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The industrial anaerobe Clostridium acetobutylicum uses polyketides to regulate cellular differentiation


Bacterial strains and media

C. acetobutylicum ATCC 824 was cultured in an anaerobic chamber (Coy Laboratory Products) containing an atmosphere of 97% nitrogen and 3% hydrogen. 2xYTG medium67 contained 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 10 g/L glucose (unless noted otherwise) with the pH adjusted to 5.2 for liquid media, and 5.8 for solid media (also containing 15 g/L agar). Clostridial growth medium (CGM)68 contained 30 g/L glucose (unless noted otherwise), 6.25 g/L yeast extract, 2.5 g/L ammonium sulfate, 1.25 g/L NaCl, 2.5 g/L asparagine, 0.95 g/L monobasic potassium phosphate, 0.95 g/L dibasic potassium phosphate, 0.5 g/L magnesium sulfate heptahydrate, 13 mg/L manganese sulfate heptahydrate, and 13 mg/L iron sulfate heptahydrate with the pH adjusted to 6.4. P2 medium69 contained 80 g/L glucose (unless noted otherwise), 1 g/L yeast extract, 2.2 g/L ammonium acetate, 0.5 g/L potassium phosphate monobasic, 0.5 g/L potassium sulfate dibasic, 0.2 g/L magnesium sulfate heptahydrate, 1 mg/L para-aminobenzoic acid, 1 mg/L thiamine hydrochloride, 10 µg/L biotin, 10 mg/L manganese sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, and 10 mg/L NaCl with the pH adjusted to 6.4. Clostridial basal medium (CBM)70 contained 10 g/L glucose, 0.5 g/L monobasic potassium phosphate, 0.5 g/L dibasic potassium phosphate, 4 g/L tryptone, 0.2 g/L magnesium sulfate heptahydrate, 10 mg/L manganese sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 1 mg/L para-aminobenzoic acid, 1 mg/L thiamine hydrochloride, and 2 µg/L biotin with the pH adjusted to 6.9. For solid CGM plates, 15 g/L agar was added. CBM-S (used for liquid sporulation assays) was identical to CBM except 50 g/L glucose was used, and 5 g/L CaCO3 was added just prior to inoculation of cultures. E. coli TOP10 (Thermo Fischer Scientific) was grown in Luria-Bertani (LB) medium at 37 °C. For the appropriate Clostridium strains, culture media was supplemented with erythromycin (Ery; 40 µg/mL for solid media, 80 µg/mL for liquid media) and/or thiamphenicol (Th; 5 µg/mL for solid and liquid media). Kanamycin (Kan; 60 µg/mL) or chloramphenicol (Cm; 25 µg/mL) were added to E. coli culture media as indicated. Clostridium and E. coli strains were maintained as 20% v/v glycerol stocks stored at −80 °C.

Plasmid construction

Oligonucleotides were provided by Integrated DNA Technologies (Supplementary Table 5). Phusion polymerase (NEB) was used for all PCR reactions. For isolation of genomic DNA from C. acetobutylicum ATCC 824, an alkaline lysis method was used71. C. acetobutylicum was cultured overnight in 2xYTG medium to stationary phase (OD600 > 2.0), at which point 10 mL of culture was centrifuged at 3500×g for 15 min (room temperature). The supernatant was discarded and the cell pellet was resuspended in 5 mL SET buffer (75 mM NaCl, 25 mM EDTA pH 8.0, 20 mM Tris-HCl, pH 7.5). Lysozyme was added to a final concentration of 2 mg/mL, and the solution was gently mixed. The mixture was incubated at 37 °C for 60 min with gentle mixing performed every 15 min. Following the incubation period, 660 µL of lysis buffer (1 M NaOH, 10% w/v SDS) was added, and the solution was thoroughly mixed. Proteinase K was added to a final concentration of 0.5 mg/mL, and the solution was incubated at 55 °C for 1 h. Following this incubation period, an equal volume of phenol:chloroform (1:1) was added, and the solution was mixed by inversion for 5 min. The solution was then centrifuged at 3500×g for 15 min (room temperature), and the upper aqueous phase was discarded by pipetting. To the organic phase, 3 M sodium acetate was added (10% v/v in final solution). To precipitate genomic DNA, 2 volumes of ethanol were added and the solution was mixed. Precipitated genomic DNA was collected using a glass hook, and washed with 70% ethanol. The washed DNA was then dried over nitrogen gas for 15 min, resuspended in low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), and dissolved by incubation at 50 °C.

