An Iterative Type I Polyketide Synthase Initiates the Biosynthesis of the Antimycoplasma Agent Micacocidin

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Hirokazu Kage,1 Martin F. Kreutzer,1 Barbara Wackler,2 Dirk Hoffmeister,2 and Markus Nett1,* 1Junior Research Group ‘‘Secondary Metabolism of Predatory Bacteria’’, Leibniz Institute for Natural Product Research and Infection Biology, Hans-Kno¨ ll-Institute, Beutenbergstrasse 11a, 07745 Jena, Germany 2Department of Pharmaceutical Biology at the Hans-Kno¨ ll-Institute, Friedrich Schiller Universita¨ t Jena, 07745 Jena, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2013.04.010

SUMMARY under macrolide therapy have been described (Averbuch et al., 2011; Cardinale et al., 2011; Itagaki et al., 2013). Micacocidin is a thiazoline-containing natural pro- Micacocidin is a new antibiotic that exerts strong inhibitory duct from the bacterium Ralstonia solanacearum effects in the nanomolar range against several Mycoplasma that shows significant activity against Mycoplasma species, including M. pneumoniae (Kobayashi et al., 1998). pneumoniae. The presence of a pentylphenol moiety In vivo efficacy after oral administration was demonstrated in distinguishes micacocidin from the structurally chicken that had been infected with M. gallisepticum (Kobayashi related siderophore yersiniabactin, and this residue et al., 2000). The thiazoline-containing natural product micacocidin is structurally closely related to the siderophore yersinia- also contributes to the potent antimycoplasma bactin (Drechsel et al., 1995), and cellular uptake studies effects. The biosynthesis of the pentylphenol moiety, suggest that it might also be involved in iron acquisition of the as deduced from bioinformatic analysis and stable producing bacterium Ralstonia solanacearum (Kreutzer et al., isotope feeding experiments, involves an iterative 2011). It is interesting that the antimycoplasma activity of mica- type I polyketide synthase (iPKS), which generates cocidin is not linked to its metal-chelating properties, as an a linear tetraketide intermediate from acyl carrier iron(III)-loaded complex exhibits the same level of activity as protein-tethered hexanoic acid by three consecu- the free ligand (Kobayashi et al., 2000). Furthermore, blockage tive, decarboxylative Claisen condensations with of the metal coordination sites by chemical modification does malonyl-coenzyme A. The final conversion into not affect the growth-inhibitory effects against Mycoplasma 6-pentylsalicylic acid depends on a ketoreductase species (Ino et al., 2001). domain within the iPKS, as demonstrated by heterol- A recent study unveiled the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes that account ogous expression in E. coli and subsequent site- for the assembly of micacocidin (Kreutzer et al., 2011). Even directed mutagenesis experiments. Our results though the domain architecture of the corresponding biosyn- unveil the early steps in micacocidin biosynthesis thesis enzymes is highly reminiscent of the yersiniabactin and illuminate a bacterial enzyme that functionally re- assembly line (Gehring et al., 1998b; Miller et al., 2002), some sembles fungal polyketide synthases. minor deviations were found to be sufficient to translate into chemical diversity (Figure 1). Both yersiniabactin and micacocidin feature a phenolic moiety in their structures, but in the case INTRODUCTION of micacocidin, the corresponding residue is further decorated with a pentyl side chain. This structural variation apparently Mycoplasma pneumoniae is one of the leading causes of com- has important consequences in terms of biological activity munity-acquired pneumonia and other respiratory disorders. (Takeda et al., 1996), fueling the interest in its biosynthetic origin. Due to the lack of a cell wall, this bacterium is naturally resistant The phenolic moiety in yersiniabactin derives from salicylic acid, against beta-lactam and glycopeptide antibiotics. Since the which was shown to be incorporated by the stand-alone AMP- pathogen also exhibits enhanced resilience against aminoglyco- dependent ligase YbtE (Gehring et al., 1998a). In the micacocidin

sides, oxazolidinones, rifampicin, and sulfonamides, therapy of pathway, the adenylation domain MicC-A1 represents the enzy- M. pneumoniae infections is restricted to a limited set of anti- matic counterpart of YbtE. However, the sequence of MicC-A1 biotics, among which macrolides are typically considered the exhibits a speciﬁcity-conferring signature that appears to be drugs of ﬁrst choice (Waites and Talkington, 2004). Of note, inconsistent with the loading of an aryl carboxylic acid (Stachel- macrolides also represent the only agents that are approved haus et al., 1999; May et al., 2002). Moreover, the presence of an for the treatment of M. pneumoniae infections in children. additional type I PKS module, which is absent in the yersiniabac- Over the past decade, the prevalence of macrolide-resistant tin assembly line, was puzzling. Intrigued by these peculiarities, M. pneumoniae strains has dramatically increased (Jacobs, we set out to resolve the molecular mechanism by which the 2012), and more recently, several cases of resistance emerging pentylphenol moiety of micacocidin is formed. The knowledge

Figure 1. Modular Biosynthesis Enzymes for the Production of Micacocidin (A) Domain architecture of the micacocidin (Mic) assembly line. Domains that likely account for the structural differences to yersiniabactin (Ybt) are highlighted in blue. Domain notation: A, adenylation; ACP, acyl carrier protein; KS, ketosynthase; AT, acyl transferase; KR, ketoreductase; C, condensation; MT, SAM-dependent methyltransferase; PCP, peptidyl carrier protein; TE, thioesterase. (B) Chemical structures of micacocidin and yersiniabactin.

