Engineering of gentiopicroside-producing yeast strain using low-energy ion implantation mediated synthetic biology

Abstract

ABSTRACTTo obtain an alternative source for the production of gentiopicroside, here genomic DNA segments of the medicinal plant Gentiana macrophylla were randomly transferred into Hansenula polymorpha by 25 KeV nitrogen ions (N+) at a dose of 2.5 × 1016 ions/cm2 under vacuum pressure of 1 × 10−3 Pa. To screen for potential gentiopicroside-producing recombinant yeast strains, geraniol 10-hydroxylase (G10H) and secologanin synthase (SLS) involved in the gentiopicroside biosynthesis pathway were used as molecular markers. Based on the conserved protein sequences of G10H and SLS, degenerate primers were designed and used for colony polymerase chain reaction (PCR). PCR-positive results for both the G10H and SLS genes were obtained in 79 out of 653 transformants by low-energy ion beam-mediated transformation. These 79 potential gentiopicroside-producing strains were further analysed by Fehling's test, thin-layer chromatography, high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. The results showed that the retention time and ion peaks of the sample from one stable recombinant strain designated as DL67 were consistent with those of the gentiopicroside standard. The corresponding gentiopicroside yield was 8.41 mg/g dry cell weight after strain DL67 was cultured for 96 h. This could offer a new starting point for the construction of recombinant yeasts for production of medicinal plant compounds.