Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min−1, comparable to the turnover rate of a truncated trimodular derivative (2.5 min−1) but slower than a bimodular derivative (21 min−1). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogs, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.

Assembly line polyketide synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Additionally, vectorial biosynthesis is enabled by the energetic coupling of polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resolution analysis of smaller PKS components and lower resolution characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megasynthases, along with a recap of key unanswered questions in the field.

Vectorial polyketide biosynthesis on an assembly line polyketide synthase is the most distinctive property of this family of biological machines, while providing the key conceptual tool for the bioinformatic decoding of new antibiotic pathways. We now show that the action of the entire assembly line is synchronized by a previously unrecognized turnstile mechanism that prevents the ketosynthase domain of each module from being acylated by a new polyketide chain until the product of the prior catalytic cycle has been passed to the downstream module from the corresponding acyl carrier protein domain. The turnstile is closed by virtue of tight coupling to the signature decarboxylative condensation reaction catalyzed by the ketosynthase domain of each polyketide synthase module. Reopening of the turnstile is coupled to the eventual chain translocation step that vacates the module. At the maximal rate of substrate turnover, one would expect the chain release step to initiate a cascade of chain translocation events that sequentially migrate back upstream, thereby repriming each module and setting up the assembly line for the next round of polyketide chain elongation.

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.