Biosynthesis of the Klebsiella oxytoca Pathogenicity Factor Tilivalline

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Biosynthesis of the Klebsiella oxytoca pathogenicity factor tilivallin: Heterologous expression, in vitro biosynthesis and inhibitor development Alexander von Tesmar, Michael Hoffmann, Antoine Abou Fayad, Stephan Hüttel, Viktoria Schmitt, Jennifer Herrmann, and Rolf Müller ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00990 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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ACS Chemical Biology

Title

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Biosynthesis of the Klebsiella oxytoca pathogenicity factor tilivallin:

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Heterologous expression, in vitro biosynthesis and inhibitor development

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Authors

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Alexander von Tesmar1 , Michael Hoffmann1, Antoine Abou Fayad1, Stephan Hüttel2, Viktoria Schmitt1, Jennifer Herrmann1, Rolf Müller1

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Contact Information

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[email protected]

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Department of Microbial Natural Products (MINS), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI) and Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany 2 Department of Microbial Drugs, Helmholtz Centre for Infection Research and German Centre for Infection Research (DZIF), partner site Hannover/Braunschweig, Inhoffenstrasse 7, 38124 Braunschweig, Germany

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Abstract

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Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxy-

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toca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous

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expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the

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respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a non-enzymatic indole

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incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the

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heterologous system was used to generate novel tilivalline derivatives by supplementation of

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respective anthranilate and indole precursors. Finally, it could be shown that salicylic and

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acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably

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by blocking the peptidyl carrier protein ThdA, pointing towards a potential application in

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combination therapy to prevent or alleviate symptoms of AAHC.

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Keywords

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Virulence factor, Antibiotic-associated colitis, TAR-assembly, heterologous expression,

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precursor-directed mutasynthesis, in vitro reconstitution of NRPS, anti-virulence

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Introduction

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Tilivalline, a pyrrolo[4,2]benzodiazepine (PBD) derivative, is produced by Klebsiella oxytoca1

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and has been identified as the causative pathogenicity factor in antibiotic associated hemorrhagic

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colitis (AAHC).2

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PBDs are a common class of natural products produced by Streptomyces species and occur with

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various substitution patterns. Compounds such as anthramycin, sibiromycin or tomaymycin can

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bind covalently to the minor grove of double stranded DNA and hence, exhibit a potent

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antineoplastic activity. In the past, a considerable effort to produce novel synthetic PBD

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derivatives has been expended, resulting in PBD conjugates such as DSB-120 and SJG-136,

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dimeric compounds with strong antitumor activity.3

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Tilivalline likewise shows activity against mouse leukemia L1210 cells 4 and has been identified

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to be causative agent for the cytotoxic effects of K. oxytoca extracts which have been initially

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described in 1989.5,6 Recently, the presence of tilivalline has been reported to be fundamental

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regarding the course of AAHC caused by K.oxytoca.2,7 AAHC is triggered by antibiotic treatment

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due to overgrowth of inherently β-lactamase resistant K. oxytoca in the colon; such a dysbiotic

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population causes bloody diarrhea and abdominal cramps 8–10

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The biosynthetic gene cluster of tilivalline reported by Schneditz et al. contains a bi-modular non-

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ribosomal peptide synthetase (NRPS) that is thought to synthesize the PBD core from an

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anthranilate and a pyrrolo precursor. This assumption is based on the finding that the tilivalline

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cluster has the same module organization as the sibiromycin and tomaymycin gene clusters.11,12

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Furthermore, during the course of the preparation of this manuscript two papers appeared which

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examined tilivalline biosynthesis by metabolite profiling, genetic deletion experiments and

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complementation experiments in K. oxytoca as well as heterologous expression of the gene

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cluster in E. coli, likewise presenting data supporting this hypothesis.13,14 Additionally, the 3 ACS Paragon Plus Environment


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tilivalline gene cluster was also identified in Xenorhabdus species, bacteria that undergo

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symbiosis with nematodes.15 We have recently reported the in vitro total biosynthesis of

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tomaymycin and characterized each biochemical step involved biochemically.16 NRPSs are

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multimodular megasynthetases that produce a diverse group of natural products by catalyzing

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peptide bonds between monomeric building blocks. Owing to the independence from the

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ribosome, NRPS building blocks are not limited to the proteinogenic amino acids. Hence,

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unusual blocks with diverse modifications are often incorporated. The minimal module for the

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chain elongation process consists of three domains: The adenylation domain (A) activates the

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substrate that subsequently is covalently tethered to the terminal thiol 4’phosphopanthein

