CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris
Abstract Background Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-08-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-019-1194-x |
| _version_ | 1818276756569718784 |
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| author | Qi Liu Xiaona Shi Lili Song Haifeng Liu Xiangshan Zhou Qiyao Wang Yuanxing Zhang Menghao Cai |
| author_facet | Qi Liu Xiaona Shi Lili Song Haifeng Liu Xiangshan Zhou Qiyao Wang Yuanxing Zhang Menghao Cai |
| author_sort | Qi Liu |
| collection | DOAJ |
| description | Abstract Background Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris. Results A non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR–Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P AOX1 UP-g2, P TEF1 UP-g1, and P FLD1 UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR–Cas9-mediated gene co-integration method in P. pastoris. Conclusions A CRISPR–Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris. |
| first_indexed | 2024-12-12T22:50:42Z |
| format | Article |
| id | doaj.art-d840b8e160d942b6a37f737f7d6a8bbe |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-12T22:50:42Z |
| publishDate | 2019-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-d840b8e160d942b6a37f737f7d6a8bbe2022-12-22T00:09:05ZengBMCMicrobial Cell Factories1475-28592019-08-0118111110.1186/s12934-019-1194-xCRISPR–Cas9-mediated genomic multiloci integration in Pichia pastorisQi Liu0Xiaona Shi1Lili Song2Haifeng Liu3Xiangshan Zhou4Qiyao Wang5Yuanxing Zhang6Menghao Cai7State Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyChinare Resources Angde Biotech Pharmaceutical Co., Ltd.State Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyState Key Laboratory of Bioreactor Engineering, East China University of Science and TechnologyAbstract Background Pichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris. Results A non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR–Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P AOX1 UP-g2, P TEF1 UP-g1, and P FLD1 UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR–Cas9-mediated gene co-integration method in P. pastoris. Conclusions A CRISPR–Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.http://link.springer.com/article/10.1186/s12934-019-1194-xPichia pastorisCRISPR–Cas9Homology directed repairMultiloci integrationMultistep enzymatic pathway |
| spellingShingle | Qi Liu Xiaona Shi Lili Song Haifeng Liu Xiangshan Zhou Qiyao Wang Yuanxing Zhang Menghao Cai CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris Microbial Cell Factories Pichia pastoris CRISPR–Cas9 Homology directed repair Multiloci integration Multistep enzymatic pathway |
| title | CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris |
| title_full | CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris |
| title_fullStr | CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris |
| title_full_unstemmed | CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris |
| title_short | CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris |
| title_sort | crispr cas9 mediated genomic multiloci integration in pichia pastoris |
| topic | Pichia pastoris CRISPR–Cas9 Homology directed repair Multiloci integration Multistep enzymatic pathway |
| url | http://link.springer.com/article/10.1186/s12934-019-1194-x |
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