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

合作研究：控制细胞生理学和酶定位以增强酵母中的油脂化学生物合成

• 批准号: 1706545
• 负责人: Ian Wheeldon
• 金额: $ 31.02万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1706545&HistoricalAwards=false
• 关键词: Collaborative; Research; Controlling; Cellular; Physiology

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.

酵母具有产生重要前体，用于生产聚合物，胶粘剂，化妆品，食品乳化剂和香水。这些产品的生物制造为可持续，安全的过程提供了无与伦比的选择性的潜力，可用于生产这两种类型的油化学物质。该项目的研究将是吸引研究生，社区学院和本科生的努力点，从事有机酸和酒精生物生产的工程酵母。 By recruiting students from under-represented backgrounds in Riverside County, California and in the rough upstate of South Carolina, this project seeks to train a divers workforce for the growing industrial biotechnology sector in the United States.The endoplasmic reticulum (ER) and peroxysome are the major sites of lipid and fatty acid modification in ologenous yeast, and the native capacity to accept overexpressed enzymes在这些细胞内的位置有限。该项目的目的是控制脂溶性yarrowia yarrowia中的ER和过氧化物体生理，以增强酶的表达，以合成油脂化学物质。我们的中心假设是，可以通过1）增加生物合成途径的催化和产量来增强ER和过氧化物体接受相关途径酶的能力，以及2）与天然脂质合成和脱位机器的共定位异源脂质化酶。该项目旨在发展基因表达的转录控制，以增殖ER和过氧化物体并增强对这些细胞器的蛋白质运输。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的基因激活和可以控制临时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，因为它们有望创建一条新的途径，以使工程生物合成途径需要细胞内定位以进行功能，从而使传统方法无法轻易访问途径的工程。代谢工程策略和基因调节工具将专门为在Y. lipolytica中应用而开发，以利用其代谢含量碳源（例如葡萄糖，Xylose，甘油和废物脂肪）的高能力，并产生高滴定剂。该提案中体现的新型生产力增强方法具有改变油化学物质的产生，其他产品通过细胞内定位最佳的产品。随之而来的对ER中酶表达的知识也将对合成和系统生物学以及扩展到较大的生物制造社区具有很高的价值。 CBET划分的细胞和生化工程计划的奖项由分子和细胞生物科学分部的系统和合成生物学计划共同资助。

项目成果

Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica

• DOI: 10.1021/acssynbio.9b00498
• 发表时间: 2020-04-17
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Ramesh, Adithya;Ong, Thomas;Wheeldon, Ian
• 通讯作者: Wheeldon, Ian

Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose

• DOI: 10.1002/biot.201700584
• 发表时间: 2018-09-01
• 期刊: BIOTECHNOLOGY JOURNAL
• 影响因子: 4.7
• 作者: Schwartz, Cory;Curtis, Nicholas;Wheeldon, Ian
• 通讯作者: Wheeldon, Ian

Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica

• DOI: 10.1016/j.ymben.2019.06.007
• 发表时间: 2019-09-01
• 期刊: METABOLIC ENGINEERING
• 影响因子: 8.4
• 作者: Schwartz, Cory;Cheng, Jan-Fang;Wheeldon, Ian
• 通讯作者: Wheeldon, Ian