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prosthetic group (PPant) on the thiolation domain (T). The condensation domain (C) catalyzes the

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peptide bond between two adjacent T domain tethered substrates. The last module usually

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contains a thioesterase (TE) or reductase (R) domain that releases the substrate chain from the

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enzyme.17–19 In addition, optional domains included in distinct modules can facilitate a variety of

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modifications such as epimerization, hydroxylation or methylation and thus increase the diversity

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of structural elements in natural products.20

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Here we present experimental evidence of the tilivalline biosynthesis using heterologous

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expression of the gene cluster in Escherichia coli and the in vitro reconstitution of the underlying

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NRPS NpsA, ThdA, and NpsB. Our data show a non-enzymatic incorporation of the tilivalline

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indole moiety by a Friedel-Crafts-like alkylation. Furthermore, the E. coli heterologous system

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was used to generate novel tilivalline derivatives in a convenient 96-well plate assay. In addition,

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we identified competitive inhibitors to block the tilivalline NRPS in the natural producer K.

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oxytoca and thus prevented toxin production in liquid culture. This finding is especially

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intriguing as tilivalline biosynthesis inhibition could serve as a valuable pathoblocker strategy in

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colitis treatment. 4 ACS Paragon Plus Environment

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Results and Discussion

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Clinical isolates of K. oxytoca produce tilivalline

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K. oxytoca (ID 1976911) was isolated from skin smear from a patient suffering from

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superinfection. The presence of the tilivalline biosynthetic gene cluster was confirmed by colony

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PCR using primers for NpsA. An initial small-scale culture of the respective isolate in liquid

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medium was extracted with ethyl acetate and fractionated into 96 well plates while a split flow

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was used to acquire LC-MS data. Eventually, the fractions were screened towards cytotoxicity

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against KB-3.1 human cervix carcinoma cells. The combined screening effort revealed one active

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fraction containing primarily a compound with 334.1550 m/z. Comparison with an authentic

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standard obtained by total synthesis revealed the detected compound as tilivalline, which was

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finally confirmed by purification using preparative HPLC and subsequent NMR analysis (Figure

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1).

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To gain further insights into the genome of K. oxytoca (ID 1976911) the Illumina sequencing

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technology was used for sequencing and the gene data were analyzed towards similarity to the

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already reported tilivalline biosynthetic gene cluster. The tilivalline biosynthetic gene cluster was

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identified using Antismash 2.021 displaying an identical locus organization as the one reported by

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Schneditz et al. The DNA sequence identity of the two clusters spanning 16.264 bp is striking

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99.4%. The lowest observed identity on protein level is 96.7% for UvrX (Table S1).

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The three genes npsA, thdA and npsB encode a two-modular NRPS including a reductive release

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domain. They share identical domain organization with the already described PBD producing

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synthetase of tomaymycin from Streptomyces.11 Similar to the tomaymycin cluster, copies of

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shikimate pathway genes and other housekeeping enzymes are present to provide the anthranilic

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acid precursor for the biosynthesis of the core structure. The homologue of the 4-nitrophenol-25 ACS Paragon Plus Environment


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monoxygenase NphA1, HmoX, is putatively responsible for the hydroxylation at position 3 of the

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anthranilic acid. Other NphA1 homologues can be found in the biosynthetic gene clusters of

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tomaymycin or sibiromycin, which both harbor a hydroxylated anthranilic acid moiety.22 The

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comparison with the tomaymycin biosynthesis further indicated a release of the formed dipeptide

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as an aldehyde followed by an immediate circularization to an imine, which reacts spontaneously

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to the respective carbinolamine by hydratation. The nature of the indole incorporation remained

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elusive as no candidate genes for the indole incorporation could be identified in the tilivalline

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biosynthetic gene cluster (Figure 1).

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Heterologous expression of the tilivalline biosynthetic gene cluster reveals non-enzymatic indole incorporation

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To generate a heterologous expression construct a transformation-associated recombination

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(TAR) based assembly of PCR amplified DNA fragments was employed (see methods section for

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details). The two operons containing the NRPS, the tailoring, and the precursor biosynthetic

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genes were PCR amplified from the K. oxytoca (ID 1976911) genomic DNA. The promoter

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cassette sequences for Ptet and PBAD as well as the vector backbone were PCR amplified while

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suitable homologous regions at the 5’-end of the primers were introduced.23 The resulting five

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fragments were assembled by TAR in Saccharomyces cerevisiae to the plasmid pCly-Til.

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Plasmids exhibiting the correct restriction pattern were verified by employing Illumina

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sequencing technology (Figure 2).