For constructing plasmid pKO_mazF_mod (which would later serve as a template for pKO_pks), primers pKON_Fo and pKON_Ro were used to PCR amplify a 5.0 kb region from the pKO_mazF template15. This step was necessary to remove a 677 bp region from the pKO_mazF backbone, which we were unable to amplify via PCR. The 5.0 kb PCR product was gel purified, digested at the 5′ and 3′ ends using PshAI, ligated to form pKO_mazF_mod, and transformed into E. coli TOP10. Transformant clones were screened by purified plasmid test digestion, and Sanger sequencing was used to confirm the sequence of the final pKO_mazF_mod clone. For constructing plasmid pKO_pks (for deletion of the pks gene from C. acetobutylicum), a 3.8 kb region containing colE1, repL, bgaR, and mazF were PCR amplified from pKO_mazF_mod using primers pKO_F and pKO_R. Additionally, primers UHR_Fo and UHR_Ro, and DHR_Fo and DHR_Ro were used to PCR amplify 1 kb regions representing the upstream and downstream homologous regions (UHR and DHR) flanking the pks gene (CA_C3355) from a C. acetobutylicum ATCC 824 genomic DNA template. The chloramphenicol/thiamphenicol resistance gene and constitutive promoter (Pptb
from C. acetobutylicum) were PCR amplified from pKO_mazF_mod using primers CMR_F and CMR_R (1.1 kb region). These four PCR products were ligated via Gibson assembly and transformed into E. coli TOP10. Transformant clones were screened by purified plasmid test digestion, and Sanger sequencing was used to confirm the sequence of the final pKO_pks clone. For constructing plasmid pAN315 (necessary for methylation of pKO_pks and pCKO_pks prior to transformation into C. acetobutylicum), a 6.4 kb region from plasmid pAN172 was amplified using primers pAN1_F and pAN1_R, and a 1.0 kb region containing the kanamycin resistance gene from vector pCOLA_Duet was amplified using primers KAN_Fo and KAN_Ro. The gel extracted PCR products were ligated via Gibson assembly and transformed in to E. coli TOP10. Transformant clones were screened by purified plasmid test digestion, and Sanger sequencing was used to confirm the sequence of the final pAN3 clone. For constructing plasmid pCKO_pks (for expression of the pks gene under the constitutive crotonase [crt] promoter from C. acetobutylicum), primers CPKS_Fo and CPKS_Ro were used to PCR amplify the 5.4 kb C. acetobutylicum ATCC 824 pks gene (CA_C3355) with appropriate overhangs for Gibson assembly. The pWIS_empty vector71 backbone (containing the constitutive crt promoter upstream of the multiple cloning site) was PCR amplified using primers pWIS_F and pWIS_R yielding a 5.0 kb product. The two gel purified PCR products were ligated via Gibson assembly and transformed into E. coli TOP10. Transformant clones were screened by purified plasmid test digestion, and Sanger sequencing was used to confirm the sequence of the final pCKO_pks clone.

For constructing plasmid pET24b_pks, the 5.4 kb C. acetobutylicum ATCC 824 pks gene (CA_C3355) was amplified from C. acetobutylicum genomic DNA using primers PKS_Fo and PKS_Ro. Doubly digested vector pET24b (EcoRI/XhoI digested) was ligated with doubly digested pks PCR product (EcoRI/XhoI digested), and the ligation product was transformed into E. coli TOP10. Transformant clones were screened by purified plasmid test digestion, and Sanger sequencing was used to confirm the sequence of the final pET24b_pks clone.

