Modular Organization of Genes Required for Complex Polyketide Biosynthesis STEFANO DONADIO, MICHAEL J. STAVER, JAMES B. MCALPINE, SUSAN J. SWANSON, LEONARD KATZ In Saccharopolyspora erythraea, the genes that govern synthesis of the polyketide portion of the macrolide antibiotic erythromycin are organized in six repeated units that encode fatt acid synthase (FAS)-like activities. Each repeated unit is designated a module, and two modules are contained in a single open reading frame. A model for the synthesis of this complex polyketide is proposed, where each module encodes a functional syn- thase unit and each synthase unit participates specifically in one of the six FAS-like elongation steps required for formation of the polyketide. In addition, genetic organi- zation and biochemical order of events appear to be colinear. Evidence for the model is provided by construc- tion of a selected mutant and by isolation of a polyketide of predicted structure. POLYKETIDES ARE A LARGE AND DIVERSE CLASS OF NATURAL products that includes antibiotics, pigments, and immunosup- pressants and have applications in medicine, agriculture, and industry (for an example, see Fig. 1). Biosynthesis of polyketides is believed to occur by a series of condensations of carbon units in a manner similar to that of long chain fatty acids (LCFA) (1). The LCFAs are formed by fatty acid synthase (FAS) through a process whereby a starter unit (commonly acetate) is condensed to the extender unit (malonate). The resulting P-keto group is then fully processed (reduced), and the cycle resumes with the condensation of a new extender unit (2). Most polyketides, however, contain structural complexities that can be accounted for by the use of different extender units at various steps and by variations in the extent of processing of the (-carbon ((3-ketoreduction, dehydration, enoylreduction). Although this complexity exists, in living orga- nisms there is believed to be a polyketide synthase (PKS) that produces only one or a few related molecular structures (3). Thus, an understanding of the biosynthesis of complex polyketides must include a description of the mechanism by which the PKS both selects the correct substrate and decides the fate of the (3 carbon at each step. The polyketide portion of macrolide antibiotics is synthesized through the condensation of short chain carbon units; for example, seven propionates in the case of erythromycin (4). 3-Hydroxy-acyl S. Donadio, M. J. Staver, and L. Katz are in the Department of Corporate Molecular Biology, Abbott Laboratories, Abbott Park, IL 60064. J. B. MeAlpine and S. J. Swanson are in the Bioactive Microbial Metabolite Project, Abbott Laboratories, Abbott Park, IL 60064. 3 MAY 1991 thioesters, which mimic hypothetical intermediates in the synthesis but not the corresponding 3-keto derivatives, are incorporated in vivo into the macrolide rings of erythromycin and tylosin, providing support for a FAS-like origin of these molecules (5). Consistent with this idea is the detection of branched-chain fatty acids in fermenta- tion broths of tylosin and mycinamicin producers (6). A FAS-like mechanism for the synthesis of the erythromycin aglycone 6-deox- yerythronolide B (6dEB) requires that (i) six methylmalonyl- coenzyme A (mmCoA) units, three of each enantiomer, are succes- sively condensed to a propionyl-CoA starter unit; (ii) that 3-ketoreduction occurs after each condensation step except step three so that a keto group is left at C-9; and (iii) that dehydration and enoyl reduction take place only after the fourth condensation to introduce a methylene at C-7 (Fig. 1). Consequently, a fill set of FAS activities (2) is required for 6dEB synthesis: acyltransferase (AT), 3-ketoacyl carrier protein synthase (KS), and acyl carrier protein (ACP) for chain elongation; (3-ketoreductase (KR), dehy- dratase (DH), and enoyl reductase (ER) for processing of the (3 carbon; and thioesterase (TE) for release and lactonization of the full-length chain. In addition, the hypothetical 6dEB PKS must also be programmed at each step to select the correct enantiomeric extender unit and to process the ( carbon to the appropriate degree. Six modules with FAS-like domains in 6dEB synthesis. The segment of the chromosome required for the formation of 6dEB in S. erythraea has been designated eryA. A 5-kb DNA fragment of eryA had been identified by its ability to restore erythromycin production when introduced into a mutant blocked in the synthesis of 6dEB. Hybridization of chromosomal DNA with this segment Fig. 1. Structure of 6-deoxy- erythronolide B. The dotted lines represent C-C bonds © formed during synthesis steps 1 through 6 by condensation be- (E) tween the starter unit propio- 2 nyl-CoA (encircled S) and the 1 7 extender units methylmalonyl- -D H CoA encircled 1 to 6. The r O stereochemistry of extender 1 units 1, 3, and 4 should be compared to that of units 2, 5, 3 5 and 6. Lactonization of the acyl I), OH chain between C-1 and C-13 , results in the formation of 6-deoxyerythronolide B. Hy- droxylation of the lactone ring O - H at C-6, followed in order by mycarose and desosamine at- tachment at hydroxyls at C-3 and C-5, respectively, results in the formation of the first bioactive com- pound, erythromycin D (4). RESEARCH ARTICLES 675 ..VE on November 27, 2013 www.sciencemag.org Downloaded from on November 27, 2013 www.sciencemag.org Downloaded from on November 27, 2013 www.sciencemag.org Downloaded from on November 27, 2013 www.sciencemag.org Downloaded from on November 27, 2013 www.sciencemag.org Downloaded from