Townsend, 2010). In the second scenario, we proposed the loading of an oxygenated decanoic acid, which would undergo a single decarboxylative thio- Claisen condensation with malonyl-CoA

to give a C12 diketide. Again, an aldol- on the biosynthesis will, in the long term, help manipulate pro- type reaction could cyclize the thioester-bound intermediate, duction and generate structural derivatives of this promising which would then be further oxidized to PSA. antibiotic. In order to distinguish between the two routes, the loading module of MicC was heterologously produced and purified RESULTS AND DISCUSSION from Escherichia coli KRX as an N-terminal hexahistidine fusion via the pET28a(+)-derived expression plasmid pHiK007 (Fig- Identification of the Starter Unit in Micacocidin ure S2; for primer sequences, see Table S1). The substrate Biosynthesis preference of the recombinant protein was tested in the ATP- Feeding studies with stable isotope precursors revealed that the [32P]-pyrophosphate exchange assay, as previously described metabolic origin of the pentylphenol moiety is dissimilar from the (Kreutzer et al., 2012). This approach revealed a clear preference salicylate building block in yersiniabactin. While the latter derives for hexanoic acid, whereas the other possible starter molecule, from shikimate metabolism (Gehring et al., 1998a), the incorpo- decanoic acid, was not activated at relevant levels (Figure 2). ration pattern of 13C-labeled acetate into micacocidin was in Further testing showed a narrow substrate tolerance of the full agreement with a polyketide origin (Figure S1 available enzyme. While pentanoic acid and heptanoic acid could still be online). It paralleled that of the plant metabolite olivetolic acid converted into their corresponding adenylate products with (4-hydroxy-6-pentylsalicylic acid), which is an important inter- low efficiency, octanoic acid already failed to give an appreciable mediate in the pathway to cannabinoids (Fellermeier et al., response. A subsequent feeding study in R. solanacearum with 2001). The biosynthesis of olivetolic acid involves a type III [1-13C]hexanoic acid led to an increased incorporation of label PKS, which forms a linear tetraketide from hexanoyl-coenzyme at C-3 of micacocidin (Figure S1), ultimately confirming this A (CoA) and three molecules of malonyl-CoA (Taura et al., short-chain fatty acid as the biosynthetic starter unit. 2009). Conversion of the tetraketide into olivetolic acid was not solved until recently, when a cyclase was found to catalyze the Heterologous Production of PSA in Escherichia coli concluding intramolecular aldol condensation (Gagne et al., To investigate whether the PKS module of MicC catalyzes the

2012). Based upon the results from the feeding studies in proposed iterative C2 chain elongation of primed hexanoic R. solanacearum, an analogous mechanism for the formation acid, a truncated enzyme including the loading and the ﬁrst of 6-pentylsalicylic acid (PSA) would be conceivable, yet the extender module (A1-ACP-KS-AT-KR-ACP) was recombinantly entire genome of the micacocidin producer is devoid of genes produced. The assembly of the respective expression vector, for type III PKSs (Salanoubat et al., 2002). Therefore, we envis- pHiK008, was accomplished by l Red-mediated in vivo cloning aged two alternative scenarios that could give rise to PSA and (Zhang et al., 2000; Figure S3). For gene expression, pHiK008 that are compatible with the experimentally observed incorpora- was introduced into E. coli BL21(DE3). Although the genome of tion of acetate units, as well as the domain organization of the E. coli harbors the genes for at least three different phosphopan- micacocidin biosynthesis enzymes. The ﬁrst scenario assumes tetheinyl transferases (PPTases), the substrate selectivity of an activation of hexanoic acid by MicC-A1 and transfer as acyl- these enzymes is known to be a bottleneck for the heterologous adenylate on to the assembly line prior to acyl chain elongation, biosynthesis of polyketide natural products (Lambalot et al., ring formation, and aromatization. It is obvious that the elonga- 1996; Pfeifer and Khosla, 2001). The micacocidin biosynthesis tion implies an iterative usage of the PKS module in MicC, locus includes a putative PPTase gene, micA, which was which is reminiscent, to a degree, of fungal type I PKSs with an hence expected to represent the cognate enzyme for the N-terminal starter unit-acyl carrier protein (ACP) transacylase posttranslational activation of the acyl and peptidyl carrier pro- domain (Crawford et al., 2006; Zhou et al., 2008; Crawford and teins in the micacocidin assembly line. High-performance liquid

