Microbial polyketide synthases : More and more prolific

Microorganisms make a wealth of unusual metabolites that have a secondary role in the organism’s ontogeny, such as self-defense, aggression, or even communication as the need arises. These compounds often have biological activity valuable to humankind (1). Nevertheless, as valuable as antibiotics and other naturally occurring drugs have been over the past 60 years, their future use is threatened by now widespread resistance to antibiotics among human, animal, and agricultural pathogens. Despite the fact that microbial metabolites are being found continually by pharmaceutical companies through targeted screening programs, fewer and fewer fundamentally new types of drugs have been found in this way during the past two decades (2). Combinatorial biosynthesis seeks to help remedy this situation through genetic engineering, the goal of which is to make new antibiotics by exploiting the microbial genes and enzymes that make these substances, thereby aiming to discover drugs that cannot be found in nature. Combinatorial biosynthesis has been especially successful with bacterial polyketide synthases (PKSs), enzymes that, in essence, polymerize simple fatty acids into a myriad of chemical structures called ‘‘polyketides’’ (3). The polyketides discovered so far number into the thousands and are usually categorized on the basis of their structures: one type resembles branched-chain fatty acids formed into large carbocyclic rings (macrolides), and another type contains two or more aromatic rings fused into polycyclic structures, although nature has invented many variations of these and other structural themes (3). Modular type I PKSs consisting of one or more large multifunctional proteins make the first type, and iterative type II PKSs, complexes of several largely monofunctional proteins, make the second type (4, 5). After the first bacterial PKS genes were cloned between 1989 and 1991, extensive structure–function investigations of both types of PKSs were carried out, and more recently, this has led to many successful attempts to produce new microbial metabolites (4–9). Recently, McDaniel et al. (10) and Shen et al. (11) report major achievements in this direction through the interchange of modules or domains among type I PKSs in one system (10) and the expression of three PKS genes from a type II PKS system (11). The basis for these seminal developments was laid through studies of the genetics of antibiotic production by the Streptomyces and related filamentous bacteria, begun by David Hopwood and other scientists nearly 20 years ago. The architecture of the type II PKSs was revealed first by analysis of the granaticin (12), tetracenomycin (13), and actinorhodin (14) PKS genes. Enzymological studies of the latter two systems carried out in the past year (15, 16) have established the features of the type II enzyme complex, as illustrated in Fig. 1B for the tetracenomycin PKS encoded by the tcmJKLMN genes. Here, a b-ketoacyl:acyl carrier protein (ACP) synthase (KS) composed of TcmK and TcmL (the latter type of protein is also known as the chain length factor or CLF) interact with a malonyl-CoA:ACP acyltransferase (MCAT), thought to be borrowed from the bacterial fatty acid synthase, and the TcmM ACP. These proteins are called the minimal PKS and select a starter unit (most often acetate, believed to be derived from decarboxylation of enzyme-bound malonate) to use with the malonate chain extender unit to form a poly-b-ketide intermediate by repetitive decarboxylative condensation of acylthioester intermediates bound to the active site of TcmK and the phosphopantetheinylated TcmM. The resulting decaketide is cyclized to Tcm F2 and then to Tcm F1 by the homodimeric TcmN and trimeric TcmI cyclases. In the tetracenomcyin system, the TcmJ protein plays an important but as yet poorly understood role in these processes. The actinorhodin system contains comparable enzymes (except for TcmJ) plus a ketoreductase (KR) that removes one of the aromatic hydroxyls by reduction of a carbonyl group in the poly-b-ketide. Work on erythromycin A biosynthesis by Leadlay (17) and Katz (18) and their coworkers led in 1990–1991 to the discovery of the remarkable properties of modular type I PKSs. The 6-deoxyerythronolide B synthase (DEBS) subunits encoded by the eryA genes, oriented 59 to 39 as shown by the pointed wedges in Fig. 1A, are large multifunctional proteins containing domains typical of fatty acid synthases: KS, AT (acyltransferase), KR, DH (dehydratase), ER (enoyl reductase), ACP, and TE (thioesterase). These are grouped into six modules that specify how each 3-carbon unit of the final product is assembled, including the choice of the starter unit (governed by the AT and ACP closest to the N terminus of DEBS1, which are known as the loading domains or module) and the chain extender unit (specified by the ATs in each module). Different combinations of KR, DH, and ER domains can create further structural diversity in the product; compare modules 3 and 4 of DEBS2, which are responsible for the C-9 carbonyl and C-7 methylene groups in the product. Colinearity between the order of the modules in the DEBS proteins and the steps of polyketide assembly, as shown in Fig. 1A, is typical of bacterial type I PKSs. Hence, once propionate is loaded onto the ACP of the loading domain and (2S)-2methylmalonate onto the second ACP of module 1, the system is set up to catalyze a decarboxylative condensation between these two substrates, after the propionate is moved to the KS domain, and then to reduce the b-carbonyl of the first intermediate formed to produce 2-methyl-3-hydroxyvalerate bound to the module 1 ACP. The same set of reactions is repeated, resulting in the formation of a 9-carbon intermediate attached to the ACP of module 2 of DEBS1, and so on, by using the catalytic activities found in each of the modules of DEBS2 and DEBS3, until the final 21-carbon product is released and cyclized (by the TE domain) as the 14-membered macrolide product (Fig. 1A). This is converted to erythromycin A by the tailoring reactions of the pathway, which can tolerate some differences in the structure of 6-deoxyerythronolide B, but not all of them that have been produced by combinatorial biosynthesis. From the outset, Katz and coworkers (18) recognized that the modular nature of DEBS coupled with the known processivity of PKSs in general possibly meant that the absence of a particular KR, DH, or ER activity would allow the product of that module to be processed by the active sites of DEBS operating subsequently to produce analogs of 6-deoxyerythronolide B. The validity of this concept was first shown by targeted reprogramming of DEBS: deleting the KR domain of eryAIII module 5 (18)