Characterization of Polyketide Synthase Machinery from the pks Island

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Characterization of Polyketide Synthase Machinery from the pks Island Facilitates Isolation of a Candidate Precolibactin Li Zha, Matthew R. Wilson, Carolyn A. Brotherton, and Emily P Balskus ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00014 • Publication Date (Web): 18 Feb 2016 Downloaded from http://pubs.acs.org on February 21, 2016

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ACS Chemical Biology

Characterization of Polyketide Synthase Machinery from the pks Island Facilitates Isolation of a Candidate Precolibactin

Li Zha‡, Matthew R. Wilson‡, Carolyn A. Brotherton, and Emily P. Balskus* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States Email: [email protected]

‡ = These authors contributed equally to this work.

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ABSTRACT: Colibactin is a human gut bacterial genotoxin of unknown structure that has been linked to colon cancer. The biosynthesis of this elusive metabolite is directed by the pks gene cluster, which encodes a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPSPKS) assembly line that is hypothesized to use the unusual polyketide building block aminomalonate. This biosynthetic pathway is thought to initially produce an inactive intermediate (precolibactin) that is processed to the active toxin. Here we report the first in vitro biochemical characterization of the PKS components of the pks enzymatic assembly line. We evaluate PKS extender unit utilization and show that ClbG, a freestanding acyltransferase (AT) from the pks gene cluster, recognizes aminomalonyl-acyl carrier protein (AM-ACP) and transfers this building block to multiple PKS modules, including a cis-AT PKS ClbI. We also use genetics to explore the in vivo role of ClbG in colibactin and precolibactin biosynthesis. Unexpectedly, production of previously identified pks-associated metabolites is dramatically increased in a ∆clbP/∆clbG mutant strain, enabling the first structure elucidation of a bithiazole-containing candidate precolibactin. This work provides new insights into the unusual biosynthetic capabilities of the pks gene cluster, offers further support for the hypothesis that colibactin directly damages DNA, and suggests that additional, uncharacterized pks-derived metabolites containing aminomalonate play critical roles in genotoxicity.


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Colibactin is a genotoxin of unknown structure produced by enterobacteria, including extraintestinal pathogenic and human gut commensal Escherichia coli strains.1,2 This metabolite is biosynthesized by the pks island, a 54-kb gene cluster that encodes a nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Figure 1A).3 Transient infection of eukaryotic cells with E. coli strains possessing the pks island (pks+) induces host-cell DNA double-strand breaks in vitro and causes chromosomal instability in vivo.3,4 Pks+ E. coli are found with increased frequency in colorectal cancer patients and influence tumor progression in animal models of colitis-associated colorectal cancer.5-8 Despite its potential relevance to cancer, the mechanism underlying colibactin’s genotoxicity remains uncharacterized as the active metabolite has eluded multiple isolation attempts.

Previously we obtained information about colibactin’s structure by characterizing biosynthetic enzymes involved in a self-resistance mechanism (Figure 1B).9 We demonstrated in vitro that colibactin biosynthesis begins with the assembly and elongation of an N-acylated D-asparagine scaffold by the NRPS modules ClbN and ClbB. We hypothesized this scaffold was processed by the remaining pks enzymes to an inactive metabolite (precolibactin) that was hydrolyzed by a periplasmic peptidase, ClbP, to release the ‘prodrug motif’ and generate the active genotoxin. Consistent with this biosynthetic logic, ClbP processed synthetic substrates containing the prodrug motif in vivo9 and pks+ E. coli ∆clbP mutants lacked genotoxicity.3 These insights have guided the isolation and structural characterization of candidate precolibactins (1–6) from pks+ E. coli ∆clbP mutants (Figure 1C), providing potential insights into colibactin’s activity.10-14 Most notably, the aza-spirocyclopropanes found in 4–6 resemble the ring systems found in DNAalkylating agents such as the duocarmycins15 and illudins,16 suggesting colibactin’s mode of action may involve the covalent modification of DNA. The structures of higher molecular weight candidate precolibactins have also been proposed based on mass spectrometry (MS) data and the 3 ACS Paragon Plus Environment


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features of the pks enzymatic assembly line.11,14 Two of these predicted structures (7, 8) contain thiazolinyl-thiazole and bithiazole heterocycles, elements of the DNA-damaging natural products phleomycin and bleomycin (Figure 1D).

