EAGER: NSF/ABF Dynamic Control of Metabolism Using Redesigned Proteins

EAGER：NSF/ABF 使用重新设计的蛋白质动态控制代谢

• 批准号: 2030221
• 负责人: Tim Whitehead
• 金额: $ 23.88万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2030221&HistoricalAwards=false
• 关键词: EAGER; NSF; ABF; Dynamic; Control

Microbes can create fuels and chemicals from biomass. Bio-based fuels help contribute to energy security in the US. One current limitation to using microbes for production is that the microbes also use the same biomass to grow. Rapid growth reduces fuel and chemical production. However, new strategies for controlling the microbial metabolism could separate growth from product formation. The objective of this project is to develop a modular control system to be applied across many different organisms. Success of this project will enhance microbial production of fuels and chemicals, thus advance the bioeconomy. The objective of this project is to evaluate a novel strategy for dynamic metabolic control. We will use advanced computational protein design and engineering to remodel T7 RNA polymerase (T7RNAP), which transcribes DNA into mRNA, such that it requires a user-defined small molecule to function. This remodeled polymerase would be constitutively expressed. This induction molecule activates the polymerase, resulting in expression of T7-specific genes. We will (i) identify inexpensive chemicals that are non-toxic and non-metabolizable for diverse non-model host organisms; (ii) deploy dynamically controlled circuits in Pseudomonas putida for production of muconate to show improved metrics of titer/yield/productivity; and (iii) demonstrate the utility of T7 RNAP circuits for other biomanufacturing-relevant organisms.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.

微生物可以从生物质中产生燃料和化学品。生物燃料有助于美国的能源安全。目前使用微生物进行生产的一个限制是微生物也使用相同的生物质来生长。快速增长减少了燃料和化学品的生产。 然而，控制微生物代谢的新策略可以将生长与产物形成分开。该项目的目标是开发一个模块化的控制系统，应用于许多不同的生物体。该项目的成功将提高燃料和化学品的微生物生产，从而促进生物经济。 本项目的目的是评估一种新的动态代谢控制策略。我们将使用先进的计算蛋白质设计和工程改造T7 RNA聚合酶（T7 RNAP），该聚合酶将DNA转录为mRNA，因此它需要用户定义的小分子才能发挥作用。这种重塑的聚合酶将组成型表达。这种诱导分子激活聚合酶，导致T7特异性基因的表达。我们将（i）确定对不同的非模型宿主生物无毒且不可代谢的廉价化学品;（ii）在恶臭假单胞菌中部署动态控制回路以生产粘康酸盐，以显示滴度/产量/生产力的改进指标;以及（iii）证明T7 RNAP电路用于其他生物制造的实用性-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

Standardizing cassette‐based deep mutagenesis by Golden Gate assembly

通过 Golden Gate 组装标准化基于盒的深度诱变

• DOI: 10.1002/bit.28564
• 发表时间: 2023
• 期刊: Biotechnology and Bioengineering
• 影响因子: 3.8
• 作者: Daffern, Nicolas;Francino‐Urdaniz, Irene M.;Baumer, Zachary T.;Whitehead, Timothy A.
• 通讯作者: Whitehead, Timothy A.

Optimization of multi-site nicking mutagenesis for generation of large, user-defined combinatorial libraries

• DOI: 10.1093/protein/gzab017
• 发表时间: 2021-02-15
• 期刊: PROTEIN ENGINEERING DESIGN & SELECTION
• 影响因子: 2.4
• 作者: Kirby, Monica B.;Medina-Cucurella, Angelica, V;Whitehead, Timothy A.
• 通讯作者: Whitehead, Timothy A.

The inner workings of an enzyme

酶的内部工作原理

• DOI: 10.1126/science.abj8346
• 发表时间: 2021
• 期刊: Science
• 影响因子: 56.9
• 作者: Baumer, Zachary T.;Whitehead, Timothy A.
• 通讯作者: Whitehead, Timothy A.

A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis

• DOI: 10.21769/bioprotoc.3697
• 发表时间: 2020-08-05
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Steiner, Paul J.;Baumer, Zachary T.;Whitehead, Timothy A.
• 通讯作者: Whitehead, Timothy A.