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Daunorubicin (DNR) and its C-14-hydroxylated derivative doxorubicin are among the most important antitumor antibiotics in current use. Both antibiotics are produced by Streptomyces peucetius through a pathway involving a type II polyketide synthase (PKS), which executes the condensation of propionyl coenzyme A (CoA), as the starter unit, and nine malonyl-CoA extender units in the production of a 21-carbon decaketide (8, 11, 12). Subsequent intramolecular aldol condensations of the decaketide and C-12 oxidation form the 21-carbon tricyclic aromatic pigment aklanonic acid (AA) (8, 11), the first identifiable polyketide intermediate in the DNR pathway (Fig. 1B). When compared with other aromatic polyketides like tetracenomycin (Tcm) C, a 20-carbon polyketide produced by a type II PKS that uses acetyl-CoA as the starter unit (Fig. 1A), the DNR system stands out in terms of its use of a distinct starter unit and possibly also a PKS-dedicated malonyl-CoA:acyl carrier protein acyltransferase (MCAT) (8, 11).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   (A) Biosynthesis of Tcm F2 from malonyl-ACP and starter unit acetyl-CoA by the Tcm PKS enzymes. (Acetyl-CoA may not be the actual substrate since formation of the starter unit by decarboxylation of malonyl-ACP has been demonstrated in vitro [1, 6].) (B) Biosynthesis of AA and UWM5 from malonyl-ACP and starter unit propionyl-CoA by the daunorubicin PKS proteins. (C) Production of the 20-carbon polyketide SEK43 from malonyl-ACP and acetyl-CoA by Dps PKS enzymes without DpsC, DpsD, and DpsY.

Compared with different sets of type II PKS genes, the cluster of DNR PKS (dps) genes contains several unique features (Fig. 2) (8, 24). Directly downstream of the genes encoding the -ketoacyl:acyl carrier protein synthase (KS) subunits are two unique genes, dpsC and dpsD, rather than an acyl carrier protein (ACP) gene, which is found in all other PKS gene clusters (Fig. 2A and B). The ACP gene, dpsG, has an atypical position within the cluster, approximately 6.8 kb upstream of the genes encoding the KS subunits (8, 24) (Fig. 2C). In addition, sequence analysis has indicated that the dpsA and dpsB genes encode the KS subunits, dpsE encodes a ketoreductase, and dpsF and dpsY each encode a cyclase enzyme. Since the dpsC and dpsD genes are unique among PKS gene clusters, we are currently investigating the role of the gene products they encode in DNR biosynthesis. On the basis of a high sequence similarity, Grimm et al. (8) proposed that the function of DpsC was analogous to that of the Escherichia coli FabH enzyme, a -ketoacyl:ACP synthase III (KS III) and a component of a type II fatty acid synthase. KS III catalyzes the condensation of acetyl-CoA with malonyl-ACP in the production of the first intermediate, acetoacetyl-ACP, in E. coli fatty acid biosynthesis. DpsD has a high similarity (48 to 50%) with the MCAT enzymes of bacterial fatty acid synthases and contains the expected active-site signature sequence (xGHSxGE) with the essential Ser residue, suggesting that it functions as an acyltransferase (8). Either or both of the DpsC and DpsD enzymes could be responsible for the starter unit specification in DNR polyketide biosynthesis (8). Using heterologous expression of two plasmids carrying the dnrI regulatory gene and dpsABCEF, dpsG, and other PKS genes in Streptomyces lividans, Grimm et al. (8) isolated and identified AA. Since the two plasmids did not carry dpsD, this result suggested that the enzyme encoded by dpsD had no specific purpose in AA biosynthesis or that a related enzyme supplanted its function in the heterologous system (8).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Physical map of Tcm, actinorhodin (ACT), and DNR PKS gene clusters. The actI-ORF1, actI-ORF2, tcmK, tcmL, dpsA, and dpsB genes encode the KS subunits; the actI-ORF3, tcmM, and dpsG genes encode the ACPs; the actVII, actIV, tcmJ, tcmN, dpsF, and dpsY genes encode the polyketide cyclases; and the actIII and dpsE genes encode ketoreductases. The dnrG gene specifies an anthraquinol oxygenase, and the dpsC and dpsD genes encode KS III-like and acyltransferase enzymes, respectively. ORF, open reading frame; //, indication that the genes are separated by several kilobases.

On the other hand, the fact that the active-site cysteine that is well conserved among KS III and thiolase enzymes is replaced with a serine in DpsC (8, 19, 24) has led to skepticism about the proposed starter unit specificity function (24). Metabolite studies by Rajgarhia and Strohl (19) and Gerlitz et al. (7), in which heterologous expression of minimal dps genes was found to result in apparent AA production in the absence of dpsC and dpsD, suggested that these genes were dispensable for the biosynthesis of AA. That is, the PKS consisting of the products of dnrG (which governs C-12 oxidation of the AA precursors [Fig. 1B]) and the dpsABEFG genes appeared to be responsible for the choice of starter unit and also the reduction, folding, and cyclization of the nascent 21-carbon decaketide to form AA (7, 19). Gerlitz et al. (7) noted that the strain also made the 20-carbon compound SEK43 (Fig. 1C). A later study by Strohl et al. (20) using a DNR-producing strain with disrupted dpsC and dpsD genes revealed that a 20-carbon anthracycline was the major metabolite produced. Taken together, these results imply that promiscuous starter unit selection can occur in the absence of dpsC and dpsD. We approached the question of starter unit specification by constructing different combinations of the DNR PKS genes in expression vectors and studied metabolite production by feeding the cultures with ¹⁴C-labeled precursors. In addition, a cell-free system was employed to synthesize ¹⁴C-labeled polyketides in vitro to study the function of dpsC and dpsD. The results of this research clearly demonstrate that dpsC is the primary genetic determinant of starter unit specificity in the biosynthesis of AA.