Knowledge gained from these isolation efforts, along with feeding and gene inactivation experiments,11,13,14 has informed a hypothesis for the biosynthesis of isolated and proposed candidate precolibactins (Figure 1E). Notably the products of several essential genes are not utilized in this proposal, including ClbD, E, F, and G. These enzymes are predicted to participate in the biosynthesis and incorporation of the unusual PKS extender unit aminomalonyl-acyl carrier protein (AM-ACP) as they are homologous to AM-synthesizing and incorporating enzymes from zwittermicin17-19 and guadinomine20 biosynthesis. The zwittermicin biosynthetic enzymes ZmaGJ produce AM-ACP from L-serine in vitro.17 AM-ACP incorporation by the zwittermicin NRPSPKS assembly line occurs at a PKS module predicted to lack a functional acyltransferase (AT) domain, and transfer of this building block to the ACP domain of ZmaA requires a trans-acting AT ZmaF (Figure 2A).19


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Figure 1. Colibactin and the pks island. (A) The pks island. (B) Colibactin biosynthesis involves the synthesis of an inactive precursor (precolibactin). (C) Structurally characterized candidate precolibactins isolated from pks+ E. coli ∆clbP mutant strains. (D) Proposed structures for candidate precolibactins identified in pks+ E. coli ∆clbP mutant strains. (E) Biosynthetic hypothesis for the production of isolated and proposed candidate precolibactins in pks+ E. coli ∆clbP strains. NRPS domains are abbreviated as C (condensation), A (adenylation), PCP (peptidyl carrier protein), E (epimerization), Cy (cyclization) and Ox (oxidase). PKS domains are



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abbreviated as KS (ketosynthase), AT (acyltransferase), KR (ketoreductase), DH (dehydratase), ER (enoyl reductase) and ACP (acyl carrier protein).

Recently, Piel and co-workers showed that NRPS ClbH, ACP ClbE, and dehydrogenases ClbD and F generate AM-ACP from L-serine in vitro in an analogous manner to the zwittermicin biosynthetic enzymes (Figure 2B).21 AM-ACP formation involved activation of L-serine by the first adenylation (A) domain of ClbH (ClbH-A1), followed by transfer to the phosphopantetheinyl (ppant) group of holo-ClbE. The resulting seryl-ClbE thioester was oxidized by dehydrogenases ClbD and ClbF to give AM-ClbE. Pks+ E. coli mutants missing any component of the AM biosynthetic machinery are not genotoxic,3 implicating AM-ACP in colibactin assembly. However, the mechanism of AM-ACP transfer to the pks assembly line and the identities of the PKS modules incorporating the extender unit have not been elucidated.

Here we characterize the extender units used by the PKS modules of the pks assembly line and investigate the role of a trans-acting AT (ClbG) in substrate selection. We find that ClbG transfers AM-ClbE to multiple PKS modules, including PKSs that lack functional AT domains (ClbC, ClbK, and ClbO) and a cis-AT PKS (ClbI). Deleting clbG greatly enhanced the production of candidate precolibactins in a clbP mutant, facilitating the isolation and structural characterization of proposed candidate precolibactin 7. The presence of a bithiazole heterocycle in this metabolite indicates colibactin may interact directly with DNA. Overall, this work provides further information about the unusual biosynthetic capabilities of the pks assembly line and supports prior evidence that as-yet uncharacterized metabolites derived from aminomalonate are critical for genotoxicity.


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Figure 2. Colibactin biosynthesis may involve the unusual PKS extender unit aminomalonyl-acyl carrier protein (AM-ACP). (A) Formation, recognition, and transfer of AM-ACP in zwittermicin biosynthesis. (B) Hypothesis for transfer of AM-ACP in colibactin biosynthesis. (C) PKS modules in the pks assembly line (AT*, partial, potentially inactive AT domain (atypical-AT)).

Results and Discussion



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The pks island encodes a mixture of cis-AT and atypical-AT PKS modules. Multi-modular PKSs are responsible for the biosynthesis of numerous bioactive natural products and secondary metabolites.22 Each PKS module contains multiple enzymatic domains that work together to catalyze selection and tethering of a malonyl extender unit to the assembly line, decarboxylative Claisen condensation, and processing of a β-ketothioester intermediate.23 While cis-AT PKS modules contain an AT domain for extender unit selection, trans-AT PKS modules use a separate freestanding AT domain. Both types of AT domains catalyze transacylation of CoA- or ACPthioesters onto a conserved active site serine, followed by transfer of this acyl-enzyme intermediate to the thiol group of the ppant arm of a holo-ACP domain on the assembly line. Contrary to most modular PKS systems, which contain exclusively cis- or trans-AT PKSs,24-26 the pks assembly line possesses two cis-AT PKS modules and three PKS modules that appear to lack a functional AT domain (Figure 2C).

