Recipient Organization
NORTHSTAR MICROBES LLC
1000 WESTGATE DR
SAINT PAUL,MN 551148612
Performing Department
(N/A)
Non Technical Summary
Growing restrictions on antibiotics and other growth promoters in the livestock industry has left an acute need for alternative supplements to enhance animal growth and prevent infection. NorthStar Microbes aims to address these issues by using synthetic biology to develop a probiotic cell factory to produce and deliver performance supplements to livestock. Engineered probiotics represent an innovative approach with several advantages to traditional chemical or fermentative synthesis methods. By employing a probiotic cell factory the supplement is produced in situ, eliminating product loss during purification and processing and overcoming the biggest problem associated with fermentation processes - inability to reach economically viable titer and rate. Furthermore, in situ production allows for delivery of less stable supplements not suitable for traditional oral delivery. The objective of this proposal is to combine state-of-the-art developments in synthetic biology to develop E. coli Nissle (EcN), a probiotic with proven gut health benefits, as a chassis to deliver high-performance supplements directly in the gut. These improvements include (i) developing a suite of promoter systems that provide desired expression in the intestinal tract, (ii) incorporating a basic metabolic structure that is conducive to high level product formation and (iii) incorporating a self-destruction switch triggered outside the GI tract as a biocontainment measure. Each of these improvements are structured as independent objectives to allow advancing the tools in parallel. Successful completion will provide a chassis for the production of supplements across a variety of species. This project is directly in line with USDA SBIR priorities to develop new technologies to increase the competitiveness of agriculturally derived products.
Animal Health Component
70%
Research Effort Categories
Basic
0%
Applied
70%
Developmental
30%
Goals / Objectives
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.
Project Methods
Objective 1: Determine optimal expression system for AGAT and GAMT enzymesIn this objective, we will evaluate three expression systems with different regulatory mechanisms to assess their ability for strong expression in the gut environment.We will clone the agat and gamt genes as an operon under the control of the three promoter systems into the E. coli expression vector pACYC184 and transformed into EcN.The resulting transformants will be evaluated under the gut physiological conditions in VL media, which mimics nutrient conditions in the chicken gut. We will measure substrate consumption and biomass to understand how each expression system affects EcN growth and assay for in vitro AGAT/GAMT activity to measure enzyme expression levels under each promoter. In addition to identifying an appropriate expression system for the AGAT and GAMT enzymes this work will also inform our choice of promoter for other exogenous genes that we introduce in our metabolic design outlined in Objective 2. We define the success metric for this objective based on a minimum requirement of 0.093 g creatine/day in chickens Metric for successIn vitro enzyme activity of 0.2 U/mg protein under physiological conditionsMilestoneSelection of a promoter for expression of the mammalian genesTimelineMonth 0 - Month 4Objective 2: Implementing the metabolic design in EcN to optimize the creatine productionCreatine production requires sufficient supply of arginine, glycine and methionine. Restricted supply of any of these precursors can constrain creatine synthesis, resulting in the production of undesirable byproducts. These precursors are also highly connected nodes in the metabolic network, involved in several reactions and their supply is tightly controlled. Previously, bacterial metabolism was engineered to produce methionine (e.g. EP2314710B1) or arginine (e.g. US7169586B2 and US7052884B2). However, the syncretic production of the three precursors requires genome-wide interrogation to identify metabolic optimization strategies.The metabolic model of EcN (iDK1463) was published in Feb-2021 which can be used as the platform to assess the flux envelopes for optimal production of creatine. To the iDK1463 model, we appended the AGAT and GAMT reactions and calculated a maximum theoretical yield of 0.15 g creatine/g glucose under anaerobic conditions (Figure below). Under the anaerobic conditions present in the gut, the model predicted producing formate, acetate and ethanol to balance redox and yield additional ATP (from acetate kinase).LEFT: Metabolic network and flux distribution correspond to maximum theoretical creatine yield under anaerobic conditions. Creatine (Cre) and its true precursors are traced, and blue texts are used to indicate the numbers of carbon or nitrogen atoms