Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris |
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| Institution: | 1. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;2. Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing 210037, China;3. Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture, Chengdu 610041, China;4. Department of Biomedical, Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221, United States;3. Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, 48109;4. Department of Molecular Sciences, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden;1. Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna, Austria;2. Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea;3. School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria;4. Austrian Centre of Industrial Biotechnology, Vienna, Austria;5. Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria;6. Metabolic Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 plus program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea;1. Department of Process Engineering, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa;2. Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa |
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| Abstract: | The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast. |
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| Keywords: | Codon optimization AOX1 and GAP promoter High cell density fed batch cultivation |
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