Biosynthesis of the Respiratory Toxin Bongkrekic Acid in ... - Cell Press

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Article Biosynthesis of the Respiratory Toxin Bongkrekic Acid in the Pathogenic Bacterium Burkholderia gladioli Nadine Moebius,1 Claudia Ross,1 Kirstin Scherlach,1 Barbara Rohm,1 Martin Roth,1 and Christian Hertweck1,2,* 1Leibniz

Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745 Jena, Germany for Natural Product Chemistry, Friedrich Schiller University, 07743 Jena, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2012.07.022 2Chair

SUMMARY

Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli l Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like b-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant. INTRODUCTION It is certainly a rare scenario when a government decides to prohibit a national specialty by law. However, in 1988 the Indonesian government responded to thousands of reported food intoxications, many of which were lethal (z10%), and attempted to officially ban tempe bongkrek from the market. Yet, with little success—as tempe bongkrek has remained a popular dish (Adams and Moss, 2008; Wood, 1998). This indigenous Southeast-Asian fermented food is based on de-oiled coconut flakes that are incubated and modified with the mold fungus Rhizopus oligosporus (Shurtleff and Aoyagi, 1979). The resulting press cake proved to be occasionally poisonous, leading to hyperglycemia followed by pronounced hypoglycemia and death in severe instances (Nugteren and Berends, 1957). Interestingly, in cases of intoxications, all investigated fungal cultures were contaminated with bacteria. Eventually, Burkholderia gladioli pathovar cocovenenans (formerly Pseudomonas cocovenenans) (Coenye et al., 1999) was identified as the producer of a poison, bongkrekic acid (BA) (Figure 1), which accounts for the lethal incidents. Structurally BA represents a highly unsaturated and unusually branched tricarboxylic fatty acid (de Bruijn et al.,

1973; Zylber et al., 1973). Toxicological analyses have revealed that BA represents one of the most potent respiratory poisons known. BA efficiently blocks the mitochondrial adenine nucleotide translocator (ANT) (Klingenberg, 2008), thus inhibiting oxidative phosphorylation, (Henderson and Lardy, 1970) with an acute toxicity of 1.41 mg kg 1 (LD50 by intravenous injection)(Lijmbach et al., 1970a). From a pharmacological point of view, however, it is interesting to note that selective blockage of the ANT could prevent unspecific pore formation in mitochondrial membranes, which may result from oxidative stress and subsequent apoptotic events. Thus, the use of the ANT as a therapeutic target with bongkrekic acid as a potent inhibitor is the matter of ongoing investigations (Belzacq and Brenner, 2003; Halestrap and Brenner, 2003; Javadov et al., 2009). In light of this, knowledge of the molecular basis of BA biosynthesis would have a 2-fold benefit. First, it could aid in understanding the regulation of toxin production and detecting potential health threats through bacterial contaminations, and second, BA biosynthesis genes could be employed for engineering BA analogs that could be developed into selective ANT inhibitors. Whereas the chemical structure of BA has been long known, only recently we succeeded in gaining insights into the biosynthetic pathway. Stable isotope labeling studies using synthetic [1,2-13C2]acetyl SNAC and simultaneous inhibition of b-oxidation showed that the BA backbone is composed of acetate units (Figure 1B), and the chain branches suggested the involvement of a polyketide synthase (Rohm et al., 2010a). Thus far, however, the genes and mechanisms involved in bacterial BA synthesis have remained elusive. Here, we report the genetic basis of bongkrekic acid biosynthesis in the pathogenic bacterium B. gladioli. For functional studies we adapted a targeted gene deletion method for Burkholderia spp. Besides regulatory factors for toxin biosynthesis, bioinformatic and functional analyses shed light on complex polyketide assembly, branching events, and stereochemical issues. Our data also indicate the involvement of a cytochrome P450 monooxygenase in a rare six-electron oxidation of a terminal methyl group, giving rise to the tricarboxylic acid. RESULTS Identification and Analysis of the Bongkrekic Acid Biosynthesis Gene Cluster To unravel the genetic basis for the biosynthetic assembly of BA we have subjected genomic DNA (gDNA) of B. gladioli pathovar

1164 Chemistry & Biology 19, 1164–1174, September 21, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology Bongkrekic Acid Biosynthesis in B. gladioli

Figure 1. Respiratory Toxin Producers and Structures (A) Microscopic picture of a coculture of Burkholderia gladioli pv. cocovenenans and Rhizopus oligosporus used for tempe bongkrek production. (B) Structures of bongkrekic acid (BA) and iso-bongkrekic acid (IBA), and stable isotope labeling pattern. MS and NMR data used for structure elucidation can be found in Figures S3A–S3J.