Electro-transformation of C. acetobutylicum

Prior to transformation into C. acetobutylicum, vector pKO_pks was co-transformed with pAN3 into E. coli TOP10 via electroporation. This procedure permitted methylation of pKO_pks necessary for overcoming the native restriction-modification system active in C. acetobutylicum72. Plasmid purification of E. coli pKO_pks/pAN3 liquid culture was performed, and the resulting plasmid mixture was used for electroporations of C. acetobutylicum using the previously published method72 which we detail here. To prepare electrocompetent cells, a single colony of C. acetobutylicum from a 2xYTG plate (more than 1 week old) was heat shocked at 80 °C for 10 min, and used to inoculate 10 mL CGM. After incubating overnight at 37 °C and reaching mid-exponential phase (OD600 0.4–0.9), 6 mL culture was used to inoculate 54 mL fresh 2xYTG. This subculture was incubated at 37 °C until reaching OD600 1.2 (~5 h), at which point the subculture was centrifuged at 3500×g for 20 min (4 °C). Following removal of the supernatant, the cell pellet was resuspended in 20 mL ice-cold electroporation buffer (EPB; 270 mM sucrose, 5 mM NaH2PO4, pH 7.4). The resuspended cells were centrifuged at 3500×g for 10 min (4 °C), the supernatant was discarded, and the cell pellet was resuspended in 20 mL ice-cold EPB. After a final pelleting by centrifugation at 3500×g for 10 min (4 °C), the supernatant was discarded, and the pellet was resuspended in 2.3 mL ice-cold EPB. This final resuspension was used as the stock of electrocompetent cells. Electro-transformations were performed by first mixing (over ice) 500 µL competent cells and ~20 µL of plasmid DNA solution (1–5 µg total) to be transformed. The mixture was transferred to a 0.4 cm electroporation cuvette, and allowed to incubate on ice for 15 min. Electroporations were performed with the following parameters: voltage, 2000 V; capacitance, 25 µF; resistance, infinite Ω. Following electroporation, samples were resuspended in 1 mL 2xYTG and transferred to a tube containing 9 mL 2xYTG. After mixing by inversion, the cultures were allowed to recover at 37 °C for 4 h. The recovery cultures were centrifuged at 3500×g for 15 min (room temperature), and the supernatant was discarded. The cell pellet was resuspended in 500 µL 2xYTG, and 100 µL was plated onto solid 2xYTG plates supplemented with the appropriate antibiotic. The plate was incubated at 37 °C for 2–3 days, and transformant colonies were subjected to PCR-based verification.

Deletion of pks gene and genetic complementation

Targeted KO of the pks gene (ca_c3355) in C. acetobutylicum ATCC 824 was achieved using the previously published method15. In detail, 5 µg of methylated pKO_pks/pAN3 plasmid mixture was transformed into C. acetobutylicum using the method described above. Following recovery in liquid 2xYTG medium for 4 h, the cell pellets were collected by centrifugation (3500×g, 15 min, room temperature), resuspended in 0.5 mL of fresh liquid 2xYTG, and 100 µL of the resuspended cell culture was plated on solid 2xYTG + 5 µg/mL Th + 40 mM β-lactose plates. Under these plating conditions, only cells which have undergone the desired double crossover homologous recombination event are expected to survive. Counterselection of the vector backbone is provided by the lactose-inducible promoter (PbgaL
), which drives the toxin gene mazF on the pKO_pks vector backbone. Following this plating procedure, roughly 10 colonies were observed on the 2xYTG + 5 µg/mL Th + 40 mM β-lactose plates. Of these 10 colonies, four were twice restreaked and subjected to colony PCR verification. Four sets of primers were used as the basis of colony PCR verification, as detailed in Supplementary Fig. 2.

