Yamamura Ei-Tora
Technical Department, Kyowa Pharma Chemical Co., Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, Japan.
J Biosci Bioeng. 2018 Jun;125(6):625-631. doi: 10.1016/j.jbiosc.2018.01.001. Epub 2018 Feb 15.
With the aim of being able to co-express multiple genes, I searched for novel compatible plasmids and isolated two plasmid species, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400. Sequencing analysis revealed that the pRET1100 plasmid is a double-stranded DNA molecule of 5444 bp with two possible open reading frames (ORFs), repT and div, and three minor ORFs. The cryptic replication protein, RepT, is not highly homologous to those from other plasmids that have been reported. The Rhodococcus-Escherichia coli shuttle vector pRET1102 was transformed into R. erythropolis JCM2895 harboring the pRE2895 plasmid. The recombinant R. erythropolis JCM2895 harbored two plasmid species. These results suggest that plasmid derivatives of pRET1100 and pRE2895 are fully compatible in R. erythropolis. I determined the minimum region of pRET1100 required for autonomous replication in R. erythropolis and constructed a high-copy plasmid, pRET1129, in R. erythropolis.
为了能够共表达多个基因，我寻找了新型兼容质粒，并从红平红球菌IAM1400中分离出两种质粒，即pRET1100和pRET1200。测序分析表明，pRET1100质粒是一个5444 bp的双链DNA分子，有两个可能的开放阅读框（ORF），即repT和div，以及三个次要的ORF。隐蔽性复制蛋白RepT与已报道的其他质粒的复制蛋白同源性不高。将红球菌-大肠杆菌穿梭载体pRET1102转化到携带pRE2895质粒的红平红球菌JCM2895中。重组红平红球菌JCM2895含有两种质粒。这些结果表明，pRET1100和pRE2895的质粒衍生物在红平红球菌中完全兼容。我确定了pRET1100在红平红球菌中自主复制所需的最小区域，并在红平红球菌中构建了一个高拷贝质粒pRET1129。
