Effects Of Acetyl-CoA Synthetase Genes Overexpression On Physiological Functions Of Saccharomyces Cerevisiae

Acetyl-CoA is one of the most important cofactors and participants hundreds of biochemical reactions, including TCA cycling, amino acids metabolism, lipids metabolism and terpene metabolism, etc. In this research, following three issues were researched:(1) acetyl-CoA synthetase genes (ACS1 and ACS2) overexpressed strains were constructed. The intracelluar acetyl-CoA content and acetyl-CoA synthetase activity were measured for both ACS1/2 overexpressed strains and control strain. Membrane lipids components, ATP contents and transcriptional changes of key genes were also detected. Tolerance of acetyl-CoA synthetase genes overexpressed strains for different concentration of ethanol stress was measured. In order to explain the reasons to ethanol tolerance for acetyl-CoA synthetase genes overexpressed strains, transcriptional changes, ATP content changes and cellular lipids components changes were measured. Followings are major works of my article.(1) PCR was used to amplify target DNA, restriction enzyme cutting and liagtion were used to construct the overexpressed shuttle vectors (pY26-TEF-GPD-ACS1, pY26-TEF-GPD-ACS2). The shuttle vectors were transformed into S. cerevisiae to construct acetyl-CoA synthetase genes overexpressed strains. Intracellular acetyl-CoA content and acetyl-CoA synthetase activity were measure by HPLC and ultraviolet spectrophotometer. The results indicated that intracellular acetyl-CoA content in ACS1 and ACS2 overexpressed strains were 2.19 and 5.02 times higher than control strain (no targeted DNA shuttle vector was transformed into S. cerevisiae), respectively. The results also suggested that ACS1 and ACS2 overexpressed strains acetyl-CoA synthetase activity were 1.62 and 1.41 times higher than control strain.(2) HPLC, GC-MS, realtime-qPCR and gene chip technologies were used to detect the physiological metabolism changes of acetyl-CoA synthetase genes overexpressed strains, including ATP content changes, membrane lipids components changes and transcriptional changes of key metabolic pathways. Compared with control strain, energy metabolism was vigrous acetyl-CoA synthetase genes overexpressed strain, especically for the strain of ACS1 overexpressed strain, intracellular ATP content is 3.93 times higher than the control strain. Fatty acids components are difference for both ACS1 and ACS2 overexpressed strains, compared to control strain, particluar for the strain of ACS1 overexpressed strain. transcriptional changes of key pathways are also different between acetyl-CoA synthetase genes overexpressed strains, such as mevalonate pathway, unsatrated fatty acids synthesis pathway and ergosterol biosynthesis pathway.(3) Methylene blue postive dyeing, HPLC, Gc-MS, realtime-qPCR and gene chip technologies were used to measure physiological metabolism changes and explain the possible reasons for ethanol tolerance discrepancy between ACS overexpressed strains and the control strain. ACS1 overexpressed strain got a significant tolerance to ethanol stress compared to control strain and ACS2 overexpressed strain. Intracellular ATP (12.4 times higher than control strain under 5% ethanol stress), unsaturated fatty acids components, ergosterol transcriptional changes are significant differences for the strain of ACS1 overexpressed strain. Thus, our results indicated that enhanced tolerance to ethanol stress for ACS1 overexpressed strain is a comprehensive reply of transcription for S. cerevisiae cell, particular for the pathways of membrane lipids metabolism and lipids biosynthesis pathway. Gene chips results also gave an additional proof for above conclution. Results for global genes transcriptional changes of S. cerevisiae under 5% ethanol stress indicated that lipids related metabolism pathway and amino acids related pathways metabolism are most significant.