Metabolic engineering of Vitamin A biosynthesis in probiotic bacteria

Diarrheal diseases kill nearly 2 million children every year. Deficient mucosal immunity is a significant contributor to susceptibility to diarrheal diseases, largely due to widespread pediatric Vitamin A deficiency. To increase Vitamin A coverage in malnourished children, we are engineering probiotic bacteria to biosynthesize Vitamin A in the gut. We hypothesize that these bacteria will deliver Vitamin A to mucosal dendritic cells in the intestine, broadly stimulating mucosal immunity and reducing the incidence and duration of diarrheal disease. To generate vitamin‐producing strains, we first assembled a gene cassette containing the necessary enzymes in E. coli . Human beta‐carotene‐15,15′‐diooxygenase was codon‐optimized for bacterial expression and inserted into a plasmid containing four enzymes derived from the bacterium Pantoea agglomerans . The final cassette was cloned into E. coli – Lactobacillus shuttle vectors for transformation of probiotic L. casei strains. As an alternative to HPLC‐based assays, a simple spectrophotometric method was developed for quantitation of Vitamin A production. If Vitamin A‐producing L. casei induce a mucosal phenotype in dendritic cells in vitro, we will assess the ameliorative effects of this novel therapy in animal models of diarrheal disease. This work is funded by a grant from the Bill & Melinda Gates Foundation through the Grand Challenges Explorations Initiative.