CAREER: Engineering redox metabolism using unnatural cofactors

职业：使用非天然辅助因子工程氧化还原代谢

• 批准号: 1847705
• 负责人: Han Li
• 金额: $ 50.22万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1847705&HistoricalAwards=false
• 关键词: CAREER; Engineering; redox; metabolism; using

Chemicals can be synthesized from renewable resources using microbes. This approach could help the chemical industry become more sustainable. A major hurdle to the widespread implementation of this strategy is low production efficiency on the part of the cells. The standard approach to overcome this has been to modify the flow of carbon through cell metabolism. In this project, the flow of electrons through the cell will be adjusted. This will be accomplished by creating artificial electron shuttle molecules, known as cofactors. This is an innovative strategy. It could greatly increase the efficiency of using microbes to manufacture chemicals. Outreach activities will be integrated with the research tasks. As a result, the project represents an attempt to (i) allow biomanufacturing to better meet the Nation's needs for energy, food, commodities, and medicine; (ii) contribute to undergraduate and graduate education in STEM; and (iii) motivate female high school students in Orange County to pursue a career in engineering.Catabolism and anabolism co-exist without interference because each has a separate redox cofactor. A third, independent redox cofactor may enable a specific pathway to be insulated from core metabolism. Increased productivity might be achieved by establishing the new redox cofactor and evolving enzymes in the product pathway to require that cofactor exclusively. This project will establish nicotinamide mononucleotide (NMN) as the third redox cofactor. This will be accomplished through a three-step experimental plan. First, high-throughput selection platforms for engineering of NMN-dependent electron circuits will be developed. Then, strains of E. coli and S. cerevisiae capable of overproduction of NMN will be constructed. Finally, NMN-dependent formate dehydrogenase will be engineered to deliver NMN reducing power independently of the host's metabolic background. Successful completion of these objectives will result in two industrially-relevant strains capable of acting as a chassis for NMN-dependent pathways and products.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.

化学品可以利用微生物从可再生资源中合成。这种方法可以帮助化学工业变得更加可持续。广泛实施该策略的一个主要障碍是电池的生产效率低。克服这个问题的标准方法是改变细胞代谢中的碳流。在这个项目中，通过电池的电子流将被调整。这将通过创建人造电子穿梭分子（称为辅因子）来实现。这是一个创新的策略。它可以大大提高利用微生物制造化学品的效率。外展活动将与研究任务结合起来。因此，该项目代表了一种尝试：(i) 让生物制造更好地满足国家对能源、食品、商品和药品的需求； (ii) 为 STEM 本科生和研究生教育做出贡献； (iii) 激励奥兰治县的女高中生从事工程职业。分解代谢和合成代谢可以不受干扰地共存，因为每种都有独立的氧化还原辅因子。第三个独立的氧化还原辅因子可能使特定途径与核心代谢隔离。通过建立新的氧化还原辅因子并在产物途径中进化酶以专门需要该辅因子，可以提高生产率。该项目将建立烟酰胺单核苷酸（NMN）作为第三种氧化还原辅因子。这将通过三步实验计划来完成。首先，将开发用于 NMN 相关电子电路工程的高通量选择平台。然后，将构建能够过量生产NMN的大肠杆菌和酿酒酵母菌株。最后，NMN 依赖性甲酸脱氢酶将被设计为独立于宿主代谢背景而提供 NMN 还原能力。成功完成这些目标将产生两种与工业相关的菌株，它们能够充当 NMN 依赖性途径和产品的基础。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值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

Aldehyde Production in Crude Lysate- and Whole Cell-Based Biotransformation Using a Noncanonical Redox Cofactor System

使用非经典氧化还原辅因子系统在粗裂解物和全细胞生物转化中生产醛

• DOI: 10.1021/acscatal.0c03070
• 发表时间: 2020-08
• 期刊: ACS Catalysis
• 影响因子: 12.9
• 作者: Richardson, Kelly N.;Black, William B.;Li, Han
• 通讯作者: Li, Han

In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO)

体内热稳定环己酮单加氧酶 (CHMO) 的高通量选择

• DOI: 10.3390/catal10080935
• 发表时间: 2020-08-01
• 期刊: Catalysts (Basel, Switzerland)
• 作者: Sarah Maxel;Linyue Zhang;Edward King;Ana Paula Acosta;R. Luo;Han Li
• 通讯作者: Han Li

Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst

在氧依赖性生物催化剂中基于生长的高通量选择 NADH 偏好

• DOI: 10.1021/acssynbio.1c00258
• 发表时间: 2021-09-17
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: Maxel, Sarah;Saleh, Samer;King, Edward;Aspacio, Derek;Zhang, Linyue;Luo, Ray;Li, Han
• 通讯作者: Li, Han

Leveraging Oxidative Stress to Regulate Redox Balance-Based, In Vivo Growth Selections for Oxygenase Engineering

利用氧化应激来调节基于氧化还原平衡的氧化酶工程体内生长选择

• DOI: 10.1021/acssynbio.0c00380
• 发表时间: 2020-11-20
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: Maxel, Sarah;King, Edward;Zhang, Yulai;Luo, Ray;Li, Han
• 通讯作者: Li, Han

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

正交糖酵解途径使非经典辅因子氧化酶能够定向进化

• DOI: 10.1038/s41467-022-35021-x
• 发表时间: 2022-11-26
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: King, Edward;Maxel, Sarah;Zhang, Yulai;Kenney, Karissa C.;Cui, Youtian;Luu, Emma;Siegel, Justin B.;Weiss, Gregory A.;Luo, Ray;Li, Han
• 通讯作者: Li, Han