Heterologous Production and Purification of a Functional Chloroform

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Heterologous production and purification of a functional chloroform reductive dehalogenase Bat-Erdene Jugder, Karl A.P. Payne, Karl Fisher, Susanne Bohl, Helene Lebhar, Mike Manefield, Matthew Lee, David Leys, and Christopher P. Marquis ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00846 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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ACS Chemical Biology

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Heterologous production and purification of a functional chloroform

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reductive dehalogenase

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Bat-Erdene Jugder†,‡, Karl A. P. Payne‡, Karl Fisher‡, Susanne Bohl†,§, Helene Lebhar†, Mike

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Manefield¶,ς, Matthew Lee¶, David Leys‡ and Christopher P. Marquis†,*

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†

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia, NSW 2052

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School of Civil and Environmental Engineering, University of New South Wales, Sydney,

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Australia, NSW 2052

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School of Chemical Engineering, University of New South Wales, Sydney, Australia,

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NSW 2052

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§

Manchester Institute of Biotechnology, University of Manchester, United Kingdom, M1 7 DN

Department of Biotechnology, Mannheim University of Applied Sciences, Mannheim, Germany

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* Corresponding Author

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ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of

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anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases

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is desirable for structural and functional studies, however there are few reports of successful

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expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred

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electron acceptor is chloroform. This study describes efforts to express recombinant reductive

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dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli

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and Bacillus megaterium. Here we report the recombinant expression of soluble and functional

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TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful

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incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis

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spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA

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was demonstrated. This is the first known report of heterologous expression and purification of a

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respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

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Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant, toxic and

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carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide

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respiring bacteria (ORB) are a potential solution to remediate organohalide contaminated sites,

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as they are capable of using these compounds as terminal electron acceptors for the generation of

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cellular energy. Central to the process are reductive dehalogenases (RDase; EC 1.97.1.8), which

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are membrane-associated enzymes that catalyze reductive dehalogenation reactions resulting in

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the generation of lesser-halogenated compounds that may be less toxic and more biodegradable.

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RDases are metalloenzymes, meaning their activity is dependent on two protein-associated

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cofactors; cobamide and iron-sulfur clusters.1-3

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Chloroform (CF), primarily used in the production of refrigerants, is a prevalent and

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recalcitrant organohalide contaminant and its improper use and disposal has resulted in

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groundwater contamination at numerous industrial sites globally.4 Its hazardous effect on the

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environment, carcinogenicity and acute toxic effects on human health has prompted

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bioremediation research towards its detoxification.5 Dehalobacter (Dhb) sp. strain UNSWDHB

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is an obligate ORB capable of respiring CF, thereby transforming it to dichloromethane (DCM).6

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Strain UNSWDHB

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respiration.7 Native TmrA was produced and purified from the membrane fraction of

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UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and

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anion exchange chromatographic purification.8 This allowed further biochemical characterization

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of the purified enzyme. However, ORB are slow growing with low cell yields. This coupled with

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non-trivial culture maintenance make routine RDase production and purification from the native

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source difficult.

produces a CF-RDase, (TmrA), which is highly expressed during CF



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The heterologous expression of respiratory RDases has been reported to be extremely

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challenging. Several efforts to heterologously express functional RDases have failed over the

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past 20 years, mainly due to the absence of one or both cofactors (a corrinoid and two Fe-S

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clusters) in the recombinant proteins.2, 3 The co-expression of solubility enhancing tags, such as

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trigger factor (TF), was reported to drive soluble expression of PceA fusion proteins from

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Dehalobacter restrictus in Escherichia coli, however, no enzymatic activity was observed.9 The

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first demonstration of functional expression of a recombinant RDase was reported for PceA from

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Desulfitobacterium hafniense Y51 produced in Shimwellia blattae, a bacterium capable of de

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novo synthesis of cobalamin.10 Co-expression of PceT, a chaperone protein, enhanced the

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overexpression of functional PceA, as demonstrated in the E. coli crude extracts. However, an

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attempt to purify the recombinant PceA failed, as the expressed protein did not bind to the Strep-

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Tag column.

