Genome Engineering Of Hansenula Polymorpha For Production Of Glycerol And Paclitaxel

Glycerin, also known as glycerol, serves as the precursor for the synthesis of derivatives, that is widely used in food, pharmaceutical formulations, chemical and nursing industry. The extensive use of glycerol, along with the new emerging added-value products from glycerol, yields a sharp increase in commercial demand for glycerol. To some degree, China has to rely on imports, and it is urgent to seek an alternative to producing glycerol to ensure a stable, efficient supply of glycerol. Microbial fermentation for production of glycerol has great advantage due to free from the limitation of raw material and green manufacturing process. The development of microbial metabolic engineering provides powerful support for constructing microbial cell factories.Paclitaxel(Paclitaxel, marketed as Taxol) is a diterpenic polyoxygenated pseudoalkaloid and a well-studied antitumor agent with application against a range of cancer types including ovarian cancer, breast cancer, colon cancer, rectal cancer, bladder cancer, lung cancer, malignant melanoma and sarcoma. Paclitaxel is currently the only one that can promote the formation of polymerized microtubules and stablize the polymerizated mircotubule resulting to block normal cell division. Paclitaxel production depended on its extraction from the bark of yew trees. However, the growth cycle of Taxus chinensis var. mairei is particularly long(typically 100-250 years) and the concentration of the taxol in Taxus chinensis is very low(average content is about 0.015%). To address the sharp contradiction between supply and demand of taxol, it is necessary to seek new sources of taxol to meet the growing clinical demand. In recent years, because of its unique mutation and transgenic mechanism, ion beam bioengineering technology as a new breeding method, has been widely used in crop improvement and microbial breeding research.Here, N+ ion beam implantation was applied to enhance Hansenula polymorpha in glycerol yield and construct the taxol-producing recombinant yeast. N+ ion beam implantation parameters were optimizated and the extraction method of yeast gneomic DNA were evaluated. Then the genomic DNA of Hansenula polymorpha was transformed into Hansenula polymorpha via low energy N+ ion implantation technology. Based on hypertonic screening method,high-yield glycerol of recombinant Hansenlua polymorpha was screened. Subsequently, using the low energy N+ implantation technology, the Taxus chinensis genome DNA was transformed into Hansenula polymorpha, and taxol-producing recombinant yeast was constructed. This study will provide a theoretical and practical basis for low energy ion implantation mediated itself or heterologous genome into yeasts. And it will also supply an alternative to the development of medicinal plant effective components source, and a new way to the protection of wild medicinal plant resources. This study will provide a new strategy framework for coping with glycerol supply in China and realize its sustainable development and protection of wild Taxus resourcesThe specific results are as follows:(1) The curve of relationship between the survival rate of Hansneula polymorpha and the dose of nitrogen ion beam implantation was studied. The optimum ion implantation conditions are as follows: the implantation energy of 25 ke V; the implantation dose of 25×101 5ion/cm2, the vacuum of 10- 3Pa, and the pulse injection time of 10 s, and the interval time of 10 s.(2) The efficient extraction of yeast genomic DNA was established. The results indcated that Li OAC/SDS/glass beads extraction combination can be used to extrcat the geomic DNA of Hansenula polymorpha.(3) Hypertonic screening method was established based on Na Cl. One high-yield glycerol-producing recombinant yeast was secreened. After shaking culture substrate for glucose production and conversion of glycerol were 70.29g/L and 53.37%, relative to the original strain improved 27.25%; subculture of eight generations of glycerol production did not change significantly.(4) An efficient screening method of taxol-producing recombinant yeast was established. First, the color reaction was used to analyze qualitatively the target strains, and then the TLC and RP-HPLC method were used for the quantitative detection. 17 recombined yeast strains were screened by the color reaction was accounting for 1.7% of the total colonies that selected randomly. Then the TLC and RP-HPLC methods were used for screening, a recombinant strain taxol production, number DL-781, deposited at CGMCC, number No.8999.(5) After the recombinant strain was culutured in YPD liquid medium for 72 h, the yield of taxol was 1.106mg/L based on RP-HPLC and the dell of dry weight was 3.98g/L; Similarly, After the recombinant strain was culutured in inorganic medium for 72 h, the yield was 1.086mg/L and the dell of dry weight was2.46g/L.Additionly, the results show that every generation paclitaxel yield was the same, indicating the heredity of the recombinant strain was stable.