Construction Of Recombinant Strains Capable Of Producing Biodiesel And Research About Lipid Production Conditions Of Saccharomyces Cerevisiae

The biodiesel is a hot research spot,its production method is that the raw material fat and the methyl alcohol or the ethyl alcohol catalysised by chemistry catalyst or biocatalyst are transformed to biodiesel.At present,the biodiesel's production needs two steps:How to acquire fat and transfer the fat to the biodiesel. If can these two steps integrate in the identical biological system,it will greatly reduce the steps and can enhance the the transform efficiency of biodiesel.Through construction of recombinant strains capable of producing biodiesel, we want to express special-purpose lipase's gene in the yeast. This may possibility directly catalysis and synthesize fatty acid ethyl ester using the fat and the ethyl alcohol that the yeast synthesis catalysised by lipase which is produced after the reorganization system transforms.Finally,we hope to obtain one new highly effective technology way to produce the biodiesel.In this article, the gene of lipase mature peptide (gene Lip2) was cloned from Candida sp.99-125 and promoter gene PGK1and terminator gene CYC1 are constructed into the recombined vector pYES2 and YEp352. Some constitutive expressions were researched about the gene Lip2 in Saccharomyces cerevsiae. Also,we optimize culture medium for lipid production of Saccharomyces cerevisiae.Firstly, the 903bp target gene Lip2 was amplified by PCR from genome DNA of Candida sp.99-125, and transformed into E.coli after TA clone. Screen and indentify the positive transformants, sequencing result showed the cloned gene sequence was same as the reported. After treated by restriction enzyme, the Lip2 fragment is inserted into the plasmid pYES2 after double digestion.Therefore,a constitutive expression system of Lip2 was constructed successfully in Saccharomyces cerevsiae.SDS-PAGE showed that target protein was expressed in vivo while almost none in vitro was detected. But it is found that the expressed lipase has almost none activity of lipase in vivo.Secondly,the 731bp promoter gene PGK1 was amplified by PCR from genome DNA of Saccharomyces cerevsiae, the 260bp terminator gene CYC1 was amplified by PCR from he plasmid pYES2 and transformed into E.coli after TA clone. Screen and indentify the positive transformants, sequencing results showed the cloned genes sequence was same as the reported. Then,we use overlap PCR to fuse Lip2 and CYC1 genes.After treated by restriction enzyme,the Lip2,CYC1 and PGK1 fragment is inserted into the plasmid YEp352 after double digestion. Therefore, a constitutive expression system of Lip2 was constructed successfully in Saccharomyces cerevsiae. SDS-PAGE showed that target protein was expressed in vivo while almost none in vitro was detected. It is found that the expressed lipase has an activity of 1.3U/ml only in vivo.Thirdly,response surface methodology (RSM) was employed to optimize culture medium for lipid production of Saccharomyces cerevisiae. According to the results of mono-factors experiment, a Plackett-Burman design was used to investigate the effects of different factors in the ferment medium, three statistically significant factors are:citric acid, CaCl2, initial pH value, and then steepest ascent procedures was employed to define optimal response region for these three factors. Finally, Box-Behnken was employed to design. The results of experiments was analyzed by RSM with Minitab15.0 soft. The optimal lipid production medium is:glucose 15%, peptone 0.2%, yeast extraction 0.4%, citric acid 0.471%, MgSO4·7H2O 0.1%,ZnSO4·7H2O 0.2%, CaCl2 0.025%, FeSO4-7H2O 0.005%, initial pH value 6.74,180r/min,30℃for 96 h.Lipid production (dry weight) after optimization is increased from 4.76% to 14.55%. The yield of lipid is increased by about 2 times.