Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate 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 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-deoxy-erythronolide 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 intermodular linker interactions (8), in the design of productive chimeric assembly lines. At the same time, the relatively broad, but not unrestricted, substrate tolerance of typical KS domains was also recognized (9). In one particularly thorough study, Menzella et al. (10) carried out a large scale assessment of the feasibility of combinatorial biosynthesis by pairwise combinations of 14 modules derived from 8 different PKS clusters, thereby allowing the in vivo characterization of 154 chimeric bimodular PKSs, with the bimodular DEBS derivative (Fig. 2) serving as the benchmark system. Approximately 50% of the resulting chimeras yielded measurable quantities of the expected polyketide product when expressed in Escherichia coli (Ͼ0.1 mg/liter culture; ϳ1% of the productivity of the benchmark DEBS system). These researchers then extended their investigation to the generation and analysis of trimodular PKSs, using the corresponding trimodular DEBS construct (DEBS modules 1, 2, and 3ϩTE) as a reference standard (11). Although this extensive study succeeded in revealing many functional PKS combinations, the low yields of the resulting engineered tetraketides suggested that inactive or low producing chimeras * This work was supported by National Institutes of Health Grants GM 087934 (to C. K.) and GM 022172 (to D. E. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. □ S This article contains supplemental Tables S1-S8 and Fig. S1 Recent biochemical investigations in our own laboratories have revealed that protein-protein recognition between ACP and KS domains during intermodular polyketide chain translocation involves a set of protein-protein interactions that are orthogonal to those implicated in intramodular polyketide chain elongation (12,13). This critical distinction has provided a critical mechanistic framework for factoring the contribution of imperfect intermodular ACP-KS interactions to the turnover efficiency of chimeric assembly line PKSs. We have also established that the KR domain of DEBS M2, which generates the enantiomeric (2R, 3S)-2-methyl-3hydroxy diketide diastereomer of the natural (2S, 3R) product of DEBS M1, has comparable specificity for the ACP domain of DEBS M1 compared with its natural ACP2 substrate (14). This finding has now allowed us to investigate the influence of the recognition of substrate stereochemistry by an acceptor KS on the overall turnover by otherwise identical chimeric PKSs. We have therefore undertaken a carefully controlled, quantitative analysis of the relative contribution of these protein-protein and protein-substrate recognition to the turnover efficiency of chimeric PKS assembly lines generated by recombining intact modules obtained from heterologous PKSs.  In DEBS M1, the KR domain then catalyzes an epimerization of the C-2 methyl group followed by diastereospecific reduction (step 5) to give the mature (2S,3R)-diketide, which is then translocated to DEBS M2 via a thioester to thiol transacylation. There it undergoes another round of chain elongation and KR-catalyzed reduction (without epimerization), followed by (TE)-catalyzed release and lactonization (steps 6 and 7).

Engineering of Chimeric Bimodular and Trimodular PKSs-
Eleven chimeric derivatives of the bimodular PKS shown in Fig.  2 were constructed in which DEBS M2 was replaced by DEBS M3, M5, or M6, or alternatively by each of six modules from the rifamycin synthase (RIFS (15)) or two modules from the rapamycin synthase (RAPS (16) ; Fig. 3). The predicted structures of each of the corresponding triketide products (structures 2-8 (17)(18)(19)) are shown in Fig. 3. To facilitate intermodular interactions between heterologous modules, each acceptor module was fused to the N-terminal docking domain of DEBS2 (Fig. 1). (The numbers in parentheses define the identity of the docking domains. For example, the benchmark bimodular derivative of DEBS consists of LDD(4), (5)M1(2), and (3)M2ϩTE, where parenthetical numbers refer to the parent DEBS modules that are normally adjacent to the corresponding N-or C-terminal docking domains). These helical docking domains mediate weak but specific non-covalent interactions between donor and acceptor modules (K D ϭ ϳ100 M) (20).
