Search

JP-2026076384-A - Modified filamentous fungi and methods for producing proteins using them

JP2026076384AJP 2026076384 AJP2026076384 AJP 2026076384AJP-2026076384-A

Abstract

[Problem] To provide a modified filamentous fungus with improved protein productivity, and a method for producing protein using the modified filamentous fungus. [Solution] An ACE3 variant is expressed, and the ACE3 variant contains Zn(II) 2C of ACE3. A modified filamentous fungus that is a variant lacking substantially all of the ys6 type DNA-binding domain. A method for producing a protein, comprising culturing the modified filamentous fungus. [Selection Diagram] None

Inventors

  • 新井 俊陽
  • 児玉 裕司

Assignees

  • 花王株式会社

Dates

Publication Date
20260511
Application Date
20260224

Claims (20)

  1. A modified filamentous fungus, The ACE3 variant manifested, The ACE3 variant is a variant in which the Zn(II)2Cys6 type DNA-binding domain region of ACE3 is deleted. The ACE3 is a polypeptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 4 or an amino acid sequence that is at least 90% identical thereto. The region of the Zn(II) 2Cys6 type DNA-binding domain corresponds to the amino acid region 120-151 of SEQ ID NO : 1. Modified filamentous fungi.
  2. The modified filamentous fungus according to claim 1, wherein the ACE3 variant lacks the region corresponding to amino acids 120-160 of SEQ ID NO: 1.
  3. The modified filamentous fungus according to claim 1, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 151 of SEQ ID NO: 1.
  4. The modified filamentous fungus according to claim 2, wherein the ACE3 variant lacks the region corresponding to amino acids 1 to 160 of SEQ ID NO: 1.
  5. The modified filamentous fungus according to any one of claims 1 to 4, wherein the ACE3 variant has a region corresponding to amino acids 260 to 701 of SEQ ID NO: 1.
  6. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to amino acids 260 to 717 of SEQ ID NO: 1.
  7. The modified filamentous fungus according to claim 6, wherein the ACE3 variant has a region corresponding to amino acids 260-723 of SEQ ID NO: 1.
  8. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to amino acids 250 to 701 of SEQ ID NO: 1.
  9. The modified filamentous fungus according to claim 8, wherein the ACE3 variant has a region corresponding to amino acids 250 to 717 of SEQ ID NO: 1.
  10. The modified filamentous fungus according to claim 9, wherein the ACE3 variant has a region corresponding to amino acids 250 to 723 of SEQ ID NO: 1.
  11. The modified filamentous fungus according to claim 5, wherein the ACE3 variant has a region corresponding to amino acids 241 to 701 of SEQ ID NO: 1.
  12. The modified filamentous fungus according to claim 11, wherein the ACE3 variant has a region corresponding to amino acids 241 to 717 of SEQ ID NO: 1.
  13. The modified filamentous fungus according to claim 1 or 2, wherein the ACE3 variant has a region corresponding to amino acids 241 to 723 of SEQ ID NO: 1.
  14. The modified filamentous fungus according to any one of claims 1 to 13, wherein the ACE3 variant lacks a region corresponding to at least seven amino acids at the C-terminus of the amino acid sequence of Sequence ID No. 1.
  15. The modified filamentous fungus according to claim 14, wherein the ACE3 variant lacks the region corresponding to the 11 amino acids at the C-terminus of the amino acid sequence of Sequence ID No. 1.
  16. A modified filamentous fungus according to any one of claims 1 to 15, wherein a gene expressing the ACE3 variant has been introduced.
  17. The modified filamentous fungus according to claim 16, wherein the gene expressing the ACE3 variant is operably linked to a regulatory region that promotes the transcription of the gene.
  18. The modified filamentous fungus according to any one of claims 1 to 17, wherein the filamentous fungus is of the genus Trichoderma.
  19. A method for producing a protein, comprising culturing a modified filamentous fungus according to any one of claims 1 to 18.
  20. The method according to claim 19, wherein the protein is cellulase and/or hemicellulase.

