# US 11,299,741 B2 · Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,741 B2 - Document ID: US11299741B2 - Status: Granted - Kind: B2 - Application number: 17/245,928 - Filed: 2021-04-30 - Published: 2021-08-19 - Granted: 2022-04-12 - Earliest claimed: 2018-06-06 - Assignees: Zymergen Inc. - Applicants: Zymergen Inc. - Inventors: Kenneth S. Bruno; Sachin Jain; Brandon Pfannenstiel; Edyta Szewczyk - Web page: https://patentgrep.com/patent/US11299741B2 ## CPC - C12N 15/80: for fungi - C07K 14/38: from Aspergillus - C12N 1/185: Saccharomyces isolates - C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)} - C12N 15/905: {in yeast} - C12R 2001/865: Saccharomyces cerevisiae - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## 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. ## Claims ### Claim 1 An engineered Aspergillus strain capable of responding to osmotic stress substantial 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. ### Claim 2 (depends on 1) The engineered Aspergillus strain 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 & 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. ### Claim 3 (depends on 2) The engineered Aspergillus strain 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. ### Claim 4 (depends on 2) The engineered Aspergillus strain 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. ### Claim 5 (depends on 2) The engineered Aspergillus strain 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. ### Claim 6 (depends on 2) The engineered Aspergillus strain 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 SLN. ### Claim 7 (depends on 1) The engineered Aspergillus strain of claim 1, wherein the engineered Aspergillus strain is capable of producing an increased level of citric add as compared to the parental strain. ### Claim 8 (depends on 1) The engineered Aspergillus strain of claim 1, wherein the engineered Aspergillus strain is capable of producing at least about 33% more citric acid as compared to the parental Aspergillus strain. ### Claim 9 (depends on 1) The engineered Aspergillus strain 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. ### Claim 10 (depends on 9) The engineered Aspergillus strain of claim 9, 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. ### Claim 11 (depends on 9) The engineered Aspergillus strain of claim 9, wherein the promoter that more weakly expresses the A. niger orthologue of the S. cererisiae sln1 gene as compared to the native promoter of the A. niger orthologue of the S. cerevisiae sln1 gene has the nucleic acid sequence of SEQ ID NO: 1 or 2. ### Claim 12 (depends on 9) The engineered Aspergillus strain of claim 9, 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. ### Claim 13 (depends on 12) The engineered Aspergillus strain of claim 12, 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. ### Claim 14 (depends on 13) The engineered Aspergillus strain of claim 13, 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. ### Claim 15 (depends on 13) The engineered Aspergillus strain of claim 13, 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. ### Claim 16 (depends on 13) The engineered Aspergillus strain of claim 13, wherein the mutated form of the one or more genes has a nucleic acid sequence selected from the nucleic acid sequence of SEQ ID NO: 5, 6, and 8. ### Claim 17 (depends on 1) The engineered Aspergillus strain 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. ### Claim 18 (depends on 1) A method for generating the engineered strain of Aspergillus of claim 1, the method comprising: transforming cells of a parental strain of Aspergillus with a nucleic acid construct comprising a heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene as compared to the A. niger orthologue of the S. cerevisiae sln1 gene of the parental strain flanked by sequence complementary to a locus for the A. niger orthologue of the S. cerevisiae sln1 gene in the genome of the parental Aspergillus strain that facilitates integration of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene into the genomic locus of the A. niger orthologue of the S. cerevisiae sln1 gene, wherein integration of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene in the transformed cells results in production of a reduced amount and/or less active form of functional S. cerevisiae sln1 orthologue of the S. cerevisiae SLN1 as compared to cells of the parental Aspergillus strain which retains a substantially similar osmotic response to the parental Aspergillus strain, thereby generating the engineered strain of Aspergillus. ### Claim 19 (depends on 18) The method of claim 18, wherein the nucleic acid construct comprises from 5′ to 3, a first portion of the sequence complementary to the locus for the A. niger orthologue of the S. cerevisiae sln1 gene, a first direct repeat sequence comprising a first copy of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene, a selectable marker gene, a second direct repeat sequence comprising a second copy of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene, and a second portion of the sequence complementary to the locus for the A. niger orthologue of the S. cerevisiae sln1 gene, wherein the direct repeats provide an unstable integration that can result in loss of the selectable marker gene. ### Claim 20 (depends on 19) The method of claim 19, wherein the nucleic acid construct is split into construct A and construct B, wherein construct A comprises from 5′ to 3′, the first portion of the sequence complementary to the locus for the A. niger orthologue of the S. cerevisiae sln1 gene, the first direct repeat sequence comprising a first copy of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene, and a first portion of the selectable marker gene, while construct B comprises from 5′ to 3′, a second portion of the selectable marker gene, the second direct repeat sequence comprising the second copy of the heterologously modified A. niger orthologue of the S. cerevisiae sln1 gene, and the second portion of the sequence complementary to the locus for the S. cerevisiae sln1 orthologue of the S. cerevisiae sln1 gene, wherein the first portion and the second portion of the selectable marker gene comprises overlapping complementary sequence. ### Claim 21 (depends on 18) The method of claim 18, wherein the heterologous modified A. niger orthologue of the S. cerevisiae sln1 gene is a single nucleotide polymorphism (SNP) containing version of the endogenous A. niger orthologue of the S. cerevisiae sln1 gene of the parental Aspergillus strain, 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. ### Claim 22 (depends on 21) The method of claim 21, 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. ### Claim 23 (depends on 21) The method of claim 21, wherein the SNP containing version of the A. niger orthologue of the S. cerevisiae sln1 gene comprises a nucleic acid sequence of SEQ ID NO: 7. ### Claim 24 (depends on 21) The method of claim 21, wherein the A. niger orthologue of the S. cerevisiae sln1 gene of the parental Aspergillus strain comprises a nucleic acid sequence of SEQ ID NO: 76. ### Claim 25 (depends on 21) The method of claim 21, 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 the A. niger orthologue of the S. cerevisiae SLN1. ### Claim 26 (depends on 18) The method of claim 18, wherein the heterologous modified A. niger orthologue of the S. cerevisiae sln1 gene comprises replacement of the native promoter of the endogenous A. niger orthologue of the S. cerevisiae sln1 gene of the parental 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. ### Claim 27 (depends on 18) The method of claim 18, 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 b selected from an amyB promoter or a manB promoter. ### Claim 28 (depends on 18) The method of claim 18, wherein the promoter that more weakly expresses the A. niger orthologue of S. cerevisiae sln1 gene as compared to the native promoter of the A. niger orthologue of S. cerevisiae sln1 gene has a nucleic acid sequence of SEQ ID NO: 1 or 2. ### Claim 29 An engineered Aspergillus 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 strain as compared to cells of die 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.