An additional eight chimeric trimodular PKSs were also engineered in an analogous manner using LDD(4), DEBS (5)M1(2), an intervening heterologous (3)Module(2), and DEBS (3)M3ϩTE as the terminal acceptor module. As before, appropriate docking domains were fused to the C-and N-terminal ends of each protein so as to enable effective proteinprotein interactions. In addition to the benchmark trimodular PKS with DEBS (3)M2(2) as the intervening module, six modules from RIFS and two modules from RAPS were also tested ( Fig. 4). The structures of each of the predicted tetraketide products (structures 9-15) are shown above each trimodular series (Fig. 4). The yield and purity of individual proteins used in this study are documented in Fig. 5.
Activity of Chimeric Bimodular and Trimodular PKSs-To test the in vitro activity of each chimeric PKS, a previously developed UV 340 spectrophotometric assay was performed using propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA in case of RIFS M2), and NADPH as substrates (21). The stoichiometric coupling between polyketide formation and NADPH consumption at steady state, in which the stoichiometric coefficient corresponds to the number of catalytically active ketoreductase domains in the assembly line, allows convenient and sensitive monitoring of the steady-state turnover rate of the multimodular PKS assembly line ( Fig. 6) (21).
For the bimodular PKSs, the reference DEBS M1ϩM2ϩTE construct showed a steady-state rate of triketide formation rate of 11.6 nmol/min/mg total protein, in good agreement with that previously reported for this system (21). All the other chimeric bimodular assembly lines exhibited significantly reduced rates of turnover, the highest being that using DEBS M6 as the surrogate acceptor module (1.4 nmol/min/mg total protein; Fig. 6A). Background NADPH consumption was measured in a control reaction containing only LDD(4) and (5) (3)ModuleϩTE as the variable acceptor. Acceptor modules were derived from DEBS, rifamycin synthase (RIFS), or rapamycin synthase (RAPS). Compatible docking domains from DEBS (depicted as black tabs or numbers in parentheses) were fused to the corresponding C-and N-terminal ends of the respective donor and acceptor modules to enhance the specificity and efficiency of intermodular chain translocation. The predicted triketide products 2-8 generated in the presence of propionyl-CoA, methylmalonyl-CoA (and malonyl-CoA, in the case of RIFS M2ϩTE), and NADPH, are shown for each chimeric module pair. All acceptor modules are specific for methylmalonyl-CoA, except for RIFS M2, which prefers malonyl-CoA but can also accept methylmalonyl-CoA.
A similar decrease in polyketide synthase activity was observed for the chimeric trimodular PKSs. The reference DEBS M1ϩM2ϩM3ϩTE construct showed a steady-state rate of tetraketide formation rate of 1.5 nmol/min/mg total protein, whereas the remaining nine chimeric trimodular PKSs were 6 -20-fold less active (Fig. 6B). The baseline rate of NADPH consumption was established by a control reaction lacking DEBS M2 and containing only LDD(4), (5)M1(2), and DEBS (3)M3ϩTE. Importantly, this control also established the extent to which aberrant intermodular chain translocation between DEBS M1 and M3 could be facilitated solely by compatible docking domains. Relative to this threshold (Fig. 6B, dashed line), only the trimodular construct harboring RIFS M2 showed detectable activity, albeit only 15% of the reference DEBS M1ϩM2ϩM3ϩTE system. The close correspondence between the turnover rates of the corresponding bimodular and trimodular PKSs argues against any potential influence of the appended TE domain on the measured relative activities of the chimeric bimodular PKSs.
The polyketide product profiles of each of the above reaction mixtures were analyzed by LC-MS after overnight incubation. As shown in Fig. 7, 6 of the 11 chimeric bimodular PKSs synthesized detectable quantities of their expected product. Although the chimeras harboring RIFS M5 and RIFS M7 showed only close to background NADPH consumption, they still produced traces of their expected triketide products in an overnight incubation. Likewise, as shown in Fig. 8, 4 of 8 chimeric trimodular PKSs yielded detectable amounts of the expected tetraketide.