The cis-AT PKS modules (ClbB and ClbI) each contain a canonical AT domain that has a GHSxG motif containing the catalytic active site serine and a HAFH motif characteristic of malonyl-CoA utilization.21 In contrast, the remaining PKSs (ClbC, ClbK, and ClbO) possess ‘deteriorated,’ potentially inactive AT domains that lack both the GHSxG motif and a fourresidue extender unit specificity motif (Figure S1). Based on structural homology predictions, Crawford and co-workers proposed that these PKS modules were inactive relics of cis-AT PKSs .10,27 These two types of modular PKSs are believed to follow different evolutionary paths; while cis-AT PKSs principally evolve by duplication of genes encoding individual modules and diversification of domain sets, trans-AT PKSs are assembled via horizontal gene transfer.24,25


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Intrigued by this combination of PKS machinery, we performed additional bioinformatic analyses of these enzymes. Multiple sequence alignment of the amino acid sequences of the ‘ATless’ PKS modules ClbC, ClbK, ClbO, and ZmaA with cis-AT and trans-AT PKSs revealed that all four enzymes harbored large, poorly aligned fragments absent in all of the trans-AT PKSs except for OzmQ, a post-NRPS trans-AT PKS containing a partial AT domain (Figure S1).28,29 We hypothesized that like OzmQ,29 these partial AT-containing enzymes evolved from type I PKSs through the degradation of a cis-AT domain. Because ClbC, ClbK, ClbO, and ZmaA contain partial AT domains that lack active site or specificity motifs, we refer to these PKS modules as atypical-AT PKSs.

While none of the PKS modules from colibactin biosynthesis have been characterized in vitro, metabolite profiles from pks mutants have provided information about their activities.13,14 The two cis-AT PKSs (ClbB and ClbI) and atypical-AT PKS ClbC are thought to incorporate malonyl-CoA into candidate precolibactins. ClbC’s reactivity is unexpected given its predicted lack of a functional AT domain, and it has been proposed that malonyl-CoA loading onto ClbC may be catalyzed by the AT from fatty acid synthesis (FabD), the cis-AT domains of ClbB or ClbI, or the freestanding AT ClbG.11 The additional atypical-AT PKSs ClbK and ClbO have not been implicated in the elongation of any assembly line intermediates and their in vivo roles are unclear. Therefore, both bioinformatic and in vivo data suggest that the PKS modules involved in colibactin biosynthesis possess unusual features, highlighting a need for in vitro biochemical characterization.

Freestanding AT ClbG recognizes the AM-ACP extender unit. We next sought to characterize the in vitro substrate specificities of the PKS components of the pks assembly line and enzymes



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involved in extender unit selection. In analogy with zwittermicin biosynthesis, we hypothesized that AM-ACP incorporation would occur at modules lacking cis-AT domains (ClbC, ClbK, and ClbO), and that AM-ACP transfer to PKSs would be mediated by ClbG, the only freestanding AT in the pks island.11,13,21 To date, in vitro and in vivo characterization of ClbG have not been reported; however a pks+ E. coli ∆clbG mutant lacked activity, suggesting that this enzyme is required for genotoxin production.3 Piel and co-workers previously proposed a role for ClbG in AM-ACP transfer and noted that in addition to the GHSxG motif, ClbG possessed an uncommon specificity motif (VPYH) that is similar to that of ZmaF (GPFH), the AM-ACP-transferring AT from zwittermicin biosynthesis.21

We began our work by investigating ClbG substrate specificity in vitro. To access AM-ACP and ClbG, we cloned, overexpressed, and purified ClbH-A1, ClbD, ClbE, ClbF, and ClbG as His6-tagged fusions (Figure S2). We established that ClbH-A1 preferentially activated L-serine using the ATP-[32P]PPi exchange assay (Figure S4). Using liquid chromatography-highresolution mass spectrometry (LC-HRMS), we confirmed that AM-ClbE was produced in assays containing ClbH-A1, ClbD-F, phosphopantetheinyl transferase Sfp,30 and all necessary cofactors. Consistent with previous reports, we detected only glycyl-ClbE, the product of AM-ClbE decarboxylation (Figure S6).17,21 These observations confirmed that all of the AM-forming enzymes were functional in vitro.

We then tested whether AM could be transferred from ClbE to ClbG using SDS-PAGE analysis and gel autoradiography. Incubating ClbG with [14C]-L-serine, ClbH-A1, ClbD-F, and all necessary cofactors resulted in a strong transfer of the [14C]-label to ClbG (Figure 3A). We also observed weaker transfer of the radiolabel when dehydrogenase ClbF was omitted. This


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observation is consistent with the activity of the homologous zwittermicin biosynthetic enzymes (ZmaF-J) and may arise from transfer of a putative aldehyde hydrate intermediate.19 Taken together, these results confirm that ClbG is a functional AT domain that recognizes AM-ClbE.

Additionally we examined the possibility that ClbG could also accept malonyl-CoA, the only PKS extender unit utilized in the synthesis of characterized and proposed candidate precolibactins. Upon incubating ClbG with 5–100 µM [14C]-malonyl-CoA, a concentration range comparable to the E. coli malonyl-CoA pool (4–90 µM),31 we consistently observed much weaker loading onto ClbG compared to AM-ACP (Figure 3B). These results strongly suggest that AM-ACP is the preferred substrate of ClbG.

