HTP genomic engineering platform for improving fungal strains

Patent metadata

Field	Value
Publication number	US-11242524-B2
Application number	US-202117207376-A
Country	US
Kind code	B2
Filing date	Mar 19, 2021
Priority date	Jun 6, 2017
Publication date	Feb 8, 2022
Grant date	Feb 8, 2022

Abstract

A HTP genomic engineering platform for improving filamentous fungal cells that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols is provided. 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. Methods for isolating clonal populations derived from individual fungal spores are also provided.

First claim

What is claimed is:

1. A method for isolating clonal populations from single fungal spores, the method comprising:
(a) dispensing discrete volumes of a liquid suspension comprising a plurality of fungal spores from a fungal strain to individual reaction areas in a substrate comprising a plurality of reaction areas, wherein each reaction area in the plurality of reaction areas comprises growth media, wherein the dispensing results in a probability that at least 90% of the individual reaction areas contain either no viable spores or no more than a single viable fungal spore from the plurality of fungal spores;
(b) culturing the dispensed single viable fungal spores in the reaction areas comprising growth media; and
(c) selecting clonal populations growing in the reaction areas, thereby isolating clonal populations from single fungal spores.

2. The method of claim 1 , further comprising screening the discrete volumes for the presence or absence of a single fungal spore in the discrete volumes, wherein only the discrete volumes containing a single fungal spore are selected for step (a).

3. The method of claim 2 , wherein the dispensing results in a probability that at least 90%, at least 95%, at least 99% or all of the individual reaction areas contain no more than a single viable fungal spore from the plurality of fungal spores.

4. The method of claim 2 , wherein the screening the discrete volumes comprises optically distinguishing the presence or absence of a single fungal spore in the discrete volumes.

5. The method of claim 4 , wherein the screening is performed using a microfluidic device capable of optically distinguishing the presence or absence of a single fungal spore in the discrete volumes.

6. The method of claim 1 , wherein the liquid suspension comprising a plurality of fungal spores from a fungal strain is a limiting dilution, wherein the dispensing of the limiting dilution results in a probability that the discrete volume of the dilution dispensed to each reaction area contains either one or no viable spore follows a Poisson Distribution, whereby greater than 90% of the reaction areas in the plurality of reaction areas contain no viable spores and greater than 90% of reaction areas that contain one or more viable spores contain only a single viable spore.

7. The method of claim 1 , wherein the reaction areas are present in a microtiter plate, wherein the microtiter plate contains 96 wells, 384 wells or 1536 wells.

8. The method of claim 1 , wherein the fungal strain is a filamentous fungal strain.

9. The method of claim 8 , wherein the filamentous fungal strain is selected from Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthora thermophila ), Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella species, teleomorphs, anamorphs, and synonyms and taxonomic equivalents thereof.

10. The method of claim 9 , wherein the filamentous fungal strain is Aspergillus niger or teleomorphs or anamorphs thereof.

11. The method of claim 10 , wherein the filamentous fungal strain possesses a non-mycelium, pellet morphology.

12. The method of claim 11 , wherein the filamentous fungal strain expresses a mutant form of an A. niger ortholog of the S. cerevisiae sln1 gene.

13. The method of claim 12 , wherein a nucleic sequence of the mutant form of the A. niger ortholog of the S. cerevisiae sln1 gene is SEQ ID NO: 13.

14. The method of claim 11 , wherein the mutant form of the A. niger ortholog of the S. cerevisiae sln1 gene is operably linked to a promoter sequence selected from SEQ ID NO: 1 and 2.

15. The method of claim 1 , wherein steps (a)-(c) are automated.

16. The method of claim 1 , wherein steps (a)-(c) are performed using automated robotics.

17. The method of claim 16 , wherein the automated robotics are in communication with one or more processors in a system, wherein the one or more processors are each in communication with one or more memories.

18. The method of claim 17 , wherein the one or more processors comprise instructions stored thereon that when executed by the one or more processors cause the system to perform steps (a)-(c).

Assignees

• Zymergen Inc

Inventors

• Sunspiral Vytas
• Fredlund Jennifer
• Abdulla Hassan
• Boccazzi Paolo
• Poust Sean
• Cleto Sara Da Luz Areosa
• Chaikind Brian
• Vaughan Dylan
• Bruno Kenneth S
• Westfall Patrick
• Szewczyk Edyta
• Rothschild-Mancinelli Kyle
• Fong Iii Arthur Muir

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · 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