Htp genomic engineering platform for improving fungal strains

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.

What is claimed: 
     
         1 . An automated high-throughput system for isolating clonal populations from single fungal host cells, the system comprising:
 (a) one or more processors; and   (b) 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:
 (i) dispense discrete volumes of a liquid suspension comprising a plurality of fungal host cells 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 fungal host cells or no more than a single viable fungal host cell from the plurality of fungal host cells; 
 (ii) culture the dispensed single viable fungal host cells in the reaction areas comprising growth media; 
 (ii) select clonal populations growing in the reaction areas, thereby isolating clonal populations from single fungal host cells; and 
   (c) automated liquid and particle handling robotics in communication with at least one of the one or more processors, wherein the robotics, upon receipt of instructions sent by the at least one of the one or more processors, perform high-throughput manipulation of liquids or particles during the isolation of the clonal populations.   
     
     
         2 . The system of  claim 2 , further comprising screening the discrete volumes for the presence or absence of a single fungal host cells in the discrete volumes, wherein only the discrete volumes containing a single fungal host cell are selected for step (i). 
     
     
         3 . The system of  claim 3 , 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 host cell from the plurality of fungal host cells. 
     
     
         4 . The system of  claim 3 , wherein the screening the discrete volumes comprises optically distinguishing the presence or absence of a single fungal host cell in the discrete volumes. 
     
     
         5 . The system of  claim 4 , wherein the screening is performed using a microfluidic device that is part of the system and is capable of optically distinguishing the presence or absence of a single fungal host cell in the discrete volumes. 
     
     
         6 . The system of  claim 5 , wherein microfluidic device is a single-cell microfluidic dispenser combined with optics. 
     
     
         7 . The system of  claim 6 , wherein the single-cell microfluidic dispenser combined with optics uses dielectrophoretic forces controlled with light to effectuate the movement of individual fungal host cells into the individual reaction areas. 
     
     
         8 . The system of  claim 7 , wherein the dielectrophoretic forces controlled with light effectuate the movement of individual fungal host cells into individual reaction areas on a chip 
     
     
         9 . The system of  claim 1 , wherein the liquid suspension comprising the plurality of fungal host cells 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 fungal host cell follows a Poisson Distribution, whereby greater than 90% of the reaction areas in the plurality of reaction areas contain no viable fungal host cells and greater than 90% of reaction areas that contain one or more viable fungal host cells contain only a single viable fungal host cell. 
     
     
         10 . The system 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. 
     
     
         11 . The system of  claim 1 , wherein the fungal host cell is a filamentous fungal host cell. 
     
     
         12 . The system of  claim 11 , wherein the filamentous fungal host cell 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. 
     
     
         13 . The system of  claim 12 , wherein the filamentous fungal host cell is  Aspergillus niger  or teleomorphs or anamorphs thereof. 
     
     
         14 . The system of  claim 13 , wherein the filamentous fungal host cell expresses a mutant form of an  A. niger  ortholog of the  S. cerevisiae  sln1 gene. 
     
     
         15 . The system of  claim 14 , wherein a nucleic sequence of the mutant form of the  A. niger  ortholog of the  S. cerevisiae  sln1 gene is SEQ ID NO: 13. 
     
     
         16 . The system of  claim 14 , wherein the mutant form of the  A. niger  ortholog of the  S. cerevisiae  sln1 gene is operably linked a promoter sequence from an  A. niger  amyB gene or an  A. niger  manB gene. 
     
     
         17 . The system of  claim 14 , 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. 
     
     
         18 . The system of  claim 1 , further comprising perturbing the genome of the fungal host cell to introduce one or more genetic variations prior to step (i). 
     
     
         19 . The system of  claim 18 , wherein the fungal host cell is either:
 (a) a parental lineage perturbed to comprise genetic variations that exist in production strains produced from the parental lineage strain, but that are not present in the parental lineage strain before the perturbing step; or (b) a production strain perturbed to comprise genetic variations that exist in a parental lineage strain from which the production strain is produced, but not present in the production strain before the perturbing step.   
     
     
         20 . The system of  claim 1 , wherein the plurality of fungal host cells are induced to sporulate prior to step (i). 
     
