Engineering Of Ginkgolides B-producing Yeast Strain Using Low-energy Ion Implantation

Ginkgolides, the main diterpenoids of Ginkgo biloba L., is a compound of pharmaceutical importance, including Ginkgolide A, Ginkgolide B, Ginkgolide C, Ginkgolide M, Ginkgolide J. Ginkgolide B, the strong inhibitory effect on the platelet activating factor (platelet activating factor, PAF) receptor, has a range of valuable pharmacological functions, such as the antithrombotic activity. Ginkgolide B is mainly isolated from Ginkgo biloba L., a perennial herb belonging to the genus Ginkgo, which is widely used as a traditional herbal drug to treat thrombus, brain stem, and rheumatism because its leaves are rich in Ginkgolide B. Recently, wild resources of Ginkgo biloba L., have been overexploited in China owing to the increasing commercial interest in Ginkgolide B as a medical treatment. What is more, the seeds of Ginkgo biloba L. are difficult to germinate and only a very tiny proportion of seedlings actually produce mature adult plants in the wild. Thus, the unsustainable use of wild resources of Ginkgo biloba L. means that the plant is at risk of becoming extinct. Thus, it is important to establish an alternative method to obtain Ginkgolide B.In this study, the ion beam-mediated gene transfer method was used to transform genomic DNA segments of the medicinal plant Ginkgo biloba L. into Hansenula polymorpha to achieve single-step whole-pathway assembly of Ginkgolide B biosynthesis in yeasts. Similarly, to screen for potential Ginkgolide B-producing recombinant yeast strains, the downstream biosynthesis genes involved in the gentiopicroside biosynthesis pathway were used as molecular markers to develop the PCR-based high-throughput screening method.The mainly results are as follows:(1) Compared with various methods, we optimize the optimal method of ginkgo extract genomic DNA. By three different methods of traditional CTAB, modified SDS method, improved CTAB method, we can conclude that the modified CTAB method can be fast and efficient for extracting ginkgo genomic DNA. It provides a strong basis of molecular studies for yeast genetic engineering.(2) We have established a PCR screening system that it makes the key enzymes in molecular screening marker based on the biosynthetic pathway of ginkgolide B. Respectively to three key enzyme GGPPS, MECPS, MECT gene as molecular markers, based on key enzymes conserved amino acid sequence to design degenerate primers, taking the Ginkgo genomic DNA and Hansenula genome as amplification template, we have got a method of the PCR high throughput screening.(3) We have made clear the parameters of the Low Energy N+ ion implantation mediated plant genomic DNA transformation to H.polymorpha..The examining results:implantation energy of 25 keV, implantation dose 25×1015 ion/cm2, the degree of vacuum 10-3 Pa, pulse 10 s, interval 10 s.(4) We have obtained a yield of ginkgolide B engineered yeast.Transformed yeast PCR screening system of early screening it was 6 strains, then by color reaction, TLC and HPLC rescreening, there were 4 strains of engineering bacteria, which has a strain with relatively high yield about 1.212mg/L.(5) Genetic stability of the DL-00627 engineering strain:No.DL-00627 for engineering strains subcultured (continuous culture 8 generations) and liquid fermentation test, extracted the fermentation product. Engineering bacteria was detected by HPLC eight consecutive generation of the desired product yield. The results showed that the yield of ginkgolide B decreased slightly, but decreased less, indicates it has a good genetic stability.