Strictly anaerobic microorganisms, such as Dehalococcoides mccartyi, play important roles in sustainable environmental biotechnologies. However, lack of direct chromosomal manipulating tools for anaerobic microbes has hindered the development of anaerobic biotechnologies. In this study, we investigated the functions of two metabolic genes from D. mccartyi str. 195 using a sulfur-reducing anaerobic bacterium – Desulfovibrio vulgaris Hildenborough (DvH) as a heterologous expression host. The vector system, pMO9075 was employed to transform, express, and identify functions of the genes from D. mccartyi str. 195. Initially, stable chromosomal deletions in DvH were constructed using both homologous recombination and CRISPR-Cas9-based genome editing. Then, two auxotrophic mutants were selected from homologous recombination with deletion of either a citrate synthase (JW9051) or a citramalate synthase (GZ9982). The pMO9075-based vectors were constructed with the candidate genes from strain 195, i.e., DET0825 and DET1614 and transformed into the auxotrophic DvH mutants. The in vivo analysis showed that the expression products of the two genes cured the auxotrophic DvH mutants. The functions of these genes were further examined using in vitro enzymatic analyses and 13C isotope tracer analysis which indicated the two genes as a R-type citramalate synthase (DET0825) and a Re-type citrate synthase (DET1614), respectively. This study demonstrated that heterologous expression of genes in DvH is an effective approach to understand metabolic genes of D. mccartyi, and this approach can potentially be used to identify a broader spectrum of functional genes of strictly anaerobic microorganisms.

Identification of functional genes from Dehalococcoides mccartyi 195 using Desulfovibrio vulgaris Hildenborough as a heterologous expression host system

2019 Spring National Meeting

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