cocovenenans (DSMZ 11318) to 454 shotgun sequencing, and remaining gaps in the putative bon gene locus were filled by PCR and Sanger sequencing (Lackner et al., 2011). Mining of the assembled contiguous regions for polyketide synthase (PKS) genes revealed an 64 kb gene locus consisting of 15 open reading frames ([ORFs], designated bonA-M, R1, and R2) (Figure 2A) flanked by housekeeping and transposase genes. All tentative functions of the deduced gene products were assigned by sequence comparisons using the NCBI BLAST and the polyketide prediction server SMART (Schultz et al., 1998) (Table 1). The deduced gene products of four ORFs (bonA-D) represent a multimodular type I PKS assembly line, and bonE-M code for typical accessory components and enzymes mediating tailoring reactions such as alkylation and oxygenation. Putative regulatory genes (bonR1-R2) are located upstream of the bonA-M genes. Interestingly, a BLAST search showed that a related gene cluster is also present in the recently sequenced, yet functionally uncharacterized, B. gladioli strain BSR3 (Seo et al., 2011). This strain has been isolated from a diseased rice sheath and its genome shows very high overall similarity with the draft genome of B. gladioli pv. cocovenenans (DSMZ 11318) in a synteny plot (Figure 2B). A BlastP analysis for all protein sequences encoded on the two gene loci indicated an identity as high as 98%–99%. Nevertheless, the PKS assembly line is encoded on only three ORFs in B. gladioli BSR3 (Figure 2). The differences in the amino acid sequences found are primarily due to single nucleotide substitutions that are more prominent in the PKS part of the gene cluster. To prove the identity of the BA biosynthesis gene cluster in B. gladioli pv. cocovenenans, we aimed at correlating toxin production with gene expression and gene inactivation experiments, respectively. Using real-time PCR we first investigated bon gene expression under BA-producing and nonproducing conditions and in parallel analyzed the culture extracts by HPLC/MS and noted a clear correlation between the production

of BA and expression of the putative bon gene cluster (Figure 2C). However, to unequivocally prove the involvement of bon genes in toxin biosynthesis a targeted deletion of biosynthetic genes was required. Targeted PKS Gene Disruption in B. gladioli Using the l Red Recombination System The targeted disruption of a predicted bon gene proved to be challenging because genetic tools that proved to be functional for related analyses in other Burkholderia spp. (Ishida et al., 2010; Leone et al., 2010; Partida-Martinez and Hertweck, 2007) were not applicable to B. gladioli. After many fruitless attempts using described methods and vectors for Burkholderia spp. we explored the l Red recombination system (Datsenko and Wanner, 2000; Doublet et al., 2008). By overlap PCR and subcloning we generated a pCR4Blunt-TOPO-based vector containing an apramycin resistance cassette (aac(3)IV) and two fragments of the bonA gene, which was introduced into B. gladioli by electroporation with the helper plasmid pKD46 (Doublet et al., 2008) carrying the l Red recombinase apparatus and a gentamicin resistance (Figure S1 available online). In this way we were able to adapt l Red recombination for B. gladioli and succeeded in inactivating bonA by scarless double crossover. The successful deletion was verified by PCR. Finally, the mutant was also confirmed by an altered phenotype. Metabolic profiling of the DbonA mutant showed that the lack of the PKS unit completely abolished the production of BA and its congener iso-bongkrekic acid (IBA) (Figure 2, lane b). In addition, a plate assay using Aspergillus nidulans as reporter strain revealed that antifungal activity of the B. gladioli extract fully relates with the presence of BA. Dissection of Complex Bongkrekic Acid Assembly on a Multimodular trans-AT PKS The clear connection of bongkrekic acid biosynthesis with the sequenced bon gene locus now allowed decoding the

Chemistry & Biology 19, 1164–1174, September 21, 2012 ª2012 Elsevier Ltd All rights reserved 1165


Figure 2. Genotype and Phenotype—Metabolic—Analysis of B. gladioli Wild-type and Mutant Strains (A) Architecture of the bon gene clusters in B. gladioli pv. cocovenenans and B. gladioli BSR3. For deduced gene functions, see Table 1. (B) Synteny dot plot showing a nucleotide-based comparison of the contigs of the draft genome of B. gladioli pv. cocovenenans DSMZ 11318 and the genome of B. gladioli BSR3. A high degree of similarity between both organisms is visible. A total of 5,576 matches were found, 393 queries were not matched. Location of the bon gene locus is indicated by a red box. (C) Bon gene expression assays. Expression of bonA in PDB (bongkrekic acid producing conditions, light gray bar) or NB medium (nonproducing conditions, dark gray bar) monitored by quantitative real-time PCR after 1 and 3 days. The rpoB gene was used as an internal standard for normalization. Relative quantification was carried out using the 2-DDCt method (left y axis). HPLC-MS monitoring of BA production in cultures in production (red graph) and nonproduction media (green graph) (right y axis). (D) HPLC monitoring of production of bongkrekic acid (BA), iso-bongkrekic acid (IBA) and deoxybongkrekic acid (DOBA), and inhibition zone assay using A. nidulans as reporter strain; (a) B. gladioli WT, (b) B. gladioli DbonA, (c) B. gladioli DbonL. Gene replacement strategy using the l RED system and confirmation of mutants can be found in Figure S1.