For generating the pks genetic complementation strain [∆pks (pCKO_pks)], electroporation of ∆pks C. acetobutylicum with a pCKO_pks/pAN3 plasmid mixture was performed as described above. Following electroporation and recovery, cultures were plated on solid 2xYTG + 5 µg/mL Th + 40 µg/mL Ery media. After twice restreaking on solid 2xYTG + 5 µg/mL Th + 40 µg/mL Ery media, potential colonies harboring pCKO_pks were screened via colony PCR using primers pCKO_F and pCKO_R.

LC-HRMS metabolomic analysis

For untargeted metabolomic comparisons of wild-type and ∆pks C. acetobutylicum, single colonies of each strain were heat shocked at 80 °C for 10 min and used to inoculate 10 mL of liquid CGM (30 g/L glucose). Following overnight incubation (stagnant) until reaching OD600 ~1.0, these cultures were used to inoculate 10 mL of liquid CGM (80 g/L glucose) with a 10% inoculum for subculturing. After ~5 h (OD600 ~1.0) of stagnant growth, the subcultures were used to inoculate quadruplicate flask fermentations (70 mL CGM, 80 g/L glucose + 5 µL Antifoam 204, 3% inoculum) agitated via magnetic stir bars. Calcium carbonate (6 g/L) was supplemented to the fermentation media for pH buffering. All culturing was performed at 37 °C. About 5 µg/mL Th was included in all cultures of ∆pks with the exception of the final fermentation culture, as certain antibiotics are known to perturb the ABE fermentation phases. Samples of fermentation broth (1 mL) from each replicate were taken during early stationary phase and extracted with 3 mL of 2:1 chloroform methanol. The mixtures were vortexed, separated via centrifugation (2700×g, 10 min), and the bottom chloroform-rich layer was transferred to a glass vial. These organic extracts were dried with nitrogen gas, resuspended in 100 µL methanol, and 10 µL was injected onto an Agilent Technologies 6520 Accurate-Mass QTOF LC-MS instrument fitted with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A linear gradient of 2–98% CH3CN (vol/vol) over 40 min in H2O with 0.1% formic acid (vol/vol) at a flow rate of 0.5 mL/min was used. The metabolomic analysis platform XCMS16 (The Scripps Research Institute) was used to compare the metabolomes of wild-type and ∆pks strains based on the quadruplicate LC-HRMS data. MS peaks unique to ∆pks (Fig. 1a) were identified using the following parameters: p-value < 0.01, fold change > 10.0, peak intensity > 5000.

Bioreactor fermentations

To compare ABE fermentation profiles of wild-type and ∆pks C. acetobutylicum, bioreactor fermentations were carried out in DASGIP Bioreactors (4 × GPI 100 Vessels, DASGIP Bioblock System) with 500 mL working volumes. Overnight cultures (10 mL CGM, 30 g/L glucose, stagnant, 34 °C) inoculated with heat shocked individual colonies of C. acetobutylicum were cultured until reaching OD600 ~1. A 10% inoculum was then used to start a subculture (30 mL P2 media, 80 g/L glucose, stagnant, 34 °C), and the subculture was incubated until reaching OD600 ~1. The 30 mL subcultures were then aseptically transferred into individual DASGIP Bioreactors pre-loaded with 500 mL P2 medium (80 g/L glucose, 100 µL Antifoam 204, 34 °C). The fermentations were allowed to proceed for 54 h with periodic sampling for optical density measurements, fermentation product analysis, and quantification of compounds 1, 2, and 3. The temperature was maintained at 34 °C throughout the fermentation, agitation was provided by stirring at 200 rpm, and the pH was maintained above 5.0 via automatic addition of 3 M NH4OH. To maintain anaerobic conditions, oxygen-free nitrogen gas was sparged at a rate of 2 sL/h for the duration of the fermentation. For quantification of compounds 1, 2, and 3, 1 mL of fermentation broth was mixed with 3 mL ethyl acetate, vortexed, separated via centrifugation (2700×g, 10 min, room temperature), and the upper organic layer isolated. The organic layer was dried by rotary evaporation, resuspended in 200 µL methanol, and 20 µL was injected onto an Agilent Technologies 6120 Quadrupole LC-MS (with DAD) instrument fitted with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A linear gradient of 2–98% CH3CN (vol/vol) over 40 min in H2O with 0.1% formic acid (vol/vol) at a flow rate of 0.5 mL/min was used. Compounds 1, 2, and 3 were identified by UV absorption (240 nm) as demonstrated in Fig. 1b, and were quantified by the integrated peak area (absorbance at 240 nm).