Role of ACP-KS Interactions at the Fusion Site in Chimeric PKSs-Because the majority of the chimeric PKS constructs shown in Fig. 6 had very low levels of activity, we sought to identify the specific step in assembly line biosynthesis at which the growing polyketide chain might have become stalled. For this purpose, radio-SDS-PAGE analysis was performed using [ 14 C]propionyl-CoA to prime the LDD. By quantifying the extent of labeling of each protein module in the presence of methylmalonyl-CoA and NADPH, the site of polyketide accumulation could be deduced. To enhance the sensitivity of the experiment and thereby allow the detection of rare intermodular chain translocation events in these low activity systems, the TE domains of each acceptor module were inactivated by active site Ser 3 Ala mutations.
As seen in Table 1, the acceptor module of the reference system (DEBS LDDϩM1ϩM2ϩTE 0 ) was rapidly labeled, reaching saturation within 3 min. In contrast, the single turnover occupancy level of the acceptor modules of representative chimeric bimodular PKSs correlated quite well with their catalytic turnover (compare Figs. 6A and Table 1). Only DEBS M6ϩTE 0 showed significant labeling, reaching 42% of the reference system after 3 min. Other acceptor modules (DEBS M3ϩTE 0 , RIFS M2ϩTE 0 , RAPS M4ϩTE 0 , and RAPS M6ϩTE 0 ) showed negligible levels of diketide occupancy on this time scale.
To ascertain the cause-effect relationship between ineffective chain translocation and turnover, we sought to enhance the rate of chain translocation in a representative chimeric PKS via site-directed mutagenesis of the ACP domain of DEBS M1. We recently carried out a detailed analysis of the structural elements that control ACP-KS recognition at the DEBS M2-M3 interface (13). Our findings revealed that the N terminus of helix I of ACP2 is the primary specificity determinant during intermodular chain translocation. By contrast, chain elonga-

Role of ACP-KS Interactions in Chimeric PKSs
16408 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 31 • JULY 29, 2016 tion within DEBS M3 was controlled by a loop connecting helices I and II of ACP3.
Based on this earlier observation, we predicted that chain translocation from DEBS M1 to M3 in a chimeric bimodular PKS would be enhanced by introducing the E23K mutation (corresponding to E1424K, based on whole module numbering) into helix I of ACP1, because a cationic residue is also found at the corresponding position of ACP2. Indeed, turnover of the E23K mutant of DEBS M1ϩM3ϩTE was increased more than 2-fold over the corresponding bimodular PKS derived exclusively from wild-type modules (0.6 Ϯ 0.05 nmol/min/mg total protein, compared with 0.3 Ϯ 0.05 nmol/min/mg total protein) under experimental conditions identical to those of Fig. 6). Taken together, these results underscore the pivotal role of ACP-KS interactions during translocation of the growing polyketide chain between heterologous PKS modules.
Role of Substrate-KS Recognition at Fusion Junctions in Chimeric PKSs-Having examined the influence of ACP-KS interactions on chain translocation at chimeric PKS junctions, we sought to interrogate the relative contribution of substrate-KS recognition on the turnover efficiency of these systems. To this end we rationalized that presentation of the enantiomer of the native (2S,3R)-2-methyl-3-hydroxy-diketide-ACP product of DEBS M1 (Fig. 2) to the downstream modules of selected chimeric bimodular PKSs from Fig. 3 would enable a controlled comparison of the relative importance of specific proteinprotein and protein-substrate recognition. Because we have recently shown that the KR domain of DEBS M2 has comparable specificity for ACP1 as for its cognate ACP2 domain (14), we engineered DEBS M1 by replacing its endogenous KR domain with the paralogous KR2 from DEBS M2. The resulting hybrid module, DEBS (5)M1-KR2 (2), was expressed as a soluble protein (Fig. 5, 2 mg/liter in E. coli) with purification behavior on an ion exchange chromatography column that was indistinguishable from most wild-type PKS modules.