2011). Two potential candidates in the R. solanacearum genome that could substitute the missing PPTase gene are RSc1067 and RSp0163, respectively. On the other hand, the loss of the TE function from micA was possibly less important than in the preceding heterologous expression experiment. In R. solanacearum, the type I TE domain from the NRPS MicH is expected to be responsible for the release of the fully assembled natural product, whereas the TE domain of MicA would merely serve an editing function to remove aberrant molecules from the enzyme complex (Miller et al., 2010). The fact that no stalled intermediates were retrieved from R. solanacearum RS7 could be attributed to the generally low micacocidin production level of the mutant.

Figure 2. Substrate Preference of the MicC Loading Module Effects of Ketoreductase Domain Inactivation on PSA Degree of exchange in the ATP-[32P]-pyrophosphate assay with various test Biosynthesis substrates. cpm, counts per minute. Bars represent SD. See also Figure S2. Ketoreductase (KR) domains of iterative type I PKSs (iPKSs) are known to act regiospecifically during polyketide chain extension, chromatography-mass spectrometry (HPLC-MS) analysis of thereby setting the substitution pattern of the final product (Hert- E. coli expression cultures confirmed that PSA is only heterolo- weck, 2009; Sun et al., 2012; Chooi and Tang, 2012). To deter- gously produced at relevant levels when micC (A1-ACP-KS- mine the role of the KR domain in MicC, we chose to mutate AT-KR-ACP) is coexpressed with micA (Figure 3). Evidence for the highly conserved Tyr residue in its active center (correspond- the production of olivetolic acid was not found. It is interesting ing to Y1991). The latter is part of a catalytic triad together with to note that the tetraketide, which had been synthesized on a Lys and Ser residues (Jo¨ rnvall et al., 1995; Oppermann et al., truncated assembly line, was readily hydrolyzed to give free 1997), and existing models suggest that the tyrosine transfers PSA. With a size of 834 amino acids and a molecular weight of a phenolic proton to the b-carbonyl oxygen atom during almost 90 kDa, the protein MicA is significantly larger than other the reduction process (Caffrey, 2003). Mutagenesis studies PPTases (Lambalot et al., 1996; Sa´ nchez et al., 2001). This size confirmed the fatal effects of amino acid substitutions at this expansion is due to the presence of a type II thioesterase (TE) site, which can result in the synthesis of lactone shunt products domain (Figure S4; Table S2) that is located N-terminal of the (Reid et al., 2003). We initially replaced Y1991 with phenylala- PPTase domain. MicA may hence provide hydrolytic activity, nine, as this amino acid should maintain the active site geometry which would contribute to the offload of the polyketide from while eliminating the required hydroxyl group for catalysis (Ding the assembly line and the concomitant regeneration of MicC. et al., 2010). Moreover, Y1991 was mutated to alanine, which would definitely shut down the KR activity in MicC. The genes Functional Analysis of MicA, a Bifunctional Protein with carrying the mentioned mutations were expressed in E. coli TE and PPTase Domains BL21(DE3) together with micA, as previously described. In To evaluate whether the release of the thioester-bound interme- both cases, no PSA could be detected in the culture superna- diate in the coexpression experiment is at least partially medi- tant of the heterologous host. Instead, the occurrence of a new ated by MicA, the serine in the active site of its TE domain was peak was observed. The high-resolution mass of this metabolite replaced with alanine by site-directed mutagenesis. Analysis of at m/z 223.0976 [M-H]À indicated a molecular formula of the MicA protein sequence had previously revealed the C12H15O4, which would be consistent with either a lactoniza- consensus sequence with the serine residue, which is respon- tion-derived pyrone or olivetolic acid. The latter possibility could sible for acyl-O-enzyme intermediate formation in TEs (Hardie be excluded after cochromatography with an authentic standard et al., 1985). As shown in Figure 3, PSA was still detected in (Figure 3). In the absence of a functional reductive domain, the the TE mutant with S417A substitution, although at drastically iPKS thus generated a linear tetraketide that is prone to intramo- lower quantities (1.5%) in comparison to the E. coli strain ex- lecular lactonization upon keto-enol tautomerism. pressing native micA. This finding supports the proposed dual function and the significance of MicA in the heterologous pro- Model for the Biosynthesis of the PSA-Derived duction of PSA. Pentylphenol Moiety in Micacocidin In order to interrogate the importance of micA for micacocidin The result from the mutation study corroborated that the KR biosynthesis, the gene was disrupted in the R. solanacearum domain of MicC is essential for the assembly of PSA. In analogy producer strain GMI1000 by allelic-exchange mutagenesis (Fig- to the biosynthesis of 6-methylsalicylic acid (MSA) in bacteria ure S5). It is surprising that micA turned out to be dispensable for and fungi, we propose that MicC-KR exclusively reduces the the assembly of the natural product, as the production was still b-keto function at the stage of the triketide intermediate. Inacti- active in the micA-defective mutant strain RS7, albeit at sig- vation of the KR domain did not halt the polyketide chain elonga- nificantly reduced levels (Figure 4). We assume that this outcome tion, suggesting an enhanced tolerance of MicC-ketosynthase is largely due to the inactivation of the PPTase function of micA, with regard to b functionality recognition. The nonreduced tetra- which was only partially complemented by redundant PPTase ketide was not converted into olivetolic acid, as expected, but genes, as described in kirromycin biosynthesis (Pavlidou et al., likely underwent lactonization to give an a-pyrone (Figure 5).

