pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium
Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unma...
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| Format: | Article |
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Frontiers Media S.A.
2020-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2020.00441/full |
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| author | Yongchuang Liu Xiangrong He Pingping Zhu Minggen Cheng Qing Hong Xin Yan Xin Yan |
| author_facet | Yongchuang Liu Xiangrong He Pingping Zhu Minggen Cheng Qing Hong Xin Yan Xin Yan |
| author_sort | Yongchuang Liu |
| collection | DOAJ |
| description | Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheSAG, which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheSAG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The Ptac promoter and the ribosome-binding site region of mmoX were employed to control pheSAG, producing the pheSAG-3 CSM. M. buryatense 5GB1C harboring pheSAG-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheSAG-3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days. |
| first_indexed | 2024-12-14T04:11:16Z |
| format | Article |
| id | doaj.art-389798ec30434af598bb34591778f449 |
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| language | English |
| last_indexed | 2024-12-14T04:11:16Z |
| publishDate | 2020-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-389798ec30434af598bb34591778f4492022-12-21T23:17:40ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-03-011110.3389/fmicb.2020.00441513145pheSAG Based Rapid and Efficient Markerless Mutagenesis in MethylotuvimicrobiumYongchuang Liu0Xiangrong He1Pingping Zhu2Minggen Cheng3Qing Hong4Xin Yan5Xin Yan6Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaKey Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaKey Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaKey Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaKey Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaKey Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, ChinaJiangsu Provincial Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing, ChinaDue to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheSAG, which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheSAG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The Ptac promoter and the ribosome-binding site region of mmoX were employed to control pheSAG, producing the pheSAG-3 CSM. M. buryatense 5GB1C harboring pheSAG-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheSAG-3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.https://www.frontiersin.org/article/10.3389/fmicb.2020.00441/fullcounterselectionpheSMethylotuvimicrobiumelectroporation-basedmarkerless mutagenesis |
| spellingShingle | Yongchuang Liu Xiangrong He Pingping Zhu Minggen Cheng Qing Hong Xin Yan Xin Yan pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium Frontiers in Microbiology counterselection pheS Methylotuvimicrobium electroporation-based markerless mutagenesis |
| title | pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium |
| title_full | pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium |
| title_fullStr | pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium |
| title_full_unstemmed | pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium |
| title_short | pheSAG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium |
| title_sort | phesag based rapid and efficient markerless mutagenesis in methylotuvimicrobium |
| topic | counterselection pheS Methylotuvimicrobium electroporation-based markerless mutagenesis |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2020.00441/full |
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