Collaborative Research: Controlling Cellular Physiology and Enzyme Localization for an Enhanced Oleochemical Biosynthesis in Yeast

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Yeast possess the capability to produce important precursors for the production of polymers, adhesives, cosmetics, food emulsifiers, and perfumes. Biomanufacturing of these products offers the potential for sustainable, safe processes with unparalleled selectivity for industrial-scale production of both these classes of oleochemicals. The research of this project will be the focal point of efforts to engage graduate, community college and undergraduate students in engineering yeast for the bioproduction of organic acids and alcohols. By recruiting students from under-represented backgrounds in Riverside County, California and in the rural upstate of South Carolina, this project seeks to train a diverse workforce for the growing industrial biotechnology sector in the United States.The endoplasmic reticulum (ER) and peroxisome are the major sites of lipid and fatty acid modification in oleaginous yeast, and the native capacity to accept overexpressed enzymes at these intracellular locations is limited. The objective for this project is to control ER and peroxisome physiology in Yarrowia lipolytica to enhance enzyme expression for the synthesis of oleochemicals. Our central hypothesis is that the catalysis and yield of biosynthetic pathways can be enhanced by 1) increasing the capacity of the ER and peroxisomes to accept relevant pathway enzymes, and 2) co-localizing heterologous lipid-modifying enzymes with the native lipid synthesis and degradation machinery. This project seeks to develop transcriptional control of gene expression to proliferate the ER and peroxisomes and enhance protein trafficking to these organelles. To enable this new metabolic engineering strategy, transcriptional controls including CRISPR-based gene activation and engineered fatty acid responsive promoters that can control temporal gene expression will be developed. These contributions are potentially transformational because they are expected to create a novel route to engineer biosynthetic pathways that require intracellular localization for function, thus enabling the engineering of pathways that are not readily accessible by traditional methods. The metabolic engineering strategy and gene regulation tools will be specifically developed for application in Y. lipolytica to exploit its high capacity to metabolize diverse carbon sources such as glucose, xylose, glycerol and waste fats, and produce high titers of oleochemicals. The novel approach to productivity enhancement embodied in this proposal has the potential to transform the production of oleochemicals, and other products best served by intracellular localization. The knowledge generated by the attendant study of enzyme expression in the ER will also be of great value to synthetic and systems biology, and by extension to the larger biomanufacturing community. The award by the Cellular 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.