Hamady Zaed Z R, Farrar Mark D, Whitehead Terence R, Holland Keith T, Lodge J Peter A, Carding Simon R
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Fermentation Biotechnology Research, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
Microbiology (Reading). 2008 Oct;154(Pt 10):3165-3174. doi: 10.1099/mic.0.2008/019109-0.
The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.
利用基因工程改造的细菌将生物活性分子直接递送至肠道，已成为一个越来越有吸引力的研究领域。由于治疗性分子的生产调控以及肠道定植方面的挑战，促使我们研究卵形拟杆菌用于这些分子的生产，这是因为它具有在结肠定植的能力以及木聚糖利用特性。在此，我们鉴定出了假定的木聚糖酶启动子。通过5'RACE分析确定了相应mRNA的5'区域，并在ATG起始密码子上游216 bp处鉴定出转录起始位点。假定的木聚糖酶启动子受木聚糖以剂量和时间依赖性方式调控，并受葡萄糖抑制。该启动子随后在作为单拷贝整合到卵形拟杆菌染色体后，用于指导编码人肠道三叶因子（TFF-3）的基因的可控表达。所得菌株在木聚糖存在的情况下产生具有生物活性的TFF-3。这些发现鉴定出了卵形拟杆菌木聚糖酶操纵子启动子，并表明它可用于在卵形拟杆菌中指导异源真核基因的木聚糖诱导型表达。
