Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production

Abstract

The present disclosure provides a microbial genomic engineering method and system for transforming, screening, and selecting filamentous fungal cells that have altered morphology and/or growth under specific growth conditions. The method and system utilize high-throughput (HTP) methods to produce filamentous fungal production strains with a desired morphological phenotype.

What is claimed: 
     
         1 . 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 engineered  Aspergillus  host strain capable of responding to osmotic stress substantially similar to a parental strain of  Aspergillus  from which the engineered  Aspergillus  strain is generated, wherein the engineered  Aspergillus  strain comprises a heterologous modification of an  Aspergillus niger  ( A. niger ) orthologue of a  Saccharomyces cerevisiae  ( S. cerevisiae ) sln1 gene that produces a reduced amount and/or less active form of the polypeptide encoded by the  A. niger  orthologue of the  S. cerevisiae  sln1 gene in the engineered  Aspergillus  strain as compared to cells of the parental  Aspergillus  strain; 
 (ii) perturbing genomes of each of the host cells to introduce a plurality of different genetic variations, thereby generating a plurality of perturbed host cells; 
 (iii) isolating individual perturbed host cells from the plurality of perturbed host cells produced in step (i) into separate reaction areas in a substrate comprising a plurality of reaction areas; 
 (iv) 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; 
 (v) 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; 
 (vi) 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 
 (vii) screening the plurality of host cells in the genetic design host cell library of step (vi) for the phenotype of step (v), 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 (v); 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)-(vii).   
     
     
         2 . The system of  claim 1 , wherein the heterologous modification comprises replacement of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene of the parental  Aspergillus  strain with a single nucleotide polymorphism (SNP) containing version of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene, wherein the SNP containing version of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene comprises a nucleotide substitution at position 814 of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene of the parental  Aspergillus  strain. 
     
     
         3 . The system of  claim 2 , wherein the SNP containing version of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene comprises a cytosine to thymine substitution at nucleotide position 814 of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene of the parental  Aspergillus  strain. 
     
     
         4 . The system of  claim 2 , wherein the SNP containing version of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene comprises the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         5 . The system of  claim 2 , wherein the  A. niger  orthologue of the  S. cerevisiae  sln1 gene of the parental  Aspergillus  strain comprises the nucleic acid sequence of SEQ ID NO: 76. 
     
     
         6 . The system of  claim 2 , wherein the SNP-containing version of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene encodes a polypeptide comprising a histidine to tyrosine substitution at amino acid position 272 of an  A. niger  orthologue of  S. cerevisiae  SLN1. 
     
     
         7 . The system of  claim 1 , wherein the heterologous modification comprises replacement of the native promoter of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene of the parental  Aspergillus  strain with a promoter that more weakly expresses the  A. niger  orthologue of the  S. cerevisiae  sln1 gene as compared to the native promoter of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene. 
     
     
         8 . The system of  claim 7 , wherein the promoter that more weakly expresses the  A. niger  orthologue of the  S. cerevisiae  sln1 gene as compared to the native promoter of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene is selected from an amyB promoter and a manB promoter. 
     
     
         9 . The system of  claim 7 , wherein the promoter that more weakly expresses the  A. niger  orthologue of the  S. cerevisiae  sln1 gene as compared to the native promoter of the  A. niger  orthologue of the  S. cerevisiae  sln1 gene comprises the nucleic acid sequence of SEQ ID NO: 1 or 2. 
     
     
         10 . The system of  claim 7 , further comprising a heterologous modification of one or more genes selected from a non-SNP containing version of the genes with nucleic acid sequences of SEQ ID NO. 77, 78, 79 and any combination thereof. 
     
     
         11 . The system of  claim 10 , wherein the heterologous modification is selected from replacement of a native promoter of the one or more genes with a promoter that weakly expresses the one or more genes as compared to the native promoter, replacement of the one or more genes with a mutated form of the one or more genes, replacement of the one or more genes with a selectable marker, and a combination thereof. 
     
     
         12 . The system of  claim 11 , wherein the promoter that weakly expresses the one or more genes as compared to the native promoter is selected from an amyB promoter and a manB promoter. 
     
     
         13 . The system of  claim 11 , wherein the promoter that weakly expresses the one or more genes as compared to the native promoter is selected from the promoter of SEQ ID NO: 1 and SEQ ID NO: 2. 
     
     
         14 . The system of  claim 11 , wherein the mutated form of the one or more genes comprises the nucleic acid sequence of SEQ ID NO: 5, 6, or 8. 
     
     
         15 . The system of  claim 1 , wherein the reduced amount and/or less active form of the polypeptide encoded by the heterologously modified  A. niger  orthologue of the  S. cerevisiae  sln1 gene results in a non-mycelium, multi-hyphal tip, pellet phenotype when grown in submerged liquid culture conditions. 
     
     
         16 . 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 engineered  Aspergillus  host strain comprising a heterologous modification of a nikA gene that produces a reduced amount and/or less active form of a polypeptide encoded by the nikA gene in the engineered  Aspergillus  host strain as compared to cells of the parental  Aspergillus  strain, wherein the heterologous modification comprises replacement of the nikA gene of the parental  Aspergillus  strain with a single nucleotide polymorphism (SNP) containing version of the nikA gene, wherein the SNP containing version of the nikA gene comprises a nucleotide substitution at position 814 of the nikA gene of the parental  Aspergillus  strain; 
 (ii) perturbing genomes of each of the host cells to introduce a plurality of different genetic variations, thereby generating a plurality of perturbed host cells; 
 (iii) isolating individual perturbed host cells from the plurality of perturbed host cells produced in step (i) into separate reaction areas in a substrate comprising a plurality of reaction areas; 
 (iv) 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; 
 (v) 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; 
 (vi) 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 
 (vii) screening the plurality of host cells in the genetic design host cell library of step (vi) for the phenotype of step (v), 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 (v); 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)-(vii).   
     
     
         17 . The system of  claim 16 , wherein the SNP containing version of the nikA gene comprises a cytosine to thymine substitution at nucleotide position 814 of the nikA gene of the parental  Aspergillus  strain. 
     
     
         18 . The system of  claim 16 , wherein the SNP containing version of the nikA gene comprises a nucleic acid sequence of SEQ ID NO: 7. 
     
     
         19 . The system of  claim 16 , wherein the nikA gene of the parental  Aspergillus  strain comprises a nucleic acid sequence of SEQ ID NO: 76. 
     
     
         20 . The system of  claim 16 , wherein the SNP-containing version of the nikA gene encodes a polypeptide comprising a histidine to tyrosine substitution at amino acid position 272 of NIKA.