Genome Engineering a Platform Approach for Biogasoline and Coproducts

我们迫切需要对与现有燃料基础设施兼容并满足未来温室气体排放政策要求的下一代碳氢化合物生物燃料进行基础研究。所提出的研究平台使用工程微生物从可再生原料生产异戊烯醇。然后使用无机催化剂将异戊烯醇转化为异辛烷或其他副产物。可持续的微生物生产异戊烯醇需要一种以化学计量产量、快速速率和高产物浓度运行的生物过程。然而，目前工程生物体中的异戊醇通量很低，而且异戊醇抑制生长和代谢的浓度远低于商业化所需的浓度。在代谢工程的前沿，通过同时和特定地操纵数十个基因来实现复杂性状的工程仍然是一个努力的领域。即使是最小的微生物基因组所编码的突变空间也是巨大的。大自然是如何有效地进入这个空间的，我们还没有完全理解，之前在这一领域的努力还没有远远超出个体基因或途径的水平。拟议的研究将通过使用新的基因组工程工具包来解决工程复杂性状的挑战。具体来说，基因组工程工具包使用了一种新的基因组工程方法，该方法结合了先进的DNA合成、重组和基因组学，可以将传统方法提高几个数量级。本研究的目标是：1)利用合理的大肠杆菌基因组工程增加对异戊烯醇的通量；2)在基因组尺度上绘制异戊烯醇耐受性突变；3)通过组合基因组工程迭代优化性能。更广泛的影响本研究提出的基本基因组工程方法在生物技术的所有领域具有广泛应用的潜力，在这些领域，复杂表型的工程是成本和生命周期分析的重要决定因素。教育活动的重点是通过科罗拉多生物精炼和生物燃料中心（C2B2）和CU-NREL可再生和可持续能源研究所（RASEI），让本科生参与生物燃料研究的综合计划。

1067730GillIntellectual MeritThere is significant need for fundamental research on next-generation hydrocarbon biofuels that are compatible with the existing fuels infrastructure and meet future greenhouse gas policy requirements. The platform for the proposed research uses engineered microorganisms to produce isopentenol from renewable feedstocks. The isopentenol can then be converted to iso-octane or other co-products using inorganic catalysts. The sustainable microbial production of isopentenol requires a bioprocess that operates at stoichiometric yields, fast rates, and high product concentrations. However, isopentenol flux in current engineered organisms is low, and isopentanol inhibits growth and metabolism at concentrations well below what is required for commercialization.The engineering of complex traits through the simultaneous and specific manipulation of dozens of genes across multiple pathways has remained an area of intense effort at the forefront of metabolic engineering. The size of mutational space encoded by even the smallest microbial genomes is immense. How nature manages to effectively access this space is not completely understood, and prior efforts in this area have not previously been extended far beyond the level of individual genes or pathways. The proposed research will address the challenge of engineering complex traits through the use of a new genome engineering toolkit. Specifically, the genome engineering toolkit uses a new approach for genome engineering that combines advanced DNA synthesis, recombineering, and genomics to potentially improve upon traditional approaches by several orders of magnitude. The objectives of the proposed research are to: 1) increase flux to isopentenol using rational genome engineering of E. coli strains, 2) map isopentenol tolerance mutations at the genome-scale, and, 3) iteratively optimize performance via combinatorial genome engineering. Broader ImpactsThe basic genome-engineering approach developed for this proposed research has potential for widespread application across all of areas of biotechnology, where engineering of complex phenotypes is an important determinant in cost and life cycle analyses. The educational activities focus on a comprehensive plan for involvement of undergraduate students in biofuels research through the Colorado Center for Biorefining and Biofuels (C2B2) and CU-NREL Renewable and Sustainable Energy Institute (RASEI).