in those metabolites. By design, we allowed lactate (Lac) as the desired by-product to meet the redox and energy constraints. RIGHT: Creatine vs. biomass production trade-off plot. The shaded regions from light to dark color are feasible creatine and growth yields subject to 0, 50, and 100% oxygen availability, respectively.The light blue area of the biomass/creatine trade-off plot (Right) shows the solution space where it is possible to produce biomass (for colonization in the gut) as well as creatine. We will aim to operate within this envelope. In the first iteration of metabolic engineering, we will eliminate genes to produce ethanol (adh) and force the co-production of acetate and/or lactate along with creatine to balance the redox. Acetic and lactic acids are known to benefit gut health and promote the natural gut microbiota.We will evaluate the metabolic designs with the engineered EcN strains cultivated in VL media and quantify their physiology by closing a carbon and redox balance. We will feed the results into the model to identify knockouts using algorithms such as OptKnock. We will discriminate between the designs based on creatine yield from substrates and the rate at which creatine is produced in order to meet the physiological threshold of at least 0.093 g creatine/day.Metric for successAchieving 85% of the theoretical yield of creatine from glucose at 3.9 mg/h1MilestoneImplement first iteration of the model-guided designTimelineMonth 0 - Month 7Objective 3: Implement Biocontainment StrategyTo control unwanted proliferation of our engineered probiotic we propose to build two kill switches that can be eventually combined and fine-tuned to reduce the number of engineered cells that escape killing outside the avian intestine.The first kill switch will allow the expression of the essential gene thyA (encoding for thymidylate synthase) only under anaerobic conditions, allowing the host probiotic to survive only in the absence of oxygen (or with an exogenously provided source of thymidine). We will replace the endogenous promoter of thyA with an FNR-regulated promoter, such as the narG promoter, which is only activated in anaerobic conditions. We will calculate the escape frequency of the PnarG-thyA EcN by growing it under anaerobic conditions then switching to aerobic conditions without supplemental thymidine and collecting samples over time to plate for viable counts. We expect that wild-type EcN will continue to exhibit aerobic grow while the PnarG-thyA EcN strain will lose viability. This strategy is widely used to prevent environmental proliferation of engineered probiotics but it is not fool-proof as organisms may survive if there is an exogenous source of thymidine [37]. Therefore, we propose to have a second layer of biocontainment.The second kill switch will be to express a toxin/antitoxin pair where the toxin gene is constitutively expressed but the antitoxin is under the control of the Cl857 repressor, which will repress its expression at temperatures below 37 °C and allow lysis by the toxin. We propose to use the MazEF toxin/antitoxin system, which can be swapped for any number of bacterial toxin/antitoxin pairs if needed. The MazF toxin is an endoribonuclease that cleaves mRNA which is lethal for the cell. The MazE antitoxin forms a stable complex with MazF, preventing it from cleaving mRNA [38]. To create a conditional kill switch we will place the mazF gene under the control of a constitutive promoter and mazE under control of the lambda phage pL promoter, which is controlled by the temperature sensitive repressor CI857. At temperatures below 37 °C (ambient environment) CI857 is a stable repressor [39] and will not allow expression of the MazE antitoxin, causing a lethal state. At temperatures above 37°C CI857 is unstable, allowing MazE expression and cell survival. The temperature of the chicken GI tract is 41 °C, which should allow robust mazE expression. CI857 will also be expressed constitutively in our system. To demonstrate the efficacy of this system we will clone pConst-CI857, pL-mazE and pConst-mazF into the E. coli expression vector pACYC184 and transform it into EcN. We will grow the cells at the permissible temperature (41°C and then shift them to a non-permissible temperature (25 °C) and collect samples over time to plate for viable counts. We expect that after the temperature down-shift we will see a loss of viability in the strain containing our kill switch compared to an empty vector control.We will target the NIH-recommended metric of attaining an escape frequency of less than one in 1x108 when the two kill switches are combined. We will validate each kill switch individually in vitro then combine the two systems and test the synergistic effect.Metric for successAn escape frequency of < 1 x 108 when kill switch 1+2 are combined under lab conditionsMilestoneIncorporation of a biocontainment mechanismTimelineMonth 4 - Month 8