Universal platform for the bioconversion of single carbon feedstocks to fuels and chemicals

人们对开发更可持续的化学品和燃料原料越来越感兴趣。单碳化合物，包括二氧化碳和甲烷，是液体燃料和化学品生产的有吸引力的替代原料，因为利用这些丰富的原料可以升级这些低价值的材料，同时减少温室气体排放。像藻类这样的光合微生物可以利用二氧化碳制造燃料，其他微生物可以代谢甲烷，但效率不高。为了实现可行的生物过程，需要在分子水平上自下而上设计一种更基本、更有效的单碳化合物代谢框架。该项目的目标是利用合成生物学的工具来设计微生物，将包括二氧化碳和甲烷在内的单碳化合物直接转化为各种化学品和燃料。与该项目相关的教育活动包括主持和指导来自休斯敦和谐科学实验室的西班牙裔中学生和高中生。这项研究将开发一个新的代谢平台，用于同化单碳化合物，包括二氧化碳或甲烷，直接在基因工程微生物中生产有机化学品和燃料。提出的工程平台定义了一种新的代谢架构，将碳固定、中心代谢和产物合成整合到一个单一的途径中。该途径使用甲酰基辅酶a作为单碳延伸单元，它绕过了生产常见代谢中间体的需要，并允许碳主链以单碳增量迭代延伸。该平台受到分解代谢α -氧化途径的启发，并使用羟酰基辅酶a裂解酶来催化甲酰基辅酶a和醛之间的C-C键形成。该工程平台由三个模块组成，包括将单个碳分子转化为延伸剂单元的激活模块、将延伸剂单元添加到碳主链上的延伸剂模块和将延伸剂模块的中间体转化为目标产物的终止模块。开发这一途径有四个研究目标。第一个目标是合理设计一种途径，允许使用1碳扩展剂单位甲酰基辅酶a迭代延长碳主链，并从该途径的中间体合成长链产物。第二个目标涉及使用分子和生化技术对途径成分和单个模块的功能组装进行体外表征。第三个目标侧重于通过微生物发酵实现产品合成途径的体内构建和表征，第四个目标侧重于野生型和工程菌株的全系统表征。为此，每个酶促步骤的功能组件将利用合成生物学工具在体内组装，以促进通过微生物发酵将单碳原料有效转化为有价值的产品。本研究提出的新的C-C键形成机制有望对单碳代谢和生物合成做出基础性和潜在的影响。

There is increasing interest in developing more sustainable feedstocks for the chemicals and fuels. One-carbon compounds, including carbon dioxide and methane, are attractive alternative feedstocks for liquid fuel and chemicals production, because utilization of these abundant feedstocks can upgrade these low-value materials and at the same time lower greenhouse gas emissions. Photosynthetic microorganisms such as algae can use carbon dioxide to make fuels, and other microorganisms can metabolize methane, but not very efficiently. A more fundamental and efficient framework for metabolism of one-carbon compounds, designed at the molecular level from the bottom up, is needed to enable a viable biological process. The goal of this project is to use the tools of synthetic biology to engineer microorganisms to convert single carbon compounds, including carbon dioxide and methane, directly into a variety of chemicals and fuels. The educational activities associated with the project include the hosting and mentoring of Hispanic middle and high school students from the Houston Harmony Science in the laboratory of the principal investigator.The research will develop a new metabolic platform for the assimilation of single carbon compounds, including carbon dioxide or methane, directly into the production of organic chemicals and fuels within genetically engineered microorganisms. The proposed engineered platform defines a new metabolic architecture that consolidates carbon fixation, central metabolism, and product synthesis into a single pathway. The pathway uses formyl-CoA as a single carbon extension unit, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single-carbon increments. The proposed platform is inspired by the catabolic alpha-oxidation pathway, and uses the enzyme hydroxyl-acyl-CoA lyase to catalyze C-C bond formation between formyl-CoA and an aldehyde. The engineered platform consists of three modules, including an activation module that converts a single carbon molecule to an extender unit, an elongation module in which the extender unit is added to a carbon backbone, and a termination module, which converts intermediates of the elongation module to target products. There are four research objectives to develop this pathway. The first objective is to rationally design a pathway allowing for the iterative elongation of a carbon backbone using 1-carbon extender unit formyl-CoA and the synthesis of longer-chain products from the intermediates of this pathway. The second objective involves the in vitro characterization of pathway components and functional assembly of individual modules using molecular and biochemical techniques. The third objective focuses on the in vivo construction and characterization of the pathway to achieve product synthesis via microbial fermentations, and the fourth objective focuses on a system-wide characterization of wild-type and engineered strains. Toward this end, functional components for each enzymatic step will be assembled in vivo by making use of synthetic biology tools to facilitate the efficient conversion of single carbon feedstocks to valuable products via microbial fermentation. The new C-C bond formation mechanism advanced by this research will be expected to make fundamental and potentially impactful contributions to one-carbon metabolism and biosynthesis.