Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris
Abstract Background The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-09-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-022-01908-z |
| _version_ | 1811211473324605440 |
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| author | Kun Zhang Xingpeng Duan Peng Cai Linhui Gao Xiaoyan Wu Lun Yao Yongjin J. Zhou |
| author_facet | Kun Zhang Xingpeng Duan Peng Cai Linhui Gao Xiaoyan Wu Lun Yao Yongjin J. Zhou |
| author_sort | Kun Zhang |
| collection | DOAJ |
| description | Abstract Background The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. Results The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. Conclusions Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds. |
| first_indexed | 2024-04-12T05:13:56Z |
| format | Article |
| id | doaj.art-7790916adff4433ab97a2a3ab3e3845c |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-12T05:13:56Z |
| publishDate | 2022-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-7790916adff4433ab97a2a3ab3e3845c2022-12-22T03:46:41ZengBMCMicrobial Cell Factories1475-28592022-09-012111910.1186/s12934-022-01908-zFusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastorisKun Zhang0Xingpeng Duan1Peng Cai2Linhui Gao3Xiaoyan Wu4Lun Yao5Yongjin J. Zhou6Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesDivision of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of SciencesAbstract Background The methylotrophic yeast Pichia pastoris is considered as an ideal host for the production of recombinant proteins and chemicals. However, low homologous recombination (HR) efficiency hinders its precise and extensive genetic manipulation. To enhance the homology-directed repair over non-homologous end joining (NHEJ), we expressed five exonucleases that were fused with the Cas9 for enhancing end resection of double strand breaks (DSBs) of DNA cuts. Results The endogenous exonuclease Mre11 and Exo1 showed the highest positive rates in seamless deletion of FAA1, and fusing the MRE11 to the C-terminal of CAS9 had the highest positive rate and relatively high number of clones. We observed that expression of CAS9-MRE11 significantly improved positive rates when simultaneously seamless deletion of double genes (from 76.7 to 86.7%) and three genes (from 10.8 to 16.7%) when overexpressing RAD52. Furthermore, MRE11 overexpression significantly improved the genomic integration of multi-fragments with higher positive rate and clone number. Conclusions Fusion expression of the endogenous exonuclease Mre11 with Cas9 enhances homologous recombination efficiency in P. pastoris. The strategy described here should facilitate the metabolic engineering of P. pastoris toward high-level production of value-added compounds.https://doi.org/10.1186/s12934-022-01908-zPichia pastorisCRISPR/Cas9Gene editingHomology-directed repairMetabolic engineering |
| spellingShingle | Kun Zhang Xingpeng Duan Peng Cai Linhui Gao Xiaoyan Wu Lun Yao Yongjin J. Zhou Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris Microbial Cell Factories Pichia pastoris CRISPR/Cas9 Gene editing Homology-directed repair Metabolic engineering |
| title | Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris |
| title_full | Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris |
| title_fullStr | Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris |
| title_full_unstemmed | Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris |
| title_short | Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris |
| title_sort | fusing an exonuclease with cas9 enhances homologous recombination in pichia pastoris |
| topic | Pichia pastoris CRISPR/Cas9 Gene editing Homology-directed repair Metabolic engineering |
| url | https://doi.org/10.1186/s12934-022-01908-z |
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