Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida.
Molecular cloning of genes encoding branched-chain keto acid dehydrogenase of Pseudomonas putida.
复制标题
恶臭假单胞菌支链酮酸脱氢酶编码基因的分子克隆。
DOI：
10.1128/jb.169.4.1619-1625.1987
复制
发表时间：
1987
影响因子：
3.2
通讯作者：
Sokatch,JR
中科院分区：
文献类型：
--
作者：
Sykes,PJ;Burns,G;Menard,J;Hatter,K;Sokatch,JR
我们通过克隆到广宿主范围载体 pKT230 中，将支链酮酸脱氢酶多酶复合物的各个亚基的结构基因克隆到恶臭假单胞菌染色体 DNA 的 7.8 KB EcoRI-SstI 限制性片段上。通过恶臭假单胞菌支链酮酸脱氢酶突变体的互补实现了在缬氨酸-异亮氨酸琼脂上生长的直接选择系统。重组质粒 pSS1-1 将野生型恶臭假单胞菌中支链酮酸脱氢酶的表达量提高了五倍。该复合物在恶臭假单胞菌(pSS1-1)中组成型表达，但在大肠杆菌HB101(pSS1-1)中可被高缬氨酸诱导表达。用 pSS1-1 转化的大肠杆菌小细胞产生的三种多肽与纯化复合物的四种多肽不匹配。为了解决这个问题，我们将 pSS1-1 中的恶臭假单胞菌 DNA 插入到 pUC18 和 pUC19 中。 pUC 衍生的质粒在大肠杆菌转录-翻译系统中用作 DNA 模板。从pUC18衍生的质粒中产生了四种具有正确分子量的多肽，表明结构基因已被克隆。由于 pUC19 衍生质粒仅产生弱条带，因此转录方向已确定。通过限制性内切酶图谱定位支链酮酸脱氢酶所有结构基因的位置和顺序。
We cloned the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenase mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain keto acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed constitutively in P. putida(pSS1-1) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells transformed with pSS1-1 produced three polypeptides which did not match the four polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-1 into pUC18 and pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.