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

生物技术可以从可再生资源中生产出大量有价值的产品。经济地制造这些产品的一个障碍是我们无法轻松地重新引导细胞新陈代谢以最大限度地提高产量。将开发新的技术，利用光和生物传感器显著提高我们控制工程微生物的能力。这一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实时通知。这种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。该项目的成果将被纳入目前教授的几门课程。向两个组织(西班牙裔激励学生的表现和成就，以及奇卡诺/拉美裔和美国原住民科学促进会)开展的外展活动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。该项目的目标是利用生物传感器和光遗传学开发新陈代谢工程的闭环控制系统。三种成分将被整合到同一酵母菌株中：(I)产生感兴趣的化学物质的代谢途径；(Ii)监测所述途径活性的生物传感器；以及(Iii)控制酶表达水平的光遗传工具。这将允许用光微调酶的浓度。这三种成分是在PI实验室的不同酵母菌株中培养出来的。第一个目标是将所有这三种成分整合到一个酵母菌株中。支链醇的生产将证明这一成功。第二个目标是使现有的对红光和红外光敏感的光遗传工具与现有的蓝光系统垂直工作。这项技术将为操作和优化发酵打开新策略的大门，例如通过使用具有反馈控制的周期性光脉冲，提供前所未有的操作、优化和自动化发酵的能力。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审查标准进行评估，被认为值得支持。

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.