Development of novel expression vectors utilizing gene promoters involved in nitrile metabolism

利用参与腈代谢的基因启动子开发新型表达载体

基本信息

批准号：
07556073
负责人：
KOBAYASHI Michihiko
金额：
$ 4.1万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

High-Mr-(H-) and low-Mr-(L-) nitrile hydratases (NHases) in Rhodococcus rhodochrous J1 were analyzed at both protein and gene levels. The H-NHase gene (nhhAB) was expressed as an active form in R.rhodochrous ATCC12674 as a host using a Rhodococcus-Escherichia coli host-vector (pK4) system, whereas it was not in E.coli.SDS/PAGE of the R.rhodochrous transformant cell-extracts has shown that the amount of alpha-and beta-subunits of H-NHase is about 50% of the total soluble protein. The 5'-upstream region of nhhAB was found to be required for nhhAB expression with the host. In this region, there are four open reading frames (nhhC,nhhD,nhhE,nhhF). Both nhhC and nhhD play positive regulatory roles in nhhAB expression. On the other hand, the 5'-upstream region from the L-NHase gene (nhlAB) was required for the amide-dependent expression of nhlBA.There are two open reading frames (nhlD and nhlC) in this region. In the process of the L-NHase formation, nhlD and nhlC play negative and positive regulatory roles, respectively.On the other hand, the 1.4 kb downstream from a nitrilase (nitA) of R.rhodochrous J1 was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis. Sequence analysis of this region revealed the existence of an open reading frame (nitR). The nitR codes for a transcriptional positive regulator in nitA expression. The transcriptional start site for nitA was mapped to a C residue located 26 bp upsteam of its translational start site.

在蛋白质和基因水平上对紫红红球菌 J1 中的高 Mr-(H-) 和低 Mr-(L-) 腈水合酶 (NHase) 进行了分析。使用红球菌-大肠杆菌宿主载体 (pK4) 系统，H-NHase 基因 (nhhAB) 在作为宿主的 R.rhodochrous ATCC12674 中以活性形式表达，而它不在大肠杆菌中表达。SDS/PAGE R.rhodochrous 转化体细胞提取物显示，H-NHase 的 α 和 β 亚基含量约为 50%。总可溶性蛋白质。发现 nhhAB 的 5'-上游区域是 nhhAB 在宿主中表达所必需的。在该区域中，有四个开放阅读框（nhhC，nhhD，nhhE，nhhF）。 nhhC 和 nhhD 在 nhhAB 表达中发挥正向调节作用。另一方面，L-NHase 基因（nhlAB）的 5' 上游区域是 nhlBA 酰胺依赖性表达所必需的。该区域有两个开放阅读框（nhlD 和 nhlC）。在L-NHase形成过程中，nhlD和nhlC分别发挥负向和正向调节作用。另一方面，R.rhodochrous J1的腈水解酶(nitA)下游1.4 kb被发现是异戊腈所必需的。腈水解酶合成的依赖性诱导。该区域的序列分析揭示了开放阅读框（nitR）的存在。 nitR 编码 nitA 表达中的转录正调节因子。 nitA 的转录起始位点被映射到位于其翻译起始位点上游 26 bp 的 C 残基。