Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation

Abstract:

The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, copper chromic salt qualitative test and RP-HPLC determination, 3 strains, l-ephedrine and d-pseudoephedrine producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO₃ as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87mg/L and d-pseudoephedrine 4.11mg/L excellular, d-pseudoephedrine 294.86mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters.

Key words:Ar⁺ implantation; mediated; Ephedra genome DNA; recombined yeast; ephedrine