In addition to evaluating the turnover efficiency of this hybrid module in the presence of DEBS LDD(4) and (3)M2ϩTE, we also measured turnover in the presence of six other acceptor PKS modules (Fig. 9). The products (Fig. 9A,  structures 16 -18) of each of these chimeric PKSs were predicted to be the corresponding (2R, 3S)-diastereomers of the previously characterized triketide lactones 2, 3, and 8. Consistent with the previously established 7-fold preference of DEBS M2 for its natural (2S,3R)-diketide substrate over the enantiomeric (2R,3S)-diketide analog (22), the bimodular construct harboring the hybrid donor module showed a comparable 7-fold reduction in turnover rate compared with its wild-type bimodular counterpart (Fig. 9B). Remarkably, none of the other bimodular PKSs containing heterologous downstream modules was significantly affected in response to the change in the configuration of the diketide intermediate (Fig. 9B), indicating that enzyme-substrate recognition is not as important as specific ACP-KS protein-protein recognition during intermodular polyketide chain translocation. To verify the predicted properties of the bimodular PKSs in Fig. 9, the corresponding triketide products were each analyzed by LC-MS (Fig. 10).

Discussion
Combinatorial assembly of modules from different assembly line PKSs is, in principle, a powerful strategy for complex molecule biosynthesis. The relative importance of protein-protein interactions and enzyme-substrate recognition in such engineered systems remains unclear, however. We have now car-

JOURNAL OF BIOLOGICAL CHEMISTRY 16409
ried an extensive in vitro analysis that addresses these issues for the first time in a systematic, carefully controlled manner.
Purification and analysis of 11 chimeric bimodular PKSs and 8 chimeric trimodular PKSs harboring modules from the erythromycin, the rifamycin, and the rapamycin synthases vividly demonstrated that most chimeric PKSs suffer severe impairment of catalytic activity (Fig. 6), notwithstanding the grafting of demonstrated compatible C-and N-terminal docking sites to their intermodular interfaces (8,23). The 10 -100-fold reduction in the catalytic efficiency and productivity between wildtype and chimeric systems was particularly striking, given that the choice of bimodular and trimodular PKS constructs was based on published reports that demonstrated measurable in vivo product forming capacity for several of these chimeras (10,11). We therefore sought to exploit the power of biochemical reconstitution to dissect the mechanistic basis for the observed loss in catalytic effectiveness.
Analysis of the occupancy of the acceptor modules in chimeric bimodular PKSs showed that in the majority of cases, polyketide chain translocation represented a serious bottleneck (Table 1). Earlier studies have highlighted a role for ACP-KS interactions in the channeling of polyketide chains between modules (13,18); this conclusion is reinforced by our observation that a Glu 3 Lys charge reversal mutation in the ACP domain of the donor module DEBS M1 improved turnover in the presence of DEBS M3ϩTE. The structural logic of these protein-protein interactions is starting to become apparent (24). We note that, because interactions between the donor ACP and acceptor KS domains have not been explicitly interrogated in the experiments summarized in Table 1, the possibility of additional unproductive and therefore catalytically silent binding between chimeric PKS modules cannot be ruled out. Indeed, an important feature of these studies is that they focus only on catalytically relevant protein-protein interactions.
To assess the relative importance of substrate-KS recognition in the same chimeric bimodular PKSs, we engineered a variant DEBS M1 donor module harboring the KR2 domain derived from DEBS M2. In place of the natural (2S, 3R)-2-methyl-3-hdyroxydiketide product of DEBS M1, this hybrid module generates the enantiomeric (2R,3S)-diketide-ACP intermedi- ate. Replacement of KR1 by KR2 was not expected to significantly alter the rate of reduction of the ACP-bound ␤-ketoacylthioester intermediate (Fig. 2) (14), thereby enabling a comparison with the corresponding bimodular PKSs harboring wild-type DEBS M1 (Fig. 9). The observed 7-fold decrease in the turnover rate of the reference system is in excellent agreement with the differences in k cat previously reported when DEBS M2ϩTE was presented with chemo-enzymatically synthesized (2S, 3R)-diketide-ACP versus (2R, 3S)-diketide-ACP (22).

