Protein production in filamentous fungal cells in the absence of inducing substrates

Patent metadata

Field	Value
Publication number	US-10876103-B2
Application number	US-201716336500-A
Country	US
Kind code	B2
Filing date	Oct 3, 2017
Priority date	Oct 4, 2016
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

Certain embodiments of the disclosure are directed to variant filamentous fungal cells, compositions thereof and methods thereof for increased production of one or more proteins of interest. More particularly, in certain embodiments, the disclosure is directed to variant filamentous fungal (host) cells derived from parental filamentous fungal cells, wherein the variant host cells comprise a genetic modification which enables the expression/production of a protein of interest (POI) in the absence of inducing substrate. In certain embodiments, a variant fungal host cell of the disclosure comprises a genetic modification which increases the expression of a variant activator of cellulase expression 3 (ace3) gene encoding an Ace3 protein referred to herein as Ace3-L.

First claim

The invention claimed is:

1. A variant filamentous fungal cell obtained from a parental Trichoderma filamentous fungal cell, the variant cell comprising an introduced polynucleotide construct encoding a functional activator of cellulase 3 (Ace3} transcription factor (TF) protein comprising at least 95% sequence identity to the Ace3 protein of SEQ ID NO: 6, wherein the variant cell produces lignocellulosic degrading enzyme in the absence of an inducing substrate relative to the parental cell, wherein the variant and parental cells are cultivated under similar conditions.

2. The variant cell of claim 1 , wherein the variant cell further produces an increased amount of a lignocellulosic degrading enzyme in the presence of an inducing substrate relative to the parental cell, wherein the variant and parental cells are cultivated under similar conditions.

3. The variant cell of claim 1 , wherein the variant cell comprises an introduced polynucleotide construct encoding a heterologous lignocellulosic degrading enzyme.

4. The variant cell of claim 1 , wherein the lignocellulose degrading enzyme is selected from the group consisting of cellulase enzymes, hemi-cellulase enzymes, or a combination thereof.

5. The variant cell of claim 3 , wherein the heterologous POI is selected from the group consisting of an α-amylase, an alkaline α-amylase, a β-amylase, a cellulase, a dextranase, an α-glucosidase, an α-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase, a lactase, a naringanase, a pectinase, a pullulanase, an acid protease, an alkali protease, a bromelain, a neutral protease, a papain, a pepsin, a peptidase, a rennet, a rennin, a chymosin, a subtilisin, a thermolysin, an aspartic proteinase, a trypsin, a lipase, an esterase, a phospholipase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, a penicillin acylase; an isomerase, an oxidoreductases, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, a peroxidase, a lyase, an aspartic β-decarboxylase, a fumarase, a histadase, a transferase, a ligase, an aminopeptidase, a carboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, an α-galactosidase, a β-galactosidase, a β-glucosidase, a laccase, a mannosidase, a mutanase, a polyphenol oxidase, a ribonuclease and a transglutaminase.

6. The variant cell of claim 1 , wherein the polynucleotide construct is integrated into the fungal cell genome.

7. The variant cell of claim 6 , wherein the polynucleotide construct is integrated into a telomere site of the fungal cell genome.

8. The variant cell of claim 6 , wherein the polynucleotide construct is integrated into a glucoamylase (gla1) gene locus of the fungal cell genome.

9. The variant cell of claim 1 , wherein the polynucleotide construct comprises a nucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 13.

10. A polynucleotide ORF encoding a functional Ace3 TF protein comprising at least 95% sequence identity to SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14.

Assignees

• Danisco Us Inc
• Vtt Technical Res Centre Of Finland Ltd

Inventors

• Ward Michael
• Luo Yun
• Bendezu Felipe Oseas
• Valkonen Mari
• Saloheimo Markku
• Aro Nina
• Pakula Tiina

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12N1/14

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

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C07K14/37

  from fungi · CPC title