Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor

Abstract Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET‐domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS‐PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.


Supplementary Figures
Supplementary Figure 1

Supplementary Tables
Supplementary Table 1  Comparison of A-domain binding pockets. A) Crystal structure of the GrsA PheA-domain from gramicidin S biosynthesis (PDB code 1amu; [1] ). The substrate is shown as sticks in magenta, the binding pocket residues in green. Residues K517 and D235 are highly conserved and bind the carboxylate and amino group of the substrate, respectively. B)-C) Homology models were prepared using the Swiss-Model server [2] with 1amu as a template. Sequence identities to the template are 25.3% (BurA-A) and 29.1% (ATRR-A [3] ). Models of the substrates have been energy minimized in Chem3D (Perkin Elmer) and manually docked into the active sites in an orientation similar to that of Phe in GrsA-A. Hence, the positioning and conformation of the substrate is only an approximation. In both models, the loop bearing D235 in GrsA-A is replaced with a shorter loop not containing an acidic residue. Instead, an acidic residue (D606 in BurA-A and E304 in ATRR-A) is found in a position where it could electrostatically interact with the sulfonium or ammonium residue of the substrate, respectively. D) Binding pocket residues of GrsA-A, BurA-A and ATRR-A. It is not clear, whether the loop carrying the residues marked in blue has been modelled reliably.

Preparation of Gene Knockout Mutants of B. thailandensis E264
An overnight preculture (0.5 mL) of B. thailandensis Pbur in LB medium (2 mL) supplemented with tetracycline (45 μg mL -1 ) was inoculated into LB medium (50 mL) supplemented with tetracycline (45 μg mL -1 ) and cultured in a 300 mL buffled Erlenmeyer flask at 30 °C with orbital shaking until an OD600 from 0.5 to 0.8 was reached. Cultured cells were centrifuged at 2,500 × g and 20 °C for 10 min. The obtained cells were washed with a 300 mM sucrose solution (3 ×) and resuspended in 300 μL sucrose solution. Subsequently, 100 μL of the washed B. thailandensis Pbur cells were subjected to electroporation (200 kV) with knockout plasmids (2-5 µg, see above). The transformed cells were precultured in LB broth (1 mL) for 4-6 h at 30 °C with shaking and then plated on either LB or nutrient agar plates with tetracycline (45 µg mL -1 ) and kanamycin (150 µg mL -1 ). After 3 days, a few positive colonies were observed and confirmed by PCR (see below) using purified genomic DNA from the respective mutants.

Heterologous Production and Purification of His6-BurB
The gene fragment burB was amplified by PCR with the primer pair burB-fw-NheI/burB-rv-HindIII using the DeepVent polymerase and the resulting amplicon was purified by the illustra GFX PCR DNA and Gel Band Purification Kit followed by subcloning into pJET1.2, generating pJET-burB. This plasmid was restricted with NheI/HindIII and the obtained gene fragment burB was ligated into NheI/HindIII-restricted pET28a (+), to yield pET28a-burB. Subsequently the plasmid was introduced into E. coli Rosetta2 (DE3) for heterologous protein expression.
E. coli Rosetta2 (DE3) pET28a-burB was cultured in LB medium (2 mL) with chloramphenicol (25 μL mL -1 ) and kanamycin (50 μL mL -1 ) overnight. These cultured cells were inoculated into LB medium (50 mL) with added chloramphenicol (25 μL mL -1 ) and kanamycin (50 μL mL -1 ) in a 300 mL baffled Erlenmeyer flask and grown at 30 °C with orbital shaking until an OD600 = 1.5 was reached. The bacterial culture was cooled in an ice bath for 20 min and IPTG (200 μM, final concentration) was added followed by further cultivation at 20 °C with orbital shaking for 18 h. The overnight cultured cells were harvested by centrifugation at 20 °C and 8,000 × g for 5 min and kept at -25 °C until usage. 15 mL lysis buffer [50 mM Tris HCl (pH 8.0), 200 mM NaCl, 2 mg mL -1 lysozyme] were added to the cells and the mixture was incubated at 37 °C for 1 h. After DNase A (5 μL) was added, the cells were lysed by usage of a sonicator (BANDELIN SONOPULS HD2200) and centrifuged at 10,000 × g and 4 °C for 30 min. The resulting supernatant was filtered through a Chromafil ® PET-45/15 MS (Macherey-Nagel) filter and subjected to a Ni-IDA agarose (Biontex) column (2 mL

Methylation of L-Methionine Through BurB
Purified His6-BurB (10 μM) was added to phosphate buffer (50 mM, pH 8.0) containing 20 mM NaCl, 1 mM L-methionine and 1 mM S-adenosylmethionine. As a control, purified BurB was heat-inactivated at 80 °C for 25 min and used in the same way. The resulting mixtures were incubated at 30 °C for 90 min. Subsequently, 50 μL of the reaction was diluted with 50 μL of a 0.5 M NaHCO3 solution and 10 μL of a 1-fluoro-2,4-dinitrobenzene (DNFB) solution (1% w/v in acetonitrile) were added. After incubation at 60 °C for 60 min the reaction mixture was quenched with 12.5 μL HCl (2 M) and diluted with 122.5 μL methanol. As a reference, reaction buffer containing 1 mM S-methylmethionine was treated in the same way. The resulting solutions were filtered through a PTFE syringe filter and subjected to HR-LCMS analysis.

