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Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of ketide units. To validate the approach, intermediates for the synthesis of trans-9,10-dehydroepothilone D were generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of epothilone analogues, thus facilitating their commercial development as therapeutic agents.

In vivo reconstitution of the dTDP-D-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea was achieved by expression of the genes in Escherichia coli. LC/MS/MS analysis of the dTDP-sugar intermediates produced by operons containing different sets of genes showed that production of dTDP-D-desosamine from dtdp-4-keto-6-deoxy-D-glucose requires only four biosynthetic steps, catalysed by MegCIV, MegCV, MegDII and MegDIII, and that MegCII is not involved. Instead, bioconversion studies demonstrated that MegCII is needed together with MegCIII to catalyse transfer of D-desosamine to 3-alpha-mycarosylerythronolide B.

A ketolide was prepared from 14,15-dehydroerythromycin A by two different routes. The first approach involving oxidation of the 3-OH of 3-descladinosyl-14,15-dehydroerythromycin A 2'-O-acetate gave unexpectedly high levels of 3,11-double oxidation. This may be due to greater formation of the 9,12-hemiketal in 14,15-dehydroerythromycin A and concomitant exposure of the 11-OH group for oxidation. NMR studies of 14,15-dehydroerythromycin A support this hypothesis, revealing a 9:1 ratio of 9-ketone to 9,12-hemiketal in CDCl3 and a 1:1 ratio in CD3OD as contrasted with the corresponding tautomer ratios of 30:1 in CDCl3, and 6: 1 in CD3OD with erythromycin A. Alteration of the 13-substituent on the erythronolide A ring from ethyl to vinyl thus favors formation of the 9,12-hemiketal. A second route to the ketolides was developed based on these findings, in which the 11-OH is eliminated prior to oxidation of the 3-OH.

Five contiguous genes in the rapamycin gene cluster, rapQONML, of Streptomyces hygroscopicus ATCC29253 were replaced with a neomycin resistance marker by double homologous recombination. The resulting strain, if fed pipecolate, produced the analog 16-O-desmethyl-27-desmethoxyrapamycin instead of rapamycin. This indicates that the P450 hydroxylase encoded by rapN is specific for C-27, and that the O-methyltransferases encoded by rapQ and rapM methylate the hydroxyl groups on C-16 and C-27. By inference, the remaining P450 hydroxylase and methyltransferase genes (rapI and rapJ) are responsible for hydroxylation of C-9 and methylation of the C-39 hydroxyl, consistent with their homology to fkbD and fkbM, respectively, in the FK506 cluster. The relatively high level of 16-O-desmethyl-27-desmethoxyrapamycin produced indicates that the reactions at C-9 and C-39 do not require previous modification of the macrolactone precursor at either C-16 or C-27.

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.

The genes for most of the biosynthesis of the kinamycin antibiotics have been cloned and heterologously expressed. Genomic DNA of Streptomyces murayamaensis was partially digested with MboI and a library of approximately 40 kb fragments in E. coli XL1-BlueMR was prepared using the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes. After transferal of these clusters to S. lividans ZX7, expression of one cluster was established by HPLC with photodiode array detection. Peaks were identified from the kin cluster for dehydrorabelomycin, kinobscurinone, and stealthin C, which are known intermediates in kinamycin biosynthesis. Two shunt metabolites, kinafluorenone and seongomycin were also identified. The structure of the latter was determined from a quantity obtained from large-scale fermentation of one of the clones.