College of Life Science and Pharmacy, Nanjing University of Technology, 210009 Nanjing, People's Republic of China.
Appl Biochem Biotechnol. 2009 Sep;158(3):571-81. doi: 10.1007/s12010-009-8597-9. Epub 2009 Mar 12.
The genomic DNA from Ephedra glauca was randomly transferred to Saccharomyces cerevisiae and Hansenula anomala by argon and nitrogen ion implantation. Through repeated subculturing and using reversed phase high-performance liquid chromatography analysis to quantify the concentrations of the secondary metabolites, l-ephedrine and d-pseudoephedrine, 12 recombinant strains of genetically stable yeast were obtained, each using glucose as a carbon source, NaNO3 as a nitrogen source and producing l-ephedrine and/or d-pseudoephedrine. After culturing in liquid medium for 72 h, extracellular l-ephedrine and d-pseudoephedrine concentrations of 18.85 and 4.11 mg/L, respectively, were detected. Using l-ephedrine and d-pseudoephedrine as the target products, the transformation efficiencies of the genomic DNA from E. glauca transferred to S. cerevisiae and H. anomala were 1.15% (1/87) and 2.13% (8/376), respectively. The addition of the amino acid, L-Phe, to culture media substantially changed the amount of l-ephedrine and/or d-pseudoephedrine produced by the recombined yeasts. However, the change in metabolite production was not consistent among strains, rising in some, while dropping to nondetectable levels in others. After random amplification of polymorphic DNA (RAPD) analysis, four RAPD primers were obtained from the initial 100 RAPD primers, each amplifying different fragments with the recombined yeast Ar_Han0458 genome. Using one primer as polymerase chain reaction primer, the result showed that the recombined yeast Ar_Han0458 genome matched E. glauca genomic DNA at 150 bp, indicating a successful transfer of genetic information, facilitated by ion implantation.
用氩气和氮气离子注入法将麻黄属植物基因组 DNA 随机转入酿酒酵母和异常汉逊酵母中。通过反复传代培养和反相高效液相色谱分析定量测定次生代谢产物 l-麻黄碱和 d-伪麻黄碱的浓度，得到了 12 株遗传稳定的酵母重组株，它们均以葡萄糖为碳源，硝酸钠为氮源，并产生 l-麻黄碱和/或 d-伪麻黄碱。在液体培养基中培养 72 h 后，分别检测到细胞外 l-麻黄碱和 d-伪麻黄碱的浓度为 18.85 和 4.11 mg/L。以 l-麻黄碱和 d-伪麻黄碱为目标产物，转入酿酒酵母和异常汉逊酵母的麻黄属植物基因组 DNA 的转化率分别为 1.15%（1/87）和 2.13%（8/376）。在培养基中添加氨基酸 L-Phe 会显著改变重组酵母产生的 l-麻黄碱和/或 d-伪麻黄碱的量。然而，代谢产物产生的变化在菌株间并不一致，有些菌株增加，而有些则降至无法检测的水平。通过随机扩增多态性 DNA（RAPD）分析，从最初的 100 个 RAPD 引物中获得了 4 个 RAPD 引物，每个引物均扩增出与重组酵母 Ar_Han0458 基因组不同的片段。使用一个引物作为聚合酶链反应引物，结果表明，重组酵母 Ar_Han0458 基因组与麻黄属植物基因组 DNA 在 150 bp 处匹配，表明遗传信息的成功转移，这得益于离子注入。
