Compositions and methods for screening microorganisms for robust dynamic metabolic control

A method of screening a plurality of genetically modified E. coli for selection of a production genetically modified E. coli, the method comprising:

• providing the plurality of distinct genetically modified E. coli, each genetically modified E. coli therein comprising:
• i. a production pathway comprising at least one production enzyme for biosynthesis of a product selected from the group:
• an amino acid, acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, 1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutryate, 3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fused alcohols, 1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, maleic acid, or poly-hydroxybutyrate;
• and
• ii. one or more synthetic metabolic valves for reducing or eliminating flux through multiple metabolic pathways within the genetically modified E. coli when the synthetic metabolic valves are induced, the one or more synthetic metabolic valves comprising:
• a) at least one silencing synthetic metabolic valve that silences gene expression of a gene selected from the group of:
• fabI, gltA, lpd, zwf, or udhA and at least one silencing synthetic metabolic valve is characterized by CRISPR interference of gene expression of a gene that is a fabI, gltA, lpd, zwf, or udhA gene and expression of a CASCADE plasmid comprising an array of guide RNA genes, or
• b) at least one proteolytic synthetic metabolic valve that controls proteolysis of a proteolyzable enzyme selected from the group of:
• fabI, gltA, lpd, zwf, or udhA;
• wherein each of the plurality of genetically modified E. coli is distinct therein by one or more of the production enzyme, the silenceable enzyme, or the proteolyzable enzyme;
• independently growing each of the plurality of genetically modified E. coli s in culture media in a growth phase;
• transitioning to a productive stationary phase, the transition comprising:
• depletion of a limiting nutrient;
• inducing the one or more synthetic metabolic valves;
• and
• activation of the production pathway;
• and
• producing the product;
• measuring the level of product produced in the productive phase for each of the plurality of genetically modified microorganisms after a period of time, and
• selecting a genetically modified E. coli from the plurality of genetically modified E. coli based on an increase in the level of product produced as compared to other microorganism within the plurality.