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Upon induction of E. coli BL21 (DE3) pCLY-Til-24 in M9 minimal medium containing

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tetracycline and L-arabinose, tilivalline was not detectable in the supernatant. However, trace

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amounts of the carbinolamine derived from 3-hydroxy anthranilic acid and

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identified by LC-MS. The heterologous system apparently lacked the required indole source

L-proline

were


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and/or enzyme for tilivalline production. Since no indole formation related genes are present in

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the tilivalline gene cluster and the E. coli tryptophan degradation pathway is repressed in minimal

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media,24 indole was supplemented to the medium at a final concentration of 5 mM. LC-MS

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analysis of the corresponding cultures then indeed confirmed the presence of tilivalline indicating

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a non-enzymatic mechanism for tilivalline formation from the imine. To facilitate indole

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generation in vivo the gene of the wild type tryptophanase TnaA of K. oxytoca was cloned tag-

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free into the pET28b expression vector. Upon induction of an E. coli transformant harboring both

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constructs, pCLY-Til-24 and pET28b-tnaA, tilivalline production was observed without any

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addition of indole (Figure 2).

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To circumvent the poor growth and low yields in E. coli transformants harboring two plasmids

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with a combined size of over 20 kb and two resistance markers, a second approach was

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established. The operon containing the core NRPS NpsA, ThdA, and NpsB was cloned entirely

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and tag-free into pET28b, which yielded a 10-fold increased tilivalline production in M9 minimal

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media supplemented with 3-hydroxy anthranilic acid, indole, and L-proline. To prove the non-

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enzymatic nature of the indole incorporation, all enzymes in the supernatant of an E. coli

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pET28b-Til-NRPS transformant grown without supplementation of indole were inactivated by

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three different methods. The supernatant containing the carbinolamine was either heat inactivated

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at 100°C for 5 minutes or supplemented with SDS or methanol to final concentrations of 1% and

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20%, respectively. Subsequently performed incubation with indole confirmed the tilivalline

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formation in all three scenarios and thus manifested the probability of a spontaneous reaction of

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the NRPS produced carbinolamine with free indole (Figure 3).

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In vitro reconstitution of the tilivalline NRPS 7 ACS Paragon Plus Environment


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To further elucidate the biosynthetic pathway of tilivalline, in vitro reconstitution of the

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underlying proteins was pursued. Thus, the three genes constituting the tilivalline NRPS, npsA,

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thdA, and npsB and the K. oxytoca tryptophanase tnaA were heterologously expressed in E. coli

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BL21 (DE3). For the NRPS enzymes an N-terminal, HRV3C cleavable 6xHis-MBP tag was used

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as it showed to enhance protein yields and solubility. The obtained NRPS proteins were purified

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to homogeneity by a three-step protocol using nickel-affinity chromatography, a reverse nickel-

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affinity chromatography step upon HRV-3C digestion to remove the tags, and a gel filtration step

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(Figure S3). TnaA was enriched by affinity chromatography using an N-terminal 6xHis-tag and

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purified to homogeneity by a final gel filtration step without removing the tag (Figure S4).

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The purified proteins NpsA, ThdA, and NpsB were used to reconstitute the biosynthesis of

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tilivalline in vitro. Upon incubation of the NRPS proteins with the substrates 3-hydroxy

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anthranilic acid, L-proline, and indole together with the cofactors ATP and NADPH, tilivalline

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formation could be observed by LC-MS. The addition of TnaA, tryptophan and the cofactor PLP

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instead of indole also restored production. The activity of TnaA was previously reconstituted in

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vitro25 (Figure S4). Trace amounts of the carbinolamine could be detected in all assays but it was

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further accumulated once the indole source was omitted. To prove the non-enzymatic nature of

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the indole incorporation, the carbinolamine was purified from the in vitro reconstitution assay on

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analytical scale. In vitro reaction with indole yielded tilivalline and a second, isobaric compound

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with a slightly different retention time (Figure 3). This additional signal is most likely owing to

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the formation epi-tilivalline, the diastereomer of tilivalline, during indole addition, since no

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enzyme controls the stereochemistry. In contrast to tilivalline, the signal of epi-tilivalline

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decreased in aqueous solution over time, while the peak area of tilivalline remained unchanged.

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This indicates degradation rather than transformation into the stable epimer.. Instability of

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epi-tilivalline also explains its absence in K. oxytoca extracts and in the heterologous system. 8 ACS Paragon Plus Environment

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Furthermore, these results hint towards a non-reversible indole addition, since otherwise

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tilivalline would over time be transformed into its diastereomer and subsequently be degraded.

