Transfer of Ephedra genomic DNA to yeasts by ion implantation.
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.
Lü
Jie
J
College of Life Science and Pharmacy, Nanjing University of Technology, 210009 Nanjing, People's Republic of China.
Jin
Xiang
X
Mao
Pei-Hong
PH
Ma
Xiang-Dong
XD
Ling
Hai-Qiu
HQ
Fan
Yong-Hong
YH
Yu
Long
L
Wu
Bao-Shan
BS
Ouyang
Ping-Kai
PK
D018744:DNA, Plant / Q000378:metabolism; D029789:Ephedra / Q000235:genetics*; D004809:Ephedrine / Q000378:metabolism; D018745:Genome, Plant; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D054199:Pseudoephedrine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D018744:DNA, Plant; D054199:Pseudoephedrine; D004809:Ephedrine