Genetic tools for the electrotroph Sporomusa ovata and autotrophic biosynthesis

ABSTRACT Sporomusa ovata is a Gram-negative acetogen of the Sporomusaceae family with a unique physiology. This anerobic bacterium is a core microbial catalyst for advanced CO 2 -based biotechnologies including gas fermentation, microbial electrosynthesis, and hybrid photosystem. Until now, no genetic tools exist for S. ovata , which is a critical obstacle to its optimization as an autotrophic chassis and the acquisition of knowledge about its metabolic capacities. Here, we developed an electroporation protocol for S. ovata . With this procedure, it became possible to introduce replicative plasmids such as pJIR751 and its derivatives into the acetogen. This system was then employed to demonstrate the feasibility of heterologous expression by introducing a functional β-glucuronidase enzyme under the promoters of different strengths in S. ovata . Next, a recombinant S. ovata strain producing the non-native product acetone both from an organic carbon substrate and from CO 2 was constructed. Finally, a replicative plasmid capable of integrating itself on the chromosome of the acetogen was developed as a tool for genome editing, and gene deletion was demonstrated. These results indicate that S. ovata can be engineered and provides a first-generation genetic toolbox for the optimization of this biotechnological workhorse. IMPORTANCE S. ovata harbors unique features that make it outperform most microbes for autotrophic biotechnologies such as a capacity to acquire electrons from different solid donors, a low H 2 threshold, and efficient energy conservation mechanisms. The development of the first-generation genetic instruments described in this study is a key step toward understanding the molecular mechanisms involved in these outstanding metabolic and physiological characteristics. In addition, these tools enable the construction of recombinant S. ovata strains that can synthesize a wider range of products in an efficient manner.