Streptomyces cholesterol oxidase, an enzyme on the first step of microbial catabolism of cholesterol, was overproduced in a secretory fashion in
Streptomyces lividans with a multi-copy shuttle vector. The gene coding for cholesterol oxidase (
choA) was modified and overexpressed in
Escherichia coli. The modified
choA gene was also used to develop promoter-probe vectors to use in enteric bacteria. Expression of the
choA gene in lactic acid bacteria might facilitate the degradation of cholesterol in dairy food. The
Streptomyces cholesterol oxidase was unexpectedly found to constitute a novel class of insecticidal proteins that may be useful for controlling pests that are resistant to
Bacillus thuringiensis toxins.
Furthermore, the
Arthrobacter simplex gene (
ksdD) encoding 3-ketosteroid-Δ
1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned and overexpressed in
S. lividans using a multi-copy shuttle vector, leading to an about 100-fold overproduction of the enzyme compared to the natural producer. Nucleotide sequence analysis revealed that
ksdD is clustered with two more genes possibly involved in steroid catabolism. Upstream of
ksdD, a gene (
ksdR) encoding a hypothetical regulatory protein that shows similarities to several regulators was found. Adjoining downstream to
ksdD in an apparent translational coupling is a gene (
ksdI) coding for a protein that would display strong similarities to 3-ketosteroid-Δ
5-isomerase. Further sequence analysis downstream of
ksdI revealed three ORFs. The deduced protein product of one of these showed significant similarities to the KsdD protein. Thus, genes involved in steroid metabolism seem to be clustered in bacteria.
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