Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853.
Appl Environ Microbiol. 1987 Jul;53(7):1470-5. doi: 10.1128/aem.53.7.1470-1475.1987.
A cellulase gene from Thermomonospora fusca coding for endocellulase E(5) was introduced into Streptomyces lividans by using shuttle plasmids that can replicate in either S. lividans or Escherichia coli. Plasmid DNA isolated from E. coli was used to transform S. lividans, selecting for thiostrepton resistance. The transformants expressed and excreted the endocellulase, but the ability to produce the endocellulase was unstable. This instability was shown to result from deletion of the endocellulase gene from the plasmid. Plasmid DNA prepared from a culture in which plasmid modification had occurred was used to transform E. coli, selecting for Amp cells, and all of the transformants were cellulase positive, showing that pBR322 and T. fusca DNA were deleted together. When a plasmid was constructed containing only T. fusca DNA in plasmid pIJ702, the transformants were more stable, and the level of endocellulase activity produced in the culture supernatant after growth on 0.2% glucose was close to the level produced by T. fusca cultures grown on 0.2% cellulose. About 50% of the total protein in the culture supernatant of the S. lividans transformant was endocellulase E(5). The enzyme produced by the S. lividans transformant was identical to pure T. fusca E(5) in its electrophoretic mobility and was completely inhibited by antiserum to E(5). Shuttle plasmids containing the E(5) gene that could replicate in Bacillus subtilis and E. coli were also constructed and used to transform B. subtilis. Again there was extensive deletion of the plasmid DNA during transformation and growth in B. subtilis. There was no evidence of E(5) activity, even in those B. subtilis transformants that retained the E(5) gene.
从嗜热真菌Thermomonospora fusca 中分离到一个纤维素酶基因，该基因编码内切纤维素酶 E(5)。通过使用既能在链霉菌属 lividans 中复制又能在大肠杆菌中复制的穿梭质粒，将该基因导入链霉菌属 lividans。从大肠杆菌中分离出的质粒 DNA 被用来转化链霉菌属 lividans，选择硫链丝菌素抗性。转化子表达并分泌内切纤维素酶，但产生内切纤维素酶的能力不稳定。这种不稳定性被证明是由于质粒上内切纤维素酶基因的缺失。从发生质粒修饰的培养物中制备的质粒 DNA 用于转化大肠杆菌，选择氨苄青霉素抗性细胞，所有转化子均为纤维素酶阳性，表明 pBR322 和 T. fusca DNA 一起缺失。当构建的质粒仅包含质粒 pIJ702 中的 T. fusca DNA 时，转化子更加稳定，在 0.2%葡萄糖上生长后培养上清液中的内切纤维素酶活性接近在 0.2%纤维素上生长的 T. fusca 培养物产生的水平。在链霉菌属 lividans 转化子的培养上清液中，约 50%的总蛋白为内切纤维素酶 E(5)。由链霉菌属 lividans 转化子产生的酶在电泳迁移率上与纯 T. fusca E(5)相同，且完全被抗 E(5) 血清抑制。还构建了可在枯草芽孢杆菌和大肠杆菌中复制的含 E(5)基因的穿梭质粒，并用于转化枯草芽孢杆菌。在转化和在枯草芽孢杆菌中生长期间，质粒 DNA 也发生了广泛缺失。甚至在那些保留 E(5)基因的枯草芽孢杆菌转化子中，也没有 E(5)活性的证据。
