Study On The DnrV And DnrX Gene In Daunorubicin Biosynthesis

Daunorubicin(DNR) and doxorubicin(DXR), two clinically important anthracyclines, are first-line chemotherapeutic agents in treatment of a variety of neoplasias and myelogenous leukemia. Doxorubicin, the C-14 hydroxylated derivative of daunorubicin, has a broader spectrum of anti-tumor activity, lower toxicity and fewer side-effects as compared with daunorubicin. To date, daunorubicin and doxorubicin are industrially produced by Streptomyces fermentation and chemical semi-synthesis, respectively.The exact function of dnrV in daunorubicin biosynthesis pathway has not yet been clarified though it is postulated that dnrV might play an important role in transformation of 13-deoxydaunorubicin into DNR, as well as DXR, by DoxA. DnrX, encoded by dnrX gene, catalyze DNR to acid-sensitive baumycin-like substances. In this study, dnrV and doxA gene were cloned and expressed in wild-type daunorubicin-producing strain Streptomyces coeruleorubidus SEPI-1482 and dnrX gene in chromosome of SIPI-1482 was knocked out by single crossover homologous recombination. The results are as follows:A DNA fragment containing 828bp full length dnrV gene was amplified by PCR from genomic DNA of Streptomyces coeruleorubidus SIPI-1482. Sequence alignment indicated that the cloned dnrV gene from SIPI-1482 had a 94.4 % homology with the published dnrV gene from S. peucetius ATCC 29050 and 100% homology with orfA from S. sp. C5 which are other two daunomycin producers. dnrV gene was subcloned into E.coli expression vectors to construct pYG980 and pYG981. After IPTG induction, an obvious band was observed by SDS-PAGE electrophoresis and was further confirmed by Western blotting. dnrV, with/out doxA gene, were cloned into two Streptomyces expression vector pYG505 and pYG907 to construct pYG974, pYG975, pYG987 and pYG988 , in which dnrV and doxA transcription were under the regulation of PermE or PsnpA. Fermentation results showed that introduction of dnrV gene alone into SIPI-1482 could increase the yield of daunorubicin to some extent, while introduction of dnrV together with doxA had the most significant effect on the production of daunorubicin.A partial dnrX gene fragment in length of 1080bp was also amplified by PCR from SIPI-1482 genomic DNA. Sequence alignment indicated that this fragment had a 94.8 % homology with the published dnrX gene from S. peucetius ATCC 29050. A 985bp DNA fragment was subcloned from this partial dnrX into an E.coli vector pYG813 to construct the disruption plasmid pYG963. After demethylation and alkaline denaturation, pYG963 were...