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US-12624376-B2 - Mutants of a filamentous fungal cell having increased productivity in the production of a polypeptide

US12624376B2US 12624376 B2US12624376 B2US 12624376B2US-12624376-B2

Abstract

The present invention relates to a mutant of a parent filamentous fungal cell, comprising: a polynucleotide encoding a polypeptide of interest; a racA gene encoding a Rho-GTPase RacA protein, wherein the racA gene is modified rendering the mutant partially or completely deficient in the production of the Rho-GTPase RacA protein; and a ras2 gene encoding a GTPase Ras2 protein, wherein the GTPase Ras2 protein is modified to produce a GTPase Ras2 variant comprising a substitution at a position corresponding to position 16 of SEQ ID NO: 11, wherein the combination of the modified racA gene and the Ras2 variant synergistically increases the productivity of the mutant in the production of the polypeptide of interest. The present invention also relates to a method of producing a polypeptide of interest with such a mutant.

Inventors

  • Gloria Muzzi-Erichsen

Assignees

  • NOVOZYMES A/S

Dates

Publication Date
20260512
Application Date
20210112

Claims (18)

  1. 1 . An isolated mutant of a parent Trichoderma cell, comprising: (a) a polynucleotide encoding a polypeptide of interest; (b) a racA gene encoding a Rho-GTPase RacA protein, wherein the racA gene is modified in the parent Trichoderma cell to produce the mutant rendering the mutant partially or completely deficient in the production of the Rho-GTPase RacA protein; and (c) a ras2 gene encoding a GTPase Ras2 protein, wherein the GTPase Ras2 protein is modified in the parent Trichoderma cell to produce a GTPase Ras2 variant comprising a substitution at a position corresponding to position 16 of SEQ ID NO: 11; wherein the combination of the modified racA gene and the Ras2 variant synergistically increases the productivity of the mutant in the production of the polypeptide of interest.
  2. 2 . The mutant of claim 1 , wherein the Rho-GTPase RacA is selected from the group consisting of: (i) a Rho-GTPase RacA protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, (ii) a Rho-GTPase RacA protein encoded by a polynucleotide comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1, and (iii) a Rho-GTPase RacA protein encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under high stringency conditions with the full-length complement of SEQ ID NO: 1.
  3. 3 . The mutant of claim 1 , wherein the GTPase Ras2 is selected from the group consisting of: (i) a GTPase Ras2 protein comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11, (ii) a GTPase Ras2 protein encoded by a polynucleotide comprising a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 10, and (iii) a GTPase Ras2 protein encoded by a polynucleotide comprising a nucleotide sequence that hybridizes under high stringency conditions with the full-length complement of SEQ ID NO: 10.
  4. 4 . The mutant of claim 1 , wherein the GTPase Ras2 variant comprises a substitution of the amino acid residue at position 16 with valine (Val).
  5. 5 . The mutant of claim 4 , wherein the GTPase Ras2 variant comprises the substitution Glyl6Val at position 16 of SEQ ID NO: 11 or corresponding to position 16 of SEQ ID NO: 11.
  6. 6 . The mutant of claim 1 , wherein the GTPase Ras2 variant has at least 85% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 11.
  7. 7 . The mutant of claim 1 , wherein the polypeptide of interest is native to the parent Trichoderma cell or the mutant thereof.
  8. 8 . The mutant of claim 1 , wherein the polypeptide of interest is heterologous to the parent Trichoderma cell or the mutant thereof.
  9. 9 . The mutant of claim 1 , wherein the polypeptide of interest is an antibody, an antigen, an antimicrobial peptide, an enzyme, a growth factor, a hormone, an immunodilator, a neurotransmitter, a receptor, a reporter protein, a structural protein, or a transcription factor.
  10. 10 . The mutant of claim 1 , wherein the polypeptide of interest is a cellulase.
  11. 11 . The mutant of claim 10 , wherein the cellulase is an endoglucanase, a cellobiohydrolase, or a beta-glucosidase.
  12. 12 . The mutant of claim 1 , wherein the polypeptide of interest is a hemicellulase.
  13. 13 . The mutant of claim 12 , wherein the hemicellulase is a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, or a glucuronidase.
  14. 14 . The mutant of claim 1 , wherein the parent filamentous fungal cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , or Trichoderma viride cell.
  15. 15 . The mutant of claim 1 , wherein the Trichoderma cell is a Trichoderma reesei cell.
  16. 16 . The mutant of claim 1 , wherein the productivity of the mutant in the production of the amount of the polypeptide of interest is increased compared to the parent Trichoderma cell.
  17. 17 . A method of producing a polypeptide of interest, comprising cultivating the mutant filamentous fungal cell of claim 1 in a medium for production of the polypeptide of interest.