programmed biosynthetic pathway. Through detection of conserved motifs in BonA-D we inferred the PKS domains and the overall architecture of the megasynthase (Figure 3A; Table 1). Our bioinformatic analysis revealed that the bon PKS is a representative of the growing family of trans-AT PKSs (Piel, 2010). In contrast to cis-AT PKS, modules of the trans-AT subgroup of type I PKS lack AT domains, and the ACP domains are loaded with malonyl units in trans by freestanding, iteratively acting AT domains (Cheng et al., 2003; Musiol et al., 2011; Piel, 2010). Typically, trans-ATs occur in pairs, one functioning as a genuine AT, whereas the other has proof-reading function (Jensen et al., 2012). Such trans-AT domains are encoded by bonJ and bonK, respectively. As to the modular architecture of the PKS, the deduced processing line shows a high degree of colinearity with the chemical structure of BA. We identified ten functional PKS modules for the ten Claisen condensations required for the assembly of the polyketide backbone. Except for few irregularities, such as split modules and tandem ACP domains, the module compositions and the presence or absence of ketoreductase (KR) and dehydratase (DH) domains correspond well with the degree of b-keto processing of the proposed intermediates. According to the structure of the polyketide backbone two full reductive cycles should take place in

modules 7 and 9, but in none of the modules any ER domains could be located. Instead, a gene putatively coding for a freestanding ER domain (BonE) with a 2-nitropropane dioxygenase (NPD)-like motif has been found adjacent to the two ECH genes. BonE shares highest sequence homology with the ER part of ThaF in thailandamide biosynthesis (Ishida et al., 2010) and belongs to the family of NAD(P)H-dependent flavin oxidoreductases. Freestanding ERs have been implicated with enoyl reduction in PUFA synthases (Kaulmann and Hertweck, 2002), highly reducing fungal PKSs (Ma et al., 2009), and bacterial trans-AT PKSs (Bumpus et al., 2008), and in analogy BonE likely acts in trans in modules 7 and 9. According to the deduced module architecture, the two methionine-derived methyl residues at C-6 and C-16 would be incorporated into the nascent chain by methyl transferase (MT) domains in PKS modules 2 and 8. In contrast, the alkyl branches at C-21 and C-3 are acetate-derived, suggesting that these alkyl side chains are introduced by b-branching, that is alkylation at the position that corresponds to the former b-keto position of a polyketide intermediate (Calderone, 2008). Indeed, the bon cluster codes for several trans-acting elements that have been correlated with such isoprenoid-like b-branching events (Calderone et al., 2006). Specifically, we identified two gene cassettes,



Table 1. Deduced Functions of the bon Biosynthesis Gene Cluster Protein

Size (aa)

Proposed Function

Closest Relative [Origin]

Identity/Similarity (%)

Accession Number

BonA

7908

PKS; domains: GNAT?, ACP, KS, ACP, ACP, KS, DH, KR, MT, ACP, KS, KR, ACP, KS, DH, KR, ACP, KS, KR, ACP, KS

Polyketide synthase BaeL [Bacillus amyloliquefaciens FZB42]

41/56

YP_001421293.1

BonB

3594

PKS; domains: DH, ACP, KS, DH, KR ACP, KS, DH, KR

Polyketide synthase SorB [Sorangium cellulosum]

41/55

ADN68477.1

BonC

1719

PKS; domains: ACP, KS, DH, KR

Polyketide synthase SorB [Sorangium cellulosum]

53/65

ADN68477.1

BonD

4172

PKS; domains: MT, ACP, ACP, ACP, KS, DH, KR, ACP, KS?

Polyketide synthase SorA [Sorangium cellulosum]

53/66

ADN68476.1

BonE

450

Enoyl reductase

2-Nitropropane dioxygenase BatK [Pseudomonas fluorescens]

53/70

ADD82952.1

BonF

416

KS (involved in b-branching)

Ketosynthase BatB [Pseudomonas fluorescens]

62/71

ADD82943.1

BonG

424

3-Hydroxy-3-methylglutaryl-CoA synthase

3-Hydroxy-3-methylglutaryl CoA synthase BatC [Pseudomonas fluorescens]

68/80

ADD82944.1

BonH

276

Enoyl-CoA hydratase

Enoyl-CoA hydratase BatD [Pseudomonas fluorescens]

58/74

ADD82945.1

BonI

276

Enoyl-CoA hydratase

Enoyl-CoA hydratase BatE [Pseudomonas fluorescens]

58/78

ADD82946.1

BonJ

337

Acyl transferase

Malonyl CoA-acyl carrier protein transacylase, SorO [Sorangium cellulosum]