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In this study, heterologous expression and purification of TmrA in a soluble and

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functional form was attempted. Initially, molecular cloning and expression studies of the enzyme

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in E. coli were undertaken. For this, the TAT signal and predicted membrane spanning regions

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were removed from the tmrA gene prior to gene synthesis and cloning; a strategy to direct

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expression of otherwise periplasmic RDases towards the cytoplasm (Figure 1A).9,

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rhamnose-inducible heterologous expression of TmrA under the control of a tunable rhaBAD

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promoter was investigated (Figure 1B). Whilst slightly tunable protein expression with

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increasing concentrations of rhamnose was observed in pD864-TmrA transformed cells, protein

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expression in general was not tightly regulated and TmrA was expressed in an insoluble form

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(Figure S1).

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First,

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Figure 1. Schematic of the heterologous expression of TmrA. (A) The region framed in red dashed line

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containing TAT signal peptide and a predicted transmembrane domain was removed prior to tmrA gene

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cloning. (B) TmrA expression in E. coli under rhamnose- and IPTG-inducible promoters in pD864 and

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pOPIN vectors, respectively. TmrA fusion constructs with solubility (SUMO, TRX, NusA, MBP and TF)

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and affinity (NHis and CHis) tags encoded in pOPIN expression vectors were constructed and

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investigated. 3C is the recognition sequence for the Human Rhinovirus 3C Protease to allow the cleavage

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of the fusion proteins. Cofactor reconstitution and refolding were attempted on pOPIN-CHis-TmrA

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construct. (C) A functional TmrA was expressed in B. megaterium containing pT7-RNAP plasmid that

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permits xylose-inducible expression of T7 polymerase. (D) Production and purification of TmrA in B.

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megaterium



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Next our attention turned to the heterologous expression of TmrA under the control of an

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IPTG-inducible system using the pOPIN vector system with a T7 inducible promoter and several

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fusion tags (Figure 1B). The co-expression of hydrophilic fusion partners has been shown to be

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an effective tool to increase the amount of soluble recombinant protein. Numerous expression

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screens were carried out and it was shown that TmrA in fusion with affinity (N- or C-terminal

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His) and solubility tags (SUMO, Trx, NusA, MBP and TF) can be overexpressed at 37°C from

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all constructs (Figure S2). However, none of our experiments yielded soluble recombinant

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protein with any of the constructs, despite attempting induction at a lower temperature, varying

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the growth media and IPTG concentration nor use of cofactor supplementation (Figure S3 and

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S4). Identical solubility tags to those used in the present study (SUMO, NusA, MBP and TF) had

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been shown previously to overexpress PceA from D. restrictus in E. coli9. However, the

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recombinant PceA was catalytically inactive, possibly due to insufficient or incorrect cofactor

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incorporation. Heterologous expression of RDases fused to solubility tags leading to insoluble

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and non-functional enzymes has also been reported elsewhere. For example, PceA from D.

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hafniense strain Y51 was investigated and overexpressed in fusion with Trx, S-tag for antibody

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recognition and N- and C-terminal His tags in E. coli.12 The fusion protein was also expressed in

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an insoluble form and denaturation followed by refolding of the fusion protein did not recover

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dechlorination activity.

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To date, no respiratory RDase in a soluble and catalytically active form has been

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heterologously expressed in E. coli. In this study, we also were not able to obtain a soluble TmrA

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in E. coli. Recently, a vinyl chloride-reducing dehalogenase VcrA from Dehalococcoides

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mccartyi strain VS was expressed in E. coli in an insoluble form but recovered from inclusion

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bodies via denaturing purification followed by chemical reconstitution of cofactors during


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protein refolding to an active form.11 Since no soluble TmrA was expressed throughout extensive

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expression studies, it was decided to investigate purification of detergent-denatured TmrA

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protein from the cellular insoluble fraction with subsequent refolding. For this, the

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pOPIN-TmrA-CHis transformant was employed due to its higher expression levels in 2YT media

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(compared to LB media) at 37°C. Five liter fermentations were undertaken with or without

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external cofactor supplementation. Adenosylcobalamin was used as a corrinoid cofactor based on

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previous reports of attempts to produce recombinant RDases.10, 11 Heterologously expressed C-

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His-tagged TmrA was captured by Ni-NTA affinity chromatography under denaturing conditions

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(8 mM urea) and subjected to several refolding methods, such as refolding by flash or slow

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dilution, by dialysis, on-column refolding and chemical cofactor reconstitution. Regardless of

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efforts undertaken in this work, using a similar approach to others for protein refolding11, no

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refolded and active TmrA could be obtained, mainly due to precipitation issues and subsequent

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protein loss.

