The Research Of Agrobacterium Tumefaciens-Mediated Transformation Of Candida Glycerinogenes

Glycerol is an important material for the light and chemical industries. Now, it is widely used in the cosmetic, toothpaste, tobacco, essence, printing and dyeing, textile, paint, pulp and paper, synthetic resin, leather, pharmaceutical, food and defense industries or as a feedstock for the production of various chemicals, more than 1,700 varieties of products. In particular, Candida glycerinogenes, a wild-type osmotolerant yeast that our laboratory possessed the independent intellectual property, is preferably used industrially in China for the glycerol production because of its high glycerol concentration, yield and recovery yield. In recent years, glycerol production by genetic engineering has received significant attention. To increase its glycerol productivity via genetic engineering, it is necessary to develop a transformation system for this industrial yeast. In this research, binary vector, pCAM3300-ura3 was constructed and transferred into Agrobacterium tumefaciens .Then to try Agrobacterium-mediated transformation system was used to transfer Saccharamyces cerevisiae successfully.Based on above researches, the plasmid pCAM3300-Zeocin was constructed and transferred into A. tumefaciens . The A. tumefaciens containing the binary vectors pCAM3300-Zeocin was co-cultured with C. glycerinogenes. Then, transformants were gained on plate containing Zeocin. PCR product demonstrated that Zeocin was integrated into C. glycerinogenes. Result indicate Zeocin gene utilized as selection marker in C. glycerinogenes and Zeocin used as selection pressure. According to the characteristic of C. glycerinogenes , researched the transformation conditions and found that at co-culture time 24 hours and cells ratio 1:500-1000 the efficiency reached 2 trasformants per 104 C. glycerinogenes cells. Transformation system of C.glycerinogenes mediated by A. tumefaciens in which Zeocin gene as selection marker was constructed, and this system can provide bases for research of C. glycerinogenes in the future. The gene of GPD1 encoding glycerol 3-phosphate dehydrogenase, the key enzyme in glycerol synthesis, was cloned from S. cerevisiae using PCR. The plasmid pCAM3300-Zeocin-GPD1 was constructed and transferred into A. tumefaciens . The transformation system was utilized to transfer GPD1 gene into C. glycerinogenes in the first time, pick the trasforment at the Zeocin plate. The obtained transformants were grown in fermentation medium contained corn steep liquor(CSL) 5 g/L, urea 2g/L, glucose 250 g/L and glycerol concentration in fermentation broth was determined at the 80 hours. A transformant which can produced higher glycerol concentration increased 12.93 % was selected and named Candida glycerinogenes-GPD1-10(C.g-G10). Compared with original st rain, at the grows balance time the rate of glycerol synthesize increased 71.7 %, the enzyme activity of Gpd1p increase 22.33 %, the glucose consumed tate increased 36.92 %, the fermentation cycle shorten about 24 h. These results suggest that GPD1 has been integrated into the genome of C. glycerinogenes and is expressed...