{"title":"高灵敏度和严格的胆碱诱导基因表达控制器的定向进化。","authors":"Yuki Yanai, Takayuki Hoshino, Yuki Kimura, Shigeko Kawai-Noma, Daisuke Umeno","doi":"10.2323/jgam.2024.05.004","DOIUrl":null,"url":null,"abstract":"<p><p>Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P<sub>T5</sub> system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":null,"pages":null},"PeriodicalIF":0.8000,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Directed evolution of highly sensitive and stringent choline-induced gene expression controllers.\",\"authors\":\"Yuki Yanai, Takayuki Hoshino, Yuki Kimura, Shigeko Kawai-Noma, Daisuke Umeno\",\"doi\":\"10.2323/jgam.2024.05.004\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P<sub>T5</sub> system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.</p>\",\"PeriodicalId\":15842,\"journal\":{\"name\":\"Journal of General and Applied Microbiology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.8000,\"publicationDate\":\"2024-11-06\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of General and Applied Microbiology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.2323/jgam.2024.05.004\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/6/17 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of General and Applied Microbiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2323/jgam.2024.05.004","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/17 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Directed evolution of highly sensitive and stringent choline-induced gene expression controllers.
Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.
期刊介绍:
JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.