A ClbE ClbH -A1, ATP ClbD, ClbF NADPH, FAD

[14C]L-Serine

ClbG ClbG

ACP

AT

AT O

S

O

O H 2N

H 2N

ACP

O

O

holo ClbE

+ + + + +

ClbH-A1 ClbE ClbD ClbF ClbG

+ + + + –

+ + + – +

+ + – + +

+ – + + +

– + + + +

kDa

ClbH-A1 ClbE ClbD ClbF ClbG [14C]-malCoA

ClbG AT O

O

AT O O

CoAS

OH O

[14C]-malonyl-CoA

HO

+ + – + +

+ – + + +

– + + + + kDa

ClbG

25 20 15

46 25-28 12

ClbE

Coomassie-stained

ClbG

+ + + – +

55-58 50 37

ClbE

B

+ + + + –

75

ClbH-A1 ClbG ClbF ClbD Sfp

HO

HO

+ + + + +

+ + + + + –

+ + + + – –

– – – – +

– – – – +

– – – – +

– – – – +

– – – – +

Phosphorimage

– – – – –

5 10 20 40 100 100

+ + + + + –

kDa

+ + + + – –

– – – – +

– – – – +

– – – – +

– – – – +

25-28 ClbE

Coomassie-stained

Figure 3. Trans-AT ClbG recognizes AM-ACP in vitro. (A)

kDa

69 55-58 46

50 ClbG 37 20 15

– – – – –

5 10 20 40 100 100

75 ClbH-A1 ClbG ClbF ClbD Sfp ClbE

– – – – +

12

Phosphorimage

14

C gel autoradiography assay

testing ClbG’s reactivity toward AM-ClbE. Assays (30 µL) contained: 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 500 µM CoA, 1 mM TCEP, 10 µM ClbE, 1.5 µM Sfp, 800 µM NAD+, 500 µM FAD, 3 µM ClbH-A1, 6 µM ClbD, 1.5 µM ClbF, 3 µM ClbG, 50 µM [14C(U)]-L11 ACS Paragon Plus Environment


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serine (145.1 mCi/mmol, Perkin Elmer), and 1.7 mM ATP. All assays were incubated at 37 ºC and were quenched after 20 min. (B)

14

C gel autoradiography assay testing ClbG’s reactivity

toward malonyl-CoA. Assays (30 µL) contained: 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 3 µM ClbG, and 5–100 µM [2-14C]-malonyl-CoA (55 mCi/mmol, American Radiolabeled Chemicals). All assays were incubated at 37 ºC and were quenched after 20 min. The AM-ACP control (Lane 1) was performed as described in (A).

Multiple PKS modules from the pks assembly line accept AM-ACP in vitro. Having characterized the enzymes involved in AM-ACP synthesis and transfer, we next cloned, overexpressed, and purified all five PKS modules from the pks gene cluster as His6-tagged fusions. The PKS module of ClbB (ClbBPKS) was excised from the full-length hybrid NRPS-PKS while all other enzymes, including hybrid PKS-NRPS ClbK, were obtained in full-length form. All enzymes were further purified by size exclusion chromatography (Figure S2) and could be successfully posttranslationally modified using Sfp and BODIPY-CoA (Figure S3).32

We determined whether each PKS module harbored a functional cis-AT domain by examining its ability to load malonyl-CoA in the absence of ClbG using SDS-PAGE and gel autoradiography. As predicted, cis-AT PKSs ClbBPKS and ClbI were labeled in the presence of [14C]-malonyl-CoA, confirming that these modules contain functional AT domains that recognize malonyl-CoA. In contrast, the atypical-AT PKSs ClbC, ClbK, and ClbO were not labeled by [14C]-malonyl-CoA (Figure S7), strongly supporting the hypothesis that these atypical-AT PKSs lack functional cis-AT domains.


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We next investigated ClbG’s ability to load malonyl-CoA and AM-ACP onto each PKS module. ClbG was unable to transfer [14C]-malonyl-CoA to any of the atypical-AT PKSs (Figure S10). In contrast, assays with in situ-generated [14C]-AM-ClbE and ClbG revealed transfer of the radiolabel to all of the atypical-PKSs (ClbC, ClbK, and ClbO) as well as the cis-AT PKS ClbI (Figure 4). We did not observe ClbG-mediated transfer of [14C]-AM-ClbE to the cis-AT PKS ClbBPKS (Figure S11). Unexpectedly, transfer of AM to ClbK was still observed without ClbG. This ‘self-acylation’, which is atypical of type I PKSs, resembles the activity of ACPs from certain type II PKS pathways, including actinorhodin and oxytetracycline biosynthesis, which recognize and load malonyl-CoA or N-acetylcysteamine β-ketothioesters without the use of an AT.33-35 Similarly, the ACP domain of ClbK might participate in AM-ACP recognition and transfer in the absence of ClbG. These results support our hypothesis that ClbG can transfer AMACP to the atypical-AT PKS modules. As previous investigations of the zwittermicin biosynthetic enzymes observed transfer of AM-ACP only to an excised ACP domain,19 this is the first demonstration of AM loading onto intact PKS modules in vitro.

