title: Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.
abstract: Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris.
authors: State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, China.
authors: Chong
authors: C
authors: Sha
authors: Xiao-Wei
authors: XW
authors: Yu
authors: Nai-Xin
authors: NX
authors: Lin
authors: Meng
authors: M
authors: Zhang
authors: Yan
authors: Y
authors: Xu
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics*; D018628:Gene Dosage; D015870:Gene Expression; D005800:Genes, Fungal; D005818:Genetic Engineering; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics*; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D008049:Lipase; D019704:Protein Disulfide-Isomerases
keywords: Chaperone PDI; Gene dosage; Lipase; Pichia pastoris