Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

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• 作者：Ziyu Dai ; Uma K. Aryal ; Anil Shukla ; Wei-Jun Qian ; Richard D. Smith ; Jon K. Magnuson ; William S. Adney ; Gregg T. Beckham ; Roman Brunecky ; Michael E. Himmel ; Stephen R. Decker ; Xiaohui Ju ; Xiao Zhang ; Scott E. Baker
• 关键词：Asparagine-linked glycosylation 3 (ALG3) ; Aspergillus niger ; Filamentous fungi ; N-linked glycosylation ; Trichoderma reesei cellobiohydrolase (Cel7A) ; Protein secretion and expression
• 刊名：Fungal Genetics and Biology
• 出版年：December, 2013
• 年：2013
• 卷：61
• 期：Complete
• 页码：120-132
• 全文大小：2212 K

文摘

Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl 伪-1,3-mannosyltransferase (also known as 鈥渁sparagine-linked glycosylation 3鈥? 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 (alg3螖) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3螖 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 alg3螖 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 alg3螖, 63% of which were secreted at higher levels in alg3螖 strain than the parent. The rCel7A expressed in the alg3螖 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.