ClbE ACP

ClbG

AT

AT

AT

PKS KS modules

O

S

S

H 2N H 2N

HO

HO

S O

O

O

AT* KS ACP

ACP

O

O H 2N

ClbC, ClbK PKS, ClbO

ClbI

ClbG

O H 2N

O

[14C]-AM-ClbE ClbG PKS

HO

+ + – + – + – + – – ClbI ClbC ClbK ClbO

O HO

+ + – + – + – + – – ClbI ClbC ClbK ClbO kDa

ClbK ClbI ClbC ClbO ClbH-A1 ClbG ClbF ClbD Sfp ClbE

kDa

250 150 PKS 100 75 50 ClbG 37 25 15 10

97 69 55-58 46 25-28 12

ClbE

Coomassie-stained

Phosphorimage

Figure 4. ClbG transfers AM-ClbE to multiple PKS modules from the pks assembly line in vitro. 14C gel autoradiography assay demonstrating ClbG-mediated transfer of AM-ClbE to cisAT (ClbI) and atypical-AT PKS modules (ClbC, full-length ClbK, and ClbO). All assays (30 µL) contained: 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 500 µM CoA, 1 mM TCEP, 13 ACS Paragon Plus Environment


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10 µM ClbE, 1.5 µM Sfp, 800 µM NAD+, 500 µM FAD, 3 µM ClbH-A1, 6 µM ClbD, 1.5 µM ClbF, 3 µM ClbG, 50 µM [14C(U)]-L-serine (145.1 mCi/mmol, Perkin Elmer), and 1.7 mM ATP, were incubated at 37 ºC, and were quenched after 20 min.

Interestingly, the in vitro extender unit specificities of several PKS modules were inconsistent with their hypothesized roles in isolated and proposed candidate precolibactin biosynthesis (Figure 1E). In agreement with the proposed pathway, the cis-AT PKSs ClbBPKS and ClbI loaded malonyl-CoA in the absence of ClbG. However, ClbI also recognized AM-ACP. Though hypothesized to use malonyl-CoA in vivo, atypical AT-PKS ClbC did not recognize this extender unit on its own or in the presence of ClbG but did accept AM-ACP. The PKS module of ClbK (ClbKPKS) and ClbO have not yet been implicated in the biosynthesis of candidate precolibactins. ClbKPKS is thought to be skipped36 and ClbO has been proposed to incorporate AM into an as-yetunidentified, higher-molecular weight pks-dependent metabolite.21 We found that both ClbKPKS and ClbO are able to accept and potentially utilize AM-ACP.

These findings raise questions regarding how PKS extender units are incorporated into the isolated and proposed candidate precolibactins and which building blocks are actually used in vivo for producing genotoxic products. Previous studies suggest that atypical-AT PKS ClbC and cis-AT PKS ClbI each incorporate one malonyl-CoA into candidate precolibactins 2–6.11,13,14 While ClbG-mediated transfer of AM-ACP to ClbC may have been anticipated based on ClbC’s lack of a cis-AT domain, transfer to ClbI was unexpected and is discussed in more detail below. Though we found ClbC unable to load malonyl-CoA in vitro in the presence or absence of ClbG, we observed weak transfer of this extender unit to ClbC when either ClbBPKS, ClbI, or the transAT domain FabD from fatty acid synthesis was included in assay mixtures (Figure S12). This


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finding suggests that multiple enzymes could work with ClbC in vivo to utilize this building block.

ClbKPKS and ClbO, atypical-AT PKS modules previously unassociated with candidate precolibactin biosynthesis, also selectively recognize AM-ACP in vitro, indicating the potential use of AM at multiple points in colibactin biosynthesis. The genes encoding the AM-ACP forming enzymes, the AM-ACP-recognizing AT ClbG, and the entire NRPS-PKS assembly line are all essential for activity.3,21 Together, these observations strongly suggest the pks island produces as-yet-unidentified AM-derived metabolite(s) that play critical roles in genotoxicity. We hypothesize the in vivo substrate specificity of this PKS machinery may depend upon the relative concentrations of extender units, the substrate preferences and/or availabilities of trans-AT partners, or the extender unit specificities of the KS domains of each module.

