Modification And Application Of Chloroplast Expression System In Chlamydomonas Reinhardtii And Research On Fermentation Technology In Xylanase Engineering Yeast

So-called "green yeast", Chlamydomonas reinhardtii is a unicellular spheroidal green alga with a single cup-shaped chloroplast, which has around 80 copies in its genome. The alga had become one of the simplest model organisms for its inexpensive cost of culture, robust metabolic capacity and rapid growth rate (approximately 6-8 h generation time). Large-scale production can be carried out because it yields much biomass more than conventional terrestrial plant (for example, corn) under illumination and CO2 supply. Furthermore, it has no toxin and complicated compound, and has too much simpler metabolic pathway than plant. In addition, the green alga is a perfect platform of recombinant protein overexpression because the cell shares some common physiological features, which can assembly protein into biological active complex by post-translational processing. For this, research and modification of efficient expression system in C.reinhardtii can not only enrich study on molecular biology of chloroplast, but also tremendously practical applications. In this research, the interest gene is VP1 antigen gene of foot-and-mouth disease virus, which resulted in substantial losses in Chinese stockbreeding. A chloroplast protein overexpression system with enhancing element of immunogenicity and a simpler transformation strategy of chloroplast was established in chlamydomonas, thus it become possible that inferior organism can be applicable to production of large-scale edible vaccine. The results as follows:1. We constructed a series of compatible plasmids, which facilitate construction of chloroplast expression plasmid with interest gene in chlamydomonas;2. A series of different expression plasmids had been introduced into recipient cell at different physiological status by electroporation. We achieve a simpler high-frequency transformation method, which provide an alternative for chloroplast transformation in C.reinhardtii. Compared to particle bombardment technology, the method has several advantages:higher transformation rate, simpler manipulation. more time-saving for transgenic line generation and lesser risk of fungal contamination. Further analysis demonstrated that degradation and regerenation of recipient cell wall have a crucial effect on chloroplast transformation by electroporation in C.reinhardtii.3. The transgenic fragment was analyzed by PCR during subculture. The results showed that transgene integrated with chloroplast genome will be lost sooner or later. However, the process can be extended for doubling time when the recipient cell is treated by 5-fluorodeoxyuridine prior to transformation. On the other hand, the existence of transgene will be prolonged a period of subculture time in rich nitrogen sources medium.4. The cholera toxin B gene and foot and mouth disease virus VP1 gene was fused with CV1 fragment, which is constructed into prokaryotic and chloroplast expression plasmids. The CV1 inclusion body expressed in E.coli was purified and immuned rabbit, which produced anti-CV1 serum. The antibody analyzed the recombinant CV1 antigen in chlamydomonas chloroplast by western blotting. The detection data of CV1 fragment in transgenic alga carried out element task for edible vaccine production using the green alga as target host, rather than higher plant.Xylanases not only play an important role on a wide array of biotechnological and industrial applications in processes such as food, animal feed, liquor-making and paper pulp bleach, but also have great potentials in the bioconversion from lignocellulosic feedstocks to fuel-grade ethanol, which have attracted tremendous interests in the past few years. In this section, an engineering pichia yeast GS115-xylCX8 secreting high-activity xylanase was carried out scale zymolysis for combinant enzyme production in the fermentor (NBS 3 L). Although the wet cell weight of the yeast cells was only 140 g/L for dissolved oxygen limitation, the yield of the recombinant xylanase were 7340 U/mL after approximately 150 h methanol inducement. The yield of the fermentor was as more than 4-fold as that of the shake flask; it is promising to produce the recombinant xylanase commercially.