The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.

Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.

B-RAF V600E ͉ hypothemycin ͉ irreversible kinase inhibitor ͉ Michael adduct R esorcylic acid lactones (RALs) are polyketide natural products with a large macrocyclic ring fused to resorcylic acid. Some RALs contain an ␣,␤-unsaturated ketone in the macrocycle, as exemplified by the cis-enone RALs hypothemycin, 5Z-7-oxozeaenol, and L-783,277 (Fig. 1). The cis-enone RALs have been shown to inhibit mammalian cell proliferation and tumor growth in animals (1-3). Furthermore, several reports have indicated that cis-enone RALs inhibit certain protein kinases, such as mitogenactivated protein (MAP) kinase (MAPK) kinase (MEK)1 (4), TGF-␤-activated kinase 1 (TAK1) (5) and platelet-derived growth factor receptor (PDGFR) (6), but not others, such as RAF, PKA, PKC, endothelial growth factor receptor (EGFR), FGF receptor, ZAP70, MEK kinase 4, and lymphoid-specific Tyr kinase p56kk (LCK) (3, 4, 6). Where tested, targets inhibited by the cis-enone RALs were not affected by trans-enone RALs or RAL analogs lacking the ␣,␤-unsaturated ketone (4, 5).L-783,277 was shown to be a potent in vitro inhibitor of MEK1, competitive with ATP, that became even more potent upon preincubation (4). The apparent time-dependent inhibition caught our attention because it is a hallmark of affinity-directed covalent bond formation between enzyme and inhibitor, and the ␣,␤-unsaturated ketone moiety is an effective Michael acceptor of protein nucleophiles, particularly Cys thiolate.Off-target inhibition by a kinase inhibitor is usually unpredictable, and assessment of complete specificity usually requires screening of the entire kinome (7). In the present work, we identified a Cys residue that is conserved in the ATP site of kinase targets reported to be inhibited by cis-enone RALs but absent from those that are not. Furthermore, a structure-bioinformatics approach revealed that this Cys is present in a subset of some 46 protein kinases in the kinome that include such important targets as mitogen receptor Tyr kinases, MEK, and ERK. We show that hypothemycin forms stable covalent adducts with this Cys residue and is highly efficacious in inhibiting growth of cells dependent on the target kinases, in particular, cells dependent on mitogen receptor RAL targets or harboring B-RAF V600E mutations that drive the ERK pathway. ResultsBioinformatics. Sequence alignment of the kinases reported to be inhibited by cis-enone RALs revealed a conserved Cys residue (Cys-166 in human ERK2) adjacent to the completely conserved Asp that is involved in binding the Mg 2ϩ complexed to ATP; kinases that were reportedly not inhibited by a RAL had no Cys residue at that position. Interrogation of the human kinome sequence database revealed that some 46 of 510 identified kinases contained the target Cys and were therefore considered candidates for RAL inhibition (Table 1).Of the 46 Cys-containing RAL targets, 38 exist in 8 evolutionarily related clusters, 7 of which lie within 5 major branches of the human kinase tree (Fig. 2) (8). The largest branch of Ϸ90 Tyr kinases contains...

To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of Ϸ5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440 -15445]. The major current impediment to the success of this tactic is the difficulty of building the Ϸ5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500-to 800-bp ''synthons'' with low error frequency by automated PCRbased gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon Ϸ5-kb segments by using only three endonucleases and ''ligation by selection.'' These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli. The chemical synthesis of genes and genomes has received considerable attention for several decades and is becoming increasingly important in the exploitation of whole-genome sequence information. The field was pioneered by Khorana and coworkers with the then-heroic total synthesis of tRNA structural genes (1, 2) and by Itakura et al. (3) with the synthesis and expression of the somatostatin gene. Since then, DNA synthesis methodology has made steady progress, with current approaches relying on the enzyme-catalyzed assembly of short, chemically synthesized oligonucleotides. Of the various methods, polymerase cycling assembly (PCA) (4) is the most widely used because of its inherent simplicity. Overlapping, complementary oligonucleotides are annealed and recursively elongated with a heat-stable DNA polymerase to ultimately yield a full-length sequence, which is amplified by conventional PCR. PCA, first reported for synthesis of the 303-bp HIV-2 Rev gene (5), has since evolved (6-8) into a widely used general method for synthesis of genes of up to Ϸ1 kb.The 1-kb size barrier was broken in 1990 by Mandecki et al. (9), who synthesized a 2.1-kb plasmid by ligation of 30 fragments, and again in 1995 when Stemmer et al. (7) reported the one-step PCA synthesis of a 2.7-kb plasmid that was purified by antibiotic selection. Smith et al. (4) assembled the 5,386 X174 bacteriophage genome from a single pool of chemically synthesized oligonucleotides by using a combination of ligation and PCA methods, but purification of the product again required biological selection. In 2002, Cello et al. (10) described a stepwise synthesis of a 7,558-bp poliov...