Fermentation analytical procedures

A spectrophotometer was used to determine cell densities by measuring the optical density at 600 nm (OD600). A Shimadzu Prominence UFLC system fitted with a Biorad Aminex HPX-87H column (300 mm × 7.8 mm) was used to analyze C. acetobutylicum fermentation broth for the concentration of glucose and fermentation products (acetate, butyrate, lactate, acetone, butanol, and ethanol). Samples of fermentation broth (1 mL) were first pelleted by centrifugation at 10,000×g for 3 min, followed by filtration of the supernatant using a 0.22 micron PVDF syringe filter. Samples of filtered supernatant (20 µL) were injected onto the UFLC system with 0.01 N sulfuric acid mobile phase flowing at 0.7 mL/min, column temperature of 35 °C, and were chromatographed for 35 min. Analytes were detected by use of a refractive index detector (used to quantify concentrations of glucose, acetate, lactate, butanol, and ethanol) and a diode array detector (used to quantify concentrations of butyrate (208 nm) and acetone (265 nm)).

RNA isolation and RNA-Seq analysis

Samples (10 mL) of fermentation broth were taken in biological triplicate from bioreactor fermentations of wild-type and ∆pks C. acetobutylicum 26 h post inoculation. The samples were centrifuged (4000×g, 10 min, 4 °C) and the pellets were resuspended and stored in RNAprotect Cell Reagent (Qiagen). Total RNA was extracted using an RNeasy Mini Kit (Qiagen) according the manufacturer’s instructions. An on-column DNase treatment was performed using DNase I (RNase-free) (NEB). RNA quality control, library construction, and library sequencing were performed by the University of California-Berkeley QB3 Functional Genomics Laboratory and Vincent J. Coates Genomic Sequencing Laboratory. RNA quality and concentration was assessed using a nanochip on an Agilent 2100 Bioanalyzer. Bacterial 16S and 23S rRNA was removed using a RiboZero Kit (Illumina). The resulting messenger RNA (mRNA) was converted to an RNA-Seq library using an mRNA-Seq library construction kit (Illumina). RNA library sequencing was performed on an Illumina HiSeq4000 with 50 bp single end reads. Sequencing reads (50 bp) were processed and mapped to the C. acetobutylicum ATCC 824 genome (NCBI accession NC_003030.1 [chromosome] and NC_001988.2 [megaplasmid]) using CLC Genomics Workbench 9.0 with default parameters. Reads that did not uniquely align to the genome were discarded. Differences in gene expression between wild-type and ∆pks C. acetobutylicum were calculated using the same software. Genes were considered differentially expressed with p-value < 0.003 (based on an unpaired t-test, n = 3) and |normalized fold change |> 2.0. False discovery rate corrections to p-values were calculated in CLC Genomics Workbench 9.0 using a published method73. Results of the RNA-Seq analysis are presented in Supplementary Data 1. STRING analysis30 was performed to determine putative protein–protein interactions between the differentially expressed genes revealed by RNA-Seq analysis.

