Genetic engineering of gut commensals: heterologous protein expression in Bifidobacterium animalis subsp. lactis...

Bifidobacterium is a key genus of the gut microbiota. Among commercially used probiotics, Bifidobacterium animalis subsp. lactis CECT 8145 has been shown to exert health benefits in metabolic health and healthy aging. Remarkably, these effects persist in its postbiotic, heat-treated form, a feature attributed to its unique lipoteichoic acid composition. However, the challenges of genetically manipulating bifidobacteria hinder the identification of the molecular basis of this functionality. In this study, we report the first successful transformation of B. animalis subsp. lactis CECT 8145, alongside reproducible and high overexpression of heterologous recombinant proteins. Our approach integrates optimized electroporation protocols, tailored vector selection, and modulation of membrane fluidity, further enhanced by polyethylene glycol as a permeabilizing agent. Membrane fluidity - driven by the ratio of unsaturated to saturated fatty acids - is shown to be a quantifiable and tunable determinant of transformation efficiency. A comparative analysis of five B. animalis strains confirmed that transformation efficiency remains strongly strain-dependent, likely reflecting differences in ecological origin. Promoter pHUP yielded the highest heterologous protein expression levels, showing that careful promoter selection can substantially increase gene expression even in strains with low transformability, with protein expression levels increasing with plasmid copy number and reaching up to 10⁴ arbitrary fluorescence units. Together, these findings establish a practical framework for the genetic engineering of recalcitrant, commensal probiotic strains such as CECT 8145. This work supports the development of next-generation probiotics and enables molecular validation of their health-promoting effects.