Screening Of Rhodotorula With High Lipid Yield And Cloning Of The Conserved Region Of NADP-dependent Malic Enzyme Gene From R. Graminis M124

Microbial oil, also known as single-cell oil, are produced by various microorganisms, such as yeast, mold, bacteria and algae, which could use carbohydrates, hydrocarbons and the general oil as carbon source to accumulate a large number of oil in the body of themselves. Early research on microbial oil mainly focused onγ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and other functional polyunsaturated fatty acids. In recent years, with the development of new bio-energy research, microbial oil has become the object of bio-energy development and utilization. Moreover, oil-producing microorganisms had characteristics of abundant resources, high in oil content, a broad spectrum of carbon utilization and huge development potential. Thus, lipid production by microorganism has become a new direction in the development of oil resources. To unearth this new oil resource is very important especially in condition of today's acute shortage of energy. Therefore, positive breeding high-yield oil strains to study the optimum conditions, and exploration the key factor in lipid accumulation from the molecular level, are of great significance.We studied the screening and identification of Rhodotorula with high lipid yield and the optimization of its lipid production fermentation conditions systematically. Then the fatty acid composition was determined. The conserved region of NADP-dependent malic enzyme gene from R. graminis M124 was amplified by PCR, which was considered as the key enzyme gene in lipid accumulation. This study provided exploring new oil-producing microorganisms resources with experimental evidence, and made preparation for the cloning of full-length NADP-ME gene. From the point of long-term development, this study would lay the foundations for further study of metabolic regulation of lipid accumulation and improving oil production capacity of oil-producing microorganisms by methods of genetic engineering. The main results are described as follows.1. Eighteen Rhodotorula strains, isolated from the natural environment, which could produce carotenoid were used to select the high lipid production strain in this study. After preliminary screening with Sudan Black dyeing technique and further screening by the method of shake-flask, strain SCAU-13 with the highest lipid content of 14.63% was screened out. The identification results showed that strain SCAU-13 was Rhodotorula graminis.2. In order to increase the production of microbial oil, ultraviolet (UV), diethyl sulfate (DES), acridine orange (ADO), UV-DES and UV-ADO had been used to induce mutagenesis of SCAU-13, then strain M124 was obtained, with the lipid content of 42.42% which was as 2.90 times as that of parent strain SCAU-13. The optimal fermentation conditions for M124 were as follows:glucose as carbon source, (NH4)2SO4 as nitrogen source,60:1 of carbon-nitrogen ratio, initial pH 6.0, peptone 1.8 g/L, yeast extraction 4 g/L, MgSO4·7H2O 0.5 g/L. Under this culture condition, R. gramini M124 accumulated lipids up to (53.67±0.17)% on a cellular biomass basis with biomass yield of (13.55±0.10) g/L, and the corresponding lipid yield reached (7.27±0.03) g/L, which had increased by 26.52%, 14.83% and 45.11%, respectively, compared with the corresponding items before optimization.3. We analyzed the fatty acid composition of the lipid from R. graminis M124 by GC-MS. The results showed that under this condition, every fatty acid in this reach could be isolated very well. And this separation procedure was fast and convenient. In the lipid from R. graminis M124,8 kinds of fatty acid were detected. In these compositions, tetradecanoate acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid and eicosanoic acid were saturated fatty acids, which accounting for 18.29% of total fatty acids, while 9-hexadecenoic acid,10-octadecenoic acid and oleic acid were monounsaturated fatty acids, which accounting for 81.71% of total fatty acids. No polyunsaturated fatty acid was found. The fatty acid composition of the lipid from R. graminis M124 was unique. What was remarkable was that 10-octadecenoic acid accounted for 80.08% of total fatty acids.4. The conserved region of two NADP-dependent malic enzyme genes from R. graminis M124 and the corresponding cDNA were both obtained. From these, we could get the conclusion that two or more genes which encode NADP-dependent malic enzyme existed in R. graminis M124. Moreover, it prepared for cloning of the full-length of NADP-ME gene by genome walking or rapid amplification of cDNA ends.