SusChEM: Development of microbial co-cultures for the production of fatty acids

提案编号：CBET - 1403815首席研究员：Cynthia H. Collins机构：Rensselaer理工学院标题：SusChEM：脂肪酸生产微生物共培养的发展在过去十年中，由于迫切需要找到柴油燃料的替代品，对脂肪酸生产的兴趣已经出现。生产脂肪酸的重组微生物有潜力显著提高生产生物柴油（即可再生柴油）的油的可用性。除了生物柴油外，脂肪酸还是一系列高价值和商品化学品的重要前体，例如用于新型可持续生物聚合物、化妆品、药品和营养保健品的单体。可持续、低成本和高产的游离脂肪酸生产的潜力怎么强调都不为过。该项目的总体目标是产生重组大肠杆菌菌株的共培养物，该菌株从葡萄糖和木糖的混合物中产生高水平的脂肪酸。为了实现这一目标，研究人员将在不同的大肠杆菌菌株之间进行分工，以便每个菌株都是葡萄糖或木糖的专家。该项目的成功完成预计将导致一种范式的转变，从目前的方法，利用优化的单一菌株有效地将处理过的生物质中发现的所有糖转化为生物燃料，到细菌群落，可以协同将简单的碳源转化为感兴趣的产品。所使用的方法和专家的发展也将有助于我们对大肠杆菌生理学的理解，因为观察到的细胞行为是多个基因功能的综合结果。在这项工作中开发的方法和菌株很可能在未来的系统中用于生产大量其他可以用作生物燃料的化合物，如烷烃和脂肪酸乙酯。该项目的目标是开发和应用集成的计算和合成生物学工具，以优化遗传易感微生物大肠杆菌的代谢通量，以从葡萄糖和木糖两种不同的碳源生物合成脂肪酸。在过去，大量的努力已经扩展到工程和优化一个单一的大肠杆菌菌株能够生物合成转化的所有单糖通常在处理的生物质为感兴趣的生物燃料。相比之下，拟议的项目旨在创建重组大肠杆菌菌株的模块化群落，其中每个成员菌株将被优化为将葡萄糖或木糖转化为脂肪酸。核心假设是，与目前使用的依赖于单一优化重组菌株的方法相比，这样的工程群落将提供竞争优势，因为它将减少每个细胞的代谢负担，减少不必要的交叉反应，并通过模块化实现灵活性。提出的系统方法将基于使用研究者开发的方法的化学计量模型的应用。这些方法将被扩展，以允许预测基因缺失和基因表达变化的所有组合，从而导致最佳的脂肪酸滴度和木糖和葡萄糖的产量。发酵数据，连同蛋白质组学数据，将进一步整合菌株和群落优化。建模和菌株开发工作将与使用启动子库的代谢途径平衡相辅相成，以便通过负责脂肪酸生物合成的代谢模块更有效地简化碳通量。本提案的最终目标将研究为改善木糖或葡萄糖转化为脂肪酸而设计的菌株组合如何影响两种碳源混合物的脂肪酸产量。该奖项由CBET部门的生物技术、生化和生物质工程项目颁发，由分子和细胞生物学部门的系统和合成生物学项目共同资助。

Proposal Number: CBET - 1403815 Principal Investigator: Cynthia H. Collins Institution: Rensselaer Polytechnic Institute Title: SusChEM: Development of microbial co-cultures for the production of fatty acids Interest in the production of fatty acids has emerged in the last ten years because of the pressing need to find alternatives for diesel fuels. Fatty acid-producing recombinant microorganisms have the potential to significantly increase the availability of oils for the production of biodiesel (i.e. renewable diesel). In addition to biodiesel, fatty acids are important precursors to a range of high value and commodity chemicals, such as monomers for new, sustainable biopolymers, cosmetics, pharmaceuticals and nutraceuticals. The potential of sustainable, low-cost and high yield production of free fatty acids cannot be overstated. The overall goal of this project is to generate co-cultures of recombinant strains of E. coli that produce high levels of fatty acids from mixtures of glucose and xylose. To achieve this goal the investigators will divide the labor between different strains of E. coli so that each strain is a specialist for either glucose or xylose. The successful completion of the proposed project is expected to lead to a paradigm shift from current methodologies that utilize single strains optimized to efficiently convert all sugars found in treated biomass to biofuels, to bacterial communities that can synergistically convert simple carbon sources to a product of interest. The approaches used and the development of the specialists will also contribute to our understanding of E. coli physiology, as observed cellular behavior is the composite outcome of the function of multiple genes. The methods and strains developed in this work are likely to be used in future systems producing a multitude of other compounds that can be used as biofuels, such as alkanes and fatty acid ethyl esters. The objective of this project is to develop and apply integrated computational and synthetic biology tools for the optimization of metabolic flux in the genetically tractable microorganism Escherichia coli for the biosynthesis of fatty acids from two different carbon sources, glucose and xylose. In the past, significant effort has been extended towards engineering and optimizing a single E. coli strain capable of biosynthetically converting all simple sugars usually found in treated biomass to a biofuel of interest. In contrast, the proposed project aims to create modular communities of recombinant E. coli strains, where each member strain will be optimized for the conversion of either glucose or xylose to fatty acids. The central hypothesis is that such an engineered community will offer a competitive advantage compared with currently used methods that rely on a single optimized recombinant strain because it will reduce metabolic burden on each cell, reduce undesired cross-reactions and will allow for flexibility through modularity. The proposed systems approach will be based on the application of stoichiometric modeling using methodologies developed by the investigators. These methodologies will be extended in order to allow for the prediction of all combinations of gene deletions and changes in gene expression that lead to optimal fatty acid titers and yields from xylose and glucose. Fermentation data, together with proteomics data, will be integrated for further strain and community optimization. The modeling and strain development efforts will be complemented with metabolic pathway balancing using a promoter library, in order to streamline more efficiently the carbon flux through the metabolic modules responsible for fatty acid biosynthesis. The final goal of this proposal will examine how a combination of strains that have been engineered for improved conversion of xylose or glucose to fatty acids affects fatty acid yield from mixtures of the two carbon sources. This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.