Figure 3. Functional Validation of micC and micA in E. coli Stacked extracted ion chromatograms for masses corresponding to the m/z of PSA (2) and the shunt pyrone (3) produced by E. coli cultures expressing

micC (A1-ACP-KS-AT-KR-ACP) (a); micC and the TE-PPTase gene micA (b); micC and the TE mutant of micA with S417A (c); micA and the KR mutant of micC with Y1991A (d); micA and the KR mutant of micC with Y1991F (e); or standard olivetolic acid (f). To distinguish 3 from olivetolic acid (4), the samples in (d) and (e) had been spiked with 1 mmol of the latter prior to HPLC-MS analysis. Identiﬁcation of the peaks was achieved by measuring the accurate mass and calculation of the chemical formula within a 5 ppm range (2: m/z À 207.1022 [M-H] ,C12H15O3; 3 and 4: m/z À 223.0979 [M-H] ,C12H15O4). t/min, retention time in minutes. See also Figure S3.

through genetic engineering of the producer strain. From a biochemical perspective, MicC is highly interesting, as it combines features from the functionally distinct partially reducing and nonreducing iterative PKSs (Cox, 2007), which are only rarely found in bacteria (Gaisser et al., 1997). The exclusive production of PSA, but not of olivetolic acid, by MicC is suggestive of regioselective ke- toreduction, as observed in partially reducing PKSs (Sun et al., 2012; Lu et al., 2011; Sthapit et al., 2004; Zhao et al., 2008). These monomodular enzymes, however, are encoded by discrete genes, and they also exhibit a conserved thioester hydrolase (TH) domain for product release (Moriguchi et al., 2010). Both features are not applicable to the PKS in MicC, even though the lack of Similar findings were reported from fungal MSA synthases, when the TH domain can be easily rationalized. Another discrepancy the NADPH binding site of their KR domains were mutated (Mor- involves the mode of initiating ketide chain formation. While iguchi et al., 2008; Richardson et al., 1999). In contrast, the native partially reducing PKSs typically use acetyl-CoA as a starter MicC assembles a correctly processed d-hydroxylated tetrake- unit, the MicC PKS is loaded via intraprotein transfer from an tide intermediate that cyclizes upon intramolecular C2-C7 aldol N-terminal ACP domain. Except for bacterial (noniterative) condensation. It is obvious that the aromatization to the PSA modular type I PKSs (Gulder et al., 2011), a related starter unit moiety requires an elimination of the d-hydroxy group. The cor- selection is only known from fungal nonreducing PKSs, which, responding dehydration reaction may occur spontaneously, as however, completely lack reductive domains (Crawford et al., proposed in the formation of MSA and islandicin, respectively 2006; Zhou et al., 2008). (Moriguchi et al., 2010; Scha¨ tzle et al., 2012). Even though the precise timing of aromatization and cyclization remain to be SIGNIFICANCE determined, the heterologous expression experiment established the minimal biochemical basis for the formation of PSA, Micacocidin is a thiazoline-containing natural product with i.e., the first two modules of holo-MicC. pronounced activity against the human pathogen Myco- In summary, MicC has been identified as 6-pentylsalicylate plasma pneumoniae. Although its biosynthetic assembly synthase and, by site-specific mutagenesis, the necessity of its line reflects the close relationship to the siderophore yersi- KR domain for correct substrate processing is provided. We niabactin, the molecular basis for the subtle, yet biologically expect that this knowledge may eventually help generate struc- important, structural differences were not clear. Biochem- tural derivatives of the antimycoplasma agent micacocidin ical analyses of the early steps in micacocidin biosynthesis

Figure 4. Metabolic Characterization of the micA Mutant Strain RS7 Chromatographic proﬁles of R. solanacearum GMI1000 (A) and the micA-deﬁcient mutant strain RS7 (B) recorded at 254 nm. The production of micacocidin is substantially reduced in the mutant strain RS7 (<1% of wild-type level), but not completely abolished. See also Figure S5.