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Summarized, the structural backbone of tilivalline is produced by a two-modular NRPS like the

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ones found in PBD producing actinomycetes. Interestingly, the indole moiety of tilivalline is

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incorporated by a non-enzymatic process spontaneously in aqueous solution. It can be assumed

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that the positive resonance effect of the ortho-hydroxyl group increases the basicity of the imine.

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The formation of an energetically favored five-membered ring including the phenolic hydrogen

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and the imine nitrogen presumably further promotes the polarization. The resulting mesomeric

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carbeniumion undergoes a Friedel-Crafts like alkylation with the electron-rich indole carbon

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(Figure 3). Since K. oxytoca is indole-positive the required free indole is provided by degrading

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tryptophan.26 The recently published results confirm the herein presented biosynthetic

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pathway.13,14 Furthermore, the high reactivity of the imine carbon is also observed for other

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PBDs, for example in the DNA-alkylation reaction of tomaymycin. Here, the missing activation

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by a ortho-hydroxyl group might allow the purification of the imine form, in contrast to

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tilivalline.

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Biological activity of Tilivalline

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Other PBDs like tomaymycin, sibiromycin, and anthramycin show promising cytotoxic activities

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and thus, have been further developed towards antitumor agents.

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action is the covalent bond formation between the imine carbon of the PBD with an amino group

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of a guanidine in the minor groove of the DNA.29 Apparently, an analogous mode-of-action for

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tilivalline is not possible since the imine-carbon of the PBD is already occupied by the indole.

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The observed cytotoxicity of synthesized tilivalline with an IC50 of 1.6 µM against KB-3.1 human

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cervix carcinoma cells raises the question what alternative mode-of-action is present.

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The underlying mode-of-



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Interestingly and in contrast to previously published data, we found that the carbinolamine (also

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termed kleboxymycin or tilimycin in other studies)13,14 is by one order of magnitude less toxic on

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KB-3.1 cells (IC50 23.6 µM). However, different cell lines have been used in the cited studies,

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making comparability of the data difficult. In our assay the DNA minor groove binder

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tomaymycin displayed pronounced cytotoxic activity with an IC50 value of 0.06 µM. Since both,

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tilivalline and its carbinolamine precursor, induce cell death at low to mid µM concentrations it is

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likely that high local concentrations in the infected host cause the observed epithelial damage in

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AAHC. Although the molecular details of tilivalline induced apoptosis remain elusive, the

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respective NRPS constitutes a promising target for anti-virulence strategies.

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Tilivalline NRPS as heterologous platform for the generation of tilivalline derivatives

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To explore the ability of the NRPS system to accept a broad spectrum of substrates, as it is

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known for the tomaymycin NRPS,16 the heterologous expression system was utilized to set up a

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96-well plate based assay for a precursor directed mutasynthesis approach (

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Table 1). Twelve different halogenated, hydroxylated, or nitrated indole derivatives were

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supplemented with 3-hydroxy anthranilic acid and L-proline and supernatants were subsequently

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analyzed by LC-MS (Figure S1). Fluoro- and hydroxyl substituted indoles were incorporated

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with comparable efficiencies to the native, unsubstituted indole. Although nitro- and bromo

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derivatives were also accepted, the signal intensities were one order of magnitude lower. The

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incorporation of 5-iodo indole was inefficient and only trace amounts of the respective tilivalline

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derivative could be detected. Furthermore, the tilivalline NRPS accepts twelve different

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anthranilic acid derivatives including halogen-, hydroxy-, methoxy-, and trifluoromethyl

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substituted ones (Figure S2). The halogenated as well as the hydroxylated and methoxylated

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anthranilic acid derivatives were readily accepted with product signal intensities comparable to 10 ACS Paragon Plus Environment

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the native tilivalline. The substitution of the 3-hydroxy group with the sterically demanding

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trifluoromethyl led to a nearly complete abolishment of production and only trace amounts of the

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respective derivative could be detected. All anthranilic acid derivatives were supplemented

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together with indole and L-proline. For all tilivalline derivatives, the emergence of a second

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signal with an approximately 100-fold lower intensity compared to the main product signal was

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observed. This indicates the formation of both epimers during indole addition and the subsequent

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degradation of the epi-tilivalline derivative, as already observed for tilivalline.

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In conclusion, all tested substrates were accepted by the NRPS system with varying overall

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efficiency. We were able to produce a variety of derivatives of this bioactive compound class,

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which opens the possibility to search for inhibitors of toxin biosynthesis in K. oxytoca liquid

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culture using competitive substrates for the A-domain NpsA.

