1990 Fiscal Year Final Research Report Summary
Genetic engineering for Cholesterol Transformation System in Streptomyces
| Project/Area Number |
01480036
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵工学
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| Research Institution | Hiroshima University |
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Principal Investigator |
MUROOKA Yosikatu Hiroshima Univ. Faculty of Engineering, Prof., 工学部, 教授 (60029882)
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| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Mituo Hiroshima Univ., Facul. of Engineering, Resh. Assoc., 工学部, 助手 (40220347)
ONO Kazuhisa Hiroshima Univ., Facul. of Engineering, Assoc. Prof., 工学部, 助教授 (10144883)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Streptomyces / Cholesterol oxidase / Transformation of cholesterol / Genetic engineering / Cytochrome P-450 |
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| Research Abstract |
Streptomyces species produce a large variety of medically important metabolites and extracellular enzymes, in particular cholesterol oxidase. Cholesterol oxidase catalyzes the oxidation of cholesterol to 4-cholesten-3-one, with the reduction of oxygen to hydrogen peroxide. The enzyme is commonly used for the enzymatic transformation of cholesterol and is alo used for quantitation of levels of cholesterol in clinical specimens.
Recently, we cloned the gene involved in the production of cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO. Cells of Streptomyces lividans carrying a plasmid that includes the choA gene overproduce extracellular Cholesterol oxidase when cultured in appropriate medium.
We sequenced the complate cholesterol oxidase gene. The sequence of the choA gene revealed that the gene of cholesterol oxidase starts from a GTG initiation codon and terminates at a TAA termination codon. In the 3'-flanking region of choA, a long palin dromic sequence, which may act as
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a transcription terminator, was found. The gene encodes a mature cholesterol oxidase of 504 amino acids with a caluculated Mr of 54,913.
The nucleotide sequence of the promoter region of the gene for choA was determined. We found an open reading frame (choP) that has a high degree of similarity to the sequence of the gene for cytochrome P-450s from heman and Pseudomonas species. We determined the product of the choP gene was cytochrome P-450-like protein.
We have cloned the chromosomal DNA, which is adjoined upstream and down stream of the choP-choA operon, by guided genomic walking method developed by us. These clones may be involved in cholesterol metabolism.
During the study on the decomposition of cholesterol and secretion of heterologous gene products in Streptomyces, we found secretory overproduction of cholesterol oxidase in Streptomyces lividans by a recombinant multi-copy shuttle vector, the results of which are potential useful for production of heterologous gene product extracellularly bu using Streptomyces host-vector system. Less
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