Impact of malonate on the metabolism and fatty acid synthesis of genetically engineered saccharomyces cerevisiae

• Main Author: Tan, Kee Yang
• Other Authors: Chen Wei Ning, William
• Format: Theses and Dissertations
• Language: English
• Published: 2015
• Subjects: DRNTU::Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/62535
• Institution: Nanyang Technological University

In view of the increasing global energy usage, biological fuel production has proved to be able to serve as a sustainable, carbon-neutral energy source compatible with current engine technology. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels and various biogases. The current range of biofuels consists primarily of microbially derived fatty acids, ethanol and plant-based biodiesel. The use of microbial systems for the production of industrially relevant compounds has been popular in the past years as a direct result of the genomics revolution. Further advances in gene regulation, protein engineering, pathway portability, synthetic biology and metabolic engineering have propelled the development of cost-efficient systems for biofuel production. Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. It is one of the main components for the initiation of the fatty acid synthesis and also acts as a building block for the elongation of fatty acid after every round of fatty acid synthesis. However, Malonyl-CoA is at a low concentration inside the cell and it is produced mainly from Acetyl-CoA through the actions of the enzyme acetyl-CoA carboxylase (ACC). As a result, it would be beneficial to find an alternative source of Malonyl-CoA and thus increasing its intracellular concentration. By doing so, the overall synthesis of the fatty acids inside the yeast should increase as well. MatB gene from the bacteria, Rhizobium leguminosarium bv trifolii encodes for a malonyl-CoA synthetase which is able to catalyze the formation of the Malonyl-CoA directly from malonate and CoA with the hydrolysis of ATP. However, results from HPLC proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is not able to uptake exogenously supplied malonate in the form of malonic acid. As such, a gene known as the mae1 gene from another species of yeast, Schizosaccharomyces pombe had been successfully cloned and transformed inside the target yeast, Saccharomyces cerevisiae. This gene encodes a dicarboxylic acid plasma membrane transporter which enables the cells to uptake exogenous malonic acid. Yeast immunofluorescence was used to detect the presence and localization of the expressed proteins in the target cells. The results had convincingly showed that the mae1 gene is successfully expressed and the expressed dicarboxylic acid transporter proteins were localized to the plasma membrane of the cells as intended. Furthermore, HPLC and LC-MS were also able to provide substantial results to show the existence of the encoded protein, which is the plasma membrane dicarboxylic acid transporter. With the correct negative controls within HPLC and LC-MS, the functional activities of the protein could also be demonstrated and verified. Therefore, the positive results from HPLC and LC-MS, together with the positive results from RT-PCR and yeast immunofluorescences, the plasma membrane dicarboxylic acid transporter was verified to be successfully expressed and functioning as intended as malonic acid was detected inside the transformed cells and having a significant impact on the proteomics of the cells as demonstrated by the LC-MS results. Being an inhibitor to the succinate dehydrogenase of the critic acid cycle in the mitochondria, malonic acid, after being transported into the yeast cells, seem to have a certain degree of toxicity displayed towards the cells. From the LC-MS results, most of the up-regulated proteins were those that were involved one way or another in the metabolism of carbohydrates to produce energy. It is also known that when the critic acid cycle was impaired due to post-mitotic aging or a result of activity from inhibitors such as malonate, alternative mechanism would be triggered to continue supply energy for the survivability of the cells. In this case, the glyoxylate cycle is activated. This is evident from the LC-MS results as the enzymes involved in the glyoxylate cycle were shown to be significantly up-regulated. Among those proteins that were down-regulated, 6-phosphogluconate dehydrogenase was decreased by around 40%. This dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate and CO2, with concomitant reduction of NADP to NADPH in the pentose phosphate. Furthermore, inositol-3-phosphate synthase, which catalyzes the chemical reaction of converting D-glucose 6-phosphate to 1D-myo-inositol 3-phosphate to form phospholipids, was also decreased by around 60%. This hinted at an energy deprived state of the cells where carbohydrates such as glucose seem to be channelled away from the other pathways and was used to increase the rate of glycolysis. Next, the MatB gene from the bacteria, Rhizobium leguminosarium bv trifolii was cloned and expressed in the yeast cells with the mae1 gene. When grown in medium containing malonic acid, the yeast cells, containing the 2 genes, were able to grow at a normal rate as compared to the wild type yeast cells. Furthermore, the toxicity due to the intake of malonate exhibited by the cells with only the mae1 gene seemed to be eliminated when growth curves were compared. Results also showed that yeast cells that contained the 2 genes were also taking in more malonate from the medium as compared to cells that only contained the mae1 gene. The increased uptake of malonate and the reduced toxicity exhibited by the cells showed that the malonate transported in were utilized and not accumulated to inhibit the citric acid cycle. Results from HPLC showed that the amount of malonate present in the cells were indeed much lower than those present in cells with only the mae1 gene. Fatty acid profiling also showed a significant increase in the amount of fatty acids produced by the cells with 2 genes as compared with wild type yeast cells and yeast cells with only the mae1 gene. Fatty acids that were typically produced by the Saccharomyces cerevisiae cells such as palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid were significantly increased and accumulated. This verified the functional expression of the matB gene and the ability of the encoded malonyl-CoA synthetase to increase the overall amount of fatty acids produced.