Regulated PepC expression

Patent metadata

Field	Value
Publication number	US-10619144-B2
Application number	US-201414913967-A
Country	US
Kind code	B2
Filing date	Aug 22, 2014
Priority date	Aug 23, 2013
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

Abstract

The present invention relates to a recombinant fungal host cell comprising at least one first polynucleotide encoding a polypeptide of interest; and one or more second polynucleotide encoding a fungal PepC protease, wherein the one or more second polynucleotide is operably linked to a regulated heterologous promoter, as well as a method for producing a polypeptide of interest, comprising cultivating said fungal host cell.

First claim

The invention claimed is:

1. A recombinant fungal host cell comprising:
(a) at least one first polynucleotide encoding a polypeptide of interest; and
(b) one or more second polynucleotides encoding a fungal PepC protease, wherein the one or more second polynucleotides are operably linked to a regulated heterologous promoter, wherein the regulated heterologous promoter is induced in the presence of nitrate and repressed in the presence of ammonium, or wherein the regulated heterologous promoter is induced in the presence of sorbitol and repressed in the absence of sorbitol, and wherein expression of the fungal PepC protease under control of the regulated heterologous promoter in the recombinant fungal host cell (1) reduces degradation of the polypeptide of interest compared to expression of PepC under control of the native promoter, (2) increases spore yield of the recombinant fungal host cell compared to a fungal host cell having the PepC protease-encoding gene deleted; and (3) increases activity of the polypeptide of interest.

2. The host cell of claim 1 , which is a filamentous fungal host cell.

3. The host cell of claim 2 , wherein the filamentous fungal host cell is selected from the group consisting of an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , and Trichoderma viride cell.

4. The host cell of claim 1 , wherein the at least one first polynucleotide is present in the chromosome of the host cell.

5. The host cell of claim 4 , wherein the at least one first polynucleotide is present in the chromosome of the host cell in two or more copies.

6. The host cell of claim 1 , wherein the polypeptide of interest is an enzyme selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase and a transferase.

7. The host cell of claim 6 , wherein the polypeptide of interest is an enzyme selected from the group consisting of an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.

8. The host cell of claim 1 , wherein the PepC protease is selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the mature sequence shown in positions 1 to 380 of SEQ ID NO: 2 or in positions 1 to 418 of SEQ ID NO: 4;
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and
(c) a fragment of the polypeptide of (a) or (b) that has protease activity.

9. The host cell of claim 1 , wherein the PepC protease is expressed with a pro-peptide comprising an amino acid sequence shown in positions −99 to −1 of SEQ ID NO: 2 or in positions −99 to −1 of SEQ ID NO: 4.

10. The host cell of claim 1 , wherein the PepC protease is expressed with a signal-peptide comprising an amino acid sequence shown in positions −115 to −100 of SEQ ID NO: 2 or in positions −115 to −100 of SEQ ID NO: 4.

11. The host cell of claim 1 , wherein the regulated heterologous promoter is induced in the presence of nitrate and repressed in the presence of ammonium.

12. The host cell of claim 1 , wherein the regulated heterologous promoter is induced in the presence of sorbitol and repressed in the absence of sorbitol.

13. A method of producing a polypeptide of interest, said method comprising the steps of:
(a) cultivating the host cell of claim 1 , under conditions for the production of the polypeptide of interest; and, optionally
(b) recovering the polypeptide of interest.

14. The fungal host cell of claim 2 , wherein the filamentous fungal host cell is selected from the group consisting of an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma cell.

15. The host cell of claim 11 , wherein the regulated heterologous promoter is a filamentous fungal nitrate reductase promoter.

16. The host cell of claim 15 , wherein the filamentous fungal nitrate reductase promoter is from an Aspergillus or a Trichoderma cell.

17. The host cell of claim 15 , wherein the filamentous fungal nitrate reductase promoter is from an Aspergillus niger, Aspergillus oryzae or Trichoderma reesei cell.

18. The host cell of claim 15 , wherein the nitrate reductase promoter is the niaD nitrate reductase promoter from Aspergillus niger, Aspergillus oryzae or Trichoderma reesei.

19. The host cell of claim 12 , wherein the regulated heterologous promoter is a filamentous fungal sorbitol transporter promoter or a sorbitol dehydrogenase promoter.

20. The host cell of claim 19 , wherein the filamentous fungal sorbitol transporter promoter or the sorbitol dehydrogenase promoter is from an Aspergillus or Trichoderma cell.

21. The host cell of claim 19 , wherein the filamentous fungal sorbitol transporter promoter or the sorbitol dehydrogenase promoter is from Aspergillus niger, Aspergillus oryzae , or Trichoderma reesei.

22. The host cell of claim 19 , wherein the filamentous fungal sorbitol transporter promoter or the sorbitol dehydrogenase promoter is the sorA or the sorb promoter from Aspergillus niger, Aspergillus oryzae , or Trichoderma reesei.

23. The host cell of claim 8 , wherein the PepC protease comprises an amino acid sequence having at least 95% sequence identity to the mature sequence shown in positions 1 to 380 of SEQ ID NO: 2 or in positions 1 to 418 of SEQ ID NO: 4.

24. The host cell of claim 8 , wherein the PepC protease comprises the

Assignees

• Novozymes As

Inventors

• Teramoto Hiroshi
• Udagawa Hiroaki
• Lehmbeck Jan
• Nielsen Michael Lynge

Classifications

• C12N9/82

  Asparaginase {(3.5.1.1)} · CPC title

• C07K14/38

  from Aspergillus · CPC title

• C12P21/02

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

• C12N9/62 Primary

  from Aspergillus · CPC title

• C12N9/18

  Carboxylic ester hydrolases {(3.1.1)} · CPC title