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.
