Engineering the secretome of Aspergillus niger for cellooligosaccharides production from plant biomass

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de Figueiredo, Fernanda Lopes ; Contesini, Fabiano Jares ; Terrasan, Cesar Rafael Fanchini ; Gerhardt, Jaqueline Aline ; Correa, Ana Beatriz ; Antoniel, Everton Paschoal ; Wassano, Natalia Sayuri ; Levassor, Lucas ; Rabelo, Sarita Candida ; Franco, Telma Teixeira ; Mortensen, Uffe Hasbro ; Damasio, Andre

• Document type: Journal article
• Source: Microbial Cell Factories; v. 23, n. 1, p. 13-pg., 2024-11-29.

Abstract

BackgroundFermentation of sugars derived from plant biomass feedstock is crucial for sustainability. Hence, utilizing customized enzymatic cocktails to obtain oligosaccharides instead of monomers is an alternative fermentation strategy to produce prebiotics, cosmetics, and biofuels. This study developed an engineered strain of Aspergillus niger producing a tailored cellulolytic cocktail capable of partially degrading sugarcane straw to yield cellooligosaccharides.ResultsThe A. niger prtT triangle strain created resulted in a reduced extracellular protease production. The prtT triangle background was then used to create strains by deleting exoenzyme encoding genes involved in mono- or disaccharide formation. Consequently, we successfully generated a tailored prtT triangle bglA triangle strain by eliminating a beta-glucosidase (bglA) gene and subsequently deleted two cellobiohydrolases and one beta-xylosidase encoding genes using a multiplex strategy, resulting in the Quintuple triangle strain (prtT triangle; bglA triangle; cbhA triangle; cbhB triangle; xlnD triangle). When applied for sugarcane biomass degradation, the tailored secretomes produced by A. niger resulted in a higher ratio of cellobiose and cellotriose compared with glucose relative to the reference strain. Mass spectrometry revealed that the Quintuple triangle strain secreted alternative cellobiohydrolases and beta-glucosidases to compensate for the absence of major cellulases. Enzymes targeting minor polysaccharides in plant biomass were also upregulated in this tailored strain.ConclusionTailored secretome use increased COS/glucose ratio during sugarcane biomass degradation showing that deleting some enzymatic components is an effective approach for producing customized enzymatic cocktails. Our findings highlight the plasticity of fungal genomes as enzymes that target minor components of plant cell walls, and alternative cellulases were produced by the mutant strain. Despite deletion of important secretome components, fungal growth was maintained in plant biomass. (AU)

• FAPESP's process: 17/26370-0 - GENETIC ENGINEERING OF FUNGAL STRAINS USING CRISPR/Cas9 MULTIPLEX TO PRODUCE TAILOR-MADE CELLULOLYTIC COCKTAILS
• Grantee: Fabiano Jares Contesini
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 15/50612-8 - An integrated approach to explore a novel paradigm for biofuel production from lignocellulosic feedstocks
• Grantee: Telma Teixeira Franco
• Support Opportunities: Research Projects - Thematic Grants
• 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: 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: 17/10083-1 - GENETIC ENGINEERING OF A FUNGAL PLATFORM TO SECRETE LIGNOCELLULOLYTIC ENZYMES FOR OLIGOSACCHARIDE PRODUCTION
• Grantee: Fabiano Jares Contesini
• Support Opportunities: Scholarships in Brazil - Post-Doctoral