Microbial strain improvement by a HTP genomic engineering platform

• US 9,988,624 B2
• Filed: 12/30/2016
• Issued: 06/05/2018
• Est. Priority Date: 12/07/2015
• Status: Active Grant

• Current Assignee: Zymergen, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Zymergen, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Serber, Zach, Dean, Erik Jedediah, Manchester, Shawn, Gora, Katherine, Flashman, Michael, Shellman, Erin, Kimball, Aaron, Szyjka, Shawn, Frewen, Barbara, Treynor, Thomas, Bruno, Kenneth S.
• Primary Examiner(s): Ketter, James S
• Application Number:
  US15/396,230
• Publication Number:
  US 20170159045A1
• Time in Patent Office: 522 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 3/0275 Interchangeable or disposable dispensing tips
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12M 1/42 Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12M 3/00 Tissue, human, animal or plant cell, or virus culture apparatus
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 41/12 of temperature controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00
• CPC Class Codes: C12N 15/00 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 Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/77 : for Corynebacterium; for Brevibacterium
• CPC Class Codes: C12N 15/80 : for fungi
• CPC Class Codes: G01N 2035/1027 : General features of the devices
• CPC Class Codes: G01N 2035/103 : using disposable tips
• CPC Class Codes: G01N 2035/1032 : Dilution or aliquotting
• CPC Class Codes: G01N 2035/1048 : using the transfer device for another function
• CPC Class Codes: G01N 2035/1053 : for separating part of the liquid, e.g. filters, extraction phase
• CPC Class Codes: G01N 2035/1058 : for mixing
• CPC Class Codes: G01N 35/00871 : Communications between instruments or with remote terminals
• CPC Class Codes: G01N 35/10 : Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices G01N35/0099 takes precedence
• CPC Class Codes: G01N 35/1002 : Reagent dispensers
• CPC Class Codes: G16B 20/00 : ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/50 : Mutagenesis
• CPC Class Codes: G16B 35/10 : Design of libraries
• CPC Class Codes: G16B 35/20 : Screening of libraries
• CPC Class Codes: G16B 40/00 : ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/20 : Supervised data analysis
• CPC Class Codes: G16B 40/30 : Unsupervised data analysis
• CPC Class Codes: G16B 5/00 : ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks

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.

Claims

1. A method for rehabilitating and improving the phenotypic performance of a production microbial strain, comprising the steps of:

a. providing a parental lineage microbial strain and a production microbial strain derived therefrom, wherein the production microbial strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, not present in the parental lineage microbial strain;

b. perturbing the genome of either the parental lineage microbial strain, or the production microbial strain, to create an initial library of microbial strains, wherein each strain in the initial library comprises a genetic variation that is unique from amongst the plurality of identified genetic variations between the parental lineage microbial strain and the production microbial strain;

c. screening and selecting individual strains of the initial library for phenotypic performance improvements over a reference microbial strain, thereby identifying genetic variations that confer phenotypic performance improvements;

d. providing a subsequent plurality of microbes that each comprise a combination of genetic variations from the genetic variations present in at least two individual microbial strains screened in the preceding step, to thereby create a subsequent library of microbial strains;

e. screening and selecting individual strains of the subsequent library for phenotypic performance improvements over the reference microbial strain, thereby identifying combinations of genetic variation that confer additional phenotypic performance improvements;

f. repeating steps d)-e) one or more times, in a linear or non-linear fashion, until a microbial strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production microbial strain, wherein each subsequent iteration creates a new library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual microbial strains of a preceding library; and

wherein the genome of at least one microbial strain of either;

the initial library or a subsequent library, comprises one or more promoters from a promoter ladder operably linked to an endogenous target gene.

2. The method according to claim 1, wherein the initial library is a full combinatorial library comprising all of the identified genetic variations between the parental lineage microbial strain and the production microbial strain.

3. The method according to claim 1, wherein the initial library is a subset of a full combinatorial library comprising a subset of the identified genetic variations between the parental lineage microbial strain and the production microbial strain.

4. The method according to claim 1, wherein the subsequent library is a full combinatorial library of the initial library.

5. The method according to claim 1, wherein the subsequent library is a subset of a full combinatorial library of the initial library.

6. The method according to claim 1, wherein the subsequent library is a full combinatorial library of a preceding library.

7. The method according to claim 1, wherein the subsequent library is a subset of a full combinatorial library of a preceding library.

8. The method according to claim 1, wherein the genome of the parental lineage microbial strain is perturbed to add one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are found in the production microbial strain.

