SusChEM: Development of a Protecting Group Toolkit for Metabolic Engineering

1605465设计活细胞作为化工厂，为各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，并使酶能够廉价、快速和清洁地催化困难的化学反应。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，经常发生不希望发生的反应来产生不想要的化学产品，或者更糟糕的是，导致对微生物生产宿主的毒性。在试管中进行的多步有机化学合成通常使用化学保护基团来控制化学底物的何时何地发生反应。这种一般的方法将通过使用天然化学基团在细胞中模拟，这些天然化学基团可以可逆地添加，以实现对细胞中化学物质的反应性的类似控制。能够催化有效保护和稳定所产生的定制产品的微生物菌株将引起代谢工程师的广泛兴趣，并将提供给科学界和工程界。设计代谢途径以可持续地生产感兴趣的分子需要在多个水平上进行设计控制。许多问题可能会限制生产率，包括代谢物毒性、非途径催化以及无法分泌最终产品。为了在DNA、RNA和蛋白质水平上引入控制，已经开发了各种合成生物学方法来应对其中的许多挑战。在这项提案中，代谢物水平的控制是这一工具包中另一项战略的目标。在合成有机化学中用来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。提出了三种生物分子剪裁基团(葡萄糖基、乙酰基和磺酰基)，以提供可逆保护，此外还具有根据改变的性质(如溶解度和膜透过性)进行剪裁的能力，以及被其他酶识别的能力，从而降低毒性。因此，反应性分子可以在受到保护时在化学上处于惰性状态，然后在需要时通过酶去保护恢复反应性。要使适当的保护基用于所需的应用，需要构建一个菌株工具箱，其中每个受保护的产品都将是稳定的。对于许多这些保护基团，需要从生产菌株中剔除几种酶，以确保小分子产品在细胞环境中的稳定性。高通量比色平板分析将被用来筛选大的基因靶标敲除矩阵。此外，至关重要的是，这些保护基团不能限制产品滴度或生产率。因此，将为每个保护反应进行合理的设计和适应增加的助熔剂。虽然乙酰化和糖基化已经有了相当高的能力，但磺化能力极低。该奖项由CBET部门的生物技术和生化工程项目共同资助，由分子和细胞生物科学部门的系统和合成生物学项目共同资助。

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.