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、食品乳化剂和香料的重要前体的能力。这些产品的生物制造为可持续、安全的工艺提供了潜力，对这两类油脂化学品的工业规模生产具有无与伦比的选择性。该项目的研究将是努力让研究生、社区学院和本科生从事工程酵母生物生产有机酸和醇的重点。通过在加州的滨江县和南卡罗来纳州的农村地区招募来自代表性不足背景的学生，该项目旨在为美国不断增长的工业生物技术部门培养多样化的劳动力。内质网（ER）和过氧化物酶体是产油酵母中脂质和脂肪酸修饰的主要场所，并且在这些细胞内位置接受过表达的酶的天然能力是有限的。本项目的目的是控制解脂耶氏酵母中的ER和过氧化物酶体生理学，以增强油脂化学品合成的酶表达。我们的中心假设是，生物合成途径的催化和产率可以通过以下方式增强：1）增加ER和过氧化物酶体接受相关途径酶的能力，2）将异源脂质修饰酶与天然脂质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ER和过氧化物酶体并增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，将开发转录控制，包括基于CRISPR的基因激活和可以控制时间基因表达的工程化脂肪酸响应启动子。这些贡献是潜在的转型，因为他们预计将创建一个新的途径，工程生物合成途径，需要细胞内定位的功能，从而使工程的途径，是不容易通过传统的方法。代谢工程策略和基因调控工具将专门开发应用于Y。因此，该公司利用其高代谢能力代谢不同的碳源，如葡萄糖，木糖，甘油和废脂肪，并产生高滴度的油脂化学品。该提案中体现的提高生产率的新方法具有改变油脂化学品和其他产品的生产的潜力，这些产品最好通过细胞内定位来服务。ER中酶表达的伴随研究所产生的知识也将对合成和系统生物学以及更大的生物制造社区具有重要价值。CBET部门的细胞和生物化学工程项目的奖项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.