Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

Abstract

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceA_Y51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.

Figure 1

HPLC analysis of Cbas extracted from D. hafniense strain DCB‐2 grown with pyruvate and ClOHPA. DMB = 5,6‐dimethylbenzimidazole, 5‐MeBza = 5‐methylbenzimidazole, Bza = benzimidazole, 5‐OHBza = 5‐hydroxybenzimidazole, 5‐OMeBza = 5‐methoxybenzimidazole (25 μM respectively).

Figure 2

HPLC analysis of Cba extracts from D. hafniense DCB‐2 cells cultivated with pyruvate and ClOHPA in the presence of purine, 5‐azabenzimidazole (5‐azaBza), or 4‐azabenzimidazole (4‐azaBza) (25 μM respectively).

Figure 3

HPLC analysis of Cba extracts from D. hafniense strain DCB‐2 cultivated with pyruvate and ClOHPA in the presence of YE that was obtained from either ¹⁵N‐enriched or unlabelled Saccharomyces cerevisiae cells or purchased. For details about the production of the ¹⁵N‐enriched YE or the unlabelled YE, see Materials and Methods section. The ¹⁵N‐enriched YE or the unlabelled YE was added to D. hafniense strain DCB‐2 cultures that contained unlabelled NH₄Cl as nitrogen source. When the purchased YE was applied, the unlabelled NH₄Cl was replaced by ¹⁵N‐labelled NH₄Cl (lower trace). The dashed line marks additional Cba signals. 1 = purinyl‐Cba, 2 = 5‐azaBza‐Cba.

Figure 4

HPLC analysis of Cba extracts from D. hafniense strain DCB‐2 cultivated with pyruvate and ClOHPA in the presence of 4,5‐dimethylimidazole (DMI) or imidazole (25 μM respectively). 1 = purinyl‐Cba, 2 = 5‐azaBza‐Cba.

Figure 5

HPLC elution profiles of Cbas extracted from S. blattae (S. b.) in comparison with Cba samples derived from D. hafniense strain DCB‐2 (D. h.) cultivated in the presence of the same Bza.

Figure 6

Enzyme activity and protein level of PceA_Y51 and DcaA heterologously produced in S. blattae cultivated in the presence of various amendments. A. The RDase activity was measured in at least two independent cultures. The standard deviation is given. PceA_Y51 was tested for the conversion of PCE, while DcaA was tested with 1,2‐dichloroethane (DCA) as substrate. B. Immunological detection of both RDases in crude extracts separated on an SDS‐PAGE (25 μg of protein was applied to each lane) with an antibody directed against the Strep‐tag.