Construction of expression plasmids. The plasmid pWHM75 (8) was used as the source of the dpsABCDEF genes. Plasmids pWHM346 (14) and pWHM555 (13) were used to obtain dpsY and dpsG, respectively. The plasmid pWHM1013, which is pGEM7zf (Promega, Madison, Wis.) containing the dpsBCDG genes, was created as described below and used as the starting point for constructing three other plasmids. A 1.35-kb PvuII fragment containing dpsD was subcloned into pGEM7zf. The SacII-PvuII fragment containing the 3' end of dpsD was then removed from this clone and replaced with a synthetic oligonucleotide linker approximately 50 bp in length that recreated the end of dpsD and added an MroI site directly behind its stop codon. The resulting dpsD gene was excised as a 940-bp BamHI-MroI fragment and ligated into pGEM7zf along with a 2.4-kb BamHI-XhoI fragment containing dpsBC and a 540-bp PinAI-KpnI fragment containing dpsG, to yield pWHM1013.

Metabolites isolated from cultures of recombinant strains. Recombinant S. lividans 1326 (10) strains each containing one of the plasmids pWHM80, pWHM1010, pWHM1011, or pWHM1012 (Table 1) were prepared by standard methods (10) and grown in 5 ml of R2YE medium (10) containing thiostrepton (40 µg/ml) in 25-ml tubes at 30°C and 280 rpm for 48 h. This seed culture was used to inoculate 50 ml of R2YE cultures in 250 baffled flasks with thiostrepton (20 µg/ml) that were grown at 30°C and 280 rpm for 20 h. To each flask, 1 µCi of [1-¹⁴C]propionic acid or [1-¹⁴C]acetate (Sigma, St. Louis, Mo.), not diluted with unlabeled carrier, was added and the cultures were grown for another 10 h. Before extraction with ethyl acetate (twice with 40 ml each time), acetic acid (0.1 ml) was added to each flask to acidify the culture. The ethyl acetate extract was dried under vacuum, and the metabolites were dissolved in methanol for further analysis. High-performance liquid chromatography (HPLC) on a C₁₈ reverse-phase HPLC column (Novapak, 4 µm in diameter, 8 by 100 mm; Waters, Midford, Mass.) was used with a gradient of acetonitrile-water-acetic acid (20:80:0.1 [vol/vol] for 2 min to 100:0:0.1 in 12 min) at a flow rate of 2 ml/min to separate the metabolites. The purified metabolites were detected with a Waters 484 variable wavelength absorbance detector and a Radiomatic Flo-One/Beta A-515 radiochromatography detector (Packard, Downers Grove, Ill.). Based on the total radioactivity recorded in the peak corresponding to the ¹⁴C-labeled product, a total incorporation of 50% into SEK43 or UWM5 was calculated. The identity of each to known compounds was verified by comparison HPLC and confirmed by liquid chromatography-mass spectrometry analysis.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   HPLC analysis of metabolites produced by S. lividans with plasmids pWHM80 (A, B, and C) and pWHM1011 (D, E, and F). Cultures were fed with [1-14C]acetic acid (A and D), [1-14C]propionic acid (B and E), or nothing (C and F). AU, absorbancy units.

Analysis of metabolites produced in vitro. Cell-free synthesis of polyketides has been successfully used to study the functions of PKS enzymes in the biosynthesis of tetracenomycin (1) and actinorhodin (6), two well-characterized bacterial aromatic polyketides. Accordingly, we employed a cell-free system to study DNR biosynthesis, in hopes of illustrating the function of the PKS enzymes involved in this biosynthetic pathway. Cultures of S. lividans strains with plasmid pWHM80, pWHM1010, pWHM1011, pWHM1012, pWHM1014, or pWHM1015 were treated as described above without the addition of ¹⁴C-labeled precursors. Cells were harvested, washed, and sonicated, and protein extracts were prepared as described earlier (1). Ammonium sulfate (504 g/liter) was added to the protein extract, and the precipitate was collected by centrifugation (25,240 × g, 20 min). The resulting pellet was dissolved in 100 mM sodium phosphate buffer (pH 7.2) with 2 mM dithiothreitol and 10% glycerol. A PD-10 column (Pharmacia) was used to desalt the solution into the same buffer at a protein concentration of 2.5 mg/ml. The complete assay solution (250 µl) contained 50 µM propionyl-CoA, 150 µM [2-¹⁴C]malonyl-CoA, 2 mM dithiothreitol, and 50 µl of protein extract in 0.1 M phosphate buffer (pH 7.5). The assay solution was incubated at 30°C for 100 min, and the reaction was terminated by the addition of 150 mg of NaH₂PO₄. The products were extracted with ethyl acetate (0.3 ml, three times), and the samples for HPLC analysis were prepared as described earlier (1). HPLC analysis of the products was done as described above.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   HPLC analysis with UV absorbance and 14C radioactivity detection of metabolites produced from malonyl-CoA by DNR PKS enzymes. (A) DnrG and DpsABEFG plus TcmN; (B) TcmJ and DpsABCDGEF. AU, absorbance units.