TABLE 1 Quantification of the occupancy of acceptor modules in chimeric bimodular PKSs
All bimodular PKSs included DEBS LDD and M1 plus the designated acceptor module. Only labeling data for the acceptor module are shown, because the upstream proteins are the same in all cases. Labeling of each acceptor module (2 M) was quantified at 1 and 3 min in the presence of both LDD (2 M) and M1 (2 M). Each measurement was performed in duplicate. The entire experiment was repeated with independently prepared proteins to verify the data trends. The TE domain of each acceptor module was inactivated by site-directed mutagenesis to abolish TE-catalyzed triketide chain release and consequent multiple turnover from this module.

Experimental Procedures
Reagents-Phusion Hot Start II polymerase was from Thermo Scientific. T4 DNA Ligase was from Invitrogen. Restriction enzymes were from New England Biolabs. All primers were synthesized by Elim Biopharm. For DNA purification, the GeneJET plasmid miniprep kit and the GeneJET gel extraction kit from Thermo Scientific were used. XL10-Gold ultracompetent cells were from Agilent; MAX Efficiency DH5␣ competent cells and One Shot BL21 (DE3) cells were from Invitrogen. All chemicals for buffer preparations were from Sigma-Aldrich. Isopropyl-␤-D-1-thiogalactopyranoside, kanamycin sulfate, and carbenicillin were from Gold Biotechnology. LB-Miller broth for cell cultures was from Fisher Scientific. Nickel-nitrilotriacetic acid affinity resin was from MC Lab. For anion exchange chromatography, the HiTrapQ column was from GE Healthcare. For all SDS-PAGE analyses, SDS-PAGE Mini Protean TGX precast gels were from Bio-Rad (4 -20% and 7.5%). For protein concentration, Amicon Ultra centrifugal filters were from Millipore. CoA, reduced NADPH, sodium propionate, propionic acid, methylmalonic acid, malonic acid, and magenesium chloride hexahydrate were from Sigma-Aldrich. ATP was from Teknova. Reducing agent tris(2-carboxyethyl )phosphine (TCEP) was from Thermo Scientific. UVette cuvettes (2 ϫ 10-mm path) were from Eppendorf. Sodium [1-14 C]-propionate was from Moravek Biochemicals.
Strains-Glycerol stocks were prepared by mixing LB medium containing 30% glycerol with the appropriate overnight culture in a 1:2 v/v ratio and were stored at Ϫ80°C. Seed cultures were inoculated with cells from the cell stock and grown overnight.
Plasmids-Plasmids harboring genes encoding individual PKS modules from different organisms were amplified from primary cosmid DNA of the corresponding PKS gene clusters (DEBS, RIFS, RAPS). Supplemental Table S1 specifies the primer sequences used for this purpose. All plasmids were constructed using the two-fragment Gibson assembly method or infusion cloning (Clontech), and their sequences were verified by DNA sequencing. Supplemental Tables S2 and S3 summarize the plasmid construction details for bimodular and trimodular PKS systems, respectively. Templates and primers used for the construction of DEBS M1-KR2 are shown in supplemental Table S5.
Site-directed Mutagenesis-To alter individual amino acid residues in expressed modules, either QuikChange site-directed mutagenesis (Agilent) or a site-directed mutagenesis strategy coupled to restriction-ligation was used. Mutagenesis primers are defined in supplemental Table S4. All mutations were verified by DNA sequencing.
