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

Patent metadata

Field	Value
Publication number	US-2016017317-A1
Application number	US-201414775025-A
Country	US
Kind code	A1
Filing date	Feb 26, 2014
Priority date	Mar 14, 2013
Publication date	Jan 21, 2016
Grant date	—

Abstract

The present invention relates to genetically modified bacteria and methods of optimizing genetically modified bacteria for the production of a metabolite.

First claim

What is claimed is:

1 . 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 has been genetically modified to include exogenous DNA encoding for an antidote to the toxin, wherein the population of microbes has been genetically modified to include exogenous DNA encoding a sensor biomolecule which when expressed regulates expression of the antidote by the microbes through a cognate nucleic acid sequence located 5′ to the antidote gene, wherein the population of microbes has been genetically modified to include exogenous DNA encoding genes to produce a metabolite binding partner of the sensor, which when produced binds to the sensor to induce expression of the antidote in a manner dependent on the concentration of the produced metabolite, and selecting a subset of microbes that produce sufficient metabolite to prevent microbe death.

2 . The method of claim 1 further comprising
genetically modifying the subset of microbes to alter genes that affect production of the metabolite directly or indirectly,
subjecting the subset of microbes to a subsequent environment of the toxin having a concentration greater than the previous environment, and
selecting a subsequent subset of microbes the produce sufficient metabolite to prevent microbe death.

3 . The method of claim 2 further comprising
repeating in sequence: (1) genetically modifying the subsequent subset of microbes by altering genes that affect the production of the metabolite, (2) subjecting the genetically altered microbes to a subsequent environment of a toxin having a concentration greater than a previous environment, and (3) selecting a further subsequent subset of microbes that produce sufficient metabolite to prevent microbe death, said repeating step resulting in optimized metabolite producing microbes.

4 . The method of claim 1 wherein the sensor biomolecule is a transcription factor, riboswitch, two-component signaling protein or a nuclear hormone receptor.

5 . The method of claim 1 wherein binding of the metabolite to the sensor regulates gene expression to induce production of the antidote in a manner dependent on the concentration of the expressed metabolite.

6 . The method of claim 5 wherein a positive selection marker is used to select the subset of microbes that produce sufficient metabolite to prevent microbe death.

7 . The method of claim 5 wherein the sensor regulates the expression of two or more antidotes independently and two or more toxins are used to select the subset of microbes that produce sufficient metabolite to prevent microbe death.

8 . The method of claim 1 wherein binding of the metabolite to the sensor regulates gene expression to induce production of the antidote in a manner dependent on the concentration of the expressed metabolite.

9 . The method of claim 8 wherein a negative selection marker is used to eliminate false positives that detoxify the microbe despite not producing sufficient metabolite.

10 . The method of claim 1 wherein the population of microbes have been genetically modified to include two or more redundant copies of the exogenous DNA encoding the sensor in order to reduce false positives.

11 . The method of claim 1 wherein the sensor also regulates its own expression through a cognate nucleic acid sequence located 5′ to the DNA sequence encoding the sensor in order to reduce false positives.

12 . The method of claim 1 wherein the degradation rate of the antidote is increased by encoding a degradation signal within the antidote in order to reduce false positives.

13 . The method of claim 2 wherein the step of genetically modifying the subset of microbes to alter genes that produce the metabolite includes multiplexed automated genome engineering.

14 . The method of claim 2 wherein the step of genetically modifying the subset of microbes includes plasmid library of pathway genes.

15 . The method of claim 2 wherein the step of genetically modifying the subset of microbes includes plasmid library of genomic fragments of any organism.

16 . The method of claim 2 wherein the step of genetically modifying the subset of microbes includes plasmid library of metagenomic sequences.

17 . The method of claim 13 wherein the multiplexed automated genome engineering includes reducing spontaneous background mutants.

18 . The method of claim 13 wherein the multiplexed automated genome engineering includes reducing spontaneous background mutants by pretreatment with a negative selector.

19 . The method of claim 1 wherein concentration of the metabolite exposed to the sensor is attenuated.

20 . The method of claim 19 wherein the concentration of the metabolite exposed to the sensor is attenuated by expressing one or more proteins to export the metabolite outside of the cell.

21 . The method of claim 19 wherein the concentration of the metabolite exposed to the sensor is attenuated by expressing one or more enzymes that convert the metabolite to another metabolite having less interaction with the sensor.

22 . The method of claim 19 wherein the concentration of the metabolite exposed to the sensor is attenuated by expressing a biomolecule that binds to the metabolite and reduces its interaction with the sensor.

23 . 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.

24 . 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 trans

Assignees

• Harvard College

Inventors

• Church George M
• Raman Srivatsan
• Taylor Noah D
• Rogers Jameson

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/1058 Primary

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12P5/02

  acyclic {(C12P5/007 takes precedence)} · CPC title

• C12N15/1079

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · CPC title

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title