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

酵母具有生产聚合物、粘合剂、化妆品、食品乳化剂和香水的重要前体的能力。这些产品的生物制造为这两类油脂化学品的工业化生产提供了具有无与伦比的选择性的可持续、安全的工艺潜力。该项目的研究将是努力吸引研究生、社区学院和本科生参与工程酵母以生物生产有机酸和醇的努力的焦点。通过招收来自加利福尼亚州河滨县和南卡罗来纳州北部农村地区的学生，该项目旨在为美国不断增长的工业生物技术部门培养一支多样化的劳动力队伍。内质网(ER)和过氧化体是产油酵母中脂肪和脂肪酸修饰的主要部位，在这些细胞内位置接受过度表达的酶的天然能力是有限的。本项目的目标是控制解脂亚罗维菌的内质网和过氧化物体生理，以提高油脂化合物合成所需的酶的表达。我们的中心假设是，可以通过以下方法提高生物合成途径的催化作用和产量：1)增加内质网和过氧化物体接受相关途径酶的能力；2)使异种脂类修饰酶与天然的脂类合成和降解机制共定位。该项目旨在开发基因表达的转录控制，以增殖内质网和过氧化物体，并加强蛋白质向这些细胞器的运输。为了实现这一新的代谢工程策略，将开发转录控制，包括基于CRISPR的基因激活和能够控制时间基因表达的工程脂肪酸反应启动子。这些贡献具有潜在的变革性，因为它们有望创造一条新的路线来设计需要细胞内定位功能的生物合成途径，从而使传统方法不容易获得的途径的工程成为可能。代谢工程策略和基因调控工具将专门为解脂耶尔森菌的应用而开发，以利用其高效代谢葡萄糖、木糖、甘油和废脂肪等多种碳源的能力，并产生高滴度的油脂化学物质。这项提议中体现的提高生产率的新方法有可能改变油脂化学品和其他最好由细胞内本地化服务的产品的生产。伴随而来的对内质网中酶表达的研究产生的知识也将对合成和系统生物学产生重要价值，并扩展到更大的生物制造社区。该奖项由CBET部门的细胞和生化工程项目共同资助，由分子和细胞生物科学部门的系统和合成生物学项目共同资助。

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.