EAGER: Engineering synthetic organelles to power formate-based microbial cell factories

工业规模的生物技术在美国是一个潜在的增长行业。一个特别令人感兴趣的领域是开发专门的细菌，这种细菌可以消耗工业废物产品，并将这些化合物转化为可用于生产燃料、塑料单体和其他重要商业化学品的分子。该项目的重点是将在普通细菌中发现的自然产生的亚细胞工厂重新利用为专门的隔室，用于将工业副产品(甲酸盐)转化为多功能分子(丙酮酸)，细菌可将其用作许多理想化合物的前体。该项目通过强调基于结构信息的生物工程，体现了一种“构建它以理解它”的方法。此外，这将为我们对细菌微室组装的理解以及是否可以通过在微室中包裹来实现酶功能的增强提供关键的测试。该项目将为早期职业科学家提供计算、结构、分子和合成生物学方面的跨学科培训。该项目包括通过基于结构的建模进行计算蛋白质和微室设计，以设计一条合成代谢途径，即PFK-PKT途径。该项目以丙酮酸甲酸裂解酶实现的理论途径为基础，丙酮酸甲酸裂解酶是一种氧敏感酶，可将合成途径限制在厌氧或微好氧条件下。为了解决这一局限性，将建立一个细胞器来包裹丙酮酸甲酸裂解酶和必要的支撑酶，并对重新调整用途的细菌微室壳的渗透性进行工程。将采取几种补充策略，包括自上而下的方法来改变自然产生的甘氨酰自由基酶微室的用途，以及自下而上的方法来构建在微室壳中包裹所需的蛋白质核心。该项目的更广泛影响包括培训本科生和博士后，他们将参加一名知名合成生物学家和一名理论家小组之间的国际合作。此外，该项目展示了基于结构合成生物学的模块化工程，这一方法有望在发展可持续化学以满足紧迫的全球需求方面发挥重要作用。

Industrial scale biotechnology is a potential growth industry in the US. One area of particular interest is the development of specialized bacteria that can consume industrial waste products and convert these compounds into molecules that can be used to produce fuels, plastic monomers and other commercially important chemicals. This project focuses on re-purposing a naturally occurring subcellular factory found in common bacteria into a specialized compartment for the conversion of an industrial byproduct (formate) into a versatile molecule (pyruvate) that can be used by bacteria as a precursor to many desirable compounds. The project exemplifies a "build it to understand it" approach, by emphasizing bioengineering based on structural information. Moreover, it will provide a crucial test of our understanding of bacterial microcompartment assembly and whether enhancement of enzyme function can be achieved by encapsulation in a microcompartment. The project will provide interdisciplinary training to early career scientists in computational, structural, molecular, and synthetic biology. The project involves computational protein and microcompartment design via structure-based modeling to engineer a synthetic metabolic pathway, the PFK-PKT pathway. The project is grounded in a theoretical pathway that can be realized with pyruvate formate lyase, an oxygen-sensitive enzyme that restricts the synthetic pathway to anaerobic or microaerophilic conditions. To solve this limitation, an organelle to encapsulate pyruvate formate lyase and the requisite supporting enzymes will be built and the permeability of the repurposed bacterial microcompartment shell engineered. Several complementary strategies will be pursued, including a top-down approach to repurpose naturally occurring glycyl radical enzyme microcompartments and a bottom-up approach to construct the requisite protein core for encapsulation in the microcompartment shell. The Broader Impacts of the project include the training of undergraduates and Postdoctoral Fellow who will take part in an international collaboration between the groups of an established synthetic biologist and a theoretician. Moreover, the project demonstrates modular engineering based on structural synthetic biology, an approach that is poised to play a significant part in the development of sustainable chemistries to meet pressing global needs.