Motamedi H, Shafiee A, Cai S J
Merck Research Laboratories, Rahway, NJ 07065, USA.
Gene. 1995 Jul 4;160(1):25-31. doi: 10.1016/0378-1119(95)00191-8.
Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.
构建了用于链霉菌中基因异源表达的整合表达载体。这些载体由一个强组成型启动子PE、一个合成核糖体结合位点、ATG起始密码子、多克隆位点、转录终止子和潮霉素抗性编码基因组成。载体还包含一个用于在大肠杆菌中繁殖的ColE1复制子和一个广宿主范围的链霉菌整合元件——小环，以指导载体在小环附着位点插入链霉菌基因组。HyR转化体在无药物选择的情况下是稳定的。通过将IncP质粒RK2的转移起始点oriT整合到这些载体中，构建了接合衍生物，并证明了从合适的大肠杆菌供体到变铅青链霉菌（Sl）的接合转移。还描述了这些载体的可能用于基因破坏以及互补的衍生物。在Sl中与载体整合拷贝共存的基于小环构建的载体的复制形式也存在，且没有任何明显的稳定性问题。通过在Sl中表达编码31 - O - 甲基转移酶的基因证明了这些载体的实用性，该酶参与免疫抑制药物FK506 31位的甲基化。
