SusChEM: Development of a Protecting Group Toolkit for Metabolic Engineering

SusChEM：代谢工程保护基团工具包的开发

• 批准号: 1605465
• 负责人: John Dueber
• 金额: $ 35万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1605465&HistoricalAwards=false
• 关键词: SusChEM; Development; Protecting; Group; Toolkit

1605465 Dueber, John E. Engineering living cells to perform as chemical factories offers a route for sustainable production of a variety of chemicals. Microbial cells can rapidly self-replicate on inexpensive substrates and make enzymes capable of catalyzing difficult chemical reactions cheaply, quickly, and cleanly. However, the cell presents a complex environment for conducting these multi-step syntheses and, accordingly, undesired reactivity often occurs to produce undesired chemical products or, even worse, result in toxicity to the microbial production host. Multi-step organic chemical syntheses conducted in test tubes often employ chemical protecting groups to gain control over when and where sites of a chemical substrate will be reactive. This general approach will be mimicked in the cell by employing natural chemical groups that can be reversibly added to achieve similar control over reactivity of a chemical in the cell. Microbial strains capable of catalyzing efficient protection and stability of the resulting tailored products will be of broad interest to metabolic engineers and will be made available to the scientific and engineering communities. Engineering metabolic pathways to sustainably produce molecules of interest requires designed control at multiple levels. Numerous problems can limit productivity, including metabolite toxicity, off-pathway catalysis, and failure to secrete the final product. Various synthetic biology approaches have been developed for introducing control at the DNA, RNA, and protein levels to address many of these challenges. In this proposal, control at the metabolite level is targeted for an additional strategy in this toolkit. The protecting group strategy used in synthetic organic chemistry to gain control over functional group reactivity will be mimicked to gain similar control over when and where a molecule is active. Three biomolecular tailoring groups (glucosyl, acetyl, and sulfonyl) are proposed to provide reversible protection in addition to the ability to tailor for altered properties such as solubility and membrane permeability as well as the recognition by other enzymes that could lower toxicity. Thus, reactive molecules can be made chemically inert while protected and then reactivity be reinstated when desired via enzymatic deprotection. Enabling the use of the appropriate protecting group for the desired application demands the construction of a toolkit of strains wherein each of these protected products will be stable. For many of these protecting groups, several enzymes will need to be knocked out of the production strain to ensure small molecule product stability in the cellular environment. A high-throughput colorimetric plate assay will be employed to screen large matrices of gene target knockouts. Furthermore, it is critical that these protecting groups do not limit product titers or production rates. Accordingly, rational engineering and adaptations for increased flux for each protection reaction will be performed. Although acetylation and glucosylation are expected to already have fairly high capacity, sulfonation capacity is extremely low. The resultant strains should prove broadly useful for a variety of metabolic engineering applications and will accordingly be shared with the community.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biosciences.

[1605465]杜伯，约翰E.把活细胞改造成化工厂，为多种化学品的可持续生产提供了一条途径。微生物细胞可以在廉价的底物上快速自我复制，并制造出能够廉价、快速、清洁地催化困难化学反应的酶。然而，细胞为进行这些多步骤合成提供了一个复杂的环境，因此，经常会发生不希望的反应性，产生不希望的化学产物，甚至更糟糕的是，对微生物生产宿主造成毒性。在试管中进行的多步骤有机化学合成通常使用化学保护基团来控制化学底物的反应时间和地点。这种一般的方法将在细胞中通过使用可以可逆添加的天然化学基团来模拟，以实现对细胞中化学物质的反应性的类似控制。微生物菌株能够催化有效的保护和稳定的定制产品将引起代谢工程师的广泛兴趣，并将提供给科学和工程界。工程代谢途径可持续地产生感兴趣的分子需要在多个水平上设计控制。许多问题会限制生产效率，包括代谢物毒性、非通路催化和不能分泌最终产物。各种合成生物学方法已经被开发出来，用于在DNA、RNA和蛋白质水平上引入控制，以解决许多这些挑战。在本建议中，代谢物水平的控制是该工具包中另一个策略的目标。在合成有机化学中用于控制官能团反应性的保护基团策略将被模仿，以获得对分子何时何地活跃的类似控制。三种生物分子裁剪基团（葡萄糖基、乙酰基和磺酰基）被提出提供可逆保护，除了能够为改变的特性（如溶解度和膜渗透性）以及其他可以降低毒性的酶的识别能力进行定制之外。因此，反应性分子可以在受到保护的同时产生化学惰性，然后在需要时通过酶解保护恢复反应性。为了在所需的应用中使用适当的保护组，需要构建一个菌株工具包，其中每个受保护的产品都是稳定的。对于许多这些保护基团，需要将几种酶从生产菌株中剔除，以确保小分子产品在细胞环境中的稳定性。高通量比色板试验将用于筛选基因靶敲除的大基质。此外，至关重要的是，这些保护基团不限制产品滴度或生产速度。因此，将进行合理的工程设计和调整，以增加每个保护反应的通量。虽然乙酰化和糖基化预计已经具有相当高的容量，但磺化容量极低。由此产生的菌株将被证明广泛适用于各种代谢工程应用，并将相应地与社区共享。该奖项由CBET部门的生物技术和生化工程项目颁发，由分子和细胞生物科学部的系统和合成生物学项目共同资助。