Heterologous Expression of Active Dehalobacter Respiratory Reductive Dehalogenases in Escherichia coli

Abstract

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, potentially because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. Here we outline a novel respiratory RDase expression system using Escherichia coli. The overexpression of E. coli's cobamide transport system, btu, and anaerobic expression conditions were found to be essential for production of active RDases from Dehalobacter-an obligate organohalide respiring bacterium. The expression system was validated on six enzymes with amino acid sequence identities as low as 28%. Dehalogenation activity was verified for each RDase by assaying cell extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of the cobamide and iron-sulfur cluster cofactors was verified; however, the precise cobalamin incorporation could not be determined due to variance between methodologies, and the specific activity of TmrA was consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCE Understanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in Escherichia coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

Keywords: Dehalobacter; Escherichia coli; anaerobic; cobalamin; enzyme purification; heterologous expression; iron-sulfur clusters; reductive dehalogenases.

FIG 1

(A) Schematic of the RDase expression system and small-scale expression tests to determine production of the active RDases. (B) Cartoon of the Btu pathway used for cobalamin incorporation in RDase expression (adapted from reference with permission [copyright 2018 American Chemical Society]). A, aerobic conditions; AN, anaerobic conditions; O/N, overnight; GC-FID, gas chromatography with flame ionization detection.

FIG 2

Reduction of chloroform to dichloromethane (DCM) by E. coli cell extracts expressing TmrA over a period of 24 h. (A) Comparison of TmrA expressed in E. coli ΔiscR with (+Btu) and without (–Btu) pBAD42-BtuCEDFB plasmid expression, and with induction under anaerobic (AN) or aerobic (A) conditions. (B) Comparison of TmrA expressed in different E. coli strains with pBAD42-BtuCEDFB co-expression and under anaerobic conditions. ACT-3 mixed culture cell extract was used as a positive control and an enzyme free negative control was used. Error bars are standard deviation between replicates (n = 6, except for ACT-3, enzyme free, and aerobic samples where n = 3).

FIG 3

Specific activity of TmrA for substrate-product transformations, where nkat is defined as the rate of product formation in nmol/s. Data for TmrA purified from the endogenous host Dehalobacter sp. UNSWDHB (striped bars) (14), and TmrA heterologously expressed and purified from E. coli (solid bars) are shown. Substrates are indicated on the left axis label and product is indicated on the right axis label. Error bars are from reported standard deviation for the literature values, and the standard deviation of purified enzyme assays (n = 3). n.a., not available.

FIG 4

(A) Specific activity of HchA for substrate-product transformations. (B) The rate of monochlorobenzene (MCB) and benzene production from purified HchA, heat killed HchA, and free cobalamin (same molarity of catalysts were used). The nkat is defined as the rate of product formation in nmol/s, this rate is normalized to mols of catalyst. Error bars are the standard deviation between assays (n = 3).

FIG 5

(A) Maximum-likelihood (ML) tree of DHB14 and DHB15 RdhA amino acid sequences (indicated by *) to show relation to the closest characterized RDases in comparison to TmrA. (B) Reduction of either PCE (top) or TCE (bottom) by DHB14 and DHB15 in E. coli cell extract, assay was done over 24 h. Error bars are the standard deviation between replicates (n = 4), except negative controls (n = 2). An amino acid alignment was built using the Geneious v8.1.9 MUSCLE plugin, and the ML tree was constructed using RAxML v8.2.12 Gamma GTR protein method with 100 bootstraps, the highest scoring tree was selected. Scale indicates average number of substitutions per amino acid site. Protein accession numbers for sequences in panel A in descending order are as follows: DHB15, AFV02698; DHB14, AFV05674; DcaA, CAJ75430; TcbA, 2821531494 (IMG ID); TcbA, WP_068882928; PceA, AAO60101; PceA, AHF10727; PceA, CAD28792; PceA, WP_011460641; TmrA, WP_034377773.