Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts
Abstract Background The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and othe...
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| Format: | Article |
| Language: | English |
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BMC
2018-10-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-018-1271-0 |
| _version_ | 1811233345361674240 |
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| author | Laiyou Wang Aihua Deng Yun Zhang Shuwen Liu Yong Liang Hua Bai Di Cui Qidi Qiu Xiuling Shang Zhao Yang Xiuping He Tingyi Wen |
| author_facet | Laiyou Wang Aihua Deng Yun Zhang Shuwen Liu Yong Liang Hua Bai Di Cui Qidi Qiu Xiuling Shang Zhao Yang Xiuping He Tingyi Wen |
| author_sort | Laiyou Wang |
| collection | DOAJ |
| description | Abstract Background The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology. Results In this study, we developed a CRISPR–Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette P ScTEF1 -TAL-P ScTPI1 -4CL-P ScTEF2 -STS were integrated into the genome. Resveratrol production was increased ~ 20 fold compared to the one copy integrant and reached 97.23 ± 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae. Conclusions An efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species. |
| first_indexed | 2024-04-12T11:19:16Z |
| format | Article |
| id | doaj.art-89f38284172242c5885d1c25bac97cc6 |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-04-12T11:19:16Z |
| publishDate | 2018-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-89f38284172242c5885d1c25bac97cc62022-12-22T03:35:26ZengBMCBiotechnology for Biofuels1754-68342018-10-0111111610.1186/s13068-018-1271-0Efficient CRISPR–Cas9 mediated multiplex genome editing in yeastsLaiyou Wang0Aihua Deng1Yun Zhang2Shuwen Liu3Yong Liang4Hua Bai5Di Cui6Qidi Qiu7Xiuling Shang8Zhao Yang9Xiuping He10Tingyi Wen11CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of SciencesCAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of SciencesAbstract Background The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology. Results In this study, we developed a CRISPR–Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette P ScTEF1 -TAL-P ScTPI1 -4CL-P ScTEF2 -STS were integrated into the genome. Resveratrol production was increased ~ 20 fold compared to the one copy integrant and reached 97.23 ± 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae. Conclusions An efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species.http://link.springer.com/article/10.1186/s13068-018-1271-0CRISPR–Cas9-assisted multiplex genome editingMarkerless multi-locus integrationMarkerless multi-copy integrationOgataea polymorphaSaccharomyces cerevisiae |
| spellingShingle | Laiyou Wang Aihua Deng Yun Zhang Shuwen Liu Yong Liang Hua Bai Di Cui Qidi Qiu Xiuling Shang Zhao Yang Xiuping He Tingyi Wen Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts Biotechnology for Biofuels CRISPR–Cas9-assisted multiplex genome editing Markerless multi-locus integration Markerless multi-copy integration Ogataea polymorpha Saccharomyces cerevisiae |
| title | Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts |
| title_full | Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts |
| title_fullStr | Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts |
| title_full_unstemmed | Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts |
| title_short | Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts |
| title_sort | efficient crispr cas9 mediated multiplex genome editing in yeasts |
| topic | CRISPR–Cas9-assisted multiplex genome editing Markerless multi-locus integration Markerless multi-copy integration Ogataea polymorpha Saccharomyces cerevisiae |
| url | http://link.springer.com/article/10.1186/s13068-018-1271-0 |
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