# US 11,028,401 B2 · Manipulation of genes involved in signal transduction to control fungal morphology during fermentation and production

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- Number: US 11,028,401 B2
- Document ID: US11028401B2
- Status: Granted
- Kind: B2
- Application number: 16/433,624
- Filed: 2019-06-06
- Published: 2019-12-12
- Granted: 2021-06-08
- 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/US11028401B2

## 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

A variant strain of Aspergillus that is genetically altered compared to a parental strain, wherein cells of the variant strain possess a genetic alteration in an osmotic response pathway gene that causes cells of the variant strain to produce a reduced amount and/or less active form of functional protein encoded by the osmotic response pathway gene as compared to cells of the parental strain when grown under submerged culture conditions,
wherein the osmotic response pathway gene is selected from an Aspergillus niger (A. niger) orthologue of a Saccharomyces cerevisiae (S. cerevisiae) sln1 gene and a Neurospora crassa (N. crassa) nik1 gene, and wherein the genetic alteration is replacement of a native promoter of the osmotic response pathway gene with a promoter that weakly expresses the osmotic response pathway gene compared to the native promoter, wherein the promoter that weakly expresses the osmotic response pathway gene as compared to the native promoter is selected from an Aspergillus amyB gene promoter and an Aspergillus manB gene promoter.

### Claim 2 (depends on 1)

The variant strain of claim 1, further comprising one or more genes of the osmotic response pathway selected from genes comprising the nucleic acid sequences of SEQ ID NO: 9, 10, 11, 12, 13 or any combination thereof, wherein each gene is operably linked to a promoter that causes cells of the variant strain to produce a reduced amount and/or less active form of functional protein encoded by the gene comprising the nucleic acid sequence of SEQ ID NO: 9, 10, 11, 12, or 13 as compared to the native promoter for the gene comprising the nucleic acid sequence of SEQ ID NO: 9, 10, 11, 12, or 13, or any combination thereof.

### Claim 3 (depends on 1)

The variant strain of claim 1, wherein the A. niger orthologue of the S. cerevisiae sln1 gene or the N. crassa nik1 gene is a non-single nucleotide polymorphism (non-SNP) containing version of a gene comprising the nucleic acid sequence of SEQ ID NO: 7.

### Claim 4 (depends on 1)

The variant strain of claim 1, wherein the osmotic response pathway gene is a mutated form of the osmotic response pathway gene.

### Claim 5 (depends on 4)

The variant strain of claim 4, wherein the mutated form of the osmotic stress response pathway gene comprises a SNP.

### Claim 6 (depends on 5)

The variant strain of claim 5, wherein the mutated form of the A. niger orthologue of the S. cerevisiae sln1 gene or the N. crassa nik1 gene comprises the nucleic acid sequence of SEQ ID NO: 7.

### Claim 7 (depends on 1)

The variant strain of claim 1, wherein the amyB gene promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

### Claim 8 (depends on 1)

The variant strain of claim 1, wherein the variant strain has a non-mycelium, pellet morphology when grown under submerged culture conditions in fermentation media as compared to a reference filamentous fungal host cell without the promoter operably linked to a gene that regulates morphology of the host cell.

### Claim 9 (depends on 1)

The variant strain of claim 1, further comprising a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 5, 6, 8, and any combination thereof.

### Claim 10 (depends on 1)

The variant strain of claim 1, wherein the manB gene promoter comprises the nucleic acid sequence of SEQ ID NO: 1.

### Claim 11

A variant strain of Aspergillus that is genetically altered compared to a parental strain, wherein cells of the variant strain possess an osmotic response pathway gene comprising a nucleic acid sequence selected from SEQ ID NO: 14 and SEQ ID NO: 76 operably linked to a manB gene promoter comprising the nucleic acid sequence of SEQ ID NO: 1 that causes cells of the variant strain to produce a reduced amount and/or less active form of functional protein encoded by the osmotic response pathway gene comprising the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 76 as compared to cells of the parental strain when grown under submerged culture conditions.

### Claim 12 (depends on 11)

The variant strain of claim 11, further comprising a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5, 6, 8 and any combination thereof.

### Claim 13 (depends on 11)

The variant strain of claim 11, further comprising a non-SNP containing version of a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8 and any combination thereof, wherein the genes are each operably linked to a promoter that causes cells of the variant strain to produce a reduced amount and/or less active form of the functional protein encoded by the non-SNP containing version of the gene comprising the nucleic acid sequence of SEQ ID NO: 5, 6, or 8 as compared to the native promoter for the non-SNP containing version of the gene comprising the nucleic add sequence of SEQ ID NO: 5, 6, or 8.

### Claim 14 (depends on 13)

The variant strain of claim 13, wherein the promoter is selected from an amyB gene promoter comprising the nucleic acid sequence of SEQ ID NO: 2 and a manB gene promoter comprising the nucleic acid sequence of SEQ ID NO: 1.

### Claim 15 (depends on 11)

The variant strain of claim 11, further comprising a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13 and any combination thereof, wherein the genes are each operably, linked to a promoter that causes cells of the variant strain to produce a reduced amount and/or less active form of functional protein encoded by the gene comprising the nucleic acid sequence of SEQ ID NO: 9, 10, 11, 12 or 13 as compared to the native promoter for the gene comprising the nucleic acid sequence of SEQ ID NO: 9, 10, 11, 12 or 13.

### Claim 16 (depends on 15)

The variant strain of claim 15, wherein the promoter is selected from an amyB gene promoter comprising the nucleic acid sequence of SEQ ID NO: 2 and a manB gene promoter comprising the nucleic acid sequence of SEQ ID NO: 1.