Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

IF 2.6 4区 生物学 Q3 MICROBIOLOGY
Rozenn Gardan, Edith Honvo-Houeto, Christine Mézange, Nathanael Jean Maillot, Aurélie Balvay, Sylvie Rabot, Luis G Bermúdez-Humarán, Philippe Langella, Véronique Monnet, Vincent Juillard
{"title":"Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in <i>Streptococcus thermophilus</i>.","authors":"Rozenn Gardan, Edith Honvo-Houeto, Christine Mézange, Nathanael Jean Maillot, Aurélie Balvay, Sylvie Rabot, Luis G Bermúdez-Humarán, Philippe Langella, Véronique Monnet, Vincent Juillard","doi":"10.1099/mic.0.001487","DOIUrl":null,"url":null,"abstract":"<p><p><i>Streptococcus thermophilus</i> holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP<sub>1358</sub>) and a transcriptional regulator (Rgg<sub>1358</sub>) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also <i>in vivo,</i> in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP<sub>1358</sub> inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in <i>S. thermophilus</i>, whose myriad applications include the delivery of therapeutic peptides or proteins.</p>","PeriodicalId":49819,"journal":{"name":"Microbiology-Sgm","volume":"170 9","pages":""},"PeriodicalIF":2.6000,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11414475/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbiology-Sgm","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1099/mic.0.001487","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

利用 Rgg 法定人数感应机制在嗜热链球菌中创建创新的重组蛋白表达系统。
嗜热链球菌有望成为生产和分泌异源蛋白质的底盘。数千年来,嗜热链球菌一直被用来发酵牛奶,它在美国具有公认安全(GRAS)的地位,在欧洲具有合格安全推定(QPS)的地位。此外,由于它的天然能力,可以很容易地进行基因改造,而且它分泌的内源性蛋白质很少,这意味着纯化目标蛋白质所需的下游处理较少,从而降低了成本。通过引入突变,使编码细菌三种主要表面蛋白酶的基因失活,可以消除异源蛋白的胞外降解。在这里,我们构建了一个诱导表达系统,该系统利用了一种多肽信息素(SHP1358)和一种参与法定人数感应调控的转录调节因子(Rgg1358)。我们通过合成荧光素酶报告蛋白,探索了完整版系统的功能,其中诱导剂由细菌自身产生。这一完整版系统不仅通过在化学定义的培养基中生长的细菌进行了评估,还通过无菌小鼠粪便中的细菌进行了体内评估。我们还通过合成荧光素酶和elafin(一种具有治疗作用的人类蛋白质)的分泌形式,测试了一个不完整的版本,其中诱导剂必须添加到培养基中。我们的结果表明,在我们的系统中,蛋白质的产生可以通过使用不同浓度的SHP1358诱导剂或其他具有封闭氨基酸序列的SHPs来调节。我们还构建了一种遗传背景,在这种背景下,系统的所有泄漏都被消除了。总之,有了这种新的可诱导表达系统,我们又增加了一套目前用于在嗜热菌中生产分泌蛋白的工具。
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
Microbiology-Sgm
Microbiology-Sgm 生物-微生物学
CiteScore
4.60
自引率
7.10%
发文量
132
审稿时长
3.0 months
期刊介绍: We publish high-quality original research on bacteria, fungi, protists, archaea, algae, parasites and other microscopic life forms. Topics include but are not limited to: Antimicrobials and antimicrobial resistance Bacteriology and parasitology Biochemistry and biophysics Biofilms and biological systems Biotechnology and bioremediation Cell biology and signalling Chemical biology Cross-disciplinary work Ecology and environmental microbiology Food microbiology Genetics Host–microbe interactions Microbial methods and techniques Microscopy and imaging Omics, including genomics, proteomics and metabolomics Physiology and metabolism Systems biology and synthetic biology The microbiome.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信