Expanding the molecular toolkit for the new gas-converting Acetobacterium wieringae strain JM

Gas fermentation is a promising way to convert CO-rich gases to chemicals. In this work we aimed to develop an electrotransformation protocol for the promising gas-converting Acetobacterium wieringae strain JM (sJM) [1], to further implement heterologous pathways for the production of new compounds from gas streams. In this matter, Clostridial species are the most genetic manipulated homoacetogens, and within the Acetobacterium genus, only A. woodii has been used. Therefore, there is a strong need and opportunity to expand the molecular toolkit for sJM and other Acetobacterium species. To achieve this, we tested reported electrotransformation procedures for A. woodii, using 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). The utilization reported electrotransformation procedures did not result in sJM transformants. Therefore, we developed a new protocol which we report to be applicable to sJM, A. wieringae DSM 1911 and A. woodii DSM 1030. The most important changes for higher expeditiously and reproducibility were the use of fixed cell density of competent cells resulting in electroporation cell-plasmid ratios of 2 to 3 (OD600.mL/g), the use of an electric field strength of 10 kV/cm, and the selection of transformants in serum anaerobic bottles with molten agar of our optimized basal media. All plasmid vectors were transformed, having pMTL83151 yielded the highest transformation efficiency in all tested Acetobacterium strains, reaching efficiencies up to 5.0 × 102, 2.0 × 103, and 5.1 × 103 transformants g-1 DNA for sJM, A. wieringae DSM 1911 and A. woodii DSM 1030, respectively. Key factors affecting the electrotransformation efficiency include cell-wallweakening using D-threonine, pH of wash buffer, field strength of the electric pulse, plasmid amount, and sucrose osmoprotection. The electrotransformation procedure and tools reported here unlock the genetic manipulation of this biotechnologically relevant sJM strain, which will be directed towards the expansion of the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol.
Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145-FEDER-000004); FCT doctoral grant PD/BD/150583/2020.
info:eu-repo/semantics/publishedVersion