| Recently, owing to its environment-friendliness, excellent quality and renewability, biodiesel has already attracted extensive attention. Microalgae are being considered to be an ideal feedstock for biodiesel due to their strong adaption to environment, fast growth, high oil yield, and not occupying of cultivated land. However, low lipid yield is still one of the key factors that restrain the large-scale industrial utilization of microalgae. Thus, cultivating microalgae species with high oil yield is of great importance. The NADPHs from malic dehydrogenation catalyzed by malic enzyme(ME) are considered as an important part of NADPH source for fatty acid synthesis. To study the effect of ME on microalgae lipid accumulation, Chlorella protothecoides were taken as the object, particle bombardment was employed to generate malic-enzyme-overexpressed genetic engineering Chlorella protothecoides strain. Furthermore, GC-MS was used to analyze the fatty acid compositions of the wild-type algae and the engineering algae, and the oil accumulation conditions of the engineering algae were further optimized through response surface methodology. The main contents and results are summarized below:1. Analyzing and successfully cloning the malic enzyme gene sequences. By analyzing and comparing the conservative sequence of malic enzyme amino acids, degenerate primers were designed, and the c DNA of Chlorella protothecoides was taken as a template, segmental me sequence was got, then employing 5'-RACE and 3'-RACE, the full-length me gene was cloned, and was further confirmed through ORF Finder and BLAST, which encodes 509 amino acid residues.2. Construction of malic-enzyme-overexpressed genetic engineering Chlorella protothecoides strain. First, expression vector p BI121-me was constructed, then with the aid of particle bombardment, p BI121-me expression vector was transformed into C. protothecoides strain. Positive clones were screened through antibiotic G418 plate, and were further validated via genomic PCR of engineering algae. Semi-quantitative PCR technology was used to analyze the transcription level of malic enzyme gene. malic enzyme protein was purified from cells broken liquid of engineering algae, and was further proved by western-bolt and mass spectrometry.3. The fatty acid compositions of the wild-type algae and the engineering algae were analyzed and compared. Soxhlet extraction method and GC-MS were employed to analyze the lipid content and fatty acid compositions of the wild-type algae and engineering algae under the condition of 10 g/L glucose as carbon source. The fatty acid compositions of the wild-type algae were C16:0(23.40%), C16:2(7.54%), C16:3(8.58%), C18:0(2.41%), C18:1(14.72%), C18:2(32.98%) and C18:3(10.37%); while those of the engineering algae were respectively C16:0(24.53%), C16:2(8.04%), C16:3(7.32%), C18:0(1.52%), C18:1(14.63%), C18:2(35.17%) and C18:3(8.79%). It can be seen that overexpression of ME had certain influence on the fatty acid compositions; however, the lipid content of the engineering algae was improved 34.7% in comparison to the wild-type algae, which attained 26.4% of dry weight of the cells.4. Lipid accumulation conditions of the engineering algae were further optimized. Through single factorial experiment of carbon and nitrogen sources, sodium acetate and sodium nitrate were confirmed to be the best carbon and nitrogen sources for lipid accumulation of the engineering algae. Response surface methodology was further utilized to optimize the lipid accumulation conditions of the engineering algae. The obtained optimum conditions were sodium acetate 54.54 g/L, sodium nitrate 651.6 mg/L, dipotassium hydrogen phosphate 194.2 mg/L. Under the optimal conditions, the lipid content of the engineering algae further increased 65.5%, reached 43.7% of the cell dry weight, and the biomass(cell dry weight) also raised 43.4% to 8.36 g/L. |
PDF Full Text Request