PKS ClbI accepts extender units from AT domains both in cis and in trans. While AM loading onto the atypical-AT PKSs by ClbG could be rationalized by their lack of a functional AT domain, it was unexpected that ClbG transferred AM to the malonyl-CoA loading, cis-AT PKS ClbI. To further probe the substrate preference of ClbI we conducted a competition experiment using radiolabeled malonyl-CoA or AM-ClbE in the presence of the alternative, unlabeled extender unit (Figure S13). The relative intensities of extender unit loading suggested that ClbI preferentially recognized AM-ACP in the presence of ClbG (Figure S13), and [14C]-AM loading did not decrease in the presence of a 30-fold excess of unlabeled malonyl-CoA (Figure 5). ClbI can therefore accept extender units from both cis- and trans-AT domains, which is unprecedented enzymatic assembly line logic. Although a trans-acting AT has been previously shown to load malonyl-CoA onto a cis-AT PKS mutant with an inactivated AT domain,37 to the best of our



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knowledge, ClbI is the first example of a wild-type cis-AT PKS preferentially accepting an extender unit supplied by a trans-acting AT in vitro.

ClbI

ClbE ACP O

vs. S

O

CoAS

AT

ClbG, ClbI

KS

ACP

OH S

O H 2N

unlabeled

O

O

H 2N

HO

O HO

[14C]-AM-ClbE AM-forming ClbG ClbI 14 [ C]-L-Ser malCoA

+ + + + –

+ + + +

+ + + +

+ + + +

+ + + +

+ + + +

3 10 20 40 100

+ + + + –

kDa

+ + + +

+ + + +

+ + + +

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ClbI ClbH-A1 ClbG ClbF ClbD

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Sfp ClbE

kDa

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ClbE

Coomassie-stained

12

Phosphorimage

Figure 5. Cis-AT PKS ClbI preferentially loads AM-ACP in vitro. 14C- gel autoradiography assay testing ClbI’s reactivity toward AM-ClbE in the presence of varying concentrations of unlabeled malonyl-CoA. All assays (15 µL) contained 50 mM Tris-HCl (pH 8.3), 200 mM NaCl, 10 mM MgCl2, 1 mM TCEP, 500 µM CoA, 5 µM of ClbI, 1.5 µM Sfp, 800 µM NAD+, 500 µM FAD, 3 µM ClbH-A1, 6 µM ClbD, 1.5 µM ClbF, 3 µM ClbG, 50 µM [14C(U)]-L-serine (145.1 mCi/mmol, Perkin Elmer), unlabeled malonyl-CoA (3–100 µM), and 1.7 mM ATP, were incubated at 37 ºC, and were quenched after 20 min.

Although these studies indicate that AM is the preferred substrate of ClbI in vitro, gene inactivation studies have suggested that ClbI incorporates a malonyl-CoA extender unit into candidate precolibactins 3–6.13,14 As for ClbC, ClbI’s in vivo substrate preference may depend on the relative cellular concentrations of malonyl-CoA, AM-ClbE, and ClbG. If AM loading is offpathway, a proofreading mechanism may exist to remove the misprimed substrates from ClbI and 16 ACS Paragon Plus Environment

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ClbC, such as hydrolysis by predicted type II thioesterase ClbQ. Because no pks-dependent metabolites isolated to date contain AM-derived structural motifs, it is still unclear if the observed AM loading onto these malonyl-CoA utilizing PKS modules is relevant to the biosynthesis of genotoxic molecules.

In vivo characterization of ClbG aids isolation of a bithiazole-containing candidate precolibactin. Given ClbG’s ability to transfer AM-ACP to PKS modules from the pks island in vitro, we sought to establish its in vivo role in colibactin and candidate precolibactin biosynthesis by profiling metabolites from E. coli DH10B strains possessing the pks island on a bacterial artificial chromosome (BAC). Comparing whole-culture methanol extracts from BACpks and BACpks∆clbG mutant strains did not reveal any identifiable AM-containing metabolites in the pks+ strain. We were also unable to identify any new, AM-containing candidate precolibactins by comparing extracts from the BACpks∆clbP and BACpks∆clbP/∆clbG mutant strains. However, we detected a number of candidate precolibactins in the ∆clbP/∆clbG mutant, including characterized molecules 1–4 and 6 (Figure S16–S20) and the proposed metabolites 7 and 8 (Figure S21–S22). The production of 1–4 and 6–8 by both the ∆clbP and ∆clbP/∆clbG mutants confirms that ClbG is not required for the biosynthesis of the candidate precolibactins identified to date and is not essential for malonyl-CoA transfer to the pks assembly line in vivo. This finding also suggests that ClbG does not supply malonyl-CoA to atypical-AT PKS ClbC in the production of 2–8 and that this activity must come from another AT acting in trans. Because clbG is essential for genotoxicity, the failure to observe loss of characterized candidate precolibactins upon deleting this enzyme further supports the hypothesis that additional, uncharacterized metabolites are critical for activity.



