SusChEM:Direct CO2 to jet fuel production in a fast growing cyanobacterium

PI姓名：Nanette R. Boyle提案编号：1604691全球对能源的需求正在上升，预计增长最快的行业之一是交通运输业。一些增加的需求可以被使用可再生电力的电动汽车所取代。 然而，航空、航运和长途卡车运输业仍将需要液体燃料。 为了解决对可持续资源中液体燃料需求的增加，光合细菌将通过基因工程将二氧化碳直接转化为柠檬烯，柠檬烯是一种可以进一步加工成喷气燃料的碳氢化合物。 柠檬烯是一类重要的生物产生的烃分子中的一种分子，称为萜类化合物，其具有作为燃料、药物和作为各种商业化学品的原料的期望性质。 该项目的目标是优化光合细菌内的代谢途径，以提高二氧化碳进入柠檬烯生产的通量。 与该项目相关的教育活动将指导一组大学生和高中生参加国际遗传工程机器（iGEM）竞赛，这是一个广为人知的有效计划，旨在促进分子生物学领域科学和社会方面的主动学习。拟议的研究将设计一种光合细菌，Synechococcus sp. PCC 7002，作为直接由二氧化碳生产单萜柠檬烯的光催化工厂。 在细菌中，所有萜类化合物都是通过甲基-4-磷酸（MEP）途径产生的。拟议的研究有三个目标，旨在加强这一途径。目的1将寻求通过工程化增加异戊烯基二磷酸（IPP）和二甲基烯丙基二磷酸（DMAP）的可用性来提高柠檬烯的生产。 这将通过用来自高产萜类化合物生物（包括毛果杨（陆生植物）和布朗葡萄球菌（藻类））的遗传物质取代已知的限速酶来实现，以规避这些限速酶的内源性调节。 目标2将进一步提高柠檬烯生产敲除一套基因，转向流量向萜类代谢，如化学计量代谢模型预测。将基于上级柠檬烯生产率选择表现最佳的菌株，并将使用同位素辅助代谢通量分析（13 C-MFA）来鉴定未知的瓶颈和副产物形成。 将通过使用野生型和表现最好的柠檬烯生产菌株的同位素辅助代谢通量分析（同位素MFA）来测量细胞内代谢通量。这将允许合理设计代谢工程策略以进一步增加MEP途径通量。目的3研究营养限制和碳库改变对柠檬烯产量的影响。 例如，缺乏合成糖原（聚球藻中主要的碳水化合物储存产物）能力的突变体可能无法感知缺氮胁迫。因此，突变体在细胞生长和分裂的情况下保持光合活性，并通过分泌有机酸（包括萜类前体）来释放能量。同位素13 C-MFA将用于确定糖原缺乏突变体中阻止过度积累的有机酸进入萜类代谢的代谢瓶颈。这些发现将建议设计工程策略，以完成从糖原到柠檬烯的流量重定向。 总的来说，拟议的工作将创建一种通过MEP途径增强碳通量的菌株，可用作生产任何萜类化合物的光合平台菌株，从而引入一种新的模式生物来生产这种广泛而多样的化学品。 平台菌株将在学术界自由分享，以加快生产菌株的开发，并增加对聚球藻的了解。

PI Name: Nanette R. BoyleProposal Number: 1604691The global demand for energy is rising, and one of the sectors projected to see the most growth is the transportation industry. Some of the increased demand can be replaced by electric vehicles running on renewable electricity. However, the aviation, shipping and long-haul trucking industries will still require liquid fuels. To address the increased demand for liquid fuels from sustainable resources, photosynthetic bacteria will be genetically engineered to convert carbon dioxide directly to limonene, a hydrocarbon which can be further processed into jet fuel. Limonene is one molecule in an important class of biologically-produced hydrocarbon molecules called terpenoids, which have desirable properties as fuels, pharmaceuticals, and as feedstocks for a variety of commercial chemicals. The goal of this project is to optimize the metabolic pathways within photosynthetic bacteria to enhance the flux of carbon dioxide into limonene production. The educational activities associated with the project will mentor a team of college and high school students to participate in the International Genetically Engineered Machines (iGEM) competition, a widely known and effective program for promoting active learning in the context of the scientific and societal aspects of the field of molecular biology.The proposed research will engineer a photosynthetic bacterium, Synechococcus sp. PCC 7002, to act as a photocatalytic factory for the production of the monoterpene limonene directly from carbon dioxide. In bacteria, all terpenoids are produced through the methylerythritol 4-phosphate (MEP) pathway. The proposed research has three objectives designed to enhance this pathway. Objective 1 will seek to improve the production of limonene by engineering the increased availability of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAP). This will be accomplished by replacing the known rate-limiting enzymes with genetic material sourced from high terpenoid-producing organisms, including Populus trichocarpa (terrestrial plant) and Botryococcus braunii (alga), to circumvent endogenous regulation of these rate-limiting enzymes. Objective 2 will further improve limonene production by knocking out a suite of genes to divert flux toward terpenoid metabolism, as predicted by stoichiometric metabolic modeling. Top-performing strains will be selected based on superior limonene productivity, and isotope assisted metabolic flux analysis (13C-MFA) will be used to identify unknown bottlenecks and side product formation. Intracellular metabolic fluxes will be measured by using isotope-assisted metabolic flux analysis (isotope MFA) of the wild type and top-performing limonene-production strains. This will allow for the rational design of metabolic engineering strategies to further increase MEP pathway flux. Objective 3 will investigate the effect of nutrient limitation and altered carbon storage ability on limonene production. For example, a mutant that lacks the ability to synthesize glycogen, the main carbohydrate storage product in Synechococcus, may not perceive nitrogen-deprivation stress. As a result, the mutant maintains photosynthetic activity in the absence of cell growth and division, and spills energy by excreting organic acids, including terpenoid precursors. Isotopic 13C-MFA will be used to identify metabolic bottlenecks in the glycogen-deficient mutant that prevent the over-accumulating organic acids from entering terpenoid metabolism. These findings will suggest design engineering strategies to complete the redirection of flux from glycogen towards limonene. Overall, the proposed work will create a strain with enhanced carbon flux through the MEP pathway that can be used as a photosynthetic platform strain for the production of any terpenoid, thus introducing a new model organism for producing this wide and diverse class of chemicals. The platform strain will be shared freely amongst the academic community to enable faster development of production strains and increased knowledge of Synechococcus.