title: A β-glucosidase hyper-production Trichoderma reesei mutant reveals a potential role of cel3D in cellulase production.
abstract: BACKGROUND
  The conversion of cellulose by cellulase to fermentable sugars for biomass-based products such as cellulosic biofuels, biobased fine chemicals and medicines is an environment-friendly and sustainable process, making wastes profitable and bringing economic benefits. Trichoderma reesei is the well-known major workhorse for cellulase production in industry, but the low β-glucosidase activity in T. reesei cellulase leads to inefficiency in biomass degradation and limits its industrial application. Thus, there are ongoing interests in research to develop methods to overcome this insufficiency. Moreover, although β-glucosidases have been demonstrated to influence cellulase production and participate in the regulation of cellulase production, the underlying mechanism remains unclear.
  
  
  RESULTS
  The T. reesei recombinant strain TRB1 was constructed from T. reesei RUT-C30 by the T-DNA-based mutagenesis. Compared to RUT-C30, TRB1 displays a significant enhancement of extracellular β-glucosidase (BGL1) activity with 17-fold increase, a moderate increase of both the endoglucanase (EG) activity and the exoglucanase (CBH) activity, a minor improvement of the total filter paper activity, and a faster cellulase induction. This superiority of TRB1 over RUT-C30 is independent on carbon sources and improves the saccharification ability of TRB1 cellulase on pretreated corn stover. Furthermore, TRB1 shows better resistance to carbon catabolite repression than RUT-C30. Secretome characterization of TRB1 shows that the amount of CBH, EG and BGL in the supernatant of T. reesei TRB1 was indeed increased along with the enhanced activities of these three enzymes. Surprisingly, qRT-PCR and gene cloning showed that in TRB1 β-glucosidase cel3D was mutated through the random insertion by AMT and was not expressed.
  
  
  CONCLUSIONS
  The T. reesei recombinant strain TRB1 constructed in this study is more desirable for industrial application than the parental strain RUT-C30, showing extracellular β-glucosidase hyper production, high cellulase production within a shorter time and a better resistance to carbon catabolite repression. Disruption of β-glucosidase cel3D in TRB1 was identified, which might contribute to the superiority of TRB1 over RUT-C30 and might play a role in the cellulase production. These results laid a foundation for future investigations to further improve cellulase enzymatic efficiency and reduce cost for T. reesei cellulase production.
authors: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China.
authors: Chengcheng
authors: C
authors: Li
authors: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China. linfengming@seu.edu.cn.|, 37 Jinxianghe Road, Xuanwu District, Nanjing, 210008, Jiangsu Province, China. linfengming@seu.edu.cn.
authors: Fengming
authors: F
authors: Lin
authors: State Key Laboratory of Medical Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
authors: Yizhen
authors: Y
authors: Li
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
authors: Wei
authors: W
authors: Wei
authors: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China.
authors: Hongyin
authors: H
authors: Wang
authors: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, 300072, People's Republic of China.
authors: Lei
authors: L
authors: Qin
authors: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
authors: Zhihua
authors: Z
authors: Zhou
authors: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, 300072, People's Republic of China.
authors: Bingzhi
authors: B
authors: Li
authors: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China.
authors: Fugen
authors: F
authors: Wu
authors: Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI, 48109, USA.
authors: Zhan
authors: Z
authors: Chen
mesh_terms: D018533:Biomass; D002244:Carbon / Q000378:metabolism; D057465:Catabolite Repression / Q000235:genetics; D002480:Cellulase / Q000096:biosynthesis* / Q000191:economics; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005285:Fermentation; D007218:Industrial Microbiology / Q000379:methods; D016296:Mutagenesis; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D002244:Carbon; D001617:beta-Glucosidase; D002480:Cellulase
keywords: Carbon catabolite repression; Cel3d; Cellulase; Hyper-production; Trichoderma reesei; β-Glucosidase