Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris
Pichia pastoris is one of the most commonly used hosts for producing heterologous proteins, whereas production levels vary depending on the protein of interest and are also regulated by regulatory factors. We conducted RNA-seq by expressing the reporter EGFP and observed significant upregulation of...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-01-01
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| Series: | Current Research in Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S259026282400008X |
| _version_ | 1827221822765006848 |
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| author | Shengyan Wang Huijia Dai Qingling Tang Yujing Yu Yaying Xie Tao Wang Yide Huang |
| author_facet | Shengyan Wang Huijia Dai Qingling Tang Yujing Yu Yaying Xie Tao Wang Yide Huang |
| author_sort | Shengyan Wang |
| collection | DOAJ |
| description | Pichia pastoris is one of the most commonly used hosts for producing heterologous proteins, whereas production levels vary depending on the protein of interest and are also regulated by regulatory factors. We conducted RNA-seq by expressing the reporter EGFP and observed significant upregulation of certain subunits (Sec61p, Sbh1p, Sss1p, Sec66p and Sec72p) of the Sec complex in the high-expression recombinant GS115 stains. The overexpression of these genes may increase the expression levels of heterogeneous proteins. In this study, the endogenous promoters of the Sec complex subunits Sbh1p, Sss1p, Sec66p and Sec72p were isolated and verified their activity using the Lac-Z reporter gene. Sss1, Sbh1, Sec66 and Sec72 were overexpressed under the control of their own promoters in Pichia pastoris, respectively. The overexpression of Sss1, Sbh1, Sec66 and Sec72 in cells was confirmed by fluorescent microscope and Western blot analysis. The α-amylase was employed to evaluate the effect of overexpression of the Sec subunits on the heterologous protein expression. The results demonstrated that the α-amylase activity increased by 16%, 58%, 16% and 17% in the strains overexpressing Sss1, Sbh1, Sec66 and Sec72, respectively. Engineering the protein translocation pathway can be an alternative to enhance heterogeneous proteins in Pichia pastoris expression system. |
| first_indexed | 2024-03-08T04:49:14Z |
| format | Article |
| id | doaj.art-0003edbff10c4e57900e30c306bf5a6d |
| institution | Directory Open Access Journal |
| issn | 2590-2628 |
| language | English |
| last_indexed | 2025-03-21T16:28:42Z |
| publishDate | 2024-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Current Research in Biotechnology |
| spelling | doaj.art-0003edbff10c4e57900e30c306bf5a6d2024-06-17T05:56:48ZengElsevierCurrent Research in Biotechnology2590-26282024-01-017100182Engineering protein translocation pathway to improve recombinant proteins in Pichia pastorisShengyan Wang0Huijia Dai1Qingling Tang2Yujing Yu3Yaying Xie4Tao Wang5Yide Huang6Engineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, ChinaEngineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, ChinaEngineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, ChinaEngineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, ChinaEngineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, ChinaShengli Clinical Medical College of Fujian Medical University, Fuzhou 350001, China; Corresponding authors.Engineering Research Center of Industrial Microbiology, College of Life Sciences, Fujian Normal University, Fuzhou 350007, China; Corresponding authors.Pichia pastoris is one of the most commonly used hosts for producing heterologous proteins, whereas production levels vary depending on the protein of interest and are also regulated by regulatory factors. We conducted RNA-seq by expressing the reporter EGFP and observed significant upregulation of certain subunits (Sec61p, Sbh1p, Sss1p, Sec66p and Sec72p) of the Sec complex in the high-expression recombinant GS115 stains. The overexpression of these genes may increase the expression levels of heterogeneous proteins. In this study, the endogenous promoters of the Sec complex subunits Sbh1p, Sss1p, Sec66p and Sec72p were isolated and verified their activity using the Lac-Z reporter gene. Sss1, Sbh1, Sec66 and Sec72 were overexpressed under the control of their own promoters in Pichia pastoris, respectively. The overexpression of Sss1, Sbh1, Sec66 and Sec72 in cells was confirmed by fluorescent microscope and Western blot analysis. The α-amylase was employed to evaluate the effect of overexpression of the Sec subunits on the heterologous protein expression. The results demonstrated that the α-amylase activity increased by 16%, 58%, 16% and 17% in the strains overexpressing Sss1, Sbh1, Sec66 and Sec72, respectively. Engineering the protein translocation pathway can be an alternative to enhance heterogeneous proteins in Pichia pastoris expression system.http://www.sciencedirect.com/science/article/pii/S259026282400008XPichia pastorisSec complexSecretion pathwayHeterologous expression |
| spellingShingle | Shengyan Wang Huijia Dai Qingling Tang Yujing Yu Yaying Xie Tao Wang Yide Huang Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris Current Research in Biotechnology Pichia pastoris Sec complex Secretion pathway Heterologous expression |
| title | Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris |
| title_full | Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris |
| title_fullStr | Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris |
| title_full_unstemmed | Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris |
| title_short | Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris |
| title_sort | engineering protein translocation pathway to improve recombinant proteins in pichia pastoris |
| topic | Pichia pastoris Sec complex Secretion pathway Heterologous expression |
| url | http://www.sciencedirect.com/science/article/pii/S259026282400008X |
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