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Salicylic acid based compounds inhibit tilivalline production in K. oxytoca

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Antibiotic resistant pathogens are an increasing threat to public health. Consequently, the

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development of alternative antibacterial strategies has become vital. The virulence of pathogens

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often depends on distinct natural products that act as toxins or siderophores.30–33 Hence, the

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underlying biosynthetic machinery has emerged as a promising antibacterial target.34,35

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Substances that are capable to block secondary metabolite pathways have less impact on the

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overall fitness of the pathogen compared to antibiotics, which usually target vital cell functions.

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Combination therapy with an anti-virulence agent and an antibacterial drug is thought to reduce

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the probability of resistance development and the problematic side effects associated with an

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antibiotic regime.36 NRPS assembly lines use adenylation domains to activate the amino acid

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substrates as acyl-AMP that remains tightly bound to the active site for downstream processing.

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Therefore, non-hydrolyzable acyl-AMP mimics such asthe acyl-sulfamoyl-adenosine (acyl-AMS) 11 ACS Paragon Plus Environment


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scaffold have been commonly used for selective inhibition of adenylating enzymes.37 Despite the

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established application in in vitro experiments, the application in liquid culture is hampered due

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to poor cell-permeability.38 Nevertheless, inhibition of the biosynthesis of salicyl-capped non-

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ribosomal-peptide-polyketide siderophores in M. tuberculosis and Y. pestis using salicyl-AMS

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has been reported where successful inhibition impaired bacterial growth under iron-limiting

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conditions.39

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Along those lines we pursued an alternative inhibition approach for the NRPS produced toxin

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tilivalline, exploring salicylic acid derivatives as potential inhibitors. Activation and transfer of

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salicylic acid, that lacks the amino group compared to the native substrate 3-hydroxy anthranilic

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acid, can block the PCP-domain to be no longer available for tilivalline biosynthesis.

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Additionally, adenylation and loading onto the PCP-domain exhibit a low substrate specificity, as

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it is known from the in vitro derivatization screening and extensive studies on the related

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tomaymycin NRPS,16 further favoring our approach.

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Initially, tilivalline production in K. oxytoca (ID 1976911) was evaluated for different

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fermentation conditions, whereby incubation for 16 h at 37 °C turned out to be most reliable.

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Tilivalline production in the presence of different potential inhibitors was subsequently quantified

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relative to each other by LC-MS. As could be predicted rationally from the biosynthetic route,

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salicylic acid derivatives lacking the amino group might be loaded to the NRPS and act as

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inhibitors of the overall reaction. Indeed, salicylic acid inhibited production completely in two

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steps with IC50 values of 0.3 µM and 43 µM. Acetylsalicylic acid showed an IC50 value of 10 µM

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for the reduction of tilivalline production to a final level of 15% (Figure 4). Both inhibitors did

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not affect bacterial growth, as controlled by measuring optical density. Acetylsalicylic acid has

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been shown to hydrolyze to salicylic acid in aqueous solution and thus probably acts as a

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prodrug.40 Furthermore, salicylic acid is known to impair bacterial cell viability at high 12 ACS Paragon Plus Environment

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concentrations, including altering gene expression and disturbing motility.41 Therefore the second

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step of the observed two-step dose model for salicylic acid is likely not result of a selective

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inhibition of tilivalline biosynthesis, but rather the consequence of overall diminished cell

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fitness.

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This result provides first evidence that salicylic and acetylsalicylic acid inhibits tilivalline

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biosynthesis in K. oxytoca liquid cultures. The low cytotoxicity values of tilivalline and the high

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production titer in liquid culture indicate a high occurrence of the toxin in the colon as well.

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Consequently, reduction of this pathogenicity factor is a promising approach to cure or at least

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reduce severity of AAHC.

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Figure 1: A Biosynthetic gene cluster of tilivalline from K. oxytoca (ID 1976911) spans 17 kb. It

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contains ten open reading frames constituting a two modular NRPS (black), the putative 3-

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hydroxylase (dark grey), genes for providing anthranilic acid (white) and regulators (light grey).

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B UV (200-600 nm) trace of the ethyl acetate extract of an K. oxytoca (ID 1976911)

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fermentation. The asterisk indicates the cytotoxic fraction harboring tilivalline. C Proposed

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biosynthesis of tilivalline: The NRPS (NpsA,ThdA,NpsB) generates the tilivalline backbone from

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3-hydroxy anthranilic acid and proline. The biochemistry of the subsequent indole incorporation

291

was elusive.

