title: Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.
abstract: BACKGROUND
  The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently.
  
  
  RESULTS
  The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose.
  
  
  CONCLUSIONS
  We report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081, People's Republic of China.
authors.forename: Xianli
authors.initials: X
authors.lastname: Xue
authors.affiliation: College of Biological Sciences and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China.
authors.forename: Yilan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Qin
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081, People's Republic of China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081, People's Republic of China. suxiaoyun@caas.cn.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Su
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing, 100081, People's Republic of China. binyao@caas.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D055326:Neosartorya / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D002475:Cellobiose; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D005947:Glucose
keywords: Cellulase; DNA assembler; Neosartorya fischeri; Trichoderma reesei; β-Glucosidase