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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.

从蛋白质和基因水平分析了红色红球菌J1的高MR-(H-)和低MR-(L-)腈水合酶(NHase)。利用红球菌-大肠杆菌宿主载体(PK4)系统，H-NHase基因(NhhAB)在红球菌-大肠杆菌宿主系统ATCC12674中以活性形式表达，而在E.coli中不表达。NhhAB的5‘-上游区域是nhhAB在宿主中表达所必需的。在这个区域有四个开放阅读框(NHHC，nhhD，nhhE，nhhf)。NHHC和nhhD对nhhAB的表达均起正向调节作用。另一方面，L氨酶基因的5‘上游区(NhlAB)是酰胺依赖的nhlBA表达所必需的，该区域有两个开放阅读框(nhlD和nhlC)。在L-氨酶的形成过程中，nhlD和nhlC分别起着正向和负向的调节作用。另一方面，研究发现，依赖异戊腈的J1菌株的氰化酶(NITA)下游的1.4kb是依赖异戊腈的氰化酶合成所必需的。对该区域的序列分析表明，存在一个开放阅读框架(NitR)。NITA表达中转录正调控因子的NitR编码。NITA的转录起始点被定位到其翻译起始点上游26个碱基的C残基。