Studies On The Mechanism Of Lipid Synthesis And Pentose Metabolism In Oleaginous Yeast

At present, glucose is the major carbon source in the synthesis of microbial oil. In order to save the of cost production and expand the scope of starting fermentation materials, seeking effective alternative carbon sources is a key direction in the oil-producing industry. Celluloses are some of the most abundant renewable resources in the world, and xylose is one of the main hydrolyzates from celluloses. Therefore, to domesticate oleaginous micro-organisms for the efficient utilization of micro-organisms has guiding significance to the development of oil industry. This study focuses on the key enzymes in the synthesis of microbial oil and the mechanism of pentose metabolism in Rhodotorula glutinis.Firstly, the author examined the crucial enzymes responsible for oil synthesis, and then selected ATP citrate lyase (ACL) for further research. The principles of cell wall cracking were thoroughly analyzed and two methods were successfully concluded for the extraction of genome in cells, using the joint effects of lysozyme and snailase, and the rapid lyophilization of liquid nitrogen respectively. The method of lysozyme-snailase gave more complete genome. To obtain the gene of ACL, homologous primers were designed through comparisons of homologous protein sequences among different species, and PCR amplification was achieved.Secondly, different utilizations of glucose, xylose and the mixture of the two by oil-producing yeast Rhodotorula glutinis were compared. As to the mixed medium, glucose consumption took priority, and after a certain delayed phase about 24h, the strain began to use xylose. The final total sugar utilization could reach 99%. Oils produced from the three types of media were separately treated with methyl esterification and analyzed through gas chromatography-mass spectrometry technique. The main components are octadecadienoic acid methyl ester and hexadecenoic acid methyl ester.Finally, the feature that Rhodotorula glutinis can utilize pentose naturally was checked. With the inspiration from sugar transport mechanism, comparisons were done by analyzing the differences of membrane protein expression between cells growing on a high concentration of glucose and a low concentration of xylose, with a view of separating the pentose transporter characteristic of induced gene expression. The results showed that a plasma membrane protein about 35kD was expressed with 0.5% xylose as carbon source, but not with 4% glucose. This indicated that the gene expression was induced by xylose but inhibited by glucose, which behaved consistently with the feature of pentose transporter at molecular levels. Therefore, the protein was determined preliminariy the right one that helped to transport pentose in Rhodotorula glutinis.