Knudsen Jan Dines, Johanson Ted, Eliasson Lantz Anna, Carlquist Magnus
Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, Getingevägen 60, SE-22100 Lund, Sweden.
Department of Systems Biology, Technical University of Denmark, Soltofts Plads, Building 223, DK-2800 Kgs. Lyngby, Denmark.
Biotechnol Rep (Amst). 2015 Jun 15;7:107-119. doi: 10.1016/j.btre.2015.06.001. eCollection 2015 Sep.
A control point for keeping redox homeostasis in during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 () gene. In this study, the possibility to steer the activity of the promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein () under the control of the promoter was used to determine the promoter activity at the single-cell level When placed in a strain background, the promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (). In contrast, the promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.
在发酵生长过程中维持氧化还原稳态的一个控制点是对3-磷酸甘油脱氢酶2（GPD2）基因转录的动态调控。在本研究中，通过将GPD2启动子置于具有不同NADH再氧化能力的菌株中，并应用不同的环境条件，研究了调控该启动子活性的可能性。利用在GPD2启动子控制下表达绿色荧光蛋白（GFP）的报告菌株进行流式细胞术分析，以确定单细胞水平的启动子活性。当置于Δgpd1菌株背景中时，GPD2启动子的活性比强组成型的3-磷酸甘油醛脱氢酶（GAPDH）高2倍。相比之下，当细胞在连续模式下以0.3 h-1的生长速率和过量氧气条件下（即通气量为2.5 vvm，搅拌速度为800 rpm）培养时，发现GPD2启动子无活性。此外，还确定了一个明确的操作窗口，在此窗口内，Δgpd1菌株可以与野生型酵母以相同的效率生长。总之，流式细胞术图谱揭示了GPD2启动子完全无活性或过度活跃的条件，这对其在未来生物技术应用（如异源基因表达的过程控制）中的实施具有重要意义。