38/55

ADN68489.1

BonK

377

Acyl transferase

Acyltransferase/oxidoreductase DszD [Sorangium cellulosum]

56/69

AAY32968.1

BonL

417

Cytochrome P450 monooxygenase

Cytochrome P450 monooxygenase [Streptomyces rochei]

37/54

NP_851448.1

BonM

269

O-methyltransferase

Methyltransferase MitM [Streptomyces lavendulae]

35/51

AAD28459.1

ACP, acyl carrier protein; DH, dehydratase; GNAT, acyl-CoA N-acyltransferase; KR, ketoreductase; KS, ketosynthase; MT, methyl transferase; PKS, polyketide synthase.

bonFG and bonHI, that code for a 3-hydroxy-3-methylglutarylCoA (HMG) synthase (HCS, BonG), as well as two enoyl-CoA hydratases (ECH, BonH, and BonI) and a freestanding KS domain (BonF) lacking the conserved active site Cys residue. The structure of BA and the acetate incorporation pattern suggest that two b-branching events could take place: one just after the first elongation of the starter unit, whereas the other one appears to coincide with chain termination. To support and verify the colinearity-based prediction and to gain further insights into obscure steps of the bon pathway, in particular the b-branching events, we performed a more detailed bioinformatic analysis of the KS domains. An intriguing feature of KS domains of transAT PKSs is that they form distinct clades according to their substrate specificities, which allows predicting structures from noncolinear PKS architectures (Nguyen et al., 2008) as well as structure-based gene targeting (Fisch et al., 2009). A phylogenetic tree was constructed that comprised all bon KS domains and over 100 KS sequences from different trans-AT systems (Figure 3B). From the cladogram substructures were predicted that match well with the proposed intermediates. KS2, KS3, and KS10 belong to a clade of KS recognizing carbon branches at either the a- or b-position. The finding that KS3 and KS10 are specific for a-methylated precursors is in agreement with the

groups incorporated in the previous elongation round. In contrast, KS4 belongs to clade (II) of KS that accept b-hydroxylated intermediates, which is in good agreement to the hydroxyl functionality predicted by the structure introduced in module 3 (KSKR-ACP). The phylogenetic analysis also revealed that KS6 is indeed a so-called KS0 that belongs to clade XIV. These KS lack the conserved HGTGT motif, including the His residue that is essential for decarboxylative condensation and therefore just channel the intermediate to the subsequent module. The unusual sequence (KS-KR-ACP-KS0XIV-DH-ACP) was found to be characteristic for members of this clade (Piel, 2010). KS7 downstream the bimodule falls into clade IX of KS accepting a,b-unsaturated substrates. In the same clade KS5 as well as KS9 and KS11 are found. The module-based structure prediction suggested that two enoylreductions of intermediates bound to modules 7 and 9 are likely carried out by the freestanding enoylreductase (ER) BonE. The full reductive cycles mediated by modules 7 and 9 are supported by the finding that the subsequent KSs have specificity for saturated intermediates. Specifically, KS8 belongs to clade V, comprising KSs recognizing saturated intermediates, and KS10 belongs to clade I, composed of KSs selecting primarily saturated substrates with carbon branches; this is also in line with the presence of an MT domain in module 9.



Figure 3. Analysis of the bon PKS (A) Deduced architecture of the bon PKS and model for BA biosynthesis. Abbreviations and details explained in the main text. (B) Neighbor-Joining phylogenetic tree of fulllength KS domains from trans-AT PKSs with 10,000 bootstrap replicates for reliability estimation. Scale bar represents 0.1 substitutions per site. Bootstrap values above 90 are shown on branches. KS from bon gene cluster are highlighted in bold. Relevant substrates for KS clades including KS from bonPKS are displayed next to the individual clades. Clade types are shown in roman numbers referring to Nguyen et al. (2008) and explained in more detail in Results. Detailed analysis of the P450 monooxygenase BonL including a multiple sequence alignment of P450 protein sequences and a Neighbor-Joining tree of P450 orthologs can be found in Figure S2.

PKS-Based Prediction of Relative and Absolute Configuration To predict the stereochemical course of b-keto processing, we investigated the specificity motifs of the KR domains (Table 2).

