{"title":"嗜热链球菌B-6自然转化过程优化及基因组编辑方法开发","authors":"Bingxin Wang, Kexiong Sun, Yukai Sun, Jingbo Wang, Yueqiu Jiang, Xipeng Liu, Jie Li, Huili Liu, Youbao Zhao, Longxian Zhang, Xinfu Zhang","doi":"10.1111/1471-0307.70065","DOIUrl":null,"url":null,"abstract":"<div>\n \n <section>\n \n <h3> Background</h3>\n \n <p><i>Streptococcus thermophilus</i> is invaluable in both dairy factory and scientific research, with diverse strains offering unique industrial advantages. Consequently, there is a high demand for efficient, user-friendly, biosafe and broadly applicable genome editing methods for this bacterium. The conventional approach based on temperature-sensitive plasmids is laborious and time-consuming, while the existing natural transformation method lacks broad applicability. The recently developed endogenous CRISPR-Cas-assisted genome editing system involves cumbersome plasmid construction.</p>\n </section>\n \n <section>\n \n <h3> Aim</h3>\n \n <p>In this study, we aimed to optimise natural transformation protocols and develop efficient, user-friendly, biosafe and broadly applicable genome editing methods for <i>S. thermophilus</i>.</p>\n </section>\n \n <section>\n \n <h3> Methods</h3>\n \n <p>We used <i>S. thermophilus</i> B-6 as a model strain. We characterised the features of the growth curves and the natural competence of this strain. Accordingly, we optimised the natural transformation protocol for this strain. We used this model strain to develop efficient, user-friendly, biosafe and broadly applicable genome editing methods.</p>\n </section>\n \n <section>\n \n <h3> Major Findings</h3>\n \n <p>By continuously implementing ComS<sub>17–24</sub> and exogenous DNAs, as well as elongating the co-incubation time, we upgraded the transformation ratio of this strain from ~10<sup>−5</sup> to ~10<sup>−2</sup>. Leveraging this improvement, we developed a rapid, simple, biosafe and plasmid-free genome editing method for <i>S. thermophilus</i>. Furthermore, we established a novel endogenous CRISPR-Cas-assisted and efficient genome editing pathway that eliminates the need for additional plasmid construction. Using these advanced techniques, we successfully generated multiple CRISPR-Cas system deleted strains and a <i>recA</i> overexpressing strain.</p>\n </section>\n \n <section>\n \n <h3> Scientific and Industrial Implications</h3>\n \n <p>Our findings contribute to better understanding the features of natural competence. Our convenient and biosafe genome editing methods will be valuable for most of the <i>S. thermophilus</i> strains and will contribute to the germplasm improvement in the dairy industry. Additionally, the mutant strains generated in this study will serve as valuable resources for future investigations regarding cellular metabolism.</p>\n </section>\n </div>","PeriodicalId":13822,"journal":{"name":"International Journal of Dairy Technology","volume":"78 3","pages":""},"PeriodicalIF":2.8000,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optimisation of natural transformation procedure and development of genome editing methods for Streptococcus thermophilus B-6\",\"authors\":\"Bingxin Wang, Kexiong Sun, Yukai Sun, Jingbo Wang, Yueqiu Jiang, Xipeng Liu, Jie Li, Huili Liu, Youbao Zhao, Longxian Zhang, Xinfu Zhang\",\"doi\":\"10.1111/1471-0307.70065\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div>\\n \\n <section>\\n \\n <h3> Background</h3>\\n \\n <p><i>Streptococcus thermophilus</i> is invaluable in both dairy factory and scientific research, with diverse strains offering unique industrial advantages. Consequently, there is a high demand for efficient, user-friendly, biosafe and broadly applicable genome editing methods for this bacterium. The conventional approach based on temperature-sensitive plasmids is laborious and time-consuming, while the existing natural transformation method lacks broad applicability. The recently developed endogenous CRISPR-Cas-assisted genome editing system involves cumbersome plasmid construction.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Aim</h3>\\n \\n <p>In this study, we aimed to optimise natural transformation protocols and develop efficient, user-friendly, biosafe and broadly applicable genome editing methods for <i>S. thermophilus</i>.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Methods</h3>\\n \\n <p>We used <i>S. thermophilus</i> B-6 as a model strain. We characterised the features of the growth curves and the natural competence of this strain. Accordingly, we optimised the natural transformation protocol for this strain. We used this model strain to develop efficient, user-friendly, biosafe and broadly applicable genome editing methods.