| Daunorubicin and doxorubicin are clinically important anthracyclines and are primarily used as first line chemotherapeutic agents in treatment of a variety of neoplasias and adult myelogenous leukemia. Doxorubicin, as the C-14 hydroxylated derivative of daunorubicin, has a broader spectrum of anti-tumor activity, a lower toxicity and fewer side-effects compared with daunorubicin. Nowadays doxorubicin is produced through chemical semi-synthesis initiated from daunorubicin, which is complicated and has low conversion efficiency with bad environment pollution. In this research work, the technology of microbial conversion was studied in order to establish an environment friendly method for the production of doxorubicin. A doxA gene encoding Daunorubicin C-14 hydroxylase was cloned from a daunorubicin-producing strain Streptomyces coeruleorubidus SIPI 1482. Some plasmids for the expression of doxA gene were constructed and transformed into S. lividans TK24 to get engineered strains for daunorubicin conversion. Conditions for the expression of doxA gene and the conversion of daunorubicin into doxorubicin in these strains were investigated. All results are described as follows:A DNA fragment containing doxA gene of approximate 1.4 kb was amplified for the fist time from SIPI 1482 strain by PCR method, but not from S. peucetius SIPI 40141. Sequence alignment indicated that the cloned doxA gene from SIPI 1482 strain was identical with that from S. sp. C5 strain, and had a 93.4 % homology with the published doxA gene from S. peucetius ATCC 29050.A novel high-copied expression vector, plasmid pYG504 was constructed for the cloning and expression of heterologous gene in Streptomyces, in which some unique restriction enzyme sites for the insertion of heterlogous genes are flanked by a promoter of melanin gene (encoding tyrosinase gene) and a fd terminator and a Shine-Dalgarno sequence is also located in the upstream of SphI site.Five plasmids for the expression of doxA gene, which were designated as pYG57, pYG502, pYG503, pYG505 and pYG506 were constructed so that the cloned doxA gene were under the control of either promoter of melanin gene or erythromycin resistant gene or their tandem promoters, and a fd terminator downstream. These plasmids were introduced into S. lividans TK24, respectively and five genetic engineering strains were constructed.The SDS-PAGE electrophoresis indicated that all engineered strains containing different expression plasmid could apparently express a 45 kD band specific for daunorubicin C-14 hydroxylase. The expression level of doxA gene was much higher when the doxA gene was under the control of promoter PermE. However, it was found that fd terminator had few effect on the expression of doxA gene. The research work involving the expression manner of doxA gene in S. lividans TK24 with plasmid pYG57 implied that the promoter PermE may control its expression constitutively, the time for maximum expression emerged from 48 to 60 h after inoculation and cultivation and the expression level was kept relatively stable, furthermore, the recombinant hydroxylase existed mostly in mycelium cell but little in broth.The bioconversion experiments of the engineered strains concluded they can convert daunorubicin into doxorubicin and an unknown product and the S. lividans TK24 bearing plasmid pYG505 strain has a relatively stronger ability to convert daunorubicin into doxorubicin among five strains. Identified by means of ultraviolet and visible spectra analysis, LC/MS analysis and 'H-NMR analysis, the converted products of daunorubicin by the engineered strain consisted of doxorubicin and 13-dihydrodaunorubicin. The former was produced by the intracellular recombinant hydroxylase and the latter by the host C-13 ketoreductase.The bioconversion conditions of daunorubicin by S. lividans TK24 with plasmid pYG57 were optimized. According to these optimized conditions, the conversion efficiency of daunorubicin is as high as 74.4 %, which is 3.1 times of that of the control before optimization, a...
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