Development of an electrotransformation protocol for the novel acetogen Acebotacterium wieringae strain JM

Abstract

Objectives: In this work we aimed to develop an electrotransformation protocol for Acetobacterium wieringae strain JM which is an attractive host for the establishment of a gas based biorefinery (1). So far only Acetobacterium woodii was modified towards the production of industrially relevant compounds within the Acetobacterium genus. Therefore, there is a strong need and interest for developing new and implementing existing genetic tools from other organisms to efficiently access and modify the metabolism of Acetobacterium species. To achieve this, we tested in Acetobacterium wieringae strain JM available molecular tools from clostridial species, namely the plasmid vectors pMTL82151, pMTL83151, pMTL84151, and pMTL84151 containing the Gram-positive replicons pBP1, pCB102, pCD6, and pIM13 with the thiamphenicol resistance gene (catP). With these tools we aimed to establish a stable, efficient, and reproducible electrotransformation protocol allowing high-level DNA transfer to Acetobacterium wieringae strain JM. Results: This work reports the development of an electrotransformation protocol for Acetobacterium wieringae strain JM. After systematic investigation of various parameters involved in the cell growth, washing, pulse delivery, outgrowth phases, and plating of the electrotransformation procedure we were able to transform strain JM with all four tested plasmid vectors, reaching efficiencies up to 2.75 x 104 transformants μg -1 DNA. Key factors affecting the electrotransformation efficiency include cell wall-weakening using D-threonine, pH of wash buffer, field strength of the electric pulse, plasmid amount, and sucrose osmoprotection. Conclusions: Acetobacterium wieringae strain JM can be efficiently electrotransformed and genetically modified with plasmid DNA containing Gram-positive replicons. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically relevant. Significance: Research efforts need to be directed toward expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study unlocks one promising acetogen for the introduction of exogenous pathways which may lead the production of biocommodities from CO/CO2.