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Major Findings</h3>\\n \\n <p>By continuously implementing ComS<sub>17–24</sub> and exogenous DNAs, as well as elongating the co-incubation time, we upgraded the transformation ratio of this strain from ~10<sup>−5</sup> to ~10<sup>−2</sup>. Leveraging this improvement, we developed a rapid, simple, biosafe and plasmid-free genome editing method for <i>S. thermophilus</i>. Furthermore, we established a novel endogenous CRISPR-Cas-assisted and efficient genome editing pathway that eliminates the need for additional plasmid construction. Using these advanced techniques, we successfully generated multiple CRISPR-Cas system deleted strains and a <i>recA</i> overexpressing strain.</p>\\n </section>\\n \\n <section>\\n \\n <h3> Scientific and Industrial Implications</h3>\\n \\n <p>Our findings contribute to better understanding the features of natural competence. Our convenient and biosafe genome editing methods will be valuable for most of the <i>S. thermophilus</i> strains and will contribute to the germplasm improvement in the dairy industry. Additionally, the mutant strains generated in this study will serve as valuable resources for future investigations regarding cellular metabolism.</p>\\n </section>\\n </div>\",\"PeriodicalId\":13822,\"journal\":{\"name\":\"International Journal of Dairy Technology\",\"volume\":\"78 3\",\"pages\":\"\"},\"PeriodicalIF\":2.8000,\"publicationDate\":\"2025-09-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International Journal of Dairy Technology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1111/1471-0307.70065\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"FOOD SCIENCE & TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Dairy Technology","FirstCategoryId":"97","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/1471-0307.70065","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"FOOD SCIENCE & TECHNOLOGY","Score":null,"Total":0}
Optimisation of natural transformation procedure and development of genome editing methods for Streptococcus thermophilus B-6
Background
Streptococcus thermophilus is invaluable in both dairy factory and scientific research, with diverse strains offering unique industrial advantages. Consequently, there is a high demand for efficient, user-friendly, biosafe and broadly applicable genome editing methods for this bacterium. The conventional approach based on temperature-sensitive plasmids is laborious and time-consuming, while the existing natural transformation method lacks broad applicability. The recently developed endogenous CRISPR-Cas-assisted genome editing system involves cumbersome plasmid construction.
Aim
In this study, we aimed to optimise natural transformation protocols and develop efficient, user-friendly, biosafe and broadly applicable genome editing methods for S. thermophilus.
Methods
We used S. thermophilus B-6 as a model strain. We characterised the features of the growth curves and the natural competence of this strain. Accordingly, we optimised the natural transformation protocol for this strain. We used this model strain to develop efficient, user-friendly, biosafe and broadly applicable genome editing methods.
Major Findings
By continuously implementing ComS17–24 and exogenous DNAs, as well as elongating the co-incubation time, we upgraded the transformation ratio of this strain from ~10−5 to ~10−2. Leveraging this improvement, we developed a rapid, simple, biosafe and plasmid-free genome editing method for S. thermophilus. Furthermore, we established a novel endogenous CRISPR-Cas-assisted and efficient genome editing pathway that eliminates the need for additional plasmid construction. Using these advanced techniques, we successfully generated multiple CRISPR-Cas system deleted strains and a recA overexpressing strain.
Scientific and Industrial Implications
Our findings contribute to better understanding the features of natural competence. Our convenient and biosafe genome editing methods will be valuable for most of the S. thermophilus strains and will contribute to the germplasm improvement in the dairy industry. Additionally, the mutant strains generated in this study will serve as valuable resources for future investigations regarding cellular metabolism.
期刊介绍：
The International Journal of Dairy Technology ranks highly among the leading dairy journals published worldwide, and is the flagship of the Society. As indicated in its title, the journal is international in scope.
Published quarterly, International Journal of Dairy Technology contains original papers and review articles covering topics that are at the interface between fundamental dairy research and the practical technological challenges facing the modern dairy industry worldwide. Topics addressed span the full range of dairy technologies, the production of diverse dairy products across the world and the development of dairy ingredients for food applications.
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