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Our focus then shifted to heterologously expressing TmrA in Bacillus megaterium, a

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bacterium known to possess de novo cobalamin biosynthesis capacity. Recently, a co-metabolic

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(non-respiratory) reductive dehalogenase, NpRdhA, from Nitratireductor pacificus strain pht-3B

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was heterologously expressed in B. megaterium.13 The C-terminal His-tagged NpRdhA was

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obtained in an active form in aerobically grown B. megaterium under a xylose-inducible

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promoter. Although the recombinant NpRdhA represents a phylogenetically distinct and aerobic

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RdhA, as opposed to respiratory RDases, it allowed further studies to reveal RDase structure and

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generation of a proposed enzymatic mechanism. In this study, we utilized the same molecular

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cloning and expression tools for B. megaterium to express the respiratory TmrA.



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The tmrA gene with BsrGI and BamHI sites and N-terminal His-tag were PCR-amplified

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and cloned into the same restriction sites of pPT7 plasmid using the In-Fusion cloning technique

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(Clontech). The pPT7 plasmid harboring the tmrA gene (pPT7-tmrA, Figure 1C) was then was

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transformed into B. megaterium MS941 protoplasts containing the pT7-RNAP plasmid that

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permits xylose-inducible expression of T7 polymerase. Xylose-inducible expression of TmrA

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was performed aerobically in TB media at 17°C. The cells were disrupted anaerobically using a

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French press (Figure 1D). Following anaerobic ultracentrifugation of cell lysate, protein SDS-

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PAGE and western blotting analyses revealed TmrA expressed in a soluble form (Figure 2A and

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B), which was further confirmed by tryptic digest and liquid chromatography tandem mass

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spectrometry (LC-MS/MS) at the UNSW Biomedical Mass Spectrometry Facility.

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Figure 2. Heterologously expressed TmrA. (A) SDS-PAGE of purification samples, including soluble

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fraction, IMAC (Ni-NTA) and ResQ column eluted proteins and Western Blot of the purified protein

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(ResQ column) with His6 Tag Monoclonal Antibody, HRP conjugate (Invitrogen). (B) UV-visible

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absorption spectra of the purified TmrA and the cobalamin extracted from the enzyme (inset). (C) Gas

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chromatogram of CF (RT=5.044 min) dechlorination reaction by the purified TmrA to produce DCM (in

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blue; RT=4.159 min) overlapped with a heat-deactivated negative control (in grey). The data represents

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the dechlorination activity after incubating the enzymatic reaction at 30°C for 5 h, during which in total of

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823 nmol DCM was produced. The peak heights (pA) for CF, DCM from the TmrA-catalyzed reaction

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and CF from the negative control are given as CF pATmrA, DCM pATmrA and CF pANC, respectively.

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To purify NHis-tagged TmrA, the soluble fraction resulting from a 15-L fermenter was

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loaded onto a Ni-NTA drip column under anaerobic conditions. The TmrA protein was eluted

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with approximately 50 mM Imidazole-containing buffer and verified by SDS-PAGE and western

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blotting (Figure 2A and B). To improve the purity of the protein preparation, an anion exchange

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ResQ column was used. For this, the Ni-NTA eluate was buffer-exchanged and loaded into the

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ResQ column aerobically using an ÄKTAexplorer. At approximately 250 mM NaCl, the TmrA

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protein was recovered from a single peak (Figure 2A and B).