have identified an unusual, iteratively acting bacterial type I Construction of the Expression Plasmid pHiK007 PKS that is integrated into a multimodular assembly line. A gene fragment of micC covering the entire loading module region was ampli- Mechanistically, the protein seems to be related to partially fied by PCR from genomic DNA of R. solanacearum GMI1000 with the primers P1 and P2 (a complete list with all primers used in this study is given in reducing iterative PKSs, which utilize programmed ketore- Table S1). The reaction (50 ml total volume) included 2 mM MgSO4, 0.2 mM duction to direct the biosynthesis toward a defined, aro- each dNTP, 5% dimethyl sulfoxide, 50 pmol each primer, and 1.25 U Pfu matic product. On the other hand, the primary amino acid DNA polymerase. Thermal cycling conditions were as follows: initial denatur- sequence, the domain organization, and also the starter ation, 5 min, 95C; amplification, 30 cycles (95C for 1 min, 65C for 1 min, unit selection of the micacocidin iPKS are clearly distinct, 72 C for 7 min); final extension, 20 min at 72 C, terminal hold at 4 C. The making this enzyme a promising candidate for further PCR product was cloned into the NdeI-HindIII site of the expression plasmid pET28a(+) (Novagen) to give pHiK007. This vector was introduced into biochemical studies. The described findings set the stage E. coli KRX (Promega) after the accuracy of the inserted nucleotide sequence for the rational engineering of more potent micacocidin had been confirmed by DNA sequencing. analogs. Furthermore, the possibility to independently reconstitute the assembly of the micacocidin precursor Expression and Purification of the Loading Module of MicC: A1-ACP 6-pentylsalicylate in E. coli may prove useful in the field of The overexpression strain was cultured at 37C in TB medium (containing m combinatorial biosynthesis. 50 g/ml kanamycin) to an optical density at 600 nm (OD600) of 0.6. To induce gene expression, 0.1% rhamnose was added to the culture. Following induction, fermentation was continued at 16C for 20 hr. After centrifugation, the EXPERIMENTAL PROCEDURES cell pellet was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 20 mM b-mercaptoethanol, 10% glycerol) and subjected to Bacterial Strains and Growth Conditions sonication. The cell debris was subsequently removed by centrifugation, and The Ralstonia solanacearum wild-type strain GMI1000 and the mutant the supernatant was loaded on to a nickel-nitriloacetic acid column. The recom- strain RS7 were grown at 30C in BG medium (15 g/l Bacto-Agar, 5g/l binant protein was eluted using lysis buffer containing increasing concentra- glucose) or modified 1/4 3 M63 minimal medium supplemented with tions of imidazole. Fractions that contained the target protein were identified 0.2% glucose and 0.02% sodium acetate (Schneider et al., 2009). The via SDS-PAGE, desalted with PD-10 columns (GE Healthcare), and concen- mutant strain RS7 was continuously kept under antibiotic selection pressure trated using Amicon ultracentrifugation devices (Millipore). The recombinant ¨ (20 mg/ml kanamycin). Escherichia coli strains were routinely grown in Luria protein was finally purified by FPLC (Amersham AKTAexplorer) using a 5 ml Hi- broth (LB) medium. For protein expression experiments, Terrific broth (TB) Trap Q HP column (GE Healthcare). The solvents used were 20 mM Tris-HCl, pH was used. 8.0, + 5% glycerol (A) and 20 mM Tris-HCl, pH 8.0, + 5% glycerol + 1 M NaCl (B). Prior to injection of the sample, the column was washed with 20 ml 100% solvent A. After injection, a linear gradient elution up to 100% of solvent B was car- Incorporation Studies of Isotopically Labeled Precursors ried out. The separation was monitored via UV absorbance at 220 and 280 nm, All feeding experiments were conducted in 5 l Erlenmeyer flasks containing as well as by conductivity. The identity of the purified protein was confirmed by 1 l of iron-deficient 1/4 3 M63 minimal medium according to a previously MALDI-TOF MS using a Bruker Ultraflex spectrometer (Bruker Daltonics). established protocol (Wackler et al., 2011). Briefly, 13C-labeled precursors were added aseptically at time of inoculation as filter-sterilized aqueous ATP-[32P]-Pyrophosphate Exchange Assay 13 13 solutions: [1- C]acetate (50 mg/l) or [1- C]hexanoate (50 mg/l). The cul- The substrate preference of MicC (A1-ACP) was determined according to an tures were shaken for 3 days at 30C. For the recovery of micacocidin, the established protocol (Kreutzer et al., 2012). A 100 ml reaction contained culture broth was extracted twice with ethyl acetate. The combined organic 80 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM ATP, 100 nM purified enzyme, layers were concentrated to dryness in vacuo, and the resulting extract was 0.1 mM [32P]-pyrophosphate, and 1 mM substrate. Reactions were initiated redissolved in 200 ml methanol. Purification was conducted on an ultra-fast by addition of enzyme and incubated at 37C for 30 min prior to quenching liquid chromatography (UFLC) system (Shimadzu) equipped with a Nucleodur with 500 ml of charcoal suspension (1% charcoal and 4.5% Na4P2O7 in 3.5% Sphinx RP column (Macherey & Nagel; 250 3 10 mm; 5 mm pore diameter). perchloric acid). Precipitate was collected with paper filter discs under vac- 13 C-labeled micacocidin was chromatographed with a linear gradient of uum. Following three consecutive washing steps with 40 mM Na4P2O7 in acetonitrile in water plus 0.1% trifluoroacetic acid (10% / 90% acetonitrile 1.4% perchloric acid (200 ml), water (200 ml), and ethanol (200 ml), the filter within 20 min; flow rate, 3 ml/min) with wavelength detection at 254 nm. For papers were added to 2.5 ml of scintillation fluid. Radioactivity was determined nuclear magnetic resonance (NMR)-based analysis, the Ga3+-complex of mi- in a Beckman Coulter LS6500 Multipurpose Scintillation Counter. All assays cacocidin was prepared. A methanolic solution of the purified natural product were run in triplicate. was treated with a 10-fold excess of Ga(III) nitrate hydrate and stirred for 12 hr at room temperature. Subsequently, NMR spectra were recorded at 300 K on Heterologous Biosynthesis of PSA a Bruker Avance III 600 MHz spectrometer using methanol-d4 as a solvent The vector to produce the first two modules of MicC (A1-ACP-KS-AT-KR-ACP) and internal standard (dH 3.31 ppm; dC 49.0 ppm). was assembled from four discrete plasmids (Figure S3). In the first step, three

