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| Author(s): |
Jaqueline Aline Gerhardt
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: | 2023-10-27 |
| Examining board members: |
André Ricardo de Lima Damásio;
Fernando Segato;
Leonardo Talachia Rosa;
Maria Celia Bertolini;
Evandro Araújo de Souza
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| Advisor: | André Ricardo de Lima Damásio |
| Abstract | |
Filamentous fungi of the genus Aspergillus have a natural ability to degrade lignocellulosic biomass. These microorganisms are capable of secreting large amounts of carbohydrate-active enzymes (CAZymes) that support their saprophytic lifestyle. Among these CAZymes, we can mention beta-xylosidases, which are classified as glycoside hydrolases (GHs). They play a crucial role in breaking down plant biomass, releasing xylose from xylooligosaccharides during the degradation of the hemicellulose portion. Furthermore, Aspergillus spp. have all the machinery for post-translational modifications (PTMs) such as proteolytic cleavage, disulfide bond formation, and protein glycosylation. This capability provides advantages in using these organisms as a host for protein production on large scale. One prevalent PTM in eukaryotic systems is asparagine-linked glycosylation or N-glycosylation. N-glycosylation of proteins is indispensable for a wide range of cellular processes, including immune responses, cellular communication, intracellular trafficking, stability, secretion, folding, and protein activity. In this study, we explored how genes associated with N-glycan synthesis impact protein production in Aspergillus nidulans, aiming to address the difficulties in producing significant amounts of recombinant protein from filamentous fungi. Our previous observations revealed that mutations in N-glycosylation sites have notable effects on thermal stability, secretion, folding, and catalytic efficiency of a recombinant GH3 beta-xylosidase (BxlB) produced in A. nidulans. Based on that, we formulated a hypothesis that the deletion of genes involved in the N-glycan pathway might influence the secretion of recombinant proteins. To test our hypothesis, we designed CRISPR/Cas9 multiplex technology to systematically knock out 14 genes associated with N-glycan assembly (alg7, alg13, alg1, alg2, alg11, rft1, alg3, alg9, alg12, alg6 and AN5748) and protein quality control (clxA, gtbA, and uggt) within A. nidulans, resulting in nine viable single mutants. Subsequently, the bxlb gene (AN8401) was transformed into each single mutant as well as into the reference strain for constitutive expression, carefully assessing BxlB secretion and enzymatic activity. The deletion of most of the target genes did not impact protein secretion or fungal growth. Intriguingly, the enzymatic activity of BxlB produced by mutant strains was significantly reduced, while BxlB secretion remained unaffected in the most part of mutants. Further exploration revealed that the simultaneous deletion of alg3 and alg9 increased the BxlB secretion, while maintaining the same kinetic parameters relative to the reference strain. In contrast, the combined deletion of alg3, alg6, and alg9 did not affect BxlB secretion but reduced catalytic efficiency by 20%, suggesting that the N-glycan inner glucose is important along the endoplasmic reticulum quality control, at least partially. In addition, the secretomes of glycomutant strains were analyzed by proteomics showing that the deletion of genes involved in N-glycan synthesis decreased the overall secretion of some CAZymes, while a mutant-specific cluster of upregulated CAZymes was also observed, suggesting truncated N-glycans favor the fold and secretion of a group of proteins, while interfering negatively in the production of another group. These data contribute to understanding the importance of N-glycans for protein folding, secretion, and function in filamentous fungi, which are widely used microbial cell factories for enzyme and other recombinant protein production. Studying the impact of gene deletions on N-glycan structures can provide valuable insights into the significance of post-translational modifications in both the overall protein secretion process and the synthesis of recombinant proteins in fungal cell factories (AU) | |
| 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 |