In canonical modular type I PKSs, the presence of a conserved LDD motif generally correlates with a D-3-hydroxy substitution (Caffrey, 2003; Hertweck, 2009), and these deductions have proven reliable (e.g., for the absolute configuration of thailandamide, another complex Burkholderia polyketide) (Ishida et al., 2012). The hydroxyl at position C-17 was predicted to result from ketoreduction by the designated domain KR2. Because KR2 lacks the conserved Asp residue (VET instead of LDD motif), the intermediate was predicted to have an L-configuration, which translates into the observed absolute configuration at C-17 (R). As to the double bond geometries, knowledge of the KR specificities is also insightful because dehydration usually occurs in an anti fashion, and consequently D-hydroxyls yield E-double bonds, whereas Z-double bonds emerge from L-hydroxyls. This correlation holds true for the predicted D-hydroxyl intermediates that are morphed into E-double bonds at C-4/5 and C-8/9, and the Z-double bond at C-18/19 could be predicted from the L-specificity of KR1. However, module 4 apparently codes for the formation of an E-double bond, but a Z-configuration has been described for C-14/15. Another inconsistency has been observed for the unusual split bimodule (modules 5 and 6), which would be suited for the introduction of a Z-double bond into the growing polyketide chain. Consequently, a Z-double bond would be expected at the position between C-12 and C-13 in BA (Figure 3A), but this is not in accord with the reported structure of BA (de Bruijn et al., 1973). As to the



Table 2. Bioinformatic Prediction of bon PKS Stereochemistry Domain(s)

KR Motif

Position/ Found

X

/ E-DB

C-20/21 (E)

GIVHSAVVLGEAPL

L

/ Z-DB

C-18/19 (Z)

Branch KR1/DH

Predicted

KR2

GVLHAAGLVETRSLL

L

(OH)

C-17 (R)

KR3/DH

GVIHAAGVLHDALLA

D

/ E-DB

C-14/15 (Z)

KR4/DH

GVLHCAGRVDAEQP

L

/ Z-DB

KR5/DH/ER GVVQGAGVLRDGLLA

/ E-DB / CH2

KR6/DH

GVIHAAGVLEDGLLP

D

KR7/DH/ ER/MT

GVVHSAIVLADRSLA

/ E-DB / CHCH3

KR8/DH

GVIHAAGLLHDGLLL

D

Branch

D

/ E-DB

D

/ E-DB

C-12/13 (E) C-10 C-8/9 (E) C-6 (S) C-4/5 (E)

x / E-DB (BA) C-3/23 (E) (BA) x / Z-DB (IBA) C-3/23 (Z) (IBA)

Inconsistency in predicted and observed stereochemistry are shown in shaded area. DH, dehydratase; ER, enoyl reductase; KR, ketoreductase.

stereochemical course of the enoyl reductions, no valid predictions could be made. Polyketide Tailoring by a Six-Electron Oxidation of a Methyl Group Finally, after the full-length polyketide has been assembled and released, it likely undergoes two enzyme-mediated tailoring reactions. The C-17 hydroxyl would be methylated by the predicted O-methyltransferase BonM, and according to stable isotope labeling experiments (Rohm et al., 2010a) we assumed that the terminal carboxyl group is generated by sequential sixelectron-oxidation of C-22 (Figure 3A). Based on the sequence analyses, the gene product of bonL could be the best candidate for catalyzing the oxygenation reaction, because BonL shows high similarity to various cytochrome P450 monooxygenases (CYPs) involved in secondary metabolic pathways. To relate BonL to the family of CYPs, a phylogenetic tree was constructed based on the BonL amino acid sequence and various functionally characterized orthologs. BonL is closely related to other CYPs that act as polyketide tailoring enzymes. To test the function of BonL in BA biosynthesis, we aimed at constructing a DbonL mutant. Again, using the l Red system we succeeded in inactivating bonL, as verified by PCR analyses (Figure S2). The growth inhibition assay using the DbonL mutant extract showed a substantially reduced antifungal activity. Furthermore, HPLC-MS analysis of the mutant’s metabolic profile revealed that the loss of bonL led to an abrogation of BA and IBA production. Instead, we noted a new peak at a higher retention time (25.4 min), suggesting a lower polarity of the metabolite compared to BA (Figure 2D, lane c), probably due to the presence of a methyl group in lieu of the carboxyl moiety. Indeed, mass spectrometry revealed that BA and the observed compound share a similar MS/MS fragmentation, and the molecular mass of 456 Da, would also support the assumption. To unequivocally determine its structure, this compound (deoxybongkrekic acid [DOBA]) was isolated from an upscaled culture (5 L). The molecular composition (C28H40O5) was deduced from HRESI-MS analysis and 1H and 13C nuclear magnetic resonance (NMR) spectra.

Figure 4. Structure of Deoxybongkrekic Acid and Key 2D NMR Connectivities MS and NMR data used for structure elucidation can be found in Figures S3B–S3J.

One-dimensional and 2D NMR data showed high similarity to those of BA confirming a related core structure. In contrast to BA, the carbon NMR of DOBA revealed the presence of only two carboxyl functions and an additional methyl carbon. HMBC coupling of the corresponding methyl protons with C-20, C-21, and C-21a established the connectivity of the methyl function and finally confirmed the proposed structure (Figures 4 and S3). As to the double bond geometry, the proposed structure of DOBA is in agreement with the configuration of BA. In 1 H NMR proton H-14 appears as triplet (J = 11 Hz), and the other protons at trans-double bonds have a diagnostic coupling constant of J = 14–16 Hz. Unfortunately, H-15 appears as a multiplet, which hampers a clear assignment. However, we observed a strong NOE contact of H-14 and H-15, and no correlation between H-15 and H-13 (Figures 4 and S3). BA production could be restored by expression of bonL in the DbonL mutant (see Supplemental Experimental Procedures). DISCUSSION In this study we have unveiled the molecular basis for the biosynthesis of the infamous toxin bongkrekic acid (BA), which is produced by B. gladioli as a contaminant in tempe bongkrek fermentation using R. oligosporus. The Burkholderia-Rhizopus interaction in the fermentation broth is reminiscent to a bacterial-fungal symbiosis producing the phytotoxin rhizoxin, the causative agent of rice seedling blight (Lackner and Hertweck, 2011; Partida-Martinez and Hertweck, 2005). It was shown that endofungal bacteria (Burkholderia rhizoxininca) of Rhizopus microsporus are the true producers of the toxin complex (Scherlach et al., 2006), and these antimitotic agents are also produced in alarmingly high concentrations in soy-based tempe (Lackner et al., 2009; Rohm et al., 2010b). As to the bacterial-fungal interaction in tempe bongkrek production it should be noted that Rhizopus and various other mold fungi are sensitive to the ´k and Behuń , 1994), as evidenced by our bacterial toxin (Subı plate assay, which is quite in contrast to the acquired rhizoxinresistance of Rhizopus bearing Burkholderia symbionts (Schmitt



et al., 2008). Furthermore, BA formation is strictly dependent on various growth conditions and is mainly observed in fatty coconut media (Adams and Moss, 2008). To gain insight into the encoded BA assembly line and regulatory factors, we have subjected total gDNA of B. gladioli to shotgun sequencing and obtained a draft genome sequence. Through gap filling we have obtained a contiguous sequence harboring a gene cluster that codes for a modular type I PKS. In this work we provided three lines of evidence for the identity of bon biosynthesis gene cluster. First, through qRT-PCR and HPLC-MS monitoring of the culture we could link bon gene expression with BA production; second, the deduced module architecture of the bon PKS is nearly colinear with the structure of the metabolite; third, selected bon gene functions were confirmed by targeted gene knock out. The latter proved to be challenging because previously employed methods and tools for Burkholderia were not workable. Whereas recently a transposon-based approach has been described for random transposon mutagenesis of B. gladioli (Somprasong et al., 2010), targeting a specific gene remained a challenge for this species. Eventually, we succeeded in deleting several bon genes using a modified l Red recombination protocol. l Red recombination is known as a powerful technique in particular for functional genetics in actinomycetes (Gust et al., 2004), and lately it was also proven to be operative in Pseudomonas aeruginosa (Lesic and Rahme, 2008; Liang and Liu, 2010). In the course of our studies, the use of l Red recombination for homologous lipase overexpression in Burkholderia cepacia (Jia et al., 2010) and for recombineering in naturally transformable Burkholderia spp. (Kang et al., 2011) has been reported. To the best of our knowledge, the bon gene mutants are the proof of principle for the use of the l Red recombination system for targeted gene deletions in B. gladioli. This is highlighted as it represents a convenient and efficient method for functional genomics of this important species. Interest in the various pathovars of B. gladioli has grown substantially during the past years as this species has been recognized as an emerging pathogen causing disease in humans, fungi, and plants (Segonds et al., 2009; Stoyanova et al., 2007). B. gladioli is a risk factor in cystic fibrosis (Kennedy et al., 2007), and numerous infections have been described in the context of lung transplantations (Hafkin and Blumberg, 2009). In agriculture, B. gladioli is known as the cause of cavity disease of the mushroom Agaricus bisporus (Largeteau and Savoie, 2010). Our finding that a bon gene locus is also present in the genome of a B. gladioli strain isolated from a diseased rice sheath suggests that BA may operate as a virulence factor in plants. Considering the high toxicity of BA it would appear plausible that the bon locus is a common trait of pathogenic B. gladioli strains, and we propose that bon genes may be used as taxonomic marker for classifying toxinogenic B. gladioli pathovars (Jiao et al., 2003). However, it should also be noted that apart from being a toxin, BA is also considered as a specific ligand of ANT and may be developed as an antiapoptotic agent. Thus, knowledge of BA biosynthesis genes will also set the basis for pathway engineering. In fact, the bioinformatic and functional analyses of the bon genes granted some unexpected insights into the assembly of the complex polyketide. On the ground of module architectures

