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Constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastoris using GAP promoter

Systems Microbiology and Biomanufacturing 2022年第3期2卷 498-506页

作者： Yun Hu Renhui Bai Shaohua Dou Zhimeng Wu ali abdulkhani Mohammad ali Asadollahi Abd El-Fatah Abomohra Fubao Sun Key Laboratory of Carbohydrate and Biotechnology and Key Laboratory of Industrial BiotechnologyMinistry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China School of Life Science and Biotechnology Dalian UniversityDalian 116622China Department of Wood and Paper Sciences and Technology Faculty of Natural ResourcesUniversity of Tehran31585-4314 KarajIran Department of Biotechnology Faculty of Biological Science and TechnologyUniversity of Isfahan81746-73441 IsfahanIran New Energy and Environmental Laboratory School of Architecture and Civil EngineeringChengdu UniversityChengdu 610106China

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and *** biochemical and enzymatic properties of the new recombinant were *** a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,*** the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of *** further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L *** high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/*** addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(***),***,TrCel5A was effectively expressed in *** under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.

关键词： Trichoderma reesei endoglucanase(Cel5A) Pichia pastoris GAP promoter Constitutive expression Fermentation optimization CMC enzyme activity

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