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规模生产提供了可持续的、安全的、无与伦比的选择性的潜力。该项目的研究将成为研究生、社区学院和本科生参与有机酸和醇生物生产工程酵母研究的重点。通过招募来自加州河滨县和南卡罗来纳北部农村地区代表性不足背景的学生，该项目旨在为美国日益增长的工业生物技术部门培养多样化的劳动力。内质网（ER）和过氧化物酶体是产油酵母脂质和脂肪酸修饰的主要部位，在这些细胞内位置接受过表达酶的天然能力是有限的。本课题的目的是控制脂质体耶氏菌的内质网和过氧化物酶体生理，以提高合成油脂化学物质的酶表达。我们的中心假设是：1)增加内质网和过氧化物酶体接受相关途径酶的能力，2)将异种脂质修饰酶与天然脂质合成和降解机制共定位，可以提高生物合成途径的催化作用和产量。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酶体，并加强蛋白质运输到这些细胞器。为了实现这种新的代谢工程策略，转录控制包括基于crispr的基因激活和工程脂肪酸响应启动子，可以控制时间基因表达将被开发。这些贡献具有潜在的变革性，因为它们有望创造一种新的途径来设计需要细胞内定位才能发挥功能的生物合成途径，从而使传统方法无法轻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开发应用于聚脂乳杆菌，以利用其代谢多种碳源如葡萄糖、木糖、甘油和废脂肪的高能力，并产生高滴度的油脂化学物质。本提案中体现的提高生产力的新方法有可能改变油脂化学品和其他最适合细胞内定位的产品的生产。内质网中酶表达的研究所产生的知识对合成生物学和系统生物学以及更大的生物制造界也有很大的价值。该奖项由CBET部门的细胞和生物化学工程项目颁发，由分子和细胞生物科学部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.