Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic， but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production， genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters， its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack ... More
Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic， but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production， genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters， its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack of DNA binding motifs did not support this， and one current analysis suggested a high likelihood of it interacting with LanD. Therefore， gene lanR1 together with lanADMIT were codon optimized and synthesized. Those genes were then cloned into an efficient dual-plasmid nisin-controlled expression system in L. lactis. The addition of the lanR1 gene exhibited toxicity in E. coli， specifically causing a shorter cell size as observed by SEM. No toxicity was observed in L. lactis. While this production system did not result in the production of a bioactive lantibiotic by L. lactis， it did successfully produce all the peptides and enzymes encoded by the original lantibiotic gene cluster from B. longum， as confirmed by LC-MS. This will now facilitate efforts into determining the proper conditions required for these enzymes to produce a bioactive lantibiotic.