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Next, UV-visible spectroscopy, was used to detect the cofactors incorporated into

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purified RDases. The heterologously expressed TmrA exhibited a broad absorbance between 400

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and 500 nm with the absorption maximum at 420 nm being indicative for the [4Fe-4S] clusters

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(Figure 2C), which is a common feature for all previously reported RDases,14-16 including the

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native version of TmrA.8 Cyanide extraction of TmrA-associated cobalamin revealed the

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absorption maxima at 361, 522 and 550 nm which is typical for cyanocobalamin (inset in Figure



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2C).14-16 A stoichiometry of 0.52 ± 0.03 mol of corrinoid per mol of TmrA was determined in the

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pink solution extracted by means of cyanolysis. The Fe content of the dehalogenase using a

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bathophenanthroline assay revealed 7.26 ± 0.48 mol of Fe/mol of protein, which is close to the

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expected value of 8.

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To confirm the catalytic activity of TmrA, a CF reductive dehalogenating activity assay

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was performed using Ti(III) citrate as the bulk electron donor and methyl viologen as an electron

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transfer mediator.8 Activity was monitored by the production of DCM. CF dechlorination

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activity was found in the soluble protein fraction (0.1 U mg of protein–1) and the Ni-NTA eluted

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protein (30.2 U mg of protein–1). The final aerobic ResQ-purified protein exhibited CF reductive

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dehalogenation activity at a specific activity of 110 U mg of protein–1 (Figure 2D and Table 1).

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This activity is 11-fold lower than the activity of native TmrA previously determined

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(1.27 × 103 U mg of protein–1).8 This might be explained by a number of factors, including the

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presence of apoenzymes that have not correctly incorporated corrinoid cofactors, the failure to

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co-express a trigger factor to facilitate correct folding or partial oxidation of enzymes during

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purification. Our data suggests that the reduced specific activity of the recombinant enzyme

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(compared to purified enzyme from UNSWDHB), can only be in part attributed incomplete

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incorporation of corrinoid, since the observed cobalamin:enzyme molar ratio is approximately

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half the expected value of 1. Heat-deactivated (incubating extracts at 80°C for 10 min)

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B. megaterium crude protein extracts and enzyme-free reactions were assayed as negative

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controls to rule out non-enzymatic CF reductive activity and no dechlorination activity was

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observed in such negative controls (Figure 2C).

Soluble fraction

Total activity (nmoles/min)

Yield (%)

Total protein (mg)

Specific activity (nmoles/min/mg)

Purification factor

278.6

100

2700.0

0.1

1


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Ni-NTA eluate Res-Q eluate

158.5 99.0

57 36

5.25 0.9

30.2 110.0

293 1066

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Table 1

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In conclusion, an extensive study to heterologously express TmrA was conducted under

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several expression conditions. Firstly, a TmrA expression study in E. coli was undertaken by

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employing several strategies to enhance soluble expression, such as removal of a TAT signal

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peptide, rhamnose or IPTG-controlled induction, co-expression with commonly used solubility-

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enhancing protein tags and expression screens under numerous conditions. Despite all the efforts

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to obtain a soluble TmrA, as well as cofactor reconstitution attempts on the denatured protein,

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E. coli expression was unsuccessful. However, using the xylose-inducible expression system in

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the corrinoid-producing B. megaterium as an expression host, soluble and functional TmrA was

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successfully expressed and purified in active form via two liquid chromatographic steps. The

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presence of both cofactors was revealed by the UV-vis spectra recorded for the purified enzyme

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and metal analysis undertaken. The purified protein exhibited CF dechlorination activity in vitro.

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As a result, a membrane-associated respiratory RDase has been heterologously expressed in a

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soluble and active form and purified for the first time.

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METHODS

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The detailed experimental procedures of tmrA gene cloning for E. coli and B. megaterium

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heterologous protein expression, solubility screenings of TmrA expressed in E. coli, production,

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denaturing purification and refolding of the TmrA-CHis variant, heterologous expression of

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TmrA in B. megaterium and its purification, protein concentration determination, SDS-PAGE

Activity, yield and purification factor of the soluble extract, Ni-NTA eluate and anion exchange fractions



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and Western blotting, UV-Vis measurement, metal analysis in the recombinant enzyme and gas

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chromatographic activity assay are provided in the Supporting Information.

