REGULATED EXPRESSION OF HETEROLOGOUS GENES IN BACILLUS-SUBTILIS USING THE TN10 ENCODED TET REGULATORY ELEMENTS
REGULATED EXPRESSION OF HETEROLOGOUS GENES IN BACILLUS-SUBTILIS USING THE TN10 ENCODED TET REGULATORY ELEMENTS
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DOI：
10.1007/bf00604933
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发表时间：
1990-09-01
影响因子：
5
通讯作者：
HILLEN, W
中科院分区：
文献类型：
--
作者：
GEISSENDORFER, M;HILLEN, W
从Tn10中获得的大肠杆菌衍生的tet调控元件已被用于构建载体，使外源基因在枯草芽孢杆菌中得到可调控、可诱导、高水平的表达。野生型tet启动子在枯草芽孢杆菌中没有活性，而一个具有改进的启动子一致序列和上游聚a块的合成突变tet序列在枯草芽孢杆菌中显示出活性。一个指示基因cat的表达可被亚致死量的四环素诱导，这表明Tet抑制蛋白和Tet操作序列是功能性的。然而，在这个结构中，诱导性和最大表达是不充分的。为了提高这些特性，在枯草芽孢杆菌衍生的强羟基启动子的-35和-10盒之间放置了一个tet操作符序列。在有tetR基因存在的情况下，该结构的诱导率约为100倍，启动子强度高，但有一定的基础表达。通过在下游放置第二个tet操作符，以降低诱导性为代价，无法检测到基础表达，从而避免了这种情况。采用单tet操作符的系统，获得了巨芽孢杆菌中葡萄糖脱氢酶的高水平诱导表达，并获得了与大肠杆菌相同水平的人单链尿激酶样纤溶酶原激活物的诱导表达。与大肠杆菌不同的是，该产物在诱导后4小时内不会降解，本文描述的调节表达载体对于从枯草芽孢杆菌培养物中生产外源基因产物非常有用。
The Escherichia coli-derived tet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes in Bacillus subtilis. While the wild-type tet promoters are inactive in B. subtilis, a synthetic mutant tet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity in B. subtilis. The expression of an indicator cat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and the tet operator sequences are functional. However, the inducibility and maximal expression are not sufficient in this construct. To improve these properties a tet operator sequence was placed between the -35 and -10 boxes of the B. subtilis-derived very strong xyl promoter. In the presence of tetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression. This is avoided by placing a second tet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility. Using the system with a single tet operator inducible expression of glucose dehydrogenase from B. megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as in E. coli. Unlike in E. coli, the product was not degraded up to 4 h after induction regulated expression vector described here should be very useful for production of foreign gene products from B. subtilis cultures.