1996 Fiscal Year Final Research Report Summary
Development of novel expression vectors utilizing gene promoters involved in nitrile metabolism
| Project/Area Number |
07556073
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KOBAYASHI Michihiko Kyoto Univ.Agr.Senior Lecturer, 農学部, 講師 (70221976)
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| Co-Investigator(Kenkyū-buntansha) |
KATAOKA Michihiko Kyoto Univ.Assist., 農学部, 助手 (90252494)
SHIMIZU Sakayu Kyoto Univ.Agr.Prof., 農学部, 教授 (70093250)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Nitrile hydratase / promoter / Gene / Induction / Expression / Amide |
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| Research Abstract |
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.
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