Figure 5. Model for the Product Formation in E. coli Coexpressing micA and micC (A1-ACP-KS-AT-KR-ACP) Route A is consistent with the initiation of micacocidin biosynthesis except for the premature ofﬂoad of the thioester-bound intermediate. Route B shows the shunt pathway of KR-mutated MicC.

overlapping gene fragments of micC were amplified by PCR with the primers crude extracts were resuspended in methanol and subjected to HPLC-electro- P3 and P4, P5 and P6, and P7 and P8. The resulting PCR products were indi- spray ionization (ESI)-MS analysis on an Exactive Orbitrap (Thermo Fisher vidually cloned into pJET1.2 (Thermo Scientific) to give pHiK008a, pHiK008b, Scientific). The chromatographic system was equipped with a Betasil 100-3 and pHiK008c, respectively. All plasmids were sequenced to verify the fidelity C18 column (150 3 2.1 mm, 3 mm pore diameter), and separation was per- of their inserts. The 1.8 kb MluI-SmaI fragment from pHiK008a was inserted formed using a gradient elution of acetonitrile in water plus 0.1% formic acid into the MluI-NruI sites of pHiK008b yielding pHiK008ab, while pHiK008c (5% / 98% acetonitrile within 15 min; flow rate, 0.2 ml/min). Extracted ions was digested with XhoI. The 1.2 kb XhoI fragment was cloned into were detected in the negative mode for the ranges m/z 206.5–207.5 (PSA, 2) pET28a(+) to give pHiK008c1. The latter was opened with NdeI-NheI and sub- and m/z 222.5–223.5 (pyrone, 3 and olivetolic acid, 4), respectively. The result- sequently merged with a pHiK007-derived plasmid, in which the HindIII site ing peaks were analyzed within a 5 ppm range for the accurate masses of 2, 3, had been removed by blunting to generate an additional NheI site. This and 4. In order to discriminate between the constitutional isomers 3 and 4, the approach yielded pHiK008c2. Finally, pHiK008ab and pHiK008c2 were com- samples were cochromatographed with 1 mmol of commercial 4 (THC Pharm). bined by l Red-mediated in vivo cloning to give pHiK008 (Zhang et al., 2000). To this end, the BglII-digested pHiK008ab and the EcoRI-HindIII- Site-Directed Mutagenesis of the TE Domain in MicA digested pHiK008c2 were used to cotransform l red-induced BW25113/ The desired S417A mutation was introduced by PCR with primers P11 and P12 pIJ790 electrocompetent cells. The overexpression host E. coli BL21(DE3) using pHiK009 as a template. This approach yielded a mutated plasmid con- (Novagen) was finally transformed with the resulting construct pHiK008. taining staggered nicks (pHiK010a), which was introduced into E. coli DH5a The PPTase gene micA was amplified by PCR from genomic DNA of after the parental DNA template had been removed by DpnI digestion. The R. solanacearum GMI1000 with the primers P9 and P10. The PCR product mutation in the active site was subsequently confirmed by DNA sequencing. was cloned into pJET1.2, yielding pHiK009a (micA). After the correct sequence To ensure that no further PCR-induced random mutation is present in the final had been confirmed by DNA sequencing, the plasmid was digested with NdeI expression plasmid, the verified 1.3 kb SfiI-EcoRV fragment of pHiK010a with and EcoRV. The resulting fragment, which contained the PPTase gene, was the S417A mutation was cloned into the original vector pHiK009. The resulting cloned into pACYCDuet-1 (Novagen) to give pHiK009 (micA). Subsequently, plasmid, pHiK010, was then used for the transformation of the E. coli E. coli BL21(DE3) harboring pHiK008 was transformed with pHiK009 to BL21(DE3) strain harboring pHiK008. generate the required strain for the coexpression experiment. For the analysis of product formation, the E. coli strains were grown at 37C in TB medium to an Inactivation of micA by Insertional Mutagenesis