Gonyol synthesis
Gonyol was prepared by following a known procedure [10] from ethyl-bromoacetate and 3-(methylthio)propionaldehyde through reformatsky reaction followed by ester hydrolysis and subsequent methylation with iodomethane.

13
C3 DMSP was synthesised according to Chambers et. al. [11] as hydrochloride from 13 C3 acrylic acid and dimethylsulfide by bubbling gaseous HCl into a solution of both educts in dichloromethane for 20 minutes and subsequent concentration in vacuo.

Stable isotope labelling of Malleicyprol with 13 C3-DMSP
B. thailandensis PburΔburI was grown in a 300 mL baffled Erlenmeyer flask filled with 100 mL MM9 medium supplemented with 45 mg L -1 tetracycline, 150 mg L -1 kanamycin and 137 mg L -1 13 C3 DMSP at 30 °C with shaking at 150 rpm for 24 h. Subsequently, the culture was extracted with ethyl acetate (2 ×), concentrated in vacuo and redissolved in methanol for LC-HRMS analysis.

Metabolite extraction and metabolomics analysis
B. thailandensis E264, B. thailandensis Pbur, B. thailandensis PburΔburA [8] , B. thailandensis PburΔburB, B. thailandensis PburΔburI, B. thailandensis PburΔburD and B. thailandensis PburΔburE were each grown in a 300 mL baffled Erlenmeyer flask filled with 100 mL MM9 medium supplemented with the appropriate antibiotic and 2% (w/v) XAD16N at 30 °C with shaking at 150 rpm for 24 h. Subsequently, the XAD16N resin was separated from the culture broth by filtration through Miracloth (Merck Millipore). The XAD16N resin was washed with water and eluted with methanol (40 mL) followed by elution with ethyl acetate (20 mL). Both eluted fractions were combined and concentrated under reduced pressure to yield supernatant extracts. For cell extracts, 50 mL of filtered culture broth was pelleted by centrifugation (6000 g, 10 min). Subsequently the pellet was resuspended in 20 mL methanol, sonicated and incubated for 1 h. Cell debris was removed by centrifugation (6000 g, 10 min) and filtration through a 0.2 μm PTFE ROTILABO syringe filter. The resulting methanol solution was concentrated in vacuo to yield cell extracts. Cell and supernatant extracts were dissolved in methanol to yield a concentration of 1.75 mg mL -1 of crude extract mass for supernatant and 1.64 mg mL -1 for cell extracts. All extracts were subjected to LC-HRMS analysis in two technical replicates for subsequent metabolomics analysis using the software Compound Discoverer (2.1, SP1) from Thermo Fisher Scientific. Additionally MM9 medium substituted with tetracycline (45 μg mL -1 ) and kanamycin (150 μg mL -1 ) was treated in the same way as mentioned above for supernatant extracts. This medium control was used to subtract medium components when supernatant extracts were analysed. Both extract types were analysed using a Metabolomics Workflow from Compound Discoverer with a Pattern Scoring node to identify sulphur-containing metabolites. All genotypes were compared to each other using differential analysis.

Conversion of DMSP to Gonyol by Expression of burA in E. coli
A 300 mL baffled Erlenmeyer flask containing 75 mL MM9 [8] liquid medium supplemented with 25 μg mL -1 chloramphenicol and 50 μg mL -1 kanamycin was inoculated with 1% of an overnight culture of E. coli Rosetta2 (DE3) expressing His8-BurA. E. coli Rosetta2 (DE3) containing an empty vector (pHis8-3-svp) was used as negative control. The cultures were grown at 37 °C with shaking at 150 rpm until an OD600 of 1.3 was reached. After cooling to 16 °C, the cultures were supplemented with 5.1 mg DMSP, heterologous protein production was induced with 0.5 mM IPTG and the resulting cultures were incubated at 16 °C with shaking at 150 rpm for 18 h. Subsequently, 1.3 g XAD16N resin was added per culture followed by incubation with shaking at 100 rpm for 30 min. The XAD16N resin was harvested from the culture broth by filtration through Miracloth (Merck Millipore), washed with water and eluted with methanol (40 mL) followed by elution with ethyl acetate (20 mL). Both eluted fractions were combined, concentrated under reduced pressure, redissolved in methanol to a