Phenotype comparison assays

Liquid sporulation assays were performed with minor modifications74. Samples were taken from biological triplicate liquid cultures after 5 days of incubation (30 mL CBM-S, 37 °C). The 20 µL samples were heat shocked (80 °C, 10 min), dilutions (101–106) were spotted on 2xYTG plates, and colonies were enumerated after 30 h of incubation (37 °C) to calculate the number of heat-resistant colony forming units (cfu/mL). For chemical complementation of ∆pks in the liquid sporulation assay, purified clostrienose (final concentration 3.5 µM) was supplemented in CBM-S cultures of ∆pks C. acetobutylicum at the time of inoculation. Since compound 3 was added as a concentrated solution in methanol, the equivalent volume of methanol (60 µL) was added to all other liquid sporulation assay cultures (wild-type and non-complemented ∆pks) to control for this effect.

For solid sporulation assays, a previously described method75 was employed with some modifications. In detail, heat shocked (80 °C, 10 min) individual colonies were cultured in liquid media (10 mL CGM, 37 °C) for 24 h. Cultures were diluted by a factor of 106 and plated on solid CBM. Sampling was conducted 1, 2, 3, 4, and 6 days following initial plating. For sampling, three individual colonies were combined and thoroughly resuspended in 60 µL of liquid CGM. 20 µL of the resuspended colony mixture was then heat shocked (80 °C, 10 min), dilutions (101–106) were spotted on 2xYTG plates, and colonies were enumerated after 30 h of incubation (37 °C) to calculate the number of heat resistant colony forming units (cfu/colony). All samples were performed as biological triplicates.

Granulose accumulation assays were performed via iodine staining53. Mid-log phase (OD600 ~0.6) liquid cultures (P2, 37 °C) were plated on solid 2xYTG medium with elevated glucose levels (50 g/L) to enable granulose production. The plates were incubated at 30 °C for 4 days, at which point they were stained by exposure to a bed of iodine crystals for 10 min. The plates were then allowed to destain for 10 min prior to imaging. For chemical complementation of ∆pks in the granulose accumulation assay, purified clostrienose (final plate concentration 4.0 µM) was embedded in solid 2xYTG plates prior to plating of ∆pks culture. Since clostrienose was added to 2xYTG plates as a concentrated solution in methanol (mixed into the molten media prior to solidification), the equivalent volume of methanol (6 µL) was added to all other 2xYTG plates used in the granulose accumulation assays to control for this effect.

Individual colonies of C. acetobutylicum cultured on CBM plates for 48 h (37 °C) were viewed and imaged using a Leica MZ16 F dissecting microscope fitted with a Leica DFC300 FX camera.

Oil spreading assays were performed as previously described76,77,78. In detail, A 400 mL glass beaker (7.5 cm inner diameter) was filled with 300 mL water, and a 10 µL droplet of paraffin lamp oil was added and allowed to spread into a thin film on the water surface over a 5 min period (~25 mm in diameter). Solutions of surfactin, purified clostrienose, and a potassium phosphate buffer control were prepared at the indicated pH and concentration (prepared in 10 mM potassium phosphate buffer). About 10 µL of sample was carefully added to the center of the oil film, and the effect on the oil film was observed. Samples with surfactant activity are expected to form stable circular clearing zones in the oil film, with more potent surfactant activity corresponding to larger clearing zones (or complete dispersion of the oil film for very-high-surfactant activity). Drop collapse assays were performed77,79,80. Circular microwells (8 mm inner diameter) within the polystyrene lid of a 96-microwell plate (12.7 × 8.5 cm) were coated with a thin layer of paraffin lamp oil by adding 2.0 µL of paraffin lamp oil to each well, and allowing the droplet to spread evenly over the microwell over a 1 h period. Samples of surfactin, purified clostrienose, and potassium phosphate control were prepared as for the oil spreading assays. About 5 µL of sample was carefully added to the center of the microwell. After 5 min, the shape and degree of spreading of the droplet was observed. While buffer controls are expected to form stable beads on the microwell surface, samples containing surfactant are expected to collapse and spread over the microwell surface, with more potent surfactant activity corresponding to a higher degree of droplet spreading.

