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Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as "asparagine-linked glycosylation 3", or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.

N-linked glycosylation, filamentous fungi, Aspergillus niger, asparagine-linked glycosylation 3 (ALG3), Trichoderma reesei cellobiohydrolase (Cel7A), protein secretion and expression

NCBI PubMed ID: 24076077
Publication DOI: 10.1016/j.fgb.2013.09.004
Journal NLM ID: 9607601
Publisher: Orlando, FL : Academic Press / Elsevier
Correspondence: Dai Z pnnl.gov>, Baker SE pnnl.gov>
Institutions: National Bioenergy Center, National Renewable Energy Laboratory, Golden, USA, Department of Chemical Engineering, Colorado School of Mines, Golden, USA, Biosciences Center, National Renewable Energy Laboratory, Golden, USA, Fungal Biotechnology Team, Chemical and Biological Processes Development Group, Pacific Northwest National Laboratory, Richland, USA, Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, USA, Bioproducts, Science Engineering Laboratory, Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Richland, USA, Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, USA