Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany.
J Biotechnol. 2012 May 31;159(1-2):12-20. doi: 10.1016/j.jbiotec.2012.02.011. Epub 2012 Feb 25.
Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele--known to cause hypersecretion in Bacillus subtilis-- resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG--known to interfere with DegU DNA-binding in B. subtilis--did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.
针对地衣芽孢杆菌 degSU 操纵子的破坏实验和 GFP 报告基因分析为 degU 上游的启动子活性提供了证据。pMutin 介导的 degU32 等位基因的同时引入——已知在枯草芽孢杆菌中引起过度分泌——导致蛋白酶活性显著增加。通过建立缺乏磷酸核糖基转移酶的 Δupp 菌株进行基于 5-氟尿嘧啶的反向选择，最终实现了 degU32 的无标记引入，进一步增强了蛋白酶，达到了过度表达 degU 的野生菌株的水平。令人惊讶的是，删除 rapG——已知在枯草芽孢杆菌中干扰 DegU DNA 结合——既不能增强野生型菌株也不能增强 degU32 菌株的蛋白酶产生。在原始菌株中，degU32 和 Δupp 反向选择的组合不仅在蛋白酶产生方面与过度分泌的野生菌株一样有效，而且还促进了旨在实现生物技术过程的生物控制和有效性的遗传菌株改良。
