Abstract
Genetic engineering of probiotics, like bifidobacteria, may improve their microbial cell factory economy. This work designed a novel shuttle plasmid pBPES, which bears exogenous appA and is stable within Bifidobacterium longum JCM 1217. Cloning of three predicted promoters into pBPES proved that all of them drive appA expression in B. longum JCM 1217. Transformation of plasmids pBPES-tu and pBPES-groEL into B. longum JCM1217 resulted in much more phytase secretion suggests Ptu and PgroEL are strong promoters. Further in vitro and in vivo experiments suggested B. longum JCM 1217/pBPES-tu degrades phytate efficiently. In conclusion, the study screened two stronger promoters and constructed a recombinant live probiotic strain for effectively phytase secretion and phytate degradation in gut. The strategy used in the study provided a novel technique for improving the bioaccessibility of phytate and decreasing phosphorus excretion.
Highlights
Bifidobacteria are ideal hosts for food-grade delivery of useful enzymes
Transformation into and successful isolation from both E. coli DH5α and B. longum JCM 1217 suggest that pBPES is a shuttle vector that can be maintained in these hosts
The segregational stability of this plasmid in B. longum JCM 1217 was further evaluated by propagation in liquid MRS without antibiotics and the recovery rate under antibiotics reaches 100% after 100 generations and surpasses 95% after 500 generations
Summary
Strong promoters can regulate high-level gene expression thereby improving the efficiency of microbial cell factories. Most of those finely defined promoters that had been used in other hosts cannot be readily applied in bifidobacteria (Sun et al, 2012). The strain reduced biofilm formation in pathogenic E. coli, reduced colitis in rats, and increased survival rate of Clostridium difficile infection in mice (Kim et al, 2012; Celiberto et al, 2017; Yun et al, 2017) If this strain was used as a host for foreign gene expression, its economy may improve as their synergistic effects
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