Improving recombinant protein secretion in Aspergillus nidulans by targeting the N-glycosylation machinery

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Gerhardt, Jaqueline Aline ; Rubio, Marcelo Ventura ; Terrasan, Cesar Rafael Fanchini ; Wassano, Natalia Sayuri ; Rodrigues, Aryadne ; Figueiredo, Fernanda Lopes de ; Antoniel, Everton Paschoal ; Contesini, Fabiano Jares ; Dias, Artur Hermano Sampaio ; Mortensen, Uffe Hasbro ; Skaf, Munir Salomao ; Damasio, Andre

• Document type: Journal article
• Source: METABOLIC ENGINEERING COMMUNICATIONS; v. 20, p. 12-pg., 2025-06-01.

Abstract

Filamentous fungi are cell factories traditionally used for enzyme production in various industrial sectors, including food and beverages, biopolymers, biofuels, and animal feed. Despite significant progress in optimizing enzyme production, challenges related to cost-effectiveness persist. Genes involved in the fungal secretory pathway have been modified to address productivity barriers, including post-translational modifications such as N-glycosylation of proteins. N-glycosylation can significantly affect protein stability, production yield, and functionality. This study investigated the isolated and combined deletion of genes involved in N-glycan assembly on protein production in Aspergillus nidulans. To test this hypothesis, we utilized CRISPR/Cas9 technology to knock out 14 genes related to N-glycan assembly (AN5888, AN11802, AN5346, AN6874, AN5725, AN7425, algC, algI, algL, algF and AN5748) and protein quality control (clxA, gtbA, and AN4623), resulting in eight viable mutants. Next, we integrated a GH3 beta-xylosidase encoding gene (bxlb; AN8401) into these mutants and the reference strain for constitutive expression and secretion. Single deletion of most target genes did not affect protein secretion and fungal growth. Interestingly, the specific activity of BxlB in the secretome of single mutants was influenced by culture time, while BxlB secretion remained unaffected. Conversely, the combined deletion of algC and algI increased BxlB secretion, whereas the kinetic parameters remained unaffected relative to the enzyme produced by the reference strain. Multiple deletions of algC, algF, and algI did not affect BxlB secretion but reduced catalytic efficiency. After analyzing the secretomes of double and triple mutant strains produced on plant biomass using mass spectrometry, we observed that these knockouts reduced the overall secretion of a specific set of carbohydrate-active enzymes (CAZymes). Other clusters were upregulated in the mutant strains, indicating severe secretome alterations. Overall, the combined deletion of algC and algI may be a promising strategy for increasing the secretion of recombinant proteins in A. nidulans while also enhancing downstream processes, such as protein purification, by reducing the protein background in the secretome of the mutant strain. (AU)

• FAPESP's process: 20/15962-6 - Genetic manipulation of transcription factors involved in the secretion of recombinant enzymes in Aspergillus nidulans
• Grantee: Everton Paschoal Antoniel
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/08293-7 - CCES - Center for Computational Engineering and Sciences
• Grantee: Munir Salomao Skaf
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 16/16306-0 - Functional and biological analyses of LPMO (Lytic Polysaccharide Monooxigenases) and accessory proteins (non-CAZymes) for degradation of sugarcane straw by filamentous fungi
• Grantee: César Rafael Fanchini Terrasan
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 19/21609-0 - Genetic engineering of Aspergillus niger for the production of a tailor-made enzymatic cocktail
• Grantee: Fernanda Lopes de Figueiredo
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 17/22669-0 - N-glycosylation and enzymes secretion in filamentous fungi
• Grantee: André Ricardo de Lima Damasio
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants
• FAPESP's process: 18/16437-2 - Deciphering the effect of N-glycosylation pathway deletion in Aspergillus nidulans enzymes secretion
• Grantee: Jaqueline Aline Gerhardt
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 22/05731-2 - BEYOND: establishing a fungal cell factory for recombinant protein production
• Grantee: André Ricardo de Lima Damasio
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 20/06151-4 - Biology of NAD+-dependent lysine deacetylases (Sirtuins) in Aspergillus fumigatus
• Grantee: André Ricardo de Lima Damasio
• Support Opportunities: Regular Research Grants