High-efficiency transformation of Bifidobacterium animalis AR668-R1 using electroporation

Bifidobacterium animalis, one of the most prevalent bacteria in the digestive tracts of humans and other mammals, is a typical addition to dairy products. The previous study reported an oxygen tolerant B. animalis AR668-R1 domesticated by adaptive laboratory evolution, which is different from most strictly anaerobic Bifidobacterium strains. However, the studies at molecular level of strain AR668-R1 were hindered due to the low electroporation efficiency. This work aims to achieve a high level of reproducibility in the electroporation-mediated transformation efficiency of AR668-R1. When the optimal parameters were the seed inoculum (OD₆₀₀ = 0.6), inoculation size (2 %), sucrose concentration (0.5 mol/L), sodium chloride concentration (0.25 mol/L), growth stage (OD₆₀₀ = 0.3), plasmid concentration (500 ng/microL), electric field intensity (15 kV/cm), and resuscitation time (3 h), the electroporation efficiency reached 3.97 × 10⁵ CFU/microg DNA, which was 79-fold higher than that of the unoptimized condition. Moreover, transcriptional analysis revealed that a series of putative competence genes (ssb, gene0596, comEC, and gene1115) in AR668-R1 were significantly upregulated after optimization. It suggested that improving transformation efficiency is attributable to the enhancement of competence gene expression. Overexpression of the above four competence genes further enhanced the transformation efficiency in AR668-R1. Specifically, comEC overexpression resulted in 2.5 times (9.78 ×10⁵ CFU/microg DNA) improvement. Furthermore, knockout of comEC resulted in a transformation efficiency 74.9-fold (5.32 ×10³ CFU/microg DNA) lower than the control, which demonstrated that the key competence gene is closely related to transformation efficiency. Together, the transformation efficiency was successfully improved in AR668-R1, which could promote extensive genetic manipulation and functional analysis in B. animalis.