Genetic Improvement Of The Oleaginous Yest And Signle Cell Oile Producity By Fermentation

In the past years, biodiesel, which is biodegradable, can be used without modifying existing engines, and produces less harmful gas emissions such as sulfur oxide has received increasing attention because of the environmental pollution and energy crisis world wide. At present the high production cost of biodiesel is a major barrier to its commercialization. Therefore, using a low cost raw material is crucial in reducing the cost of biodiesel production.In our previous study, it was found that Rhodotorula mucilaginosa TJY15a isolated from the marine fish Synechogobius hasta could accumulate a large amount of oil from glucose and hydrolysate of cassava starch. In this study, we found that R. mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C16:0, C18:1 and C18:2, especially C18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.In order to directly convert inulin and the extract of Jerusalem artichoke tubers into single cell oil by R.mucilaginosa TJY15a and avoid mixing the cells of R. mucilaginosa, the cells of the mutant M-30 were immobilized. Then the immobilized cells were co-cultured with the free cells of the oleaginous yeast R. mucilaginosa TJY15. It was found that the immobilized inulinase-producing cells of P. guilliermondii M-30 can produce 169.3 U/mL of inulinase activity while the free cells of the same yeast strain only produces 124.3 U/mL of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells are co-cultivated with the free cells of R. mucilaginosa TJY15a, R. mucilaginosa TJY15a can accumulate 53.2% oil from inulin in its cells and cell dry weigh reaches 12.2 g/L. Under the similar conditions, R. mucilaginosa TJY15a can accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reaches 12.8 g/L within 48 h. When the co-cultures are grown in 2-L fermentor, R. mucilaginosa TJY15a can accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weigh reaches 19.6 g/L within 51 h. About 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers is C16:0, C18:1 and C18:2.In order to convert inulin and inulin-containg materias into single cell oil by one step fermentation, the gene of exo-inulinase cloned from Kluyveromyces marxianusCBS6556 was expressed in the oleaginous yeast Y. lipolytica ACA-DC50109. First, the INU1 gene was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. Then, the inulinase activity of different transformants was determined. The activity of the inulinase by the transformant Z31 of the transformants obtained was found to be 41.7 U/mL after cells growth for 78h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g/Lwithin 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g/L within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g/L within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C16:0, C18:0 and C18:1, especially C18:1 (58.5%).