformation of Glycyrrhiza uralensis genomic DNA into Saccharomyces cerevisiae with glycyrrhizic acid as the target product and nitrogen (N +) and argon (Ar +) influx. Methods Genomic DNA of Ural licorice was randomly transformed into Hansenula anomala by N + and Ar + injection. The transformed yeast was subcultured by bengal and liquid, and then qualitatively identified by acetic anhydride-concentrated sulfuric acid and RP-HPLC , The amount of glycyrrhizic acid and glycyrrhetic acid detected in the recombinant yeast culture solution. Results Five strains of recombinant yeast were obtained by biosynthesis of glycyrrhizic acid and / or glycyrrhetinic acid. Liquid 96h, the highest amount of glycyrrhizic acid 114.49mg / L, 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid maximum amount of 0.56 and 0.81mg / L in their culture broth by RP-HPLC. TLC test found that one of the recombinant yeast culture medium contains an unknown red component. CONCLUSION: The ion-implantation-mediated transformation of Glycyrrhiza uralensis gene-level DNA can provide a strain of microorganism capable of producing artificial metabolites such as glycyrrhizin.