9. The method according to claim 1, wherein the genome of the production microbial strain is perturbed to remove one or more of the identified single nucleotide polymorphisms, DNA insertions, or DNA deletions, which are not found in the parental lineage microbial strain.

10. The method according to claim 1, wherein perturbing the genome comprises utilizing at least one method selected from the group consisting of:

random mutagenesis, targeted sequence insertions, targeted sequence deletions, targeted sequence replacements, and combinations thereof.

11. The method according to claim 1, wherein steps d)-e) are repeated until the phenotypic performance of a microbial strain of a subsequent library exhibits at least a 10% increase in a measured phenotypic variable compared to the phenotypic performance of the production microbial strain.

12. The method according to claim 1, wherein steps d)-e) are repeated until the phenotypic performance of a microbial strain of a subsequent library exhibits at least a one-fold increase in a measured phenotypic variable compared to the phenotypic performance of the production microbial strain.

13. The method according to claim 1, wherein the improved phenotypic performance of step f) is selected from the group consisting of:

volumetric productivity of a product of interest, specific productivity of a product of interest, yield of a product of interest, titer of a product of interest, and combinations thereof.

14. The method according to claim 1, wherein the improved phenotypic performance of step f) 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, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, primary extracellular metabolite, secondary extracellular metabolite, intracellular component molecule, and combinations thereof.

15. The method according to claim 1, wherein said production microbial strain is a prokaryote.

16. The method according to claim 1, wherein said production microbial strain is from a genus selected from the group consisting of:

Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.

17. The method according to claim 1, wherein said production microbial strain is Corynebacterium glutamicum.

18. The method according to claim 1, wherein said production microbial strain is Corynebacterium glutamicum, and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine.

19. The method according to claim 1, wherein said production microbial strain is a eukaryote.

20. The method according to claim 1, wherein said production microbial strain is from a genus selected from the group consisting of:

Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium, and Volvariella.

21. The method according to claim 1, wherein said production microbial strain is Aspergillus niger.

22. The method according to claim 1, wherein said production microbial strain is Aspergillus niger, and wherein the improved phenotypic performance of step f) is increased or more efficient production of citric acid.

23. The method according to claim 1, wherein said production microbial strain is Corynebacterium glutamicum

and the one or more promoters are selected from the group of promoters of SEQ ID NOs:

1-8.

24. The method according to claim 1, wherein said production microbial strain is Corynebacterium glutamicum, and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine

and the one or more promoters are selected from the group of promoters of SEQ ID NOs:

1-8.

25. The method according to claim 1, wherein said production microbial strain is Corynebacterium glutamicum, and wherein the improved phenotypic performance of step f) is increased or more efficient production of lysine

and the endogenous target gene is selected from the group consisting of:

PTS, zwf, pgi, tkt, fbp, ppc, pyc, aspB, ask, asd, dapA, dapB, dapD, cg0931, dapE, dapF, ddh, lysA, and lysE, and the one or more promoters are selected from the group of promoters of SEQ ID NOs;

1-8.

26. A system for rehabilitating and improving the phenotypic performance of a production microbial strain, the system comprising:

one or more processors; and

one or more memories operatively coupled to at least one of the one or more processors and having instructions stored thereon that, when executed by at least one of the one or more processors, cause the system to;

a. provide a parental lineage microbial strain and a production microbial strain derived therefrom, wherein the production microbial strain comprises a plurality of identified genetic variations selected from single nucleotide polymorphisms, DNA insertions, and DNA deletions, not present in the parental lineage microbial strain;

b. perturb the genome of either the parental lineage microbial strain, or the production microbial strain, to create an initial library of microbial strains, wherein each strain in the initial library comprises a genetic variation that is unique from amongst the plurality of identified genetic variations between the parental lineage microbial strain and the production microbial strain;

c. screen and select individual strains of the initial library for phenotypic performance improvements over a reference microbial strain, thereby identifying genetic variations that confer phenotypic performance improvements;

d. provide a subsequent plurality of microbes that each comprise a combination of genetic variations from the genetic variations present in at least two individual microbial strains screened in the preceding step, to thereby create a subsequent library of microbial strains;

e. screen and select individual strains of the subsequent library for phenotypic performance improvements over the reference microbial strain, thereby identifying combinations of genetic variation that confer additional phenotypic performance improvements; and

f. repeat steps d)-e) one or more times, in a linear or non-linear fashion, until a microbial strain exhibits a desired level of improved phenotypic performance compared to the phenotypic performance of the production microbial strain, wherein each subsequent iteration creates a new library of microbial strains, where each strain in the new library comprises genetic variations that are a combination of genetic variations selected from amongst at least two individual microbial strains of a preceding library.