Gregg K, Cooper C L, Schafer D J, Sharpe H, Beard C E, Allen G, Xu J
Institute of Biotechnology, University of New England, Armidale, Australia.
Biotechnology (N Y). 1994 Dec;12(13):1361-5. doi: 10.1038/nbt1294-1361.
We isolated the fluoroacetate dehalogenase gene (H1), from Moraxella species strain B, and placed it under the transcriptional control of a 154 bp fragment of the erm gene promoter. The promoter/gene construct was attached to the Butyrivibrio fibrisolvens shuttle vector pBHerm, and the resulting dehalogenase expression plasmid (pBHf) was transferred to B. fibrisolvens OB156 by electroporation. The erm gene promoter directed expression of dehalogenase activity in both E. coli and B. fibrisolvens OB156. Cell-free lysates of the genetically modified OB156 defluorinated 10.6 nmol fluoroacetate/min/mg protein. Growing cultures of OB156 were able to detoxify fluoroacetate in the culture medium, at the rate of 9.9 nmol/min/mg. Plasmid pBHf was retained by 100% of OB156 cells after 500 generations of non-selective culture. The restriction pattern of pBHf remained unchanged after extensive non-selective growth and host bacteria continued to produce active dehalogenase. The construction of rumen bacteria that are able to detoxify an important natural poison supports the feasibility of using genetically modified rumen bacteria to aid animal production.
我们从莫拉克斯氏菌属菌株B中分离出氟乙酸脱卤酶基因（H1），并将其置于erm基因启动子154 bp片段的转录控制之下。将启动子/基因构建体连接到溶纤维丁酸弧菌穿梭载体pBHerm上，然后通过电穿孔将所得的脱卤酶表达质粒（pBHf）转移到溶纤维丁酸弧菌OB156中。erm基因启动子在大肠杆菌和溶纤维丁酸弧菌OB156中均指导脱卤酶活性的表达。转基因OB156的无细胞裂解物以10.6 nmol氟乙酸/分钟/毫克蛋白质的速度进行脱氟。生长的OB156培养物能够以9.9 nmol/分钟/毫克的速度对培养基中的氟乙酸进行解毒。经过500代非选择性培养后，100%的OB156细胞保留了质粒pBHf。经过大量非选择性生长后，pBHf的限制图谱保持不变，宿主细菌继续产生活性脱卤酶。构建能够解毒一种重要天然毒物的瘤胃细菌，支持了使用转基因瘤胃细菌辅助动物生产的可行性。
