Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans.
We examined the expression of choB, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Escherichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and choB was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when ChoB protein was fused with the NH2-terminal portion of LacZ protein. In contrast, choB with more than 256-bp of the 5'-flanking region was efficiently expressed in S. lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of choB exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH2-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
T
T
Ohta
K
K
Fujishiro
K
K
Yamaguchi
T
T
Uwajima
K
K
Aisaka
M
M
Hasegawa
D001709:Biotechnology; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics*; D002789:Cholesterol Oxidase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013302:Streptomyces / Q000235:genetics
D004269:DNA, Bacterial; D002789:Cholesterol Oxidase