Development of a counterselectable seamless mutagenesis system in lactic acid bacteria
Abstract Background Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient...
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| Format: | Article |
| Language: | English |
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BMC
2017-07-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-017-0731-8 |
| _version_ | 1811263504633561088 |
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| author | Yongping Xin Tingting Guo Yingli Mu Jian Kong |
| author_facet | Yongping Xin Tingting Guo Yingli Mu Jian Kong |
| author_sort | Yongping Xin |
| collection | DOAJ |
| description | Abstract Background Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. Results The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. Conclusions As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment. |
| first_indexed | 2024-04-12T19:46:32Z |
| format | Article |
| id | doaj.art-63834d074e9e4e05b93653cebc0ac52e |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-12T19:46:32Z |
| publishDate | 2017-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-63834d074e9e4e05b93653cebc0ac52e2022-12-22T03:18:57ZengBMCMicrobial Cell Factories1475-28592017-07-0116111310.1186/s12934-017-0731-8Development of a counterselectable seamless mutagenesis system in lactic acid bacteriaYongping Xin0Tingting Guo1Yingli Mu2Jian Kong3State Key Laboratory of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Shandong UniversityAbstract Background Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. Results The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 l-lactate dehydrogenase promoter (Pldh) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. Conclusions As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 could be used in the wild-type host cells without pretreatment.http://link.springer.com/article/10.1186/s12934-017-0731-8Lactic acid bacteriaTemperature-sensitive plasmidSeamless mutagenesisCounterselectable markerpheS |
| spellingShingle | Yongping Xin Tingting Guo Yingli Mu Jian Kong Development of a counterselectable seamless mutagenesis system in lactic acid bacteria Microbial Cell Factories Lactic acid bacteria Temperature-sensitive plasmid Seamless mutagenesis Counterselectable marker pheS |
| title | Development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| title_full | Development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| title_fullStr | Development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| title_full_unstemmed | Development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| title_short | Development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| title_sort | development of a counterselectable seamless mutagenesis system in lactic acid bacteria |
| topic | Lactic acid bacteria Temperature-sensitive plasmid Seamless mutagenesis Counterselectable marker pheS |
| url | http://link.springer.com/article/10.1186/s12934-017-0731-8 |
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