Research On Doxorubicin Biotransformation And Disruption Of DauU Gene

Doxorubicin, a C-14 hydroxylated derivative of daunorubicin, is primarily used as first line chemotherapeutic agent in treatment of a variety of neoplasias and adult myelogenous leukemia. Compared with daunorubicin, doxorubicin has a broader spectrum of anti-tumor activity, lower toxicity and fewer side-effects. The world market of doxorubicin exceeded two hundreds of millions of dollars. Currently, doxorubicin is produced by semi-synthesis which is initiated from daunorubicin. The relative low productivity and high pollution make it possible to study on enzymatic biotransformation and fermentation production of doxorubicin, which is the essential way to friendly environment and technology advancement. In this research, two methods were studied for the production of doxorubicin. One was optimization on microbial conversion system by means of choosing different promoters and/or introducing DNR/DXR resistance gene into the host to increase the yield of doxorubicin. The other was disruption of dauU gene responsible for a shunt pathway in the daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482 so as to explore the possibility of direct production of doxorubicin by fermentation.Results are described as follows:doxA gene coding for daunorubicin C-14 hydroxylase was amplified by PCR from Streptomyces coeruleorubidus SIPI-1482 genomic DNA and was cloned into a E.coli expression vector pET32a. The expressed protein was confirmed by western blotting and it showed that most of the fusion protein was inclusion body. Thus, the process of renaturation including washing, dissolving, refolding and purification of inclusion body were studied and a single band of purified protein was obtained. In vitro biotransformation of doxorubicin was studied in E.coli by adding co-factor regeneration system. The result showed E.coli is not a suitable host due to uncertain impact factor in the system.doxA gene containing a strong ribosome binding site GGAGG was PCR amplified from SIPI-1482 strain. A gene encoding SnpR and snpA promoter sequence were also amplified from SIPI-1482 strain. Sequence alignment indicated that the cloned doxA gene and the snpA promoter from SIPI-1482 were identity with that from S. sp. C5, while snpR from SIPI-1482 had a 99.4% DNA homology and 96.5% amino acid homology with the published sequence from S. sp. C5, respectively.Three novel expression plasmids pYG908, pYG915, pYG927 were constructed for the cloning and expression of doxA in Streptomyces lividans so that the transcription of doxA was...