  18. 18 . The method of claim 17 , further comprising recovering the polypeptide of interest from the cultivation medium.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a 35 U.S.C. 371 national application of PCT/EP2021/050464, filed Jan. 12, 2021, which claims priority or the benefit from U.S. Provisional Application Ser. No. 62/965,752, filed Jan. 24, 2020. The contents of these applications are fully incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing that was submitted as an txt file named 15108-US-PCT Corrected SQ ST25.txt (created on May 15, 2025, containing 78,280 bytes), which is incorporated herein by reference. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to mutant filamentous fungal cells with increased productivity in the production of polypeptides. Description of the Related Art Filamentous fungi are widely used for producing enzymes and other biologicals for a variety of industrial applications. The productivity of a filamentous fungal cell in the production of a polypeptide of interest is dependent upon several factors, such as carbon source, nitrogen source, secretion, pH, temperature, and dissolved oxygen. In particular, the carbon source can determine which genes for secreted enzymes are induced and/or repressed and their production rates. The carbon source acts through transcription factors and their associated promoters that are either activated or repressed depending on the level of the carbon source. It is reported that the racA gene plays a key role in hyphal growth and hyphal branching in filamentous fungi and that racA gene inactivation contributes to increase protein production because of enhanced secretion potential (Chen et al., 2016, Biotech 6: 214; Fitz et al., 2019, Fungal Biol. Biotechnology (2019), 6: 16; Fielder et al., 2018, Microb. Cell. 17: 95; Virag, 2007, Molecular Microbiology 66: 1579-1596). It is also reported that the Ras GTPase ras2 gene is important for regulating morphogenesis and cellulase expression in Trichoderma reesei where the ras2G16V constitutively active variant increases cellulase gene transcription in T. reesei under inducing conditions, especially cellulase genes under control of the Xyr1, Ace2, Cre1 and Ace1 transcription factors (Zhang et al., 2012, PLOS One 7: e48786). The present invention provides mutants of a filamentous fungal cell for increasing the productivity of the filamentous fungal cell in the production of a polypeptide of interest where the combination of a modified racA gene and a Ras2 variant synergistically increases the productivity of the mutant. SUMMARY OF THE INVENTION The present invention relates to an isolated mutant of a parent filamentous fungal cell, comprising: (a) a polynucleotide encoding a polypeptide of interest;(b) a racA gene encoding a Rho-GTPase RacA protein, wherein the racA gene is modified in the parent filamentous fungal cell to produce the mutant rendering the mutant partially or completely deficient in the production of the Rho-GTPase RacA protein; and(c) a ras2 gene encoding a GTPase Ras2 protein, wherein the GTPase Ras2 protein is modified in the parent filamentous fungal cell to produce a GTPase Ras2 variant comprising a substitution at a position corresponding to position 16 of SEQ ID NO: 11; wherein the combination of the modified racA gene and the GTPase Ras2 variant synergistically increases the productivity of the mutant in the production of the polypeptide of interest. The present invention also relates to a method of producing a polypeptide of interest, comprising cultivating such a mutant filamentous fungal cell in a medium for production of the polypeptide of interest, and optionally recovering the polypeptide of interest. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a map of plasmid pGMEr263. FIG. 2 shows a map of plasmid pGMEr263-racA-proto3. FIG. 3 shows a map of plasmid pGMEr263-ras2G16V-proto. FIG. 4 shows a comparison of T. reesei strains O83E59, O83E58, and O838XE and parent T. reesei strain O44N7J vs. day 7 total protein values in a “box plot” representation. The black boxes represent the spread of data samples between the first and the third quartiles. The white lines in the middle represent the median value. FIG. 5 shows a comparison of T. reesei strains O83E59, O83E58, and O838XE and parent T. reesei strain O44N7J vs day 7 beta-glucosidase activity values in a “box plot” representation. The black boxes represent the spread of data samples between the first and the third quartiles. The white lines in the middle represent the median values. FIG. 6 shows a comparison of T. reesei strains O83E59, O83E58, and O838XE and parent T. reesei strain O44N7J vs day 7 total feed (total grams of feed used in the fermentation run) in a “box plot” representation. The black boxes represent the spread of data samples between the first and the third quartiles. The white lines in the middle represent the median values. DEFINITIONS Acetylxylan esterase: The term “acetylxylan esterase” means a carboxylesterase (EC 3.1.1