     
         21 . An automated high-throughput system for screening a host cell library for a phenotype, the system comprising:
 (a) one or more processors;   (b) 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:
 (i) provide an initial genetic design host cell library containing a plurality of different host cells, each host cell having the same genomic strain background, except for an artificially introduced genetic variation; 
 (ii) dispense individual host cells from the initial genetic design host cell library of step (i) into separate reaction areas in a substrate comprising a plurality of reaction areas, wherein the dispensing results in a probability that at least 90% of the separate reaction areas contain either no host cells or no more than a single isolated individual host cell from the plurality of different host cells; 
 (iii) culturing the isolated individual host cells in the separate reaction areas, thereby generating a plurality of isolated clonal host cell populations from the isolated individual host cells; 
 (iv) screening the plurality of isolated clonal host cell populations for the phenotype, wherein at least one of the one or more memories operatively coupled to the one or more processors has instructions stored thereon that, when executed by at least one of the one or more processors, cause the system to identify the phenotype; 
 (v) providing a subsequent plurality of host cells that each comprise a unique combination of genetic variations, said genetic variations selected from the artificially introduced genetic variations present in at least two individual host cell populations screened in a preceding step, to thereby create a subsequent genetic design host cell library; and 
 (vi) screening the plurality of host cells in the genetic design host cell library of step (v) for the phenotype of step (iv), wherein at least one of the one or more memories operatively coupled to the processor has instructions stored thereon that, when executed by at least one of the one or more processors, cause the system to identify the phenotype of step (iv); and 
   (c) automated liquid and particle handling robotics in communication with at least one of the one or more processors, wherein the robotics, upon receipt of instructions sent by the at least one of the one or more processors, perform high-throughput manipulation of liquids or particles during steps (i)-(vi).   
     
     
         22 . The system of  claim 21 , wherein the host cells of the initial genetic design host cell library of step (i) are either:
 (a) parental lineage host cells artificially engineered to comprise genetic variations that exist in production host cells produced from the parental lineage host cells, but that are not present in the parental lineage host cells before they are engineered; or   (b) production host cells artificially engineered to comprise genetic variations that exist in parental lineage host cells from which the production host cells are produced, but not present in the production host cells before they are engineered.   
     
     
         23 . The system of  claim 21 , wherein the artificially introduced genetic variation is from a library selected from the group consisting of a promoter swap microbial strain library, a SNP swap microbial strain library, a start/stop codon microbial strain library, an optimized sequence microbial strain library, a terminator swap microbial strain library, and any combination thereof. 
     
     
         24 . The system of  claim 21 , wherein the memory operatively coupled to at least one of the one or more processors has instructions stored thereon that, when executed by the processor, cause the system to:
 (vii) repeat steps (iv)-(vi) one or more times, in a linear or non-linear fashion, until a host cell exhibits a desired improvement in the phenotype of step (iv), wherein each subsequent iteration creates a new genetic design host cell library comprising individual host cells harboring genetic variations that are a combination of genetic variation selected from amongst at least two individual host cells of a preceding genetic design host cell library.   
     
     
         25 . The system of  claim 21 , wherein the dispensing comprises optically distinguishing the presence or absence of single isolated individual host cells in the discrete volumes using a microfluidic device that is part of the system and is capable of optically distinguishing the presence or absence of single isolated individual host cell in the discrete volumes. 
     
     
         26 . The system of  claim 25 , wherein microfluidic device is a single-cell microfluidic dispenser combined with optics. 
     
     
         27 . The system of  claim 26 , wherein the single-cell microfluidic dispenser combined with optics uses dielectrophoretic forces controlled with light to effectuate the movement of individual host cells into the individual reaction areas. 
     
     
         28 . The system of  claim 21 , wherein the host cell is a filamentous fungal cell. 
     
     
         29 . The system of  claim 28 , wherein the filamentous fungal cell is  Aspergillus niger  or teleomorphs or anamorphs thereof. 
     
     
         30 . The system of  claim 28 , wherein the plurality of different filamentous fungal host cells are induced to sporulate prior to step (ii).