title: Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris.
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 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/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.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China. Electronic address: fubaosun@jiangnan.edu.cn.
authors.forename: Fubao Fuelbiol
authors.initials: FF
authors.lastname: Sun
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing 210037, China.
authors.forename: Renhui
authors.initials: R
authors.lastname: Bai
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture, Chengdu 610041, China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Yang
authors.affiliation: Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, Nanjing Forestry University, Nanjing 210037, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture, Chengdu 610041, China. Electronic address: hejing@caas.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: He
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Chundi
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Biomedical, Chemical and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221, United States.
authors.forename: Maobing
authors.initials: M
authors.lastname: Tu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003062:Codon / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
chemical_list: D003062:Codon; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: AOX1 and GAP promoter; Codon optimization; High cell density fed batch cultivation; Pichia pastoris; Trichoderma reesei cellobiohydrolase II (Cel6A)