SusChEM: Development of a Protecting Group Toolkit for Metabolic Engineering

1605465约翰·迪伯将活细胞改造成化学工厂，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、清洁地催化困难的化学反应。然而，细胞呈现用于进行这些多步合成的复杂环境，并且因此，经常发生不期望的反应性以产生不期望的化学产物，或者甚至更糟地，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位点何时和何处将是反应性的。这种一般方法将通过采用天然化学基团在电池中模拟，所述天然化学基团可以可逆地添加以实现对电池中化学品的反应性的类似控制。能够催化所得到的定制产品的有效保护和稳定性的微生物菌株将引起代谢工程师的广泛兴趣，并将提供给科学和工程界。设计代谢途径以可持续地产生感兴趣的分子需要在多个水平上进行设计控制。许多问题会限制生产力，包括代谢物毒性、非途径催化和无法分泌最终产物。已经开发了各种合成生物学方法用于在DNA、RNA和蛋白质水平引入控制以解决许多这些挑战。在本提案中，代谢物水平的控制是本工具包中的一项额外策略的目标。在合成有机化学中用于获得对官能团反应性的控制的保护基策略将被模拟以获得对分子何时和何处是活性的类似控制。提出了三种生物分子剪裁基团（葡糖基、乙酰基和磺酰基），以提供可逆的保护，此外还能够剪裁改变的性质，如溶解性和膜渗透性，以及通过其他酶的识别，从而降低毒性。因此，可以使反应性分子在受保护时化学惰性，然后在需要时通过酶促脱保护恢复反应性。使合适的保护基团能够用于所需的应用需要构建菌株的工具包，其中这些受保护的产物中的每一种都是稳定的。对于许多这些保护基团，需要从生产菌株中敲除几种酶，以确保小分子产物在细胞环境中的稳定性。将采用高通量比色板测定来筛选基因靶点敲除的大矩阵。此外，至关重要的是，这些保护基团不限制产物滴度或生产速率。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。尽管乙酰化和葡糖基化预期已经具有相当高的能力，但磺化能力极低。由此产生的菌株应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。CBET部门的生物技术和生物化学工程计划的这一奖项由分子和细胞生物科学部门的系统和合成生物学计划共同资助。

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.