Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris
Pichia pastoris, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the P. pastoris cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-g...
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KeAi Communications Co., Ltd.
2024-12-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X24000917 |
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| author | Shupeng Ruan Yuxin Yang Xinying Zhang Guanjuan Luo Ying Lin Shuli Liang |
| author_facet | Shupeng Ruan Yuxin Yang Xinying Zhang Guanjuan Luo Ying Lin Shuli Liang |
| author_sort | Shupeng Ruan |
| collection | DOAJ |
| description | Pichia pastoris, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the P. pastoris cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in P. pastoris using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10–100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of P. pastoris, such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of P. pastoris. This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of P. pastoris. This may lead to improved efficiency of genetic engineering in P. pastoris. |
| format | Article |
| id | doaj-art-a28f5efde6a84c2eb2cf955a7f967977 |
| institution | Kabale University |
| issn | 2405-805X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj-art-a28f5efde6a84c2eb2cf955a7f9679772024-12-15T06:16:03ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2024-12-0194759765Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastorisShupeng Ruan0Yuxin Yang1Xinying Zhang2Guanjuan Luo3Ying Lin4Shuli Liang5Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, ChinaGuangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, ChinaGuangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, ChinaGuangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, ChinaGuangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Corresponding author. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China; Corresponding author.Pichia pastoris, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the P. pastoris cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in P. pastoris using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10–100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of P. pastoris, such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of P. pastoris. This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of P. pastoris. This may lead to improved efficiency of genetic engineering in P. pastoris.http://www.sciencedirect.com/science/article/pii/S2405805X24000917P. pastorisCRISPR-Cpf1Integration sitesMetabolic engineering |
| spellingShingle | Shupeng Ruan Yuxin Yang Xinying Zhang Guanjuan Luo Ying Lin Shuli Liang Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris Synthetic and Systems Biotechnology P. pastoris CRISPR-Cpf1 Integration sites Metabolic engineering |
| title | Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris |
| title_full | Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris |
| title_fullStr | Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris |
| title_full_unstemmed | Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris |
| title_short | Screening and characterization of integration sites based on CRISPR-Cpf1 in Pichia pastoris |
| title_sort | screening and characterization of integration sites based on crispr cpf1 in pichia pastoris |
| topic | P. pastoris CRISPR-Cpf1 Integration sites Metabolic engineering |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X24000917 |
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