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Figure 2: A Schematic display of the TAR assembly of the five PCR generated DNA fragments

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to yield the heterologous expression construct pCLY-Til. B Two different heterologous

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expression systems for tilivalline in E. coli BL21 (DE3) have been generated. Simultaneous

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expression of pCLY-Til and pET28b-tnaA or indole supplementation led to successful restoration

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of tilivalline production. Expression of the construct pET28b-NRPS while supplementation of all 15 ACS Paragon Plus Environment


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three precursors indole, 3-hydroxy anthranilic acid, and L-proline restored tilivalline production

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as well. C LC-MS analysis of the different heterologous systems shows the successful production

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of tilivalline in M9 minimal media. E. coli BL21 (DE3) pCly-Til-24 produces tilivalline when

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indole is either produced in the cell by the tryptophanase construct pET28b-tnaA (I) or

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supplemented in the culture media (II). E. coli BL21 (DE3) pET28b-NRPS produces tilivalline

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when the substrates are supplemented (III). Synthesized (V) and purified tilivalline from K.

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oxytoca (ID 1976911) (IV) were used as standard.

306 307


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308 309

Figure 3: A Formation of tilivalline and epi-tilivalline can be detected upon incubation of indole

310

with the purified PBD precursor in the carbinolamine form (I). PBD-precursor was purified from

311

the in vitro reconstitution of NpsA, ThdA and NpsB without indole supplementation (II).

312

Synthesized tilivalline was used as a standard (III). EIC traces for tilivalline (black, 334.1500 ±

313

0.002 m/z) and carbinolamine (grey, 235.10772 ± 0.002 m/z) are shown. B LC-MS derived EICs

314

prove formation of tilivalline in the supernatant of induced E. coli BL21 (DE3) pET28b-NRPS

315

cultures in M9 minimal media (IV) upon addition of indole (I). Inactivation of enzymes by heat

316

(HI) (II) or methanol (III) did not impair product formation. Synthesized tilivalline was used as

317

standard (V) C Theoretical reaction mechanism for indole incorporation include the iminium

318

transition state stabilized by the acidic phenolic hydrogen in ortho-position. Tilivalline is formed

319

after Friedel-Crafts-like alkylation of nucleophilic indole at the carbeniumion takes place. Non-

320

enzymatic incorporation leads to loss of stereochemical control and both epimers, tilivalline and

321

epi-tilivalline are formed, while the latter proved instable and degrades over time. 17 ACS Paragon Plus Environment


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322

Table 1: Mutasynthesis of tilivalline derivatives by supplementing E. coli BL21 (DE3) pET28b-

323

NRPS fermentations with anthranilic acid or indole derivatives. Incorporation efficiency was

324

estimated by comparing LC-MS derived EIC signal intensities compared to tilivalline production:

325

similar production (+++), one magnitude less (++), little (+) and trace amount (tr).

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327 328

Figure 4: Salicylic acid and acetylsalicylic acid inhibit tilivalline biosynthesis in K. oxytoca (ID

329

1976911) liquid culture. Experiments were carried out in triplicates. Mean values and standard

330

deviations are given. A two-step and one-step dose-response model was used to fit salicylic acid

331

and acetylsalicylic acid data, respectively.

332



333

Methods

334

Sequencing of strain K. oxytoca (ID 1976911)

335

The strain K. oxytoca (ID 1976911) was obtained from Dr. Alexander Halfmann, Saarland

336

University Medical Center, Homburg, Germany. Prior sequencing, presence of the tilivalline

337

gene cluster was confirmed by successfully amplifying npsA using the primer pair NpsA-

338

FP/NpsA-RP designed for protein purification. Draft genome sequence of Klebsiella oxytoca (ID

339

1976911) was obtained using Illumina sequencing technology in cooperation with the Genome

340

Analytics group at the Helmholtz Centre for Infection Research (Braunschweig, Germany). Raw

341

sequencing data obtained from the MiSeq platform comprised 2,849,072 paired-end reads with

342

the length of 250 bp each. This raw data was assembled into contigs with Abyss-pe assembler

343

software42, version 1.9.0. The resulting 192 contiguous sequences (contigs) were mapped to the

344

reference sequence of a close relative genome of Klebsiella oxytoca MGH 28 (GenBank

345

accession code: KI535631) in Geneious software43, verision 9.0.5. The resulting draft genome

346

circular scaffold of 5,720,468 bp was used for the downstream analysis. The cluster sequence will

347

be made available at GenBank upon acceptance of the manuscript.

