Methods For Selecting Microbes From A Diverse Genetically Modified Library to Detect and Optimize the Production of Metabolites

A method of selecting a subset of microbes for the production of a metabolite comprising

• placing a population of microbes in an environment of a toxin,
• wherein the population of microbes have been genetically modified to include exogenous DNA encoding for an antidote to the toxin,
• wherein the population of microbes have been genetically modified to include exogenous DNA encoding a sensor which when expressed regulates production of the antidote by the microbes,
• wherein the population of microbes have been genetically modified to include exogenous DNA encoding genes that produce a metabolite binding partner of the DNA binding molecule, which when expressed binds to the sensor to induce production of the antidote in a manner dependent on the concentration of the expressed metabolite,
• repeatedly genetically modifying the microbes to alter genes that produce the metabolite, subjecting the microbes to negative selection,
• transforming surviving microbes with a plasmid comprising genes completing the metabolic pathway to produce the metabolite,
• selecting microbes including the plasmid, and
• selecting a subset of microbes that produce sufficient metabolite to prevent microbe death.

A method of eliminating false positives and selecting only high producing microbes by a toggled selection scheme

• wherein a positively-and-negatively selectable marker is chosen as the antidote (example tolC gene)
• wherein the last or last few genes of the pathway to be optimized are cloned on a plasmid, called pathway completion plasmid, such that the final product metabolite cannot be made in the absence of the plasmid, and thus not triggering the sensor to make antidote
• wherein diversity is generated around the remaining targets by MAGE or other methods in a strain containing sensor and antidote but the pathway completion plasmid
• wherein the diverse population of microbes is subjected to negative selection to eliminate mutants the constitutively express the antidote thereby eliminating the false positives wherein the pathway completion plasmid is now transformed into microbes that survive the negative selection which now completes the pathway to produce the final product metabolite
• wherein the selection mode is now toggled to positive selection enabling enrichment of high producing microbes.