The polyketide-derived macrolactone of the antibiotic erythromycin is made through successive condensation and processing of seven three-carbon units. The fourth cycle involves complete processing of the newly formed 13-keto group (.3keto reduction, dehydration, and enoyl reduction) to yield the methylene that will appear at C-7 of the lactone ring. Synthesis of this molecule in Saccharopolyspora erythraea is determined by the three large eryA genes, organized in six modules, each governing one condensation cycle. Two amino acid substitutions were introduced in the putative NAD(P)H binding motif in the proposed enoyl reductase domain encoded by eryAMi. The metabolite produced by the resulting strain was identified as A6"7-anhydroerythromycin C resulting from failure ofenoyl reduction during the fourth cycle ofsynthesis ofthe macrolactone. This result demonstrates the involvement of at least the enoyl reductase from the fourth module in the fourth cycle and indicates that a virtually complete macrolide can be produced through reprogramming of polyketide synthesis.A wide variety of natural compounds, exhibiting antibacterial, antihelminthic, antitumor, and immunosuppressive activities, contain a polyketide-derived skeleton. Biosynthesis of polyketides is mechanistically equivalent to formation of long-chain fatty acids (1), where the fatty acid synthase (FAS) condenses the extender unit malonate with the starter unit acetate and the resulting (3-keto group undergoes three processing steps, 3-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. The C4 chain is elongated through repeated addition of two carbon atoms (derived from malonate) and fully processed at each cycle, until the proper length of a symmetrical chain has been reached. Many polyketides, in contrast, retain ketone, hydroxyl, or olefinic functions and contain methyl or ethyl side groups interspersed along an acyl chain of length comparable to that of common fatty acids. This asymmetry in structure implies that the polyketide synthase (PKS), the enzyme system responsible for formation of these molecules, although mechanistically equivalent to FAS, must somehow be programmed to produce the correct molecular structure.The current model (2) for biosynthesis of complex polyketides (defmed as compounds whose synthesis requires each FAS-like cycle to be usually different from the previous one) is exemplified, for the erythromycin aglycone DEB (Fig. 1) teins-EryAl, EryAII, and EryAIII-encoded by eryA (Fig. 2). Thus, the noniterative processive synthesis ofasymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities of the domains contained in each SU.The involvement of a distinct enzymatic activity in each synthesis step implies that a modification affecting a single activity should perturb only the corresponding step. Such modifications offer the potent...

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

A complex of 18-membered macrolide antibiotics has been discovered in the fermentation broth of strain AB718C-41. The producing culture, isolated from a soil sample collected in Hamden,Connecticut, was identified as a strain of Dactylosporangium aurantiacum and was designated D. aurantiacum subsp. hamdenensis subsp. nov. The antibiotic complex was produced in a NewBrunswick 150-liter fermentor using a mediumconsisting of glucose, soybean oil, soybean flour, beef extract and inorganic salts. Several of the antibiotics were active against sensitive and multiple antibiotic-resistant strains of pathogenic Gram-positive bacteria.

Determination of the mechanism of action of FK506and cyclosporin A has yielded new molecular targets involved in signal transduction during T cell activation. A commontarget of FK506 and cyclosporin A is inhibition of activation of the NFATtranscription factor, for which a specific binding region is present in the promoter of the IL-2 gene. A reporter gene assay has been used to screen for agents that interfere with this early step in T cell activation. Simple aromatic compoundsthat block NFAT-dependent transcription and showin vitro immunosuppressiveactivity were isolated from the broth and mycelia of two Streptomyces sp. fermentations. The compounds were active at concentrations that were not directly cytotoxic.