Methods of producing doxorubicin

#Daunomycin and doxorubicin are clinically important chemotherapeutic agents. Daunomycin is used primarily to treat adult myelogenous leukemia. Doxorubicin is widely used to treat a variety of neoplasias, making it the more valuable of the two anticancer drugs. The world wide market for doxorubicin is estimated to exceed $156 million. As of 1984, the wholesale price for doxorubicin was estimated to be $1,370,000 per kilogram.

#While daunomycin is synthesized by several species of Streptomyces, doxorubicin is biologically synthesized by only one strain, a mutant strain of Streptomyces peucetius, called S. peucetius subsp. caesius which is available from the American Type Culture Collection under Accession number 27952.

#The alternative in vitro laboratory synthesis of doxorubicin is difficult. The in vitro synthesis of doxorubicin is a process involving multiple steps and resulting in a poor yield, with a lack of stereospecificity in several of the synthetic steps, producing forms which are difficult to separate.

#FIG. 1 is a restriction map of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster which shows the position of doxA within the cluster. Abbreviations for restriction endonuclease sites are as follows: "B" represents BamHI; "Bg" represents BglII; "C"represents ClaI; "E" represents EcoRI; "K" represents KpnI; "P" represents PstI; "S" represents SstI; and "X" represents XhoI;

#FIG. 2 is a detailed restriction map of part of the daunomycin biosynthesis gene cluster from Streptomyces sp. strain C5. Abbreviations for restriction endonuclease sites are as follows: "B" represents BamHI; "Bg" represents BglII; "E" represents EcoRI; "K" represents KpnI; "P" represents PstI; "S" represents SstI; "Sp" represents SphI;

#FIG. 3A and 3B is a nucleotide sequence of the 3196 base pair KpnI- SstI DNA fragment from Streptomyces sp. strain C5 containing the doxA gene. The deduced amino acid sequence of the daunomycin C-14 hydroxylase is given below the nucleotide sequence. Potential ribosome binding sites, designated "rbs" are identified, as are significant restriction endonuclease sites. The sequences and deduced products of the 3' end of orfl, all of orfA, and the 5' end of dauI are also shown;

#A novel method for producing doxorubicin from daunomycin has been developed which employs genetically engineered host microorganisms that contain and express the gene doxA. Preferably, doxA is cloned into a plasmid which is then inserted into a host microorganism, preferably a bacterial host, more preferably Streptomyces, most preferably Streptomyces lividans to provide a transformed host. The doxA gene encodes daunomycin C-14 hydroxylase which catalyzes the hydroxylation of daunomycin and 13-dihydrodaunomycin at C-14 to form doxorubicin. Thus, expression of doxA in the transformed host using a plasmid which contains the doxA gene enables the transformed host to convert daunomycin to doxorubicin.

#Daunomycin is also known as daunorubicin; doxorubicin is also known as 14-hydroxydaunomycin and adriamycin. The structure of daunomycin is shown below: ##STR1##

#The structure of doxorubicin is shown below: ##STR2## Cloning and Analysis of the DoxA Gene