• KSBB Journal
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• v.23 no.1
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• pp.44-47
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• 2008

Anthracycline antibiotics doxorubicin (DXR) is clinically important cancer therapeutic agent produced by Streptomyces peucetius. DXR result by further metabolism of rhodomycin D (RHOD) and require a deoxy-sugar component for their biological activity. In this study, production of TDP-L-daunosamine and its attachment to ${\varepsilon}$-rhodomycinone (RHO) to generate RHOD has been achieved by bioconversion in Streptomyces venezuelae that bears eleven genes. S. peucetius seven genes (dnmUTJVZQS) were transformed by plasmid and S. venezuelae two genes desIII, IV and two more S. peucetius drrA, B genes were integrated into chromosomal DNA. To generate the feeding substrate RHO, 6L S. peucetius grown on agar plate was harvested, extracted with organic solvent and then purified using preparative HPLC. Recombinant S. venezuelae grown on agar plate containing RHO was harvested and its n-butanol soluble components were extracted. The glycosylated product of aromatic polyketide RHO using heterologous host S. venezuelae presents the minimal information for TDP-L-daunosamine biosynthesis and its attachment onto aglycone. Moreover, the structure of auxiliary protein, DnrQ, was predicted by fold recognition and homology modeling in this study. This is a general approach to further expand of new glycosides of antitumor anthracycline antibiotics.

• Microbiology and Biotechnology Letters
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• v.25 no.6
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• pp.580-585
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• 1997

We selected two mutants namely strain D5 and Nu23 by mutagenesis of anthracycline producing Streptomyces: the former is an $\varepsilon$-rhodomycinone overproducing mutant selected from Streptomyces sp. C5, a baumycin producer and the latter, a blocked mutant of early pathway for doxorubicin biosynthesis obtained from Streptomyces peucetius ATCC 27952, a doxorubicin producer. The mutant strain Nu23 does not produce anthracycline metabolites but retains the most of enzyme activities converting aklavinone to doxorubicin and the mutant strain D5 produced $\varepsilon$-rhodomycinone at a level of 150 $\mu$g/ml. These strains were grown separately in NDYE medium and each was mixed at day 3 by equal volume of culture broth but the quantity of doxorubicin produced was far below an estimation based on the level of $\varepsilon$-rhodomycinone normaly produced by the strain D5. On the other hand doxorubicin was reached at maximum level after 4 days in the mixed culture condition which was composed of culture broth of strain D5 grown for 6 day and that of strain Nu23 grown for 3 day. It was turned out that the growth of mutant strain D5 was inhibited by the accumulation of daunorubicin and doxorubicin in mixed culture broth, which cause the limitation of $\varepsilon$-rhodomycinone.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.595-595
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• 2003

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.15-22
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• 1998

The dnrF gene, responsible for conversion of aklavinone to $\varepsilon$-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin gene cluster of Streptomyces peucetius subsp. caesius ATCC 27952, close to drrAB, one of the anthracycline resistance genes. To characterize the enzymatic properties of the aklavinone 11-hydroxylase, the dnrF gene was overexpressed in Escherchia coli. The pET-22(+) plasmid which has the T7 promoter under the control of lacUV5 gene was used for the overexpression of the dnrF gene, and the recombinant plasmid pET213 that contains the dnrF gene linked to the T7 promoter of pET-22b(+) was introduced into the E. coli BL2l. When the expression of the dnrF gene was induced by IPTG at the final concentration of 1 mM, the induced protein could be detected in SDS-PAGE only in insoluble precipitate. The insoluble protein was electroeluted from the gel and used for the preparation of antiserum in mice. Various culture conditions were tested to maximize the expression of the aklavinone 11-hydroxylase in soluble form. The enzymatic activity was checked by the bioconversion experiment, and the protein was confirmed by the SDS-PAGE and the Western blot analysis. From the analysis of the data, it was concluded that the culture induced with IPTG at the final concentration of 0.02 mM at 37$^{\circ}C$ yielded the best productivity of active form of enzyme.