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ASSOCIATED CONTENT

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Supporting Information Available: Detailed experimental procedures, including gene cloning,

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protein expression and purification, protein refolding and protein characterization are available

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as Supplementary Information. This material is available free of charge via the internet at

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http://pubs.acs.org.

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AUTHOR INFORMATION

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Corresponding Author

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*Phone: +612 93853898, E-mail: [email protected]

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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We acknowledge the PEF at the University of Queensland, Australia for cloning the tmrA gene

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into pOPIN vectors with solubility tags. We also acknowledge the University of New South

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Wales (UNSW) for scholarship and travel support for BJ. DL is a Royal Society Wolfson

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Research Merit Award holder.

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REFERENCES

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1. Leys, D., Adrian, L., and Smidt, H. (2013) Organohalide respiration: microbes breathing chlorinated molecules, Philos. Trans. Soc., B 368, 20120316.


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2. Jugder, B. E., Ertan, H., Lee, M., Manefield, M., and Marquis, C. P. (2015) Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides, Trends Biotechnol. 33, 595-610. 3. Jugder, B. E., Ertan, H., Bohl, S., Lee, M., Marquis, C. P., and Manefield, M. (2016) Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation, Front. Microbiol. 7, 249. 4. Koenig, J., Lee, M., and Manefield, M. (2015) Aliphatic organochlorine degradation in subsurface environments, Rev.Environ. Sci. Bio/Technol. 14, 49-71. 5. ASTDR. (2015) Toxological profile for chloroform. http://www.atsdr.cdc.gov/ToxProfiles/tp6-c4.pdf 2015., (accessed 1 April, 2017). 6. Wong, Y. K., Holland, S. I., Ertan, H., Manefield, M., and Lee, M. (2016) Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration, Environ. Microbiol. 18, 3092-3105. 7. Jugder, B. E., Ertan, H., Wong, Y. K., Braidy, N., Manefield, M., Marquis, C. P., and Lee, M. (2016) Genomic, transcriptomic and proteomic analyses of Dehalobacter UNSWDHB in response to chloroform, Environ. Microbiol. Reports 8(5), 814-824. 8. Jugder, B. E., Bohl, S., Lebhar, H., Healey, R. D., Manefield, M., Marquis, C. P., and Lee, M. (2017) A bacterial chloroform reductive dehalogenase: purification and biochemical characterization, Microb. Biotechnol. 10(6),1640-1648 9. Sjuts, H., Fisher, K., Dunstan, M. S., Rigby, S. E., and Leys, D. (2012) Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus, Protein Expression Purif. 85, 224-229. 10. Mac Nelly, A., Kai, M., Svatos, A., Diekert, G., and Schubert, T. (2014) Functional heterologous production of reductive dehalogenases from Desulfitobacterium hafniense strains, Appl.Environ. Microbiol. 80, 4313-4322. 11. Parthasarathy, A., Stich, T. A., Lohner, S. T., Lesnefsky, A., Britt, R. D., and Spormann, A. M. (2015) Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS, J. Am. Chem. Soc. 137, 3525-3532. 12. Suyama, A., Yamashita, M., Yoshino, S., and Furukawa, K. (2002) Molecular characterization of the PceA reductive dehalogenase of Desulfitobacterium sp. strain Y51, J.Bacteriol. 184, 3419-3425. 13. Payne, K. A., Quezada, C. P., Fisher, K., Dunstan, M. S., Collins, F. A., Sjuts, H., Levy, C., Hay, S., Rigby, S. E., and Leys, D. (2015) Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation, Nature 517, 513-516. 14. Neumann, A., Wohlfarth, G., and Diekert, G. (1996) Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans, J. Biol.Chem. 271, 16515-16519.



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15. Schumacher, W., Holliger, C., Zehnder, A. J., and Hagen, W. R. (1997) Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus, FEBS Lett. 409, 421-425. 16. Christiansen, N., Ahring, B. K., Wohlfarth, G., and Diekert, G. (1998) Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense, FEBS Lett. 436, 159-162.


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Soluble and active expression of a recombinant reductive dehalogenase 342x195mm (150 x 150 DPI)


Heterologous Production and Purification of a Functional Chloroform

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