Erythromycin lactone , biosynthesi

Although cell-free synthesis of 6-dEB was achieved, kinetic studies demonstrated that the process was very inefficient [34], The low rate of biosynthesis was likely due to the fact that the association of the three DEBS proteins in vitro was suboptimal. To simplify in vitro synthesis and to facilitate mechanistic analysis, a truncated version of the erythromycin PKS was created. The protein, DEBS 1-TE, was engineered by relocating the thioesterase (TE) from the end of DEBS 3 to the terminus of DEBS 1 (Fig. 9a,b) [35], In vivo, this bimodular construct synthesizes two triketide lactone products, one derived from propionate as a starter unit, and the other from acetate. 

In the presence of its natural substrates, propionyl-CoA, methylmalonyl-CoA, and NADPH, DEBS 1-TE was initially shown to catalyze the formation of lactone in a cell-free system [36]. Concomitant work on a similar bimodular system called DEBS 1+TE (Fig. 9c) [37] in a cell-free extract and with partially purified protein, demonstrated that it too was competent for biosynthesis of the triketide lactone [33], These experiments set the stage for more rigorous investigation of mechanistic aspects of erythromycin biosynthesis. 

Figure 9 Construction of bimodular polyketide synthases, (a) Chromosomal repositioning of the thioesterase domain from the C-terminus of module 6 to the end of module 2 in the erythromycin PKS leads to production of triketide lactones and the disruption of erythromycin biosynthesis, (b) DEBS 1-TE contains a fusion within the ACP domains of modules 2 and 6. In Saccharopolyspora erythraea and Streptomyces coelicolor the construct produced both propionate and acetate-derived lactones, (c) DEBS 1+TE contains a fusion between ACP2 and the thioesterase domain. In S. coelicolor, the protein biosynthesized the same lactones.

Figure 13 Stereochemistry of chain extension in erythromycin biosynthesis. DEBS 1-TE uses only the (25)-isomer of methylmalonyl-CoA to generate both a D- and an L-center in its lactone product. The D-methyl stereochemistry corresponds to condensation of (25)-methylmalonyl-CoA with retention of configuration, while the L-methyl corresponds to condensation with inversion.

Figure 19 Altering the starter unit specificity of DEBS 1-TE. A hybrid PKS was constructed by replacing the erythromycin loading domains with those of the avermectin PKS. The resulting mini-PKS produced lactones incorporating the natural starter units of erythromycin biosynthesis, acetate and propionate, as well as those characteristic of avermectin, isobutyrate, and 2-methylbutyrate.

The biosynthesis of erythromycin can be divided into two phases (Scheme 1). In the first constructive phase of the pathway a set of key enzymes, collectively known as the polyketide synthase (PKS), assembles the typical polyketide chain by sequential condensation of one unit of propionyl-CoA and six units of methylmalonyl-CoA 6. The initially formed chain is cyclised to give the first macrocychc lactone (macrolide) intermediate 6-deoxyerythronolide B 7 [6,7]. In the second phase 6-deoxyerythronohde B is elaborated by a series of tailoring enzymes which carry out regiospecific hydroxylations, glycosylations and a methylation (of an added sugar residue) to give finally erythromycin A. The core polyketide structure is generated by the PKS in phase one, but the later steps of phase two are essential to produce active antibiotics. 

The avermectin PKS loading module has been used to generate a hybrid PKS system by replacing the loading module of DEBS 1-TE. The new hybrid PKS produced new hybrid polyketide (triketide lactones) which incorporated the isobutyrate and 2-methylbutyrate starter acids of avermectin biosynthesis, as well as the normal acetate and propionate starter units of erythromycin biosynthesis [51]. 

Additionally, altering starter units has been successful in erythromycin biosynthesis. Replacement of the loading domain of DEBS with the loading domain from the avermectin PKS, which has a broad specificity for branched-chain acyl units, led to the incorporation of branched starter units into products [82]. This was accomplished in S. codicolor with both a DEBSl-tTE construct that resulted in the production of triketide lactone analogues derived from acetate, propionate, isobutyrate, and 2-methylbutyrate (24—27), and in the complete DEBS pathway, that resulted in products with the corresponding starter units introduced to the macrolide core. 

Tylosin [46-49] (Fig. 6), produced by Streptomyces fradiae, was one of the first antibiotics for which a comprehensive set of blocked mutants was isolated. They were used to help define the biosynthetic pathway. Whereas erythromycin is a 14-membered macrolide, tylosin has a 16-membered lactone structure. The gene for the final step of biosynthesis was cloned by reverse genetics from the protein sequence of the enzyme [46], and specific segments of surrounding DNA were found to complement other classes of blocked mutants. The formation of tylosin aglycon, protylonolide, is involved in five polyketide synthases. 

Another successful approach for blocking the intramolecular decomposition illustrated in Fig. 2 involved inhibition of the dehydration step leading to the anhydrohemiketal by replacement of the C-8 proton of erythromycin with fluorine. Preparation of flurithromycin (8-fluoroerythromycin, see Fig. 4) has been achieved by both chemical and biochemical methods. Addition of 8(S)-8-fluoroerythronolide A to a mutant strain of Streptomyces erythreus blocked in biosynthesis of its endogenous lactone (erythronolide) yielded the desired fluorinated erythromycin [40]. This technique of mutasynthesis has been further employed for the production of other fluorinated derivatives of erythromycin [40, 41]. Fluorination of different 8,9-anhydro-6,9-hemiketal derivatives of erythromycin by chemical means with reagents such as trifluoromethyl hypo-fluorite or perchloryl fluoride (with subsequent reduction of the N-oxide) has also been reported [42, 43]. 

FIGURE 25.16 An outline of the pathway forthe biosynthesis of erythromycin A. One propionate and six methylmalonate units are first assembled into the macrocyclic lactone 6-deoxyerythronolide B, which is then hydroxylated, glycosylated by two different sugars, hydroxylated again, and finally methylated. 

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