CAREER: Closed-loop control of engineered metabolism using biosensors and optogenetics

职业：利用生物传感器和光遗传学对工程代谢进行闭环控制

• 批准号: 1751840
• 负责人: Jose Avalos
• 金额: $ 52.48万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1751840&HistoricalAwards=false
• 关键词: CAREER; Closed; loop; control; engineered

Biotechnology can produce a vast number of valuable products from renewable sources. One barrier to making these products economically is our inability to easily redirect cellular metabolism to maximize production. New technologies to significantly improve our ability to control engineered microorganisms, using light and biological sensors, will be developed. This strategy will enable feed-back controls, in which light signals used to fine-tune enzyme concentrations are informed by fluorescence signals from biosensors in real time. The approach will allow the development of entirely new technologies to control, optimize, and operate fermentations, as well as improve our basic understanding of how enzyme concentrations affect product formation. The results of this project will be incorporated into several courses currently taught. Outreach activities to two organizations with which the PI has been involved previously (Hispanics Inspiring Student's Performance and Achievement, and Society for the Advancement of Chicano/Hispanics and Native Americans in Science) will promote STEM-related careers among under-represented groups and encourage their participation in related research. The objective of this project is to develop a closed-loop control system for metabolic engineering using biosensors and optogenetics. Three components will be integrated in the same yeast strain: (i) a metabolic pathway to produce a chemical of interest; (ii) a biosensor to monitor the activity of said pathway; and (iii) optogenetic tools to control enzyme expression levels. This will allow fine-tuning of enzyme concentrations using light. These three components have been developed in separate yeast strains in the PI's lab. The first objective is to integrate all three components in a single yeast strain. Success will be demonstrated by the production of branched chain alcohols. The second objective is to adapt an existing optogenetic tool sensitive to red and infrared light to work orthogonally with the existing blue light system. This technology will open the door to new strategies to operate and optimize fermentations, such as by using periodic light pulses with feedback controls, providing unprecedented capabilities for operating, optimizing, and automating fermentations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

生物技术可以从可再生资源中生产大量有价值的产品。经济地制造这些产品的一个障碍是我们无法轻松地重新定向细胞代谢以最大限度地提高产量。将开发新技术，利用光和生物传感器，大大提高我们控制工程微生物的能力。这种策略将实现反馈控制，其中用于微调酶浓度的光信号由来自生物传感器的荧光信号以真实的时间通知。 该方法将允许开发全新的技术来控制，优化和操作发酵，并提高我们对酶浓度如何影响产品形成的基本理解。该项目的成果将纳入目前教授的几门课程。对PI以前参与过的两个组织开展外联活动（西班牙裔学生赞扬学生的表现和成就，和促进奇卡诺人/西班牙裔和美洲原住民科学协会）将在代表性不足的群体中促进STEM相关职业，并鼓励他们参与相关研究。该项目的目标是开发一个封闭的，使用生物传感器和光遗传学的代谢工程回路控制系统。三种组分将整合在同一酵母菌株中：（i）产生感兴趣的化学品的代谢途径;（ii）监测所述途径的活性的生物传感器;和（iii）控制酶表达水平的光遗传学工具。这将允许使用光来微调酶浓度。这三种成分已经在PI实验室的不同酵母菌株中开发出来。第一个目标是将所有三种成分整合到单一的酵母菌株中。成功将通过支链醇的生产来证明。第二个目标是使现有的对红光和红外光敏感的光遗传学工具与现有的蓝光系统正交工作。该技术将为操作和优化发酵的新策略打开大门，例如通过使用带有反馈控制的周期性光脉冲，为操作、优化和自动化发酵提供前所未有的能力。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Critical Roles of the Pentose Phosphate Pathway and GLN3 in Isobutanol-Specific Tolerance in Yeast

• DOI: 10.1016/j.cels.2019.10.006
• 发表时间: 2019-12-18
• 期刊: CELL SYSTEMS
• 影响因子: 9.3
• 作者: Kuroda, Kouichi;Hammer, Sarah K.;Avalos, Jose L.
• 通讯作者: Avalos, Jose L.

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold

• DOI: 10.1038/s41467-020-17837-7
• 发表时间: 2020-08-13
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Carrasco-Lopez, Cesar;Zhao, Evan M.;Avalos, Jose L.
• 通讯作者: Avalos, Jose L.

Light-Controlled Fermentations for Microbial Chemical and Protein Production

用于微生物化学和蛋白质生产的光控发酵

• DOI: 10.3791/63269
• 发表时间: 2022
• 期刊: Journal of Visualized Experiments
• 作者: Hoffman, Shannon M.;Lalwani, Makoto A.;Avalos, José L.
• 通讯作者: Avalos, José L.

Light-based control of metabolic flux through assembly of synthetic organelles

• DOI: 10.1038/s41589-019-0284-8
• 发表时间: 2019-04
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Evan M. Zhao;Nathan Suek;Maxwell Z. Wilson;E. Dine;N. Pannucci;Zemer Gitai;J. Avalos;Jared E. Toettcher-Jared-E

The bright frontiers of microbial metabolic optogenetics

微生物代谢光遗传学的光明前沿

• DOI: 10.1016/j.cbpa.2022.102207
• 发表时间: 2022
• 期刊: Current Opinion in Chemical Biology
• 影响因子: 7.8
• 作者: Wegner, Scott A.;Barocio-Galindo, Rachel M.;Avalos, José L.
• 通讯作者: Avalos, José L.