A novel and widespread class of ketosynthase is responsible for the head-to-head condensation of two acyl moieties in bacterial pyrone biosynthesis

The biosynthesis of photopyrones, novel quorum sensing signals in Photorhabdus, has been studied by heterologous expression of the photopyrone synthase PpyS catalyzing the head-to-head condensation of two acyl moieties. The biochemical mechanism of pyrone formation has been investigated by amino acid exchange and bioinformatic analysis. Additionally, the evolutionary origin of PpyS has been studied by phylogenetic analyses also revealing homologous enzymes in Pseudomonas sp. GM30 responsible for the biosynthesis of pseudopyronines including a novel derivative. Moreover this novel class of ketosynthases is only distantly related to other pyrone-forming enzymes identified in the biosynthesis of the potent antibiotics myxopyronin and corallopyronin.

product were both digested with restriction enzymes BamHI and HindIII and ligated using the T4 DNA Ligase. This mixture was then used to transform E. coli DH10B by electroporation (1250 V). After plasmid extraction the obtained plasmid was verified by sequencing at SeqIT GmbH (Germany, Kaiserslautern). The verified plasmid was used to transform E. coli BL21 (DE3) Star by electroporation. The construction of pCom10_pyrS was performed using the Gibson assembly method [3]. Therefore the vector was amplified via PCR using the oligonucleotides pCom10_Fw and pCom10_Rev. PyrS was amplified using the oligonucleotides pyrS_pCom_Fw and pyrS_pCom_Rev and pCOLA_pyrS as template, the oligonucleotides were previously modified with a 30 bp 3' overhang which are homologues to the amplified pCom10 product. Both products were then incubated with the Gibson assembly mix for 1 h at 50 °C. This mixture was then used to transform E. coli DH10B by electroporation as described earlier. The plasmid was obtained by using the extraction protocol described previously.

Electrotransformation of Pseudomonas strains. Pseudomonas putida KT2440
and Pseudomonas sp. GM30 were grown over night at 30 °C in liquid LB media. To prepare cells for electro transformation 2 mL of fresh liquid LB media were inoculated with an overnight culture (1:100) and were then grown for 3 hours at 30 °C. The cells were then centrifuged and washed twice with cold water. Centrifuged cells were then resuspended in 50 L cold water and cells were kept on ice. 1 L of plasmid was used to transform Pseudomonas strains by electroporation (2500 V).
Analytical scale culture extraction. In order to detect photopyrone production in the wildtype and mutant strains by means of HPLC/MS, 20 mL of liquid LB-medium, containing the appropriate resistant markers, were inoculated with an overnight culture to an optical density of OD 600 = 0.05 and cultivated for 3.5 h at 37 °C. Then S4 0.01 mM isopropyl--D-thiogalactopyranoside (IPTG) (Fermentas) and 2% Amberlite TM XAD16 (Sigma-Aldrich) were added to the culture, which was cultivated for 48 h at 16 °C. For detection of pseudopyronines 20 mL of liquid LB-medium, containing the appropriate resistant marker and 2% Amberlite TM XAD16, were inoculated with an overnight culture (1:100). Expression was induced with addition of 0.05% (v/v) dicyclopropyl ketone. The Pseudomonas containing cultures were then incubated for 72 h at 30 °C. After 48 h again 0.05% (v/v) of dicyclopropyl ketone was added. Cultures were harvested by centrifugation (4000 rpm, 10 min, 18 °C) followed by removal of the supernatant. Amberlite TM XAD16 resins were extracted with 30 mL of methanol and incubated for 1 h under constant rotation followed by a filtration step (Folded Filters (Quality), grade: 3 m/N, Munktell) to remove cells and resins. The elution step was repeated once with 10 mL of methanol. The methanol extract was then concentrated to dryness using a rotary evaporator. The solid residue was redissolved in 2 mL of methanol and a 1:10 dilution was analyzed by means of HPLC/MS. Extracts were analyzed using a Dionex UltiMate 3000 system coupled to a Bruker Daltonik AmaZon X mass spectrometer, a RP18-column (50 mm × 2.1 mm × 1.7 m; Waters GmbH) and an acetonitrile/0.1% formic acid in H 2 O gradient, ranging from 5 to 95% in 22 min at a flow rate of 0.6 mL/min. The production of ppyS mutants of 4 was calculated against standard concentrations of the main compound photopyrone D (4) produced by wildtype ppyS. The retention time of 4 under these conditions was 10.5 min.
Preparative extraction and purification. For the isolation of compounds 9-11 from Pseudomonas sp. GM30, the strain was cultivated in 6 L of LB-medium, with an addition of 2% Amberlite TM XAD16 for 3 days at 30 °C. Cultures were harvested by centrifugation (4000 rpm, 10 min, 18 °C) followed by removal of the supernatant.

S5
Amberlite TM XAD16 resins were extracted with methanol and incubated for 1 h under constant rotation followed by a filtration step to remove cells and resins. The methanol extract was then concentrated to dryness using a rotary evaporator. The solid residue (4.6 g) was redissolved in 10 mL of water, then 20 mL of ethyl acetate was added and the mixture was shacked in a separating funnel. This step was repeated two times with the addition of 20 mL of ethyl acetate and separation. The NMR. 1D and 2D nuclear magnetic resonance (NMR) spectra for purified compounds were recorded on a Bruker DRX 500 spectrometer using deuterated dimethyl sulfoxide as solvent and internal standard.  [4] searches in the PDB [5], to identify the most similar available structure in the PDB. This resulted in the identification of OleA (sequence identity 27%, E-value 1e-14, PDB: 3S21) from Xanthomonas campestris for PpyS and OleA (sequence identity 37%, E-value 4e-10, PDB: 3S21) for PyrS-. These template structures were used to create a sequence alignment applying the ClustalW algorithm [6]. The homology models were generated using the Homology Modelling Tool integrated in MOE 2012.10 (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Canada) and the ClustalW sequence alignment was imported. A series of ten models was created, for further processing the one with the highest packing quality score was chosen and energy minimized applying the AMBER12EHT (integrated in MOE) force field. All figures showing protein structures in this work, were created using MOE.

HR-ESI-MS.
Docking. Protein-ligand docking calculations were carried out using the program GOLD (version 5.2) [7] using the empirical scoring function for advanced proteinligand docking CHEMPLP [8]. For each docking study the result with the highest docking score is shown in this work.
Phylogenetic analysis. A PHYML [9] tree (50 bootstraps) was calculated using a ClustalW alignment (gap opening: 10; gap extension: 0.1), which was generated using the collected ketosynthases. For visualization and calculation of the alignment as well as the PHYML tree the Geneious software (Biomatters Ltd., New Zealand) was used. Table S1: Strains used in this work.

Strain
Genotype Reference Invitrogen Pseudomonas sp.

GM30
Wildtype [11] Pseudomonas putida      represented as spheres, this residue is predicted to be involved in the dimerization of PpyS by interacting with Asp137. The mutation of Arg121 led to a complete loss of photopyrone production. Furthermore Glu330 is shown in cyan spheres, this residue was mutated as a control, which should not influence the photopyrone production as indeed shown in Figure S1.