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Unexpectedly, comparing the relative amounts of candidate precolibactins generated by the ∆clbP and ∆clbP/∆clbG mutants revealed a significant enhancement in production by the ∆clbP/∆clbG strain. Specifically, LC-MS analysis showed 20-, 6-, and 3-fold increases in

metabolites 3, 4, and 6, respectively (Figure S18–S20). Additionally, proposed candidate precolibactin 7 (m/z = 796.3521 [M+H]+) was enriched 36-fold (Figure 6A). There are several potential explanations for the increased levels of these metabolites, including an overall enhancement of productivity from the pathway, the accumulation and hydrolysis of biosynthetic intermediates that are substrates for aminomalonate utilizing enzymes, and the enhanced production of shunt metabolites derived from off-pathway intermediates. Concentrations of the hydrolyzed prodrug motif were similar in pks+ and ∆clbG mutant strains, suggesting that deleting clbG does not dramatically alter overall pathway flux (Figure S15). Therefore, we hypothesize that the increase in candidate precolibactins observed in the ∆clbP/∆clbG mutant results from onand/or off-pathway intermediates accumulating without AM-ACP utilization.

We decided to utilize the ∆clbP/∆clbG mutant to access proposed candidate precolibactin 7 for structural characterization. Qian and co-workers recently predicted a structure for 7 using MS2 fragmentation analysis.14 Interestingly, this structure is similar to that suggested for candidate precolibactin 8 (m/z = 816.3783 [M+H]+).11 The potential presence of bithiazole and thiazolinylthiazole heterocyclic ring systems in these proposed structures could further support the hypothesis that colibactin directly targets duplex DNA.38,39 However, these metabolites have eluded structural characterization due to their instability and low isolation yields from ∆clbP mutants (~0.1 mg of 7 from 200 L of culture).11,14


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We obtained approximately 0.5 mg of purified metabolite 7 from 48 L of BACpks∆clbP/∆clbG E. coli. High-resolution mass measurement (m/z = 796.3521, [M+H]+) gave a molecular formula of C39H53N7O7S2, which indicated 7 contained 17 degrees of unsaturation. The structure of 7 was elucidated using MS2 fragmentation analysis along with one-(1H) and two-dimensional NMR spectroscopy (gHSQCAD, gCOSY, and gTOCSY). While the 1H spectrum of 7 in CD3OD-d4 was remarkably similar to that of the thiazole containing candidate precolibactin 6,40 an overlay of both spectra showed an additional proton resonance at δ 8.16 ppm (s, 1H) indicating the presence of another thiazole ring (Figure 6B). The observed resonances at δ 8.28 ppm (s, 1H) and δ 8.16 ppm (s, 1H) in the 1H spectrum of 7 also resembled the reported shifts for the protons of the bithiazole ring system of bleomycin (δ 8.25 and 8.13 ppm, CD3OD-d4).41 Notably, this effort provides the first confirmation that the pks enzymatic assembly line can construct a bithiazole heterocycle.

Figure 6. Metabolite profiling of a ∆clbP/∆clbG mutant enables the isolation and structural characterization of a bithiazole-containing candidate precolibactin. (A) Overlay of extracted ion chromatogram (EIC) traces for candidate precolibactin 7 in ∆clbP and ∆clbP/∆clbG mutant strains. (B) Overlay of 6.5–9.0 ppm 1H NMR spectral region of candidate precolibactins 6 and 7



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reveals the presence of a bithiazole heterocycle. (C) Structural features of candidate precolibactin 7 suggest that colibactin may interact with DNA.

Our structural characterization of 7 provides additional information about colibactin biosynthesis. In particular, the presence of the bithiazole ring system supports the hypothesis that ClbK’s NRPS module catalyzes the condensation of L-cysteine with a PCP domain-tethered thiazoline intermediate followed by cyclization to afford a bis-thiazoline heterocycle (Figure 1E).11,14 ClbK’s oxidase domain could then convert the resulting bis-thiazoline intermediate to the corresponding bithiazole-thioester, as has been proposed for bleomycin biosynthesis.42 Intramolecular Knoevenagal condensation, pyridone formation, and thioester hydrolysis could then afford 7. The timing of these condensation reactions relative to product release from the assembly line is unclear.