348

Isolation of tilivalline

349

Preparative HPLC was performed with a Waters Autopurifier System (APS) equipped with a

350

Waters XBridge C18 150 x 4.6 mm, 5 µm dp analytical column for method development at 1.0

351

mL/min flow rate. Preparative separations were performed with a XBridge C18 150 x 19 mm, 5

352

µm dp column at 25 mL/min flow rate. Both used (A) water + 0.1 % FA and (B) acetonitrile +

353

0.1 % FA as solvent system. Elution of an 800 µL sample injection started with a 3 min isocratic

354

step at 5 % B, a linear increase to 40 % B within 25 min, a steep increase to 95 % B in 2 min,

355

followed by a 3 min plateau at 95 % prior to reequilibration to initial conditions. The overall run

356

time was 34 min while 40 fractions were collected in a period from 19 to 22 min. Fractions were 20 ACS Paragon Plus Environment

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357

combined based on MS data and yielded tilivalline in high purity without any further purification

358

efforts.

359

TAR assembly of the tilivalline biosynthetic gene cluster

360

PCR fragments of the vector backbone, promoter cassettes, tilivalline NRPS and tailoring operon

361

were amplified using primers with suitable 40bp 5’overhangs (Table S2). TAR assembly in S.

362

cerevisiae was conducted as described previously.23 Colony PCR targeting fragment borders was

363

used to preselect yeast colonies (Table S2). Positive clones were grown in selective leucine

364

deficient media overnight and subsequently the plasmid DNA was isolated. Plasmid isolation by

365

alkaline lysis from E. coli Gbred transformants and subsequent restriction digest was used to

366

identify correct constructs. Isolation attempts with E. coli Dh10b or HS916 cells yielded low

367

DNA concentration. Plasmids were subsequently verified by Illumina sequencing.

368

Heterologous expression of the tilivalline biosynthetic gene cluster

369

E. coli BL21 (DE3) pCly-Til-24 (or 28) cells were grown overnight in LB media at 30°C.

370

Expression in 10 mL M9 minimal media with 2% glycerol as sole carbon source at 30°C for 16 h

371

was induced by addition of 2% (w/v) arabinose and 5 ng/mL tetracycline after cell density

372

reached OD600=0.6. Subsequently the filtrated supernatant was forwarded to LCMS analysis.

373

TnaA was coexpressed in pET28b using the same protocol but with additional induction with 0.1

374

mM IPTG. Supplementation with indole was carried out simultaneously with induction with a

375

final concentration of 1mM.

376

Heterologous expression of the tilivalline NRPS

377

The operon harboring the tilivalline NRPS was cloned into pET28b using suitable primers

378

allowing for tag-free expression. E. coli BL21 (DE3) pET28b-NRPS cells were grown overnight

379

in LB media at 30°C. Expression in M9 minimal media with 2% glucose as sole carbon source at 21 ACS Paragon Plus Environment


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30°C was induced by addition of 0.1 mM IPTG, 1mM indole, 1mM 3-hydroxy anthranilic acid

381

and 1mM L-proline after cell density reached OD600=0.6. After incubation for 16h the filtrated

382

supernatant was forwarded to LCMS analysis. Synthetic biology studies were carried out by

383

substituting indole or 3-hydroxy anthranilic acid with the respective derivative. Initial

384

fermentation was carried out in 10 mL volume. Feeding studies were carried out in 150 µL

385

volume using a 96-well flat bottom plate.

386

Protein Purification of NpsA, ThdA, and NpsB

387

The respective gene was inserted into a petM44 expression vector with an N-terminal 6xHis-

388

MBP tag using according primers (restriction site in bold):

389

NpsA-FP

AAAAACCATGGCAACGCATTCAGCATA

390

NpsA -RP

AAAAAAAGCTTTTACACCTGCTCCAGTAAAGAATTT

391

ThdA-FP

AAAAACCATGGCAGACAACGTTGAGCAA

392

ThdA -RP

AAAAAAAAGCTTTTAGAGCTTTTGCCGTTGCC

393

NpsB-FP

AAAAACCATGGCACCATCATTCAATAGCG

394

NpsB-RP

AAAAAGGATCCCTAGACAATTTCTTTCTGCCGCT

395

The respective construct was expressed in E. coli BL21 (DE3) cells (500 mL LB, 0.1 mM IPTG,

396

16°C, 16h). The cell pellet was resuspended in lysis buffer (150 mM NaCl, 25 mM Tris, 40 mM

397

Imidazole, 1 mM TCEP, pH 7.5), sonicated and centrifuged. The supernatant was loaded onto a

398

gravity flow column containing Ni-NTA loaded sepharose, washed with lysis buffer, and

399

subsequently eluted in one step (250 mM Imidazole). The tag was cleaved using HRV3C

