Effects Of AMP Metabolism On Physiological Function Of C.glabrata

C. glabrata ATCC 55 was employed as a model system in this study. To analyze the influences of AMP metabolic genes on physiological function, intracellular ATP level, efficiency of TCA cycle and intracellular microenvironment under acetic acid stress was analyzed, with the method of molecular biotechnology and cell physiology. The main results were described as follows:(1) Cgamd1Δ, Cgade12Δ, Cgade13Δ and Cgade12ade13Δ were constructed from the original strain C. glabrata ATCC 55. Analysis showed that the intracellular ATP level of Cgamd1Δ was increased by 15.38%, while that of Cgade12Δ, Cgade13Δ, Cgade12ade13Δ were decreased by 7.69%, 2.56% and 5.12%, compared with that of ATCC 55 in YNB medium. The same tendency was found during fermentation process. Controlling DO concentration at 20%, the intracellular ATP level of Cgamd1Δ was increased by 27.28% while that of Cgade12Δ, Cgade13Δ, Cgade12ade13Δ were decreased by14.54%, 5.74% and 10.47%, compared with that of ATCC 55 at early period of fermentation.(2) During fermentation process, metabolism efficiency of mutants was decreased compared with that of ATCC 55. The pyruvic acid production intensities of Cgamd1Δ, Cgade12Δ, Cgade13Δ, Cgade12ade13Δ were decreased by 37.50%, 15.00%, 75.00%, 77.50% and the glucose consumption rate was decreased by 20.37%, 15.43%, 34.57%, 37.04%. Further analysis of Cgamd1Δ showed that the pyruvic acid productivity and the yield of dry cell weight on glucose was increased by 31.40% and 35.71% which indicate that carbon flow was prefer to pyruvic acid production. The physiological mechanism was studied and we found that the enzymes activities and intermediate metabolites concentration of TCA cycle were decreased, which reduced the efficiency of the TCA cycle. AS a result, the intracellular pyruvic acid level was increased by 103.64% compared with that of ATCC 55. For Cgade13Δ and Cgade12ade13Δ, the cell dry weight on glucose concentration was increased by 21.42% and 18.57%, respectively. However, the pyruvic acid productivity was decreased by 67.44% and 71.51%, indicating that carbon flow was prefer to cell growth. Study of its physiological mechanism showed that the increased key enzymes activity and intermediate metabolites concentration of TCA cycle enhanced the efficiency of the TCA cycle.(3) Differences of cell growth ability between ATCC 55 and mutants were more obviously in YNB-acetic acid medium than that in YNB-pyruvic acid and YNB-malic acid medium. The cell concentration of Cgamd1Δ was decreased by 55.91% while that of Cgade12Δ, Cgade13Δ, Cgade12ade13Δ were increased by 31.56%, 16.44% and 12.71%, compared with that of ATCC 55. Analysis of intracellular microenvironment under acetic acid stress showed that the deletion of CgAMD1 affected the intracellular microenvironment. The intracellular pH, H+-ATPase activity, and cell membrane integrity of Cgamd1Δ were decreased by 7.34%, 5.61%, 30.63%, while the ROS production and cell membrane electric potential were increased by 53.93% and 148.03%, compared with that of ATCC 55. However, the deletion of CgADE12, CgADE13 improved the intracellular environment under acetic acid stress. Compared with ATCC 55, the intracellular pH, cell membrane integrities and cell membrane electric potentials of Cgade12Δ, Cgade13Δ, Cgade12ade13Δ were increased, while the ROS productions were decreased by.59.67%, 16.98% and 19.51%...