Bacterial Cell Culture and Protein Purification-All proteins were expressed and purified using similar protocols. For holoproteins (where the ACP domain is post-translationally modi-  supplemental Table S8. fied with a phosphopantetheine arm), E. coli BAP1 cells (25) were used as the host. All proteins contained a C-terminal His 6 tag for purification. Cultures were grown on a 1-liter scale at 37°C to an A 600 of 0.6. Protein production was induced with 0.25 mM isopropyl-␤-D-1-thiogalactopyranoside, whereupon the temperature was adjusted to 18°C, and the cells were grown for another 16 h. The cells were harvested by centrifugation at 4420 ϫ g for 20 min and lysed by sonication in a buffer consisting of 50 mM sodium phosphate, 10 mM imidazole, 450 mM NaCl, and 10% glycerol, pH 7.6. The cell debris was removed by centrifugation at 25,000 ϫ g for 1 h. The supernatant was added to nickelnitrilotriacetic acid-agarose resin (2 ml of resin/liter of culture), and the resulting slurry was incubated at 4°C for 1 h. Thereafter, the mixture was applied to a Kimble-Kontes Flex column and washed with the above lysis buffer (25 column volumes). Additional washing was performed with 12.5 column volumes of wash buffer (50 mM phosphate, 25 mM imidazole, 300 mM NaCl, 10% glycerol, pH 7.6). Proteins were eluted in two steps (each with 6 column volumes). The first elution step utilized 75 mM phosphate, 150 mM imidazole, 40 mM NaCl, 10% glycerol, pH 7.6, whereas the second elution step was performed with 75 mM phosphate, 500 mM imidazole, 40 mM NaCl, 10% glycerol, pH 7.6.
Further purification was performed by anion exchange chromatography on an ÄKTA FPLC system using a HiTrapQ column. Buffer A contained 50 mM phosphate, 10% glycerol, pH 7.6, whereas buffer B contained 50 mM phosphate, 500 mM NaCl, 10% glycerol, pH 7.6. Protein concentrations were measured with the BCA protein assay kit (Thermo Scientific). Samples were stored as aliquots at Ϫ80°C until further use.
PKS Enzymatic Assays-The stoichiometric relationship between polyketide production and NADPH consumption in a reconstituted enzyme system has been previously verified, thereby enabling the use of a UV assay procedure to monitor PKS turnover (21). Reactions were performed on a 70-l scale and contained 400 mM sodium phosphate (pH 7.2), 5 mM TCEP, 10 mM MgCl 2 , 1 mM CoA, and 8 mM ATP. Methylmalonate/ malonate (1 mM) was converted to racemic methylmalonyl-CoA using the enzymes MatB (2 M) and methylmalonyl-CoA epimerase (4 M) (26). Propionyl-CoA was synthesized from propionate using PrpE (1 M) (27). The concentration of these enzymes and cofactors was selected to assure that the acyl-CoA supply was not limiting the rate of product formation. Reactions were initiated by the addition of PKS proteins (4 M each) along with a mixture of propionate (0.5 mM), methylmalonate/ malonate (1 mM), and NADPH (0.5 mM). The rate was monitored at 340 nm over 20 min. For product analysis by LC-MS, reactions were quenched with ethyl acetate, extracted twice with 450 l, dried in vacuo, and stored at Ϫ20°C.
Liquid Chromatography-Mass Spectrometry Analysis of Polyketides-Dried samples were reconstituted in 100 l of methanol, separated on a Gemini-NX C18 column (Phenomenex, 5 m, 2 ϫ 100 mm) connected to an Agilent 1260 HPLC over a 28-min linear gradient of acetonitrile from 3% to 95% in water, and subsequently injected into a 6520 Accurate-Mass QTOF mass spectrometer. Reduced and unreduced triketides and tetraketide products were located by searching for the theoretical m/z for the [ (2), and 2 M of each acceptor module in the presence of 400 mM sodium phosphate, pH 7.2, 5 mM TCEP, and 0.75 mM NADPH. By minimizing the amount of labeled propionyl-CoA in the reaction mixture, nonspecific transfer of the radiolabel directly from the LDD to an acceptor module was minimized. At specified time points, reactions were quenched by addition of Laemmli buffer, and samples were separated via 7.5% SDS-PAGE at 200 V for 44 min. The gel was washed with water for 5 min and stained with Simply-Blue TM SafeStain (Invitrogen) for 10 min. Following destaining with water for 5 min, the gel was mounted on a filter paper and dried in vacuo for 2 h using a Bio-Rad 543 gel dryer. The dried gel was imaged for 1 h lane by lane to quantify 14 C on individual protein bands using a Rita Star TLC Analyzer (Raytest). Peaks were integrated to quantify the radiolabel bound to each protein.