OD600 of 0.6. Expression of micC and the PPTase gene was induced by A 2.0 kb gene fragment of micA was ampliﬁed by PCR from R. solanacearum addition of 1 mM isopropyl 1-thio-b-D-galactopyranoside. Afterward, the incu- GMI1000 genomic DNA with primers P13 and P14. The PCR product was bation was continued at 16C overnight. To enable the recovery of acidic blunt-end cloned into pJET1.2 to give pHiK011a. After the correct sequence metabolites (e.g., PSA), the resulting culture broths were adjusted to pH 2 prior had been conﬁrmed by DNA sequencing, the sacB gene was cloned into to extraction with ethyl acetate. After evaporation of the organic solvent, the pHiK011a to yield pHiK011b. The latter was used to transform E. coli

BW25113 harboring pIJ790 (Gust et al., 2003). Insertion of a kanamycin-resis- Caffrey, P. (2003). Conserved amino acid residues correlating with ketoreduc- tance gene into the micA sequence was achieved by l Red-mediated recom- tase stereospecificity in modular polyketide synthases. ChemBioChem 4, bination. For this purpose, the antibiotic resistance cassette was amplified 654–657. from pAphA-3 by PCR with the primer pair P15 and P16, generating extensions Cardinale, F., Chironna, M., Dumke, R., Binetti, A., Daleno, C., Sallustio, A., homologous to the target micA sequence at either end of the marker. E. coli Valzano, A., and Esposito, S. (2011). Macrolide-resistant Mycoplasma pneu- BW25113/pIJ790/HiK011b was transformed with the PCR product following moniae in paediatric pneumonia. Eur. Respir. J. 37, 1522–1524. induction of l Red genes with 10 mM L-arabinose at 30 C for 3 hr. After recov- Chooi, Y.-H., and Tang, Y. (2012). Navigating the fungal polyketide chemical ery in Super Optimal Broth with Catabolite repression (SOC) medium (2% tryp- space: from genes to molecules. J. Org. Chem. 77, 9933–9953. tone, 0.5% yeast extract, 20 mM glucose, 10 mM NaCl, 2.5 mM KCl, 10 mM Cox, R.J. (2007). Polyketides, proteins and genes in fungi: programmed nano- MgCl2) and a 1 hr incubation at 37 C, the transformants were spread on to LB agar plates containing 50 mg/ml kanamycin, followed by incubation machines begin to reveal their secrets. Org. Biomol. Chem. 5, 2010–2026. overnight at 37C. Integration of the marker in the plasmid DNA by homolo- Crawford, J.M., and Townsend, C.A. (2010). New insights into the formation of gous recombination yielded the micA disruption plasmid pHiK011, which fungal aromatic polyketides. Nat. Rev. Microbiol. 8, 879–889. was isolated and verified by PCR analysis. For the transformation of Crawford, J.M., Dancy, B.C.R., Hill, E.A., Udwary, D.W., and Townsend, C.A. R. solanacearum GMI1000, cells were grown in 1/4 3 M63 medium supple- (2006). Identification of a starter unit acyl-carrier protein transacylase domain mented with 0.2% glucose and 0.4% glycerol to an optical density at 580 in an iterative type I polyketide synthase. Proc. Natl. Acad. Sci. USA 103, nm (OD580) of 0.5. After centrifugation, the harvested cells were successively 16728–16733. washed with sterile Milli-Q water and ice-cold 10% glycerol. Electroporation Ding, W., Lei, C., He, Q., Zhang, Q., Bi, Y., and Liu, W. (2010). Insights into bac- was carried out in a 0.2 cm gap cuvette using a Bio-Rad GenePulser II set to terial 6-methylsalicylic acid synthase and its engineering to orsellinic acid syn- U m 200 ,25 F, and 2.5 kV. SOC medium was immediately added to the shocked thase for spirotetronate generation. Chem. Biol. 17, 495–503. cells, which were then incubated at 30C for 3 hr. The resulting culture was Drechsel, H., Stephan, H., Lotz, R., Haag, H., Za¨ hner, H., Hantke, K., and Jung, spread on to LB agar containing 25 mg/ml kanamycin, followed by incubation G. (1995). Structure elucidation of yersiniabactin, a siderophore from highly at 30C. The mutation was verified by colony PCR (Figure S5). Micacocidin virulent Yersinia strains. Liebigs Ann. 1995, 1727–1733. production of the micA-defective strain RS7 was analyzed on an HPLC-diode Fellermeier, M., Eisenreich, W., Bacher, A., and Zenk, M.H. (2001). array detector (Agilent 1100 series) equipped with a C8 column (Zorbax Eclipse 13 XDB, 150 3 4.6 mm, 5 mm pore diameter) and coupled to a mass-selective Biosynthesis of cannabinoids. Incorporation experiments with C-labeled detector with ion trap (MSD Trap; Agilent) operating in alternating ionization glucoses. Eur. J. Biochem. 