HTP genomic engineering platform

Patent metadata

Field	Value
Publication number	US-11208649-B2
Application number	US-202117308528-A
Country	US
Kind code	B2
Filing date	May 5, 2021
Priority date	Dec 7, 2015
Publication date	Dec 28, 2021
Grant date	Dec 28, 2021

Abstract

The present disclosure provides a HTP microbial genomic engineering platform that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. The HTP genomic engineering platform described herein is microbial strain host agnostic and therefore can be implemented across taxa. Furthermore, the disclosed platform can be implemented to modulate or improve any microbial host parameter of interest.

First claim

What is claimed is:

1. An automated high-throughput genomic engineering system for manufacturing a promoter swap host cell library, comprising:
a) a processor; and
b) a memory operatively coupled to the processor and having instructions stored thereon that, when executed by the processor, cause the system to:
i) manufacture an initial promoter swap host cell library by engineering the genome of each host cell in a plurality of base host cells to comprise one or more promoters from a promoter ladder operably linked to a target gene present in the base host cell to thereby create a plurality of promoter:gene target combinations,
wherein said promoter ladder comprises a plurality of promoters exhibiting different expression profiles in the base host cell, and
wherein the initial promoter swap host cell library comprises at least two different promoters from the promoter ladder; and

c) automated liquid and particle handling robotics in communication with the processor, said robotics performing high-throughput manipulation of liquid or particles added to, or removed from, cultures comprising the host cells, upon receipt of instructions sent by the processor during the manufacture of the initial promoter swap host cell library.

2. The system of claim 1 , wherein the memory operatively coupled to the processor has instructions stored thereon that, when executed by the processor, cause the system to:
ii) identify, based on screening and selecting individual host cells of the initial promoter swap host cell library, a promoter:gene target combination that confers a desired phenotype.

3. The system of claim 1 , wherein the memory operatively coupled to the processor has instructions stored thereon that, when executed by the processor, cause the system to:
ii) manufacture a subsequent promoter swap host cell library by engineering the genome of each host cell in a plurality of host cells to comprise at least two promoter:gene target combinations from at least two individual host cells from the initial promoter swap host cell library.

4. The system of claim 1 , wherein the initial promoter swap host cell library comprises at least one host cell with a 10% level increase in a phenotypic variable compared to that of the base host cell.

5. The system of claim 4 , wherein the phenotypic variable is selected from the group consisting of: increased volumetric productivity of a product of interest, increased specific productivity of a product of interest, increased yield of a product of interest, increased titer of a product of interest, and combinations thereof.

6. The system of claim 4 , wherein the phenotypic variable is increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, protein, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

7. The system of claim 1 , wherein the initial promoter swap host cell library comprises at least one host cell with a one-fold level increase in a measured phenotypic variable compared to that of the base host cell.

8. The system of claim 7 , wherein the phenotypic variable is selected from the group consisting of: increased volumetric productivity of a product of interest, increased specific productivity of a product of interest, increased yield of a product of interest, increased titer of a product of interest, and combinations thereof.

9. The system of claim 7 , wherein the phenotypic variable is increased or more efficient production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, protein, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

10. The system of claim 3 , wherein the subsequent promoter swap host cell library comprises at least one host cell with a 10% level increase in a phenotypic variable compared to that of a host cell in the initial promoter swap host cell library.

11. The system of claim 10 , wherein the phenotypic variable is selected from the group consisting of: increased volumetric productivity of a product of interest, increased specific productivity of a product of interest, increased yield of a product of interest, increased titer of a product of interest, and combinations thereof.

12. The system of claim 10 , wherein the phenotypic variable is production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, protein, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

13. The system of claim 3 , wherein the subsequent promoter swap host cell library comprises at least one host cell with a one-fold level increase in a measured phenotypic variable compared to that of a host cell in the initial promoter swap host cell library.

14. The system of claim 13 , wherein the phenotypic variable is selected from the group consisting of: increased volumetric productivity of a product of interest, increased specific productivity of a product of interest, increased yield of a product of interest, increased titer of a product of interest, and combinations thereof.

15. The system of claim 13 , wherein the phenotypic variable is production of a product of interest, said product of interest selected from the group consisting of: a small molecule, enzyme, protein, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

16. The system of claim 3 , wherein the memory operatively coupled to the processor has instructions stored thereon that, when executed by the processor, cause the system to:
ii) identify, based on screening and selecting individual host cells of the subsequent promoter swap host cell library, a promoter:gene target combination that confers a desired phenotype.

17. The system of claim 1 , wherein at least one of the host cells in the initial promoter swap host cell library comprises a genetic variation selected from the group consisting of: a single nucleotide polymorphism, DNA insertion, or DNA deletion not present in the base host cell.

18. The system of claim 3 , wherein at least one of the host cells in the subsequent host cell library comprises a genetic variation selected from the group consisting of: a single nucleotide polymorphism, DNA insertion, or DNA deletion not present in a host cell in the initial promoter swap host cell library.

19. The system of claim 1 , wherein the initial promoter swap host cell library comprises at least three different promoters from the promoter ladder.

20. The system of claim 1 , comprising: one or more robotic arms in communication with the processor.

21. The system of claim 1 , comprising: integrated thermal cyclers in communication with the processor.

22. The system of claim 1 , comprising robotics in communication with the processor, said robotics comprising: plate handlers for the positioning of microplates, plate sealers, plate piercers, automated lid handlers adapted to remove and replace

Assignees

• Zymergen Inc

Inventors

• Serber Zachariah
• Dean Erik Jedediah
• Manchester Shawn
• Gora Katherine
• Flashman Michael
• Shellman Erin
• Kimball Aaron
• Szyjka Shawn
• Frewen Barbara
• Treynor Thomas
• Bruno Kenneth S

Classifications

• G01N35/00871

  Communications between instruments or with remote terminals · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• B01L2300/18

  Means for temperature control · CPC title

• G16B35/10

  Design of libraries · 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