Purification and in vitro analysis of PKS protein

To purify the His6-tagged PKS protein for in vitro analysis, pET24b_pks was transformed into E. coli BAP1. A single colony was inoculated into 10 mL LB + 50 µg/mL kanamycin (Kan) for overnight growth at 37 °C. About 7 mL of overnight culture was used to inoculate 700 mL LB + 50 µg/mL Kan, and the culture was shaken at 240 rpm and 37 °C until reaching OD600 of 0.5. After icing the culture for 10 min, isopropyl thio-β-d-galactoside (IPTG) was added to a final concentration of 0.1 mM to induce protein expression, and the culture was incubated at 16 °C for 16 h. The cells were harvested by centrifugation (5500×g, 4 °C, 20 min), resuspended in 25 mL lysis buffer (25 mM HEPES, pH 8.0, 0.5 M NaCl, 5 mM imidazole), and lysed by homogenization over ice. Cell debris was removed by centrifugation (17,700×g, 4 °C, 60 min), and Ni-NTA agarose resin was added to the supernatant (2 mL/L culture). The mixture was nutated at 4 °C for 1 h, loaded onto a gravity flow column, and the PKS protein was eluted with increasing concentrations of imidazole in Buffer A (20 mM HEPES, pH 8.0, 1 mM DTT). Purified PKS protein was concentrated and buffer exchanged into Buffer A + 10% glycerol using a Vivaspin Centrifugal Concentrator. Aliquots of purified PKS protein were aliquoted and flash frozen in liquid nitrogen. The approximate protein yield was 5 mg/L (203 kDa).

A PKS loading assay with 14C-labeled substrate contained, in a total volume of 15 µL, 4 mM ATP, 2 mM MgCl2, 1 mM TCEP, 0.5 mM CoA, 2-14C-malonic acid (0.1 µCi), 10 µM MatB, 17 µM PKS, and 50 mM HEPES, pH 8.0. After 2 h of incubation at 25 °C, samples were quenched by adding an equal volume of 1× SDS sample buffer. Following SDS–PAGE analysis with a 4–15% TGX gel (Criterion), the gel was dried for 2 h at 50 °C and exposed on a storage phosphor screen (20 × 25 cm; Molecular Dynamics) for 5 days. Radiolabeled protein was imaged on a Typhoon 9400 phosphorimager (Storage Phosphor mode, 50 μm resolution; Amersham Biosciences).

For in vitro product assays (50 µL) of the PKS protein, 8 µM of PKS was incubated with malonyl-CoA (2 mM) in phosphate buffer (100 mM, pH 7.0) at room temperature for 2 h to generate compound 4, identified by comparison to a chemical standard. NADPH (2 mM) was added to the assay to generate compounds 5 and 6, both of which were identified by comparison to chemical standards. Following incubation, the assay mixtures were extracted twice with 100 µL of 99% ethyl acetate/1% acetic acid (v/v). The organic extracts were dried and resuspended in 100 µL of methanol, and 10 µL was injected onto an Agilent Technologies 6120 Quadrupole LC-MS (with DAD) instrument fitted with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). A linear gradient of 2–98% CH3CN (vol/vol) over 40 min in H2O with 0.1% formic acid (vol/vol) at a flow rate of 0.5 mL/min was used. LC-HRMS analysis of the assay extracts was performed on an Agilent Technologies 6520 Accurate-Mass QTOF LC-MS instrument fitted with an Agilent Eclipse Plus C18 column (4.6 × 100 mm) using the same solvent gradient and flow rate described above.

Data availability

RNA-seq data generated in this study have been deposited in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-6019. All other relevant data are available from the authors upon request.



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