Warren Travis K, Lund S Amanda, Jones Kevin F, Hruby Dennis E
Siga Technologies, Inc., 4575 SW Research Way, Suite 230, Corvallis, OR 97333, USA.
Can J Microbiol. 2007 Mar;53(3):417-26. doi: 10.1139/W07-004.
An active area of research in the development of Streptococcus gordonii for use as a bacterial commensal vector involves the identification and utilization of strong promoters for high-level expression of heterologous products. Escherichia coli plasmid vectors containing different streptococcal promoters often fail to become established in E. coli for unknown reasons. Therefore, it is desirable at times to transform S. gordonii, which is naturally competent, with small quantities of nascently ligated DNA without using E. coli first to amplify or screen the product. By comparing the efficiency of two methods used to induce competence in S. gordonii, it was shown that the use of a synthetic competence stimulating peptide substantially enhanced plasmid uptake by S. gordonii. We amplified the amylase-binding protein (abpA) promoter from the S. gordonii genome and, using a synthetic peptide to induce competence, directly introduced plasmid DNA containing this promoter into S. gordonii as an unamplified product of ligation. This plasmid facilitated abundant secretion of a heterologous product by S. gordonii. By assessing the levels of heterologous product secreted by two plasmid constructs, it was possible to evaluate the relative strength of two native promoters.
将戈登氏链球菌开发为细菌共生载体的一个活跃研究领域涉及鉴定和利用强启动子以实现异源产物的高水平表达。含有不同链球菌启动子的大肠杆菌质粒载体常常因不明原因无法在大肠杆菌中稳定存在。因此，有时需要直接用少量新连接的DNA转化天然感受态的戈登氏链球菌，而不先利用大肠杆菌对产物进行扩增或筛选。通过比较两种用于诱导戈登氏链球菌感受态的方法的效率，结果表明使用合成的感受态刺激肽可显著增强戈登氏链球菌对质粒的摄取。我们从戈登氏链球菌基因组中扩增出淀粉酶结合蛋白（abpA）启动子，并利用合成肽诱导感受态，将含有该启动子的质粒DNA作为未扩增的连接产物直接导入戈登氏链球菌。该质粒促进了戈登氏链球菌大量分泌异源产物。通过评估两种质粒构建体分泌的异源产物水平，可以评估两个天然启动子的相对强度。
