Mutation Breeding Of Yeast With High Lipid And Carotenoid Production

At present, the demand for grease continually grow along with population increase, but the yield of animal and vegetable grease is not enough. Microbial oil, the replacer of animal and vegetable oils and a new resource, contains a lot of unsaturated fatty acids and is very helpful for improving human health. It is also used as a new raw material to prepare biodiesel. Thus, exploiting microbial oil possess the important realistic significance for production of health food and biodiesel.Carotenoid, a kind of fat-soluble pigments, is widely distributed in the nature and has many important biological functions, such as anti-cancer, oxidation resistance and resistance to disease of heart head blood-vessel. At present, carotenoids is widely applied to the food, feed, health care products and cosmetics industries, and attracting more and more attention. Although many researchers had paid great attention on how to improve the production and quality of carotenoids, the problem remains.In this study, we selected the Rhodotorula mucilaginosa(KC8) as a starting strain, which can product lipid and carotenoids, and improved its yield of lipid and carotenoids by a series of methods such as mutation and gene engineering. Furthermore, we also investigated the impact of tetoconazole on the yield of carotenoids.The main research findings include the following aspects:(1) To screen strains of higher lipids production, Atmospheric and room temperature plasma(ARTP) was used to induce mutation of the starting strain. Firstly, the strain Y3 with the 2.38 g/L lipid yield after 96 hours flask fermentation was obtained from 300 mutant strains. Next, we optimized the fermentation conditions. The result showed that the lipid productivity can reach 3.96 g/L under the optimized conditions: glucose as carbon source,(NH4)2SO4 as nitrogen source, ratio of carbon to nitrogen at 90:1, initial p H 6.5, incubated at 28℃ for 96 h shaking fermentation. GC-MS analysis indicated that similarly to vegetable oil, the fatty acids from strain Y3 were mainly composed of palmic acid, oleic acid,linoleic acid, stearic acid, eicosenoic acid and tetracosanoic acid.(2) To increase the carotenoids production of KC8, a series of mutation methods, including ARTP,NaNO2, UV light, combination of NaNO2 and UV light, were applied. Finally, K 4 was obtained from 800 mutant strains. The yield of carotenoids by K4 was 14.47 mg/L mg/L, which was 1.67 times of the starting strain, KC8. High performance liquid chromatography(HPLC) analysis indicated that the main componentof carotenoids was β-carotene.(3) To increase carotenoids production of the K4, we tried to overexpress the key enzymes(HMG CoA reductase) to increase the amount of carotenoids precursor(IPP). At last, a strain G1 with a highly pigment production was obtained. The carotenoid yield of strain G1 reached 17.02 mg/L and increased by17.9% when compared with that of the strain, K4. HMG CoA reductase gene was firstly amplified from the saccharomyces cerevisiae genome, and two sequences of 5.8S- ITS rDNA were amplified from the Rhodotorula mucilaginosa genome. In order to overexpress HMG CoA reductase, the vector pPIC-2rDNA-G-H-C was composed of GDP promoter, rDNA and HMG Co A reductase gene, and transformed into the yeast K4 by electric conversion.(4) Ketoconazole was a kind of ergosterol inhibitors. In addition, we found that the addition of ketoconazole during the carotenoids fermentation can affect the carotenoids production. Furthermore, the strain G1 had the highest yield of carotenoids(19.14 mg/L) by adding ketoconazole(28 mg/L).