Wang G, Manns D C, Guron G K P, Churey J J, Worobo R W
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
J Dairy Sci. 2014 Jul;97(7):4120-6. doi: 10.3168/jds.2014-8196. Epub 2014 May 2.
The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.
芽孢杆菌细菌素thurincin H对多种食源性病原体，如单核细胞增生李斯特菌，以及乳制品腐败细菌，特别是乳制品中常见的不同芽孢杆菌属细菌，具有广泛的抑制活性谱。此前，我们构建了3种质粒，用于在工程化的天然产生菌苏云金芽孢杆菌SF361thnH(-)中同源表达天然thurincin H。由于天然产生菌苏云金芽孢杆菌SF361染色体上的结构基因thnA1、thnA2和thnA3发生了框内缺失，该宿主缺乏thurincin H的产生。先前构建的表达载体是通过将天然thurincin H启动子、3个（或1个）结构基因拷贝以及天然（或Cry蛋白）终止子克隆到大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315中构建而成的。在本研究中，构建了3种相应的表达载体（pGW134、pGW135和pGW136），用于在同一宿主中表达重组thurincin H-His6，其中每个结构基因的C末端融合了一个6-组氨酸标签。产生的细菌素产量较低，这表明His标签可能会对thurincin H-His6前肽翻译后的后续翻译后修饰或输出过程产生负面影响。此外，为了过表达天然thurincin H，构建了另外2种质粒（pGW137和pGW138），它们由依赖芽孢形成的Cry蛋白双启动子BtI和BtII、thnA1结构基因以及thurincin H天然或Cry蛋白终止子组成。然而，在Luria肉汤平板上产量较低，在芽孢形成平板上则没有产量。有可能由于芽孢形成阶段不期望的生化和生理变化，产生的thurincin H前肽没有被正确修饰或输出到细胞外环境中。
