Expression The CgGPD1 Genes For Key Enzyme Of Glycerol Synthesis In Escherichia Coli And 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 dyeing, textile, paint, leather, paper, synthetic resin, pharmaceutical, food and defense industries and as a feedstock for the production of various chemicals. Compared with the traditional methods, the zymotechnics has its own adventage in the synthesis of glycerol. Candida glycerinogenes WL2002-5 is a excellent strain for glycerol production and its intellectual property rights belongs to our lab, the yeast has several useful properties, such as tolerance to high glucose concentrations, rapid growth and ability to accumulate high extracellular glycerol concentration. It has been applied to the glycerol industrial.The study shows that glycerol-3-phosphate dehydrogenase is the key enzyme of glyceol synthesis , the most direct way to increase the glycerol production is to overexpress the gene of GPDH -GPD1. The gene of glycerol-3-phosphate dehydrogenase CgGPD1 from Candida glycerinogenes was cloned using PCR on the base of previous research, and constructed binary vector with different copies of CgGPD1: pCAM3300-zeocin-CgGPD1,pCAM3300- zeocin-CgGPD1-CgGPD1 and pCAM3300-zeocin-CgGPD1-CgGPD1-CgGPD1. The vectors were transformed in Escherichia coli JM109 respectively. The expression of GPDH of the transformants containing different copies of CgGPD1 under different concentration of NaCl and glucose were researched.The efficiency of the general way to transform the Candida glycerinogenes is very low, because Candida glycerinogenes is a industrial strain, its cell wall is typical.Using the way of Agrobacterium tumefaciens-mediated transformation, the tranformants with one copy CgGPD1 were screened and norminated Candida glycerinogenes-CgGPD1(C.g-G).The transformant with high glycerol production were screened and norminated C.g-G8, the experiments show that the insert of the gene CgGPD1 didn't change the growth of C.g-G8, the rate of glucose consumption of C.g-G8 is faster than C.g; the glycerol production of C.g-G8 is 12.08 % higher than C.g; and also, the fermentaion time of C.g-G8 is 6 hours shorter than C.g; the rate of glycerol synthesis of C.g-G8 is 20.49 % quicker than C.g. During the fermentation, the activity of glycerol-3-phosphate dehydrogenase of C.g-G8 maintains a high level as the C.g,, but it is higher than C.g, it indicates that the gene CgGPD1 is transfered into C. glycerinogenes and expressed successfully.