Description

This invention relates to a modified filamentous fungus and a method for producing proteins using the same. Filamentous fungi are plant polysaccharide degrading fungi that produce various cellulases and hemicellulases. Among them, Trichoderma is attracting attention as a microorganism for the production of cellulase-based biomass degrading enzymes because it can produce cellulase and hemicellulase simultaneously and in large quantities. For industrial microbial culture, carbon sources should ideally be inexpensive and soluble. Conventionally, Glucose is commonly used as a carbon source in microbial culture. On the other hand, inducible substances are sometimes necessary for the production of enzymes and other proteins by microorganisms. For example, inducible substances are generally essential for cellulase production by microorganisms. In Trichoderma, the expression of major cellulase genes such as cbh1, cbh2, egl1, and egl2 is induced by inducible substances such as cellulose and cellobiose (Non-Patent Literature 1). When inducible substances are not used, for example, when glucose is used as the sole carbon source, saccharifying enzymes are generally hardly produced in Trichoderma. As for microbiological protein production methods using inducible substances, a cellulase production method using Avicel, a microcrystalline cellulose, is known. Furthermore, a cellulase production method using soluble lactose without cellulose (Patent Document 1) and a method for inducing cellulase production by reacting Trichoderma-derived cellulase (including β-glucosidase, endoglucanase, and cellobiohydrolase) with glucose at high temperatures to synthesize inducible sugars such as sophorose and gentiobiose from glucose are disclosed (Patent Document 2). However, cellulose substrates are expensive and mostly insoluble, placing a burden on industrial processes, making their use in industrial applications difficult in terms of cost and equipment. Cellulase production using other inducible sugars still suffers from disadvantages in terms of cost and process load. Therefore, in order to create microorganisms capable of expressing cellulase and xylanase without the use of inducible substances by modifying transcription factors, the mechanism of cellulase expression is being analyzed. XYR1 and ACE2 are positive transcription factors involved in cellulase-induced expression in Trichoderma. ACE3, HAP2/3/5, etc. have been reported (Non-Patent Literature 2). ACE3 is a transcription factor that regulates the promoters of major cellulases such as cbh1. It binds to the promoter via the Zn(II) 2Cys6 type DNA-binding domain (DBD) located at amino acids 120-160 on the N-terminal side (Non-Patent Literature 3). Non-Patent Literature 3 also suggests that ACE3 and XYR1 interact to regulate the cellulase gene expression of Trichoderma riseyi. Patent Document 3 and Non-Patent Document 4 describe the use of tre7751 in Trichoderma risey. Methods are disclosed for increasing and decreasing the productivity of cellulases, etc., by increasing and decreasing the expression of the 3 (ACE3) gene. Patent documents 4 and 5 disclose the N-side Z n(II) 2 Cys 6 retains all six cysteine molecules of the DNA-binding domain and has 7 at the C-terminus. It has been reported that filamentous fungi that upexpress a modified ACE3 lacking 17 amino acids have improved cellulase expression even in the absence of inducible substances. Non-patent document 5 also describes a filamentous fungus that upexpresses the C-terminally deficient ACE3 and co-expresses either the wild-type or A824V mutant of XYR1. However, the effect of co-expression of XYR1 on cellulase expression in this filamentous fungus was only slightly observed in the presence of the inducible substance and not in the absence of the inducible substance. Non-patent document 6 reports that xylanase deregulation occurs and cellulase production increases in Trichoderma with the XYR1 A824V mutation. Non-patent document 7 reports that It has been reported that combining the V821F mutation in XYR1 with enhanced ACE2 expression in Trichoderma strains improves protein productivity in media using glucose or sucrose as a carbon source. Patent No. 6169077Patent No. 5366286U.S. Patent Publication No. 9512415International Public Gazette No. 2018/067599 Curr Genomics, 2013, 14:230-249BMC Genomics, 2015, 16:326J Biol Chem, 2019, doi:10.1074/jbc.RA119.008497Biotech Biofuels, 2014, 7:14Biotech Biofuels, 2020, 13:137Biotech Biofuels, 2013, 6:62Biotech Biofuels, 2017, 10:30 Structure of the ACE3 variant expressed by the modified filamentous fungus.Structure of the ACE3 variant expressed by the modified filamentous fungus.Effect of DBD deficiency of ACE3 on protein productivity: (A) The values on the vertical axis represent the relative protein productivity of modified filamentous fungi expressing the ACE3 variant, and the horizontal axis (1, 2, 3) represents three modified filamentous fungal strains expressing the same ACE3 v