Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase

Abstract

Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

“…Hence, a host organism for heterologous production had to be found that provided both an adequate amount of Cbas and iron-sulfur clusters for RDase assembly. In recent years, the Cba-producing gammaproteobacterium Shimwellia blattae (Lawrence and Roth, 1996) and the Gram-positive Bacillus megaterium (Wolf and Brey, 1986) were successfully applied (Mac Nelly et al, 2014;Payne et al, 2015;Kunze et al, 2017;Jugder et al, 2018). B. megaterium synthesizes the standard-type B 12 cofactor (5,6-dimethylbenzimidazolyl-Cba), and S. blattae produces pseudo-B 12 (adeninyl-Cba) de novo (Wolf and Brey, 1986;Mac Nelly et al, 2014).…”

Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli

“…UNSWDHB. It was chosen because it was previously expressed in its active form in B. megaterium (35). TmrA was cloned without its TAT signal peptide into the p15TV-L expression vector.…”

“…One advantage is higher protein yields in E. coli . From 1 L of culture, we were able to obtain approximately 2 mg of TmrA whereas to get 0.9 mg of TmrA at a similar purity, 15 L of B. megaterium was required (35). Furthermore, the RDase expression vectors can be commercially produced in typical pET vectors while those for B. megaterium and S. blattae generally have to be manually cloned (33, 35, 36).…”

“…Cobalamin was quantified using two separate methods, in both cases cyanocobalamin at a concentration range of 0.1-10 µM (final concentration) was used for a standard curve. The first method was used as previously described with some modifications (35, 44, 56). The standard samples and protein samples (50 μL) were mixed with 15 mM KCN to a volume of 750 µL and were heated at 80°C for 10 min.…”

“…Most typical heterologous hosts, such as E. coli , neither produce cobamides de novo nor import the molecule under basal conditions. Since the crystal structures, there have been a couple successful reports of catalytically active RDase expression all using hosts S. blattae and B. megaterium , both with full cobamide biosynthetic pathways (35, 36). However, less conventional hosts tend to have lower protein yields and have less standardized protocols.…”