Optimisation of natural transformation procedure and development of genome editing methods for Streptococcus thermophilus B-6

IF 2.8 2区农林科学 Q3 FOOD SCIENCE & TECHNOLOGY

International Journal of Dairy Technology Pub Date : 2025-09-09 DOI:10.1111/1471-0307.70065

Bingxin Wang, Kexiong Sun, Yukai Sun, Jingbo Wang, Yueqiu Jiang, Xipeng Liu, Jie Li, Huili Liu, Youbao Zhao, Longxian Zhang, Xinfu Zhang

{"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}

引用次数: 0

Abstract

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 ComS_17–24 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.

Abstract Image

查看原文本刊更多论文

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

嗜热链球菌在奶牛场和科研中都具有不可估量的价值，其菌株种类繁多，具有独特的工业优势。因此，对这种细菌的高效、用户友好、生物安全且广泛适用的基因组编辑方法有很高的需求。传统的基于温度敏感质粒的方法耗时费力，而现有的自然转化方法缺乏广泛的适用性。最近开发的内源性crispr - cas辅助基因组编辑系统涉及繁琐的质粒构建。在本研究中，我们旨在优化自然转化方案，开发高效、用户友好、生物安全且广泛适用的嗜热葡萄球菌基因组编辑方法。方法以嗜热链球菌B-6为模型菌株。我们描述了该菌株的生长曲线特征和自然能力。因此，我们对该菌株的自然转化方案进行了优化。我们利用该模型菌株开发高效、用户友好、生物安全且广泛适用的基因组编辑方法。通过连续导入ComS17-24和外源dna，延长共孵育时间，将菌株的转化率从~10−5提高到~10−2。利用这一改进，我们开发了一种快速、简单、生物安全且无质粒的嗜热链球菌基因组编辑方法。此外，我们建立了一种新的内源性crispr - cas辅助的高效基因组编辑途径，无需额外的质粒构建。利用这些先进的技术，我们成功地生成了多个CRISPR-Cas系统缺失菌株和一个recA过表达菌株。科学和工业意义我们的发现有助于更好地理解自然能力的特征。我们的便捷和生物安全的基因组编辑方法将对大多数嗜热链球菌菌株有价值，并将有助于乳制品行业的种质改良。此外，本研究中产生的突变菌株将为未来细胞代谢研究提供宝贵的资源。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

International Journal of Dairy Technology 工程技术-食品科技

CiteScore

7.00

自引率

4.50%

发文量

审稿时长

12 months

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.