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

PI姓名：Nanette R. BoyleProposal Number: 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行业仍将需要液体燃料。为了解决对可持续资源中液体燃料日益增长的需求，光合细菌将通过基因工程将二氧化碳直接转化为柠檬烯，这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碳氢化合物分子萜类中的一种分子，萜类分子作为燃料、药品和各种商业化学品的原料具有理想的特性。该项目的目标是优化光合细菌的代谢途径，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和高中学生参加国际基因工程机器（iGEM）竞赛，这是一个广为人知且有效的项目，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，聚球菌sp. pcc7002，作为光催化工厂，直接从二氧化碳中生产单萜烯柠檬烯。在细菌中，所有萜类化合物都是通过甲基赤藓糖醇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。目标1将寻求通过工程增加二磷酸异戊烯基（IPP）和二磷酸二甲基烯基（DMAP）的可用性来改善柠檬烯的生产。这将通过用来自高萜类产生生物的遗传物质取代已知的限速酶来实现，包括毛杨（陆生植物）和布朗杆菌（藻类），以绕过这些限速酶的内源性调节。目标2将通过敲除一组基因来进一步提高柠檬烯的产量，从而将通量转向萜类代谢，正如化学计量代谢模型所预测的那样。将根据柠檬烯的高产率选择表现最好的菌株，并使用同位素辅助代谢通量分析（13C-MFA）来确定未知的瓶颈和副产物的形成。将利用同位素辅助代谢通量分析（同位素MFA）对野生型和表现最好的柠檬烯生产菌株进行细胞内代谢通量测量。这将允许代谢工程策略的合理设计，以进一步增加MEP途径通量。目的3研究营养限制和碳储存能力改变对柠檬烯生产的影响。例如，一个突变体缺乏合成糖原的能力，糖原是聚球菌中主要的碳水化合物储存产物，它可能无法感知氮剥夺胁迫。结果，突变体在没有细胞生长和分裂的情况下保持光合作用活性，并通过排泄有机酸（包括萜类前体）来泄漏能量。同位素13C-MFA将用于识别糖原缺陷突变体的代谢瓶颈，以防止过度积累的有机酸进入萜类代谢。这些发现将建议设计工程策略来完成从糖原到柠檬烯的通量重定向。总的来说，这项工作将通过MEP途径创造一种具有增强碳通量的菌株，这种菌株可以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化的化学物质。该平台菌株将在学术界自由共享，以便更快地开发生产菌株并增加对聚珠球菌的了解。

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.