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| Author(s): |
Marcelo Ventura Rubio
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2018-07-12 |
| Examining board members: |
André Ricardo de Lima Damásio;
Maria de Lourdes Teixeira de Moraes Polizeli;
Marcos Silveira Buckeridge;
Thamy Lívia Ribeiro Corrêa
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| Advisor: | André Ricardo de Lima Damásio; Fabio Marcio Squina |
| Abstract | |
The industrial enzymes market covers a wide variety of applications such as personal care, food industries, biofuels, biopolymers, among others. The main bottleneck for using enzymatic cocktails at large scale is the high-cost, which increases the bioproducts final value. Thus the bioproducts cost can be significantly reduced by improving the yield of enzymes production by molecular biology of fungal strains and/or by improving enzymes efficiency by protein engineering. Filamentous fungi are the main producers of industrial enzymes due to the great enzymatic repertoire and the high levels of protein secretion. The genus Aspergillus includes microorganisms that naturally degrade lignocellulosic biomass by secreting large amounts of carbohydrateactive enzymes (CAZymes). The capacity of Aspergillus to perform post-translational modifications such as proteolytic cleavage, disulfide bond formation and glycosylation, provides an additional advantage to their use as hosts for heterologous protein production. However, the overexpression of target proteins overloads the Nglycosylation pathway and folding mechanisms resulting in the accumulation of unfolded or misfolded proteins. Misfolded proteins are directed to degradation, consequently reducing the secretion yield. Furthermore, the position and the number of N-glycans attached to proteins can influence their secretion and functional properties. Aiming to minimize the cost of enzymes production, Aspergillus nidulans was used as a model organism to study the effect of N-glycosylation in the secretion of industrial enzymes. A proteomics approach identified 265 N-glycoproteins secreted by A. nidulans grown on xylan and pretreated sugarcane bagasse. CAZymes corresponded to more than 50% of the secretome and 182 N-glycosylated sites were validated by LC-MS/MS. In order to investigate the influence of N-glycosylation in protein secretion by A. nidulans, a beta-xylosidase (BxlB) was selected as target protein due to its high secretion level during growth on xylan. beta-xylosidases are glycoside hydrolases that assist plant biomass degradation by releasing xylose from xylooligosaccharides and/or xylobiose. Seven N-glycosylation sites were predicted in the BxlB and five were validated by LC-MS/MS. Glycomutants were designed in order to investigate the influence of glycosylation on beta-xylosidase secretion and function. The deglycosylated mutant (BxlBDeglyc) showed similar results regarding enzyme secretion and activity compared to the wild-type (BxlBwt). Interestingly, a partially glycosylated mutant (BxlBN1;5;7) showed increased activity and secretion levels. On the other hand, the mutant BxlBCC, in which the glycosylation context was changed by the design of four new N-glycosylation sites, was expressed but not secreted in A. nidulans. BxlBwt, BxlBDeglyc and BxlBN1;5;7 showed similar secondary structure, although the mutants had lower melting temperature compared to the wild-type. Moreover, an additional BxlB glycomutant maintaining only two N-glycosylated sites (BxlBN5;7) showed improved catalytic efficiency. This study showed the influence of Nglycosylation on BxlB function and production in A. nidulans, reinforcing that proteins glycoengineering is a promising tool to enhance thermal stability, secretion and enzymatic activity. Our report may also support N-glycosylation modification in CAZymes to biotechnological applications (AU) | |
| FAPESP's process: | 13/24988-5 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases |
| Grantee: | Marcelo Ventura Rubio |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |