A Probiotic Cell Factory for Production & Delivery of Supplements

The major, overall goal of this project to create an engineered probiotic to deliver the dietary supplement creatine to livestock to improve performance. In phase I we will focus on building the engineered probiotic, usingE. coliNissle,and testing feasbility.Objective 1: Determine the optimal expression system for the creatine biosynthetic enzymes, AGAT and GAMT, in E. coliNissle.In this objective we will test several well-characterized expression systems (promoters, regulators etc) to drive expression of the two biosynthetic enzymes required to produce creatine. For each system builtwe will test expression of the genes of interest and effect on growth of the organism in conditions that mimic the intestinal tract. We define success for this metric based on the minimum requirement for f 0.093 grams of creatine per day inchickens.Objective 2: Implementing the metabolic design inE. coliNissle to optimize creatine productionCreatine production requires the amino acid precursors arginine, glycine and methionine. We have used a genome-wide metabolic model forE. coliNissle to identify gene targets that will increase pools of these precursors. In this objective we will systematically create gene deletions to improve flux through our desired pathways and test the creatine production of the resulting strains in conditions that mimic the intestinal tract. We will descriminate between the designs based on creatine yield and the rate at which creatine is produced in order to reach the pysiological threshold of at least 0.093 grams per day.Objective 3: Implement a biocontainment strategyIn order to prevent unwanted proliferation of a GMO probiotic, incorporating a biocontainment strategy is critical. In this objective we will build a kill switch that will cause the modifiedE. coliNissle strain to self-destruct outside of the gut. We have conceived of two layers of this kill switch: one that will allow expression of an essential gene only under anaerobic conditions and another that will express a lethal toxin at temperatures below 37C. These kill switches will be evaluated by growing strains in conditions mimicking the intestinal tract then bringing the strains into non-permissive (environmental) conditions and evaluating survivial. Success for this metric will be measured by the escape frequencyenvironmental conditions: aiming for the NIH guideline of <1x108 when both kill switches are combined.