Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-09-29 DOI:10.1021/acssynbio.5c00095

Sai Akhil Golla, Mona Abo-Hashesh, Dev Gupta, Yilan Liu, Radhakrishnan Mahadevan

{"title":"Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.","authors":"Sai Akhil Golla, Mona Abo-Hashesh, Dev Gupta, Yilan Liu, Radhakrishnan Mahadevan","doi":"10.1021/acssynbio.5c00095","DOIUrl":null,"url":null,"abstract":"Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.9000,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Synthetic Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acssynbio.5c00095","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.

查看原文本刊更多论文

基于模型的qCRISPRi电路代谢途径动态控制优化。

代谢工程能够实现可持续的化学生产，但往往会增加代谢负担，降低细胞活力和生产力。动态控制策略，如基于群体感应（QS）的电路，可以通过响应细胞密度自主调节基因表达来减轻这些影响。在本研究中，我们研究了一个qs调控的CRISPR干扰（qCRISPRi）电路对代谢途径的动态控制，重点研究了泄漏表达和调控严格性的作用。利用数学建模和实验相结合的方法，我们评估了启动子泄漏和LuxR严格性如何影响关键的开关特性，包括最大基因表达、开关密度、折叠抑制和转换时间。我们的研究结果表明，dCas9的高泄漏表达降低了开关密度，并过早地抑制了GFP，而高泄漏的LuxR变体通过减少泄漏和实现更清晰的转换来提高开关精度。这些模型预测在大肠杆菌中得到了实验验证，证实了LuxR的严格性提高了动态电路的性能。总之，这项工作为优化基于质量的调节系统提供了一个定量框架，并为在代谢工程中实施动态控制提供了可推广的设计见解。

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

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.