Collaborative Research: Overproduction and Harvesting of Methylated Triterpenes in a Heterologous Host

面对不断减少的化石燃料储量，高度重视制定能够从可再生资源中生产可行的运输燃料替代品的战略。即使最近美国国内天然气产量大幅增加，可再生燃料仍然是缺乏可比自然资源的国家的优先事项，因此是美国生物技术的一个潜在增长领域。与完成这项任务相关的挑战是广泛的，这一努力的成功最终取决于在生物来源中产生良好的目标分子的能力，以及从来源中有效地分离和回收该分子的能力。这项提案的重点将有两个：1)在细菌宿主中生产一种燃料状化合物，以及2)设计、筛选和实施生物相容性材料，以有效地将该化合物从细菌中分离出来。作为一家以本科生为主的机构的提议，这种直接和相关的替代能源合作方法将有助于促进位于犹他州偏远西南角的迪克西州立大学(DSU)化学与生物学接口本科生研究项目的发展。这个农村社区由不同背景的学生组成，他们将通过拟议的研究计划，与互补大学的现有项目进行合作。化学部的生命过程化学计划和NSF-EPSCoR将资助Rico Del Sesto博士(犹他州迪克西州立大学)，Blaine Pfeifer博士(大学)。纽约州布法罗)和安德鲁·科皮施(北亚利桑那大学)通过多种途径解决上述问题。首先，将合成新的萃取剂，以有效地将生物燃料产品从生物宿主系统中隔离出来。要生产的萃取剂将适用于产生相关异戊二烯化合物的任何宿主，但它们将在具有异源生产平台的本提案中特别使用。已知的C32-灰球菌烯生物合成途径将被移植到大肠杆菌中。特别是，建议的大肠杆菌宿主已经被设计成支持高水平的异戊二烯类化合物的生产(达到紫杉醇前体紫杉二烯的g/L水平)。此外，新的异源系统将受到一系列现成的工程和表征策略的影响，以优化C32-葡萄球虫的生产。因此，整个生产过程将得到一个异源生产宿主的补充，以及新的提取技术的引入。除了通过大肠杆菌高水平生产C32-葡萄球菌素的明确应用目标外，拟议的计划还致力于类异戊二烯生物合成、萃取剂合成和提取潜力的基础知识，以及对这一应用的成功至关重要的细胞萃取剂动力学。因此，该提案同时具有科学和工程方面的特点，以解决一个有意义和及时的问题。推动该提案的协作团队及其集体专门知识也反映了该提案的应用主题和基本主题，以支持和支持拟议的整个生产过程。

In the face of dwindling fossil fuel reserves, a high priority has been placed on the development of strategies capable of producing viable replacements for transportation fuels from renewable resources. Even with a dramatic recent increase in domestic natural gas production in the U.S., renewable fuels remain a priority for countries that lack comparable natural resources, and thus represent a potential area for growth for U.S. biotechnology. The challenges associated with accomplishing this task are extensive, and success in this endeavor ultimately depends upon the ability to generate a good yield of a target molecule in a biological source and an efficient isolation and recovery of the molecule from the source. The focus of this proposal will be twofold: 1) the production of a fuel-like compound in a bacterial host, and 2) the design, screening, and implementation of biocompatible materials to efficiently isolate the compound from the bacteria. As a proposal from a predominantly undergraduate institution, this collaborative approach to an immediate and relevant alternative energy source will help catalyze the development of an undergraduate research program in chemistry at the interface with biology at Dixie State University (DSU), located in the remote southwest corner of Utah. This rural community is comprised of students with diverse backgrounds who, through the proposed research plans, will engage in collaborations with established programs at complementary universities. With this award, the Chemistry of Life Processes Program in the Chemistry Division and NSF-EPSCoR are funding Drs. Rico Del Sesto (Dixie State University, Utah), Blaine Pfeifer (Univ. Buffalo, NY) and Andrew Koppisch (Northern Arizona Univ.) to address the issues above through multiple approaches. First, new extractants will be synthesized for the purpose of efficient sequestration of biofuel products from biological host systems. The extractants to be produced will be applicable to any host generating relevant isoprenoid compounds, but they will particularly be utilized in this proposal with a heterologous production platform. The known biosynthetic pathway for C32-botryococcene will be transplanted into E. coli. In particular, the proposed E. coli host has been engineered to support high-level production of isoprenoid compounds (reaching g/L levels of the Taxol precursor taxadiene). Furthermore, the new heterologous system will be subjected to a range of readily-available engineering and characterization strategies to optimize production of C32-botryococcene. The overall production process will therefore be complemented by a heterologous production host, and the novel introduction of extraction techniques. In addition to a clear applied goal of high level C32-botryococcene production through E. coli, the proposed plan is dedicated to fundamental knowledge of isoprenoid biosynthesis, extractant synthesis and extraction potential, and the cellular-extractant dynamic that is crucial for the success of this application. As such, the proposal features both science and engineering towards a meaningful and timely problem. The applied and basic themes of the proposal are also mirrored by the collaborative team driving this proposal and their collective expertise available to support and enable the overall production process proposed.