A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris
The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite...
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| Format: | Article |
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KeAi Communications Co., Ltd.
2023-09-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X23000480 |
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| author | Xiang Wang Yi Li Zhehao Jin Xiangjian Liu Xiang Gao Shuyuan Guo Tao Yu |
| author_facet | Xiang Wang Yi Li Zhehao Jin Xiangjian Liu Xiang Gao Shuyuan Guo Tao Yu |
| author_sort | Xiang Wang |
| collection | DOAJ |
| description | The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in P. pastoris. Firstly, we improved the HR rates of P. pastoris through knocking out the non-homologous-end-joining gene (Δku70) and overexpressing HR-related proteins (RAD52 and RAD59), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes PpURA3 as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter (scFUR4) to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in P. pastoris. The systems will contribute to P. pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization. |
| first_indexed | 2024-03-12T11:02:32Z |
| format | Article |
| id | doaj.art-768b8ea4277f46128da0150b7a4319c2 |
| institution | Directory Open Access Journal |
| issn | 2405-805X |
| language | English |
| last_indexed | 2024-03-12T11:02:32Z |
| publishDate | 2023-09-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj.art-768b8ea4277f46128da0150b7a4319c22023-09-02T04:31:52ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2023-09-0183445451A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastorisXiang Wang0Yi Li1Zhehao Jin2Xiangjian Liu3Xiang Gao4Shuyuan Guo5Tao Yu6Center for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; University of the Chinese Academy of Sciences, Beijing, 100049, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; University of the Chinese Academy of Sciences, Beijing, 100049, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, ChinaCenter for Materials Synthetic Biology, CAS Key Laboratory of Quantitative Engineering Biology of CAS, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academic of Science, Shenzhen, 518055, ChinaCenter for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; Corresponding author.Center for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China; Corresponding author.The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in P. pastoris. Firstly, we improved the HR rates of P. pastoris through knocking out the non-homologous-end-joining gene (Δku70) and overexpressing HR-related proteins (RAD52 and RAD59), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes PpURA3 as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter (scFUR4) to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in P. pastoris. The systems will contribute to P. pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.http://www.sciencedirect.com/science/article/pii/S2405805X23000480Pichia pastorisUracil auxotrophCRISPR/Cas9Recyclable selective markerHomology directed repair |
| spellingShingle | Xiang Wang Yi Li Zhehao Jin Xiangjian Liu Xiang Gao Shuyuan Guo Tao Yu A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris Synthetic and Systems Biotechnology Pichia pastoris Uracil auxotroph CRISPR/Cas9 Recyclable selective marker Homology directed repair |
| title | A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris |
| title_full | A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris |
| title_fullStr | A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris |
| title_full_unstemmed | A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris |
| title_short | A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris |
| title_sort | novel crispr cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple step metabolic rewriting in pichia pastoris |
| topic | Pichia pastoris Uracil auxotroph CRISPR/Cas9 Recyclable selective marker Homology directed repair |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X23000480 |
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