- Molecular Cloning and Analysis of the Gene for P-450 Hydroxylase from Pseudonocardia autotrophica IFO 12743
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Jung-Mee Kim , Younmie Jin , Chang-Gu Hyun , Jong-Hee Kim , Hong-Sub Lee , Dae-Kyung Kang , Dae-Jung Kang , Tae-Yong Kim , Joo-Won
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J. Microbiol. 2002;40(3):211-218.
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 Abstract
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A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D_3 into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450_Sca2 protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Streptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D_3 to its hydroxylated forms.
- Streptomyces griseus HH1, An A-factor Deficient Mutant, Produces Diminished Level of Trypsin and Increased Level of Metalloproteases
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Jung-Mee Kim , Soon-Kwang Hong
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J. Microbiol. 2000;38(3):160-168.
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Abstract
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A-factor is a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. To identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by S. griseus IFO 13350 and its A-factor deficient mutant strain, S. griseus HH1, as well as S. griseus HH1 transformed with the afsA gene were studied. In general, S. griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of S. griseus IFO 13350 was greatly enhanced more than twice compared with that of S. griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of S. griseus HH1 was greatly enhanced more than twice compared with that of S. griseus IFO 13350, and this observation was reversed in the presence of thiostreptone. However, S. griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between S. griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-aminopeptidase activity was 2 times higher in S. griseus HH1 than in strain IFO 13350. S. griseus HH1 harboring afsA showed a similar level of enzyme activity, however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morphological differentiation, the formation of aerial mycelium and spores was delayed by two or three days.
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