268, 1596–1604. mode. Separation was achieved with a linear gradient of methanol in Gagne, S.J., Stout, J.M., Liu, E., Boubakir, Z., Clark, S.M., and Page, J.E. water plus 0.1% formic acid (10% / 90% methanol within 15 min; flow (2012). Identification of olivetolic acid cyclase from Cannabis sativa reveals a rate, 1 ml/min) with wavelength detection at 254 nm. unique catalytic route to plant polyketides. Proc. Natl. Acad. Sci. USA 109, 12811–12816. Construction of the KR Domain Mutant Strains Gaisser, S., Trefzer, A., Stockert, S., Kirschning, A., and Bechthold, A. (1997). Site-directed mutagenesis was performed on the truncated micC gene in Cloning of an avilamycin biosynthetic gene cluster from Streptomyces virido- pHiK008c1. The desired mutations were introduced by PCR using the primers chromogenes Tu¨ 57. J. Bacteriol. 179, 6271–6278. P17 and P18 for the Y1991A mutant or P19 and P18 for the Y1991F mutant. Gehring, A.M., Mori, I., Perry, R.D., and Walsh, C.T. (1998a). The nonribosomal Following the PCR reaction, the methylated parental DNA template was peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersi- digested with DpnI. The remaining circular, nicked double-stranded DNA car- niabactin, an iron-chelating virulence factor of Yersinia pestis. Biochemistry a rying the mutation was used to transform E. coli DH5 . After the correct 37, 11637–11650. sequences had been confirmed by DNA sequencing, the plasmids were Gehring, A.M., DeMoll, E., Fetherston, J.D., Mori, I., Mayhew, G.F., Blattner, digested with HincII to obtain the 0.5 kb nucleotide sequences that contained F.R., Walsh, C.T., and Perry, R.D. (1998b). Iron acquisition in plague: modular the mutated region. These fragments were cloned into the corresponding re- logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis. Chem. Biol. gion of pHiK008 to give pHiK012 (Y1991F) and pHiK013 (Y1991A), which 5, 573–586. were used in the heterologous expression experiments. Gulder, T.A.M., Freeman, M.F., and Piel, J. (2011). The catalytic diversity of multimodular polyketide synthases: natural product biosynthesis beyond SUPPLEMENTAL INFORMATION textbook assembly rules. Top. Curr. Chem. http://dx.doi.org/10.1007/ 128_2010_113. Supplemental Information includes five figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.chembiol.2013. Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003). PCR-tar- 04.010. geted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. ACKNOWLEDGMENTS USA 100, 1541–1546. Hardie, D.G., Dewart, K.B., Aitken, A., and McCarthy, A.D. (1985). Amino acid Financial support was generously provided by the Bundesministerium fu¨ r sequence around the reactive serine residue of the thioesterase domain of Bildung und Forschung (GenoMik Transfer Programme, Grant No. 0315591A rabbit fatty acid synthase. Biochim. Biophys. Acta 828, 380–382. to M.N.) and a postdoctoral fellowship from the Deutsche Forschungsgemein- Hertweck, C. (2009). The biosynthetic logic of polyketide diversity. Angew. schaft-funded graduate school JSMC to H.K. We gratefully acknowledge A. Chem. Int. Ed. Engl. 48, 4688–4716. Perner (Hans-Kno¨ ll-Institute Jena) for numerous HR-ESI-MS measurements. Ino, A., Kobayashi, S., Ueda, K., Hidaka, S., and Hayase, Y. (2001). Structure- activity relationship of the antimycoplasma antibiotic micacocidin—a prelimi- Received: March 15, 2013 nary study. J. Antibiot. 54, 753–756. Revised: April 8, 2013 Itagaki, T., Suzuki, Y., Seto, J., Abiko, C., Mizuta, K., and Matsuzaki, Y. (2013). 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