Biosynthesis of doxorubicin

Doxorubicin (DXR) is a 14-hydroxylated version of daunorubicin, the immediate precursor of DXR in its biosynthetic pathway. Daunorubicin is more abundantly found as a natural product because it is produced by a number of different wild type strains of streptomyces. In contrast, only one known non-wild type species, streptomyces peucetius subspecies cesius ATCC 27952, was initially found to be capable of producing the more widely used doxorubicin. This strain was created by Arcamone et al. in 1969 by mutating a strain producing daunorubicin, but not DXR, at least in detectable quantities. Subsequently, Hutchinson's group showed that under special environmental conditions, or by the introduction of genetic modifications, other strains of streptomyces can produce doxorubicin. His group has also cloned many of the genes required for DXR production, although not all of them have been fully characterized. In 1996, Strohl's group discovered, isolated and characterized dox A, the gene encoding the enzyme that converts daunorubicin into DXR. By 1999, they produced recombinant Dox A, a Cytochrome P450 oxidase, and found that it catalyzes multiple steps in DXR biosynthesis, including steps leading to daunorubicin. This was significant because it became clear that all daunorubicin producing strains have the necessary genes to produce DXR, the much more therapeutically important of the two. Hutchinson's group went on to develop methods to improve the yield of DXR, from the fermentation process used in its commercial production, not only by introducing Dox A encoding plasmids, but also by introducing mutations to deactivate enzymes that shunt DXR precursors to less useful products, for example baumycin-like glycosides. Some triple mutants, that also over-expressed Dox A, were able to double the yield of DXR. This is of more than academic interest because at that time DXR cost about $1.37 million per kg and current production in 1999 was 225 kg per annum. More efficient production techniques have brought the price down to $1.1 million per kg for the non-liposomal formulation. Although DXR can be produced semi-synthetically from daunorubicin, the process involves electrophilic bromination and multiple steps and the yield is poor. Since daunorubicin is produced by fermentation, it would be ideal if the bacteria could complete DXR synthesis more effectively. Doxorubicin (DXR) is a 14-hydroxylated version of daunorubicin, the immediate precursor of DXR in its biosynthetic pathway. Daunorubicin is more abundantly found as a natural product because it is produced by a number of different wild type strains of streptomyces. In contrast, only one known non-wild type species, streptomyces peucetius subspecies cesius ATCC 27952, was initially found to be capable of producing the more widely used doxorubicin. This strain was created by Arcamone et al. in 1969 by mutating a strain producing daunorubicin, but not DXR, at least in detectable quantities. Subsequently, Hutchinson's group showed that under special environmental conditions, or by the introduction of genetic modifications, other strains of streptomyces can produce doxorubicin. His group has also cloned many of the genes required for DXR production, although not all of them have been fully characterized. In 1996, Strohl's group discovered, isolated and characterized dox A, the gene encoding the enzyme that converts daunorubicin into DXR. By 1999, they produced recombinant Dox A, a Cytochrome P450 oxidase, and found that it catalyzes multiple steps in DXR biosynthesis, including steps leading to daunorubicin. This was significant because it became clear that all daunorubicin producing strains have the necessary genes to produce DXR, the much more therapeutically important of the two. Hutchinson's group went on to develop methods to improve the yield of DXR, from the fermentation process used in its commercial production, not only by introducing Dox A encoding plasmids, but also by introducing mutations to deactivate enzymes that shunt DXR precursors to less useful products, for example baumycin-like glycosides. Some triple mutants, that also over-expressed Dox A, were able to double the yield of DXR. This is of more than academic interest because at that time DXR cost about $1.37 million per kg and current production in 1999 was 225 kg per annum. More efficient production techniques have brought the price down to $1.1 million per kg for the non-liposomal formulation. Although DXR can be produced semi-synthetically from daunorubicin, the process involves electrophilic bromination and multiple steps