400

protease during overnight dialysis against SEC buffer (150 NaCl, 25 Tris, pH 7.5) at 4 °C, and

401

removed by a second Ni-NTA chromatography step. After passing through a Superdex 200 16/60

402

pg column (GE Healthcare Life Sciences) the protein was concentrated using a 30 kDa (NpsA 22 ACS Paragon Plus Environment

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403

and NpsB) or 10 kDa (ThdA) cutoff filter and stored at -80 °C in 10% glycerol. Protein purity

404

was determined by SDS-PAGE. Protein concentration was determined spectrophotometrically

405

upon determining the respective extinction coefficient from the amino acid sequence using the

406

PROTPARAM webserver (http://web.expasy.org/protparam/).44

407 408

In Vitro Reconstitution of tilivalline NRPS

409

NpsA, ThdA and NpsB (0.4 µM each) were incubated with the respective cofactors (1 mM ATP,

410

0.5 mM NADPH) and substrates (0.5 mM Proline, 3-OH-anthranilic acid, indole) in reaction

411

buffer (150 mM NaCl, 10 mM MgCl2, 50 mM Tris-HCl, pH 7.5) in a total volume of 20 µL at

412

room temperature for 4h. The mixture was dried in a vacuum condenser and subsequently

413

resuspended in 20 µL water:acetonitrile (1:1), centrifuged and submitted to LC-MS analysis.

414

In Vitro Reconstitution of tilivalline NRPS and TnaA

415

2 µM TnaA and 0.4 µM each of NpsA, ThdA and NpsB were incubated with the respective

416

cofactors (0.1 mM PLP, 1 mM ATP, 0.5 mM NADPH) and substrates (0.5 mM Prolin, 3-OH-

417

anthranilic acid and tryptophan) in reaction buffer (150 mM NaCl, 10 mM MgCl2, 50 mM Tris-

418

HCl, pH 7.5) in a total volume of 20 µL at 37°C for 2.5h. The mixture was dried in a vacuum

419

condenser and subsequently resuspended in 20 µL water:acetonitrile (1:1), centrifuged and

420

submitted to LC-HRMS analysis.

421

Determination of Cytotoxicity

422

KB-3.1 human cervix carcinoma cells were obtained from the German Collection of

423

Microorganisms and Cell Cultures (Deutsche Sammlung für Mikroorganismen und Zellkulturen,

424

DSMZ) and were cultured under conditions recommended by the depositor. Cells were seeded at

425

6 x 103 cells per well of 96-well plates in 180 µL complete medium and treated with compounds 23 ACS Paragon Plus Environment


Page 24 of 29

426

in duplicate in serial dilution after 2 h of equilibration. After 5 d incubation, 20 µL of 5 mg mL-1

427

MTT (thiazolyl blue tetrazolium bromide) in PBS was added per well and it was further

428

incubated for 2 h at 37°C. The medium was then discarded and cells were washed with 100 µL

429

PBS before adding 100 µL 2-propanol/10 N HCl (250:1) in order to dissolve formazan granules.

430

The absorbance at 570 nm was measured using a microplate reader (Tecan Infinite M200Pro),

431

and cell viability was expressed as percentage relative to the respective solvent control. IC50

432

values were determined by sigmoidal curve fitting.

433

Inhibition of tilivalline production in K. oxytoca (ID 1976911)

434

Cryo-cultures of K. oxytoca (ID 1976911) were streaked out on TSA-S plates and incubated at

435

37 °C overnight and subsequently stored at 4°C until use. Inhibition of tilivalline production was

436

screened in 8 mL TSB media inoculated from single colonies in flasks without baffles. The

437

inhibitor was added to its final concentration using DMSO stock solutions. Cultures were

438

incubated for 16 h at 37 °C and 100 rpm. 10 mL ethyl acetate was added and shaking was

439

continued for additional 30 minutes. 1 mL of the organic phase was dried and resuspended in

440

40 µL acetonitrile:water (1:1), centrifuged and submitted to LCMS analysis. Experiments were

441

carried out in triplicates.

442 443

Supporting Information

444

Sequence homologies, TAR primer sequences, tilivalline NMR data, tilivalline derivatives MS

445

data, NpsA, NpsB and ThdA SDS-PAGE, TnaA in vitro reconstitution, supplemental methods

446

(purification and in vitro reconstitution of TnaA, analytical methods), synthetic procedures. This

447

material is available free of charge via the Internet at http://pubs.acs.org.

448

References 24 ACS Paragon Plus Environment

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TOC 78x37mm (300 x 300 DPI)

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Biosynthesis of the Klebsiella oxytoca Pathogenicity Factor Tilivalline

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