and KS-based phylogeny the chain elongation, b-keto processing and timing of chain branching events could be deduced. Accordingly, the molecule is assembled in a nearly colinear fashion, initiated by GNAT-mediated PKS loading (Gu et al., 2007), followed by ten chain elongations. Two isoprenoid-like b-branching events take place at the beginning and at the end of chain assembly. The first branching seems to yield an isopentenyl moiety after decarboxylation and double bond shift. In contrast, the terminal branching leaves the C2 unit intact and generates an acrylate side chain that is reminiscent of bryostatin substructures (Sudek et al., 2007). Notably, syn or anti eliminations during b-branching could account for the occurrence of the E/Z-isomers BA and IBA. It should be highlighted that no designated off-loading domain could be identified in the deduced PKS assembly line. Because b-branching appears to be the final step in chain assembly, one may conceive that branching and chain termination coincide, a scenario that is currently unparalleled. Whereas the constitution of the polyketide backbone is in line with the PKS domain analysis, comparison of predicted and reported configurations of the BA structure posed some riddles. Generally, the absolute configuration of the hydroxyl-substituted C-17 and the geometries of most double bonds are in full accord with the deduced KR specificity. However, we noted peculiar deviations in predicted and observed double bond geometries for the C-14 diene stretch. The deduced KR specificity in conjunction with the typical DH-mediated anti elimination would call for the opposite order of E- and Z-double bonds. Because the NMR-based resolution of double bond geometries in polyunsaturated compounds may be ambiguous, one might question the published structure of BA. It may be noted that the structure of BA was initially wrongly assigned (Lijmbach et al., 1970b), but the revised structure (de Bruijn et al., 1973) and the relative and absolute configuration (Zylber et al., 1973) has been confirmed by numerous total syntheses (Corey and Tramontano, 1984; Francais et al., 2010, 2011; Sato et al., 2009; Shindo et al., 2004). Even so, E/Z- and Z/E-isomers are not easily discriminated from each other, and future synthetic and analytical studies may be needed to address this particular issue. In recent years, stereochemical analyses of complex polyketides are more and more often supported or even facilitated using bioinformatics, as demonstrated for etnangien (Menche et al., 2008), thuggacins (Bock et al., 2008), chivosazole (Janssen et al., 2007), and thailandamide (Ishida et al., 2012). The in silico dissection of the BA pathway and the configurational analysis underline the power of bioinformatics deductions, yet also indicates a potential caveat regarding the double bond geometries. In addition to stable isotope labeling and bon PKS analysis, the proposed polyketide assembly is supported by the isolation and structural elucidation of a key pathway intermediate from an engineered B. gladioli strain. We selected a candidate gene (bonL) coding for a cytochrome P450 monooxygenase that could be involved in a polyketide tailoring reaction. We successfully generated a DbonL mutant producing a bongkrekic acid derivative. The full structure elucidation by MS, 1D, and 2D NMR revealed that this compound bears a methyl group in place of the C-22 carboxyl moiety. Consequently, C-22 is derived from C-2 of the priming acetate, which is in line with the 13C2-acetate labeling experiments, and the C-1 carboxyl constitutes the



COOH terminus of the polyketide chain. It is intriguing to find that BonL is a CYP that catalyzes the oxidation of the polyketide terminus, and it appears that BonL is involved in a six-electron oxidation. CYPs are known to catalyze a variety of oxidation reactions (Werck-Reichhart and Feyereisen, 2000), mainly resulting in hydroxyl groups or epoxides. Considering the large number of functionally characterized cytochrome P450 monooxygenases, it is surprising to find that only very few cytochrome P450 enzymes catalyze multiple oxidation steps in biosynthetic pathways. Interestingly, the oxidation of methyl groups to carboxylic acids has been primarily observed in plant and fungal terpene biosynthetic pathways. Rare examples include CYPs involved in gibberellic acid biosynthesis in Arabidopsis thaliana (ent-kaurene-oxidase) (Helliwell et al., 1999) and Gibberella fujikuroi (P450-1) (Rojas et al., 2001). Functionally related CYPs mediate the oxidation of amorpha-4,11-diene to artemisinic acid in Artemisia annua (CYP71AV1) (Ro et al., 2006; Teoh et al., 2006), and participate in the biosynthesis of resin acid in conifers (abietadienol/abietadienal oxidase PtAO) (Ro et al., 2005). The biotransformation of pentalenene to pentalen-13-al in Streptomyces avermitilis by the CYP PtlI is a rare example of a similar process in bacterial terpene biosynthesis (Quaderer et al., 2006). In contrast, CYPs catalyzing sequential methyl oxidations in complex polyketide pathways are scarce. From deletion of a CYP450-encoding gene, fscP, in the candicidin/ FR-008 biosynthesis gene locus it was inferred that the pathway involves oxidation of a methyl branch to a carboxylate residue (Chen et al., 2009). Borrelidin biosynthesis in Streptomyces parvulus Tu¨4055 employs a CYP (BorI) that is capable of oxidizing a methyl group to an aldehyde, which may be further oxidized to the carboxylic acid in a shunt pathway (Olano et al., 2004). The multifunctional CYP AurH from Streptomyces thioluteus, mediates a rare sequential hydroxylation-heterocyclization sequence (Richter et al., 2008), and point mutations result in regiodivergent, multiple oxidations of an allylic methyl group to the carboxyl via alcohol and aldehyde intermediates (Zocher et al., 2011). We assume that BonL oxidizes the C-22 methyl of BA in a similar fashion, yet no partially oxidized intermediates could be detected by MS. Nonetheless, BonL is a fresh entry in the small family of multifunctional CYPs. It is one of the very few carboxylate-forming CYPs in polyketide pathways, and to our knowledge, the first one that is involved in the oxidation of a carbon chain terminus. Thus, it is an important addition to the currently available enzymatic toolbox for polyketide tailoring. Finally, from a natural product perspective Burkholderia spp. are rather neglected organisms (Winter et al., 2011). Compared to the more prolific bacterial producers of biologically active secondary metabolites, such as actinomycetes and myxobacteria, only few natural products from Burkholderia spp. are known. To date the only examples of polyketide metabolites from this genus that have been discovered and linked to a gene cluster include rhizoxins from Burkholderia rhizoxinica (Ishida et al., 2010; Leone et al., 2010; Partida-Martinez and Hertweck, 2007), thailandamide (Nguyen et al., 2008) and bactobolins from Burkholderia thailandensis (Seyedsayamdost et al., 2010), and enacyloxins from Burkholderia ambifaria (Mahenthiralingam et al., 2011). The BA biosynthetic machinery is an important addition to the growing family of Burkholderia modular PKS assembly