The structure of 7 also informs proposals regarding the activity of colibactin (Figure 6C). It has been previously hypothesized that the aza-spirocyclopropane ring systems of characterized candidate precolibactin 4 and proposed metabolite 8 may undergo ring opening in the presence of nucleophiles such as DNA.11-14 The aza-spirocyclopropane in metabolite 7 is embedded in a dramatically different scaffold from that proposed for 4 and 8, likely altering its reactivity. As it is still unknown whether any metabolite derived from a ∆clbP mutant actually represents a precursor to a genotoxin, the relevance of these two distinct electrophilic warheads for colibactin’s biological activity is unclear. The bithiazole motif found in 7 is also observed in the DNA damaging metabolite bleomycin and several myxothiazol metabolites.43 Planar bithiazole rings are thought to intercalate into duplex DNA by making π–stacking interactions within the purine-pyrimidine backbone.44 Thus, in combination with the aza-spirocyclopropane group, the


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heterocyclic ring system of 7 provides further support for the hypothesis that colibactin directly targets DNA.

Conclusions. In summary, we have acquired new insights into the biosynthesis of the bacterial genotoxin colibactin. By characterizing the PKS modules from the pks assembly line, along with the essential AM-ACP forming and incorporating enzymes, we have shown that AT ClbG recognizes AM-ACP and transfers this unusual extender unit to the ACP domains of PKS modules ClbC, ClbO, ClbK, and ClbI. The acceptance of AM-ACP by multiple modules suggests that AM may be incorporated more than once into colibactin’s scaffold. This logic would be distinct from the zwittermicin and guadinomine pathways, which each use AM-ACP at a single point in biosynthesis.17-20 The biosynthetic enzymes that make and utilize unusual extender units have attracted attention due to their potential application for natural product diversification via combinatorial biosynthesis.45 Before this work only two AM-utilizing PKS modules were associated with known biosynthetic pathways.17-20 Our efforts therefore greatly expand the AMutilizing biosynthetic machinery available to synthetic biologists, providing multiple new tools for PKS engineering efforts.

In the context of examining AM-ACP utilization, we have obtained further evidence that the pks enzymatic assembly line employs unusual biosynthetic logic. Previous studies revealed several uncommon features of the colibactin NRPS-PKS, including the incorporation of aminocyclopropane carboxylic acid and malonyl-CoA utilization by both cis-AT and atypical-AT PKS modules.11-14 Using an in vitro biochemical approach, we obtained evidence that this assembly line not only has the potential to use the rare extender unit AM-ACP, but also may incorporate this building block in unusual ways, including ClbG-mediated transfer to the cis-AT



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PKS module ClbI and self-acylation by atypical AT-PKS ClbK. These findings highlight the ability of biochemical studies to reveal events that are not predictable from bioinformatics and underscore the need for caution when using canonical assembly line logic for structure prediction.

Our in vivo studies of trans-AT ClbG not only revealed that this essential enzyme is not required for the synthesis of the pks metabolites characterized to date but also facilitated the isolation and complete structural characterization of the first bithiazole-containing candidate precolibactin. Confirming that this structural feature can be synthesized by the pks island provides further insights into colibactin’s activity. However, these in vivo experiments raise serious questions about the biological relevance of 7 and the other characterized and proposed candidate precolibactins. As ClbG mediates AM-ACP transfer to the assembly line, and both this enzyme and the AM-ACP synthesizing machinery are essential for activity, we propose that AM-ACP incorporation is critical for constructing genotoxic metabolites. Notably, the presence of positively charged AM-derived amino substituents could enhance colibactin’s affinity for DNA by providing favorable electrostatic interactions with the negatively charged phosphate backbone.

Finally, this work further highlights the utility of combining in vitro biochemical approaches and in vivo metabolite profiling to obtain structural information about natural products that are challenging to characterize. Insights gained from this strategy have previously enabled the isolation of candidate precolibactins from ∆clbP mutants.10-14 The information gained from these in vitro and in vivo studies will inform additional experiments aimed at identifying new AMderived pks metabolites. Deciphering the structures and biosynthesis of these elusive molecules will be critical for improving our knowledge of colibactin’s structure and activity.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at: TBD. Details of sequence alignments, protein production, biochemical assays, metabolite isolation, and structure elucidation. Supplementary Figures and Tables.

AUTHOR INFORMATION Corresponding Author *Phone: (617) 496-9921. E-mail: [email protected]. Author Contributions ‡These authors contributed equally. ACKNOWLEDGMENT We thank A. Sieg for help with cloning, F. Rubino, Y. Qiao, and K. Schaefer (Kahne Lab, Harvard University, Cambridge, MA) for help with gel autoradiography, H. Nakamura and Y. Jiang for helpful discussions, G. Heffron (Harvard Medical School, Boston, MA) for assistance with NMR experiments, and S. Trauger, J. Wang, and G. Byrd (Small Molecule Mass Spectrometry Facility, Harvard University, Cambridge, MA) for help with LC−MS analyses. We acknowledge financial support from the Damon Runyon-Rachleff Innovation Award, the Smith Family Award for Excellence in Biomedical Research, the Packard Fellowship for Science and Engineering, and the Searle Scholar Award.

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