and the yield is poor. Since daunorubicin is produced by fermentation, it would be ideal if the bacteria could complete DXR synthesis more effectively. The anthracycline skeleton of doxorubicin (DXR) is produced by a Type II polyketide synthase (PKS) in streptomyces peucetius. First, a 21-carbon decaketide chain (Fig 1. (1)) is synthesized from a single 3-carbon propionyl group from propionyl-CoA, and 9 2-carbon units derived from 9 sequential (iterative) decarboxylative condensations of malonyl-CoA. Each malonyl-CoA unit contributes a 2-carbon ketide unit to the growing polyketide chain. Each addition is catalyzed by the 'minimal PKS' consisting of an acyl carrier protein (ACP), a ketosynthase (KS)/chain length factor (CLF) heterodimer and a malonyl-Coa:ACP acyltransferase(MAT). (refer to top of Figure 10. This process is very similar to fatty acid synthesis, by fatty acid synthases and to Type I polyketide synthesis. But, in contrast to fatty acid synthesis, the keto groups of the growing polyketide chain are not modified during chain elongation and they are not usually fully reduced. In contrast to Type I PKS systems, the synthetic enzymes (KS, CLF, ACP and AT) are not attached covalently to each other, and may not even remain associated during each step of the polyketide chain synthesis. After the 21-carbon decaketide chain of DXR is completed, successive modifications are made to eventually produce a tetracyclic anthracycline aglycone(without glycoside attached). The daunosamine amino sugar, activated by addition of Thiamine diphosphateTDP, is created in another series of reactions. It is joined to the anthracycline aglycone and further modifications are done to produce first daunorubicin then DXR.There are at least 3 gene clusters important to DXR biosynthesis: dps genes which specify the enzymes required for the linear polyketide chain synthesis and its first cyclizations, the dnr cluster is responsible for the remaining modifications of the anthracycline structure and the dnm genes involved in the amino sugar, daunosamine, synthesis. Additionally, there is a set of 'self resistance' genes to reduce the toxic impact of the anthracycline on the producing organism. One mechanism is a membrane pump that causes efflux of the DXR out of the cell (drr loci). Since these complex molecules are only advantageous under specific conditions, and require a lot of energy to produce, their synthesis is tightly regulated. Doxorubicin is synthesized by a specialized polyketide synthase. The initial event in DXR synthesis is the selection of the propionyl-CoA starter unit and its decarboxylative addition to a two carbon ketide unit, derived from malonyl-CoA to produce the five carbon B-ketovaleryl ACP. The five carbon diketide is delivered by the ACP to the cysteine sulfhydryl group at the KS active site, by thioester exchange, and the ACP is released from the chain. The free ACP picks up another malonate group from malonyl-CoA, also by thioester exchange, with release of the CoA. The ACP brings the new malonate to the active site of the KS where is it decarboxylated, possibly with the help of the CLF subunit, and joined to produce a 7 carbon triketide, now anchored to the ACP (see top of Figure 1). Again the ACP hands the chain off to the KS subunit and the process is repeated iteratively until the decaketide is completed. In most Type II systems the initiating event is delivery by ACP of an acetate unit, derived from acetyl-CoA, to the active site of the ketosynthase (KS) subunit of the KS/CLF heterodimer. The default mode for Type II PKS systems is the incorporation of acetate as the primer unit, and that holds true for the DXR 'minimal PKS'. In other words, the action of KS/CLF/ACP (Dps A, B and G) from this system will not produce 21-carbon decaketides, but 20-carbon decaketides instead, because acetate is the “preferred” starter. The process of specifying propionate is not completely understood, but it is clear that it depends on an additional protein, Dps C, which may be acting as a ketosynthase or acyltransferase selective for propionyl-CoA, and possibly Dps D makes a contribution. A dedicated MAT has been found to be dispensable for polyketide production under in vitro conditions. The PKS may 'borrow' the MAT from its own fatty acid synthase and this may be the primary way ACP receives its malonate group in DXR biosynthesis. Additionally, there is excellent evidence that 'self-malonylation' is an inherent characteristic of Type II ACPs. In summary, a given Type II PKS may provide its own MAT (s), it may borrow one from FAS, or its ACP may “self-malonylate”.