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.

生物技术可以利用可再生资源生产大量有价值的产品，经济地生产这些产品的一个障碍是我们无法轻松地改变细胞新陈代谢以最大限度地提高产量，从而显着提高我们使用光和生物传感器控制工程微生物的能力。该策略将实现反馈控制，其中来自生物传感器的荧光信号实时通知用于微调酶浓度的光信号，该方法将允许开发全新的技术来控制、优化、并进行发酵，如以及提高我们对酶浓度如何影响产物形成的基本了解，该项目的结果将被纳入目前教授的几门课程中，该课程是 PI 之前参与过的两个组织的活动（西班牙裔激励学生的表现和成就）。奇卡诺/西班牙裔和美洲原住民科学促进会）将在代表性不足的群体中促进与 STEM 相关的职业，并鼓励他们参与相关研究。​该项目的目标是开发闭环控制使用生物传感器和光遗传学的代谢工程系统将三个组件集成到同一酵母菌株中：（i）产生感兴趣的化学物质的代谢途径；（ii）监测所述途径的活性的生物传感器； PI实验室在不同的酵母菌株中开发了控制酶表达水平的光遗传学工具，这将允许对酶浓度进行微调。成功将会是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智力优势和更广泛的影响进行评估，被认为值得支持审查标准。

项目成果

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: 2022-01
• 期刊: Journal of Visualized Experiments
• 作者: Hoffman, Shannon M.;Lalwani, Makoto A.;Avalos, José L.
• 通讯作者: Avalos, José L.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering

酵母代谢工程动态控制快速光遗传学电路的设计和表征

• DOI: 10.1021/acssynbio.0c00305
• 发表时间: 2020-12-18
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: Zhao, Evan M.;Lalwani, Makoto A.;Lovelett, Robert J.;Garcia-Echauri, Sergio A.;Hoffman, Shannon M.;Gonzalez, Christopher L.;Toettcher, Jared E.;Kevrekidis, Ioannis G.;Avalos, Jose L.
• 通讯作者: Avalos, Jose L.

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: 2022-12
• 期刊: Current Opinion in Chemical Biology
• 影响因子: 7.8
• 作者: Wegner, Scott A.;Barocio;Avalos, José L.
• 通讯作者: Avalos, José L.

¡Viva la mitochondria!: harnessing yeast mitochondria for chemical production

线粒体万岁！：利用酵母线粒体进行化学生产

• DOI: 10.1093/femsyr/foaa037
• 发表时间: 2020-09
• 期刊: FEMS Yeast Research
• 影响因子: 3.2
• 作者: Duran, Lisset;López, José Montaño;Avalos, José L
• 通讯作者: Avalos, José L

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

通过组装合成细胞器对代谢通量进行光控制

• DOI: 10.1038/s41589-019-0284-8
• 发表时间: 2019-04-16
• 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
• 通讯作者: Jared E. Toettcher