lines. Notably, all of the previously studied pathways involve hybrid PKS-nonribosomal peptide synthetases (NRPSs), and the bon PKS represents, to our knowledge, the first genuine PKS system. SIGNIFICANCE The respiratory toxin bongkrekic acid caused countless health-threatening and even lethal intoxications after consumption of the Southeast-Asian specialty tempe bongkrek. Whereas it was long known that the toxin producer is a contaminant of the fungus used for fermentation, the genetic basis for BA biosynthesis has remained obscure. Through whole-genome sequencing of Burkholderia gladioli pv. cocovenenans we identified the gene locus for a giant trans-AT PKS and accessory enzymes catalyzing assembly the polyunsaturated tricarboxylic acid. For functional analyses we successfully adapted l Red recombination and provide the proof of principle for targeted gene deletions in B. gladioli. This protocol is of great value because B. gladioli pathovars are the cause of onion soft rot, mushroom cavity disease, and emerging risk factors for patients suffering from cystic fibrosis and lung transplantations. In this context, it is noteworthy that a strain that infected rice plants harbors an intact bon gene cluster. From a biosynthetic point of view, unexpected insights are granted into the stereocontrolled assembly of BA by a thiotemplate. Such multifunctional enzymes are underexplored in Burkholderia spp., whereas the bon PKS represents now a characterized genuine PKS in this genus. Dissection of the bon PKS revealed the pathway to the unusual bifurcated fatty acid structure, suggesting an unprecedented scenario where b-branching and chain termination coincide. Through isolation of deoxybongkrekic acid from a CYP mutant we inferred an unparalleled six-electron oxidation of the methyl terminus of a complex polyketide chain. Considering that BA is one of the most potent respiratory toxins known, these insights are of significance for pinpointing toxinogenic B. gladioli pathovars in the context of food processing, agriculture and medicine. On pharmacological grounds knowledge of the molecular basis for BA biosynthesis paves the way for engineering specific ligands for adenine nucleotide translocator, a promising target for antiapoptosis and chemotherapy. EXPERIMENTAL PROCEDURES See Supplemental Experimental Procedures.

ACCESSION NUMBERS The EMBL accession number for the bon biosynthetic gene cluster sequence reported in this paper is JX173632.

SUPPLEMENTAL INFORMATION Supplemental Information includes three figures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi. org/10.1016/j.chembiol.2012.07.022.



ACKNOWLEDGMENTS We are grateful for financial support by the Pakt fu¨r Wissenschaft und Innovation and the Jena School for Microbial Communication (JSMC). We thank A. Perner for MS analyses and H. Heinecke for NMR measurements, B. Doublet (Paris) for the gift of plasmid pKD46, and G. Lackner for assistance in bioinformatics analyses. N.M. analyzed genome sequences; N.M. and B.R. performed phylogenetic and bioinformatic analyses; C.R., N.M., and B.R. conducted functional gene analyses; B.R. optimized culture conditions; M.R. conducted fermentations; C.R., K.S., and N.M. isolated and analyzed compounds; all authors analyzed data; and C.H. designed research and wrote the manuscript.

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Received: May 31, 2012 Revised: July 20, 2012 Accepted: July 24, 2012 Published: September 20, 2012

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