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

面对日益减少的矿物燃料储备，高度优先考虑的是制订能够用可再生资源生产可行的运输燃料替代品的战略。尽管最近美国国内天然气产量大幅增加，但可再生燃料仍然是缺乏可比自然资源的国家的优先选择，因此代表了美国生物技术的潜在增长领域。与完成这项任务相关的挑战是广泛的，这项努力的成功最终取决于在生物源中产生良好产量的目标分子的能力，以及从源中有效分离和回收分子的能力。本提案的重点将是双重的：1)在细菌宿主中生产类似燃料的化合物，2)设计，筛选和实施生物相容性材料，以有效地从细菌中分离化合物。作为一个以本科生为主的机构的提议，这种直接和相关的替代能源的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）化学与生物学界面的本科研究项目的发展。这个农村社区由不同背景的学生组成，通过拟议的研究计划，他们将与互补大学的既定项目合作。有了这个奖项，化学部门的生命过程化学项目和NSF-EPSCoR将资助博士。Rico Del Sesto（犹他州迪克西州立大学），Blaine Pfeifer（纽约州布法罗大学）和Andrew Koppisch（北亚利桑那大学）通过多种方法解决上述问题。首先，将合成新的萃取剂，以有效地从生物宿主系统中分离生物燃料产品。将要生产的萃取剂将适用于任何产生相关类异戊二烯化合物的宿主，但在本提案中，它们将特别用于异源生产平台。将已知的c32 -芽孢杆菌的生物合成途径移植到大肠杆菌中。特别是，拟议的大肠杆菌宿主已被改造成支持高水平生产类异戊二烯化合物（达到g/L的紫杉醇前体杉二烯水平）。此外，新的异源体系将受到一系列易于获得的工程和表征策略的影响，以优化c32 -芽孢杆菌的生产。因此，整个生产过程将由异源生产宿主和新引入的提取技术加以补充。除了通过大肠杆菌生产高水平c32 -芽孢杆菌的明确应用目标外，该计划还致力于类异戊二烯生物合成、萃取剂合成和萃取潜力的基础知识，以及对该应用成功至关重要的细胞萃取剂动态。因此，该提案的特点是科学和工程都朝着一个有意义和及时的问题。提案的应用和基本主题也反映在推动该提案的协作团队和他们的集体专业知识中，以支持和实现所提议的整个生产过程。

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.