Xylan degradation in filamentous fungi and bacteria

Vasconcelos Pereira, Gabriel

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Description

Title

Xylan degradation in filamentous fungi and bacteria

Author(s)

Vasconcelos Pereira, Gabriel

Issue Date

2016-12-09

Director of Research (if dissertation) or Advisor (if thesis)

Cann, Isaac

Committee Member(s)

• Mackie, Roderick
• Ridlon, Jason

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• Xylan degradation
• Filamentous fungi
• Heterologous expression
• Biofuels
• Bacteria
• Bacteroides
• Human gut
• Fiber degradation

Abstract

Biochemical characterization of two recombinant gh10 family glycosyl hidrolases and differential expression in two model filamentous fungi Polysaccharides from plant cell walls are the most abundant biomass on Earth and are important resource for biofuel production. The main components of plant cell walls are cellulose, hemicellulose and lignin. Hemicellulose is the second most abundant component of renewable biomass and as arabinoxylan, is mainly composed of xylose and arabinose. Filamentous fungi, such as Trichoderma reesei, produce gram-per-liter levels of glycoside hydrolases (GHs). GH enzymes are required for hydrolysis of the glycosidic bonds present in complex polysaccharides, releasing fermentable sugars. In this project, we investigated the biochemical characteristics of two endoxylanases in two model filamentous fungi, Neurospora crassa and Aspergillus nidulans. Putative endoxylanase genes from N. crassa (ncu05924) and A. nidulans (an1818) were expressed homologously and heterologously in both filamentous fungi. Here, we demonstrate that A. nidulans was able to expressed and secrete at the same levels, both the recombinant homologous (AN1818) and heterologous (NCU05924) proteins, while N. crassa expressed the recombinant homologous protein at 26-fold more than the recombinant heterologous protein. All 4 endoxylanases had similar optimal pH (~5.8) and temperature (50 to ~55°C), similar secondary structures, and comparable glycosylation patterns. High performance liquid chromatography (HPLC) was used to identify the end products released by each enzyme from xylan substrates. The specific activity of AN1818 was ~50% higher than NCU05924 on different model xylans. Xylan degrading enzymes from human colonic bacteroides intestinalis1 Many human diets contain arabinoxylan, and the ease of genome sequencing coupled with reduced cost have led to unraveling the arsenal of genes utilized by the colonic Bacteroidetes to depolymerize this polysaccharide. The colonic Bacteroidetes with potential to ferment arabinoxylans include Bacteroides intestinalis. In this study, we analyzed the hydrolytic activities of members of a xylan degradation cluster encoded on the genome of Bacteroides intestinalis DSM 17393. Here, it is demonstrated that a cocktail of the xylanolytic enzymes completely hydrolyze arabinoxylans found in human diets. Fascinatingly, this bacterium and other relatives have evolved and secrete a unique bifunctional endoxylanase/arabinofuranosidase in the same polypeptide. The bifunctional enzyme and other secreted enzymes attack the polysaccharides extracellularly to remove the side-chains, exposing the xylan backbone for cleavage to xylo-oligosaccharides and xylose. These end products are transported into the cell where a β-xylosidase cleaves the oligosaccharides to fermentable sugars. While our experiments focused on B. intestinalis, it is likely that the extracellular enzymes also release nutrients to members of the colonic microbial community that practice cross-feeding. The conservation of the genes characterized in this study in other colonic Bacteroidetes alludes to a conserved strategy for energy acquisition from xylans, a component of human diets.

Graduation Semester

2016-12

Type of Resource

text

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Copyright and License Information

Copyright 2016 Gabriel V. Pereira

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