Conversion of CO2 into valuable products: Regulatory engineering of cyanobacteria to direct carbon flux into desired reactions

将二氧化碳转化为有价值的产品：蓝藻的调控工程将碳通量引导至所需的反应

• 批准号: 513290319
• 负责人: Professor Dr. Karl Forchhammer
• 金额: --
• 依托单位: Lehrstuhl Organismische Interaktionen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/513290319?language=en
• 关键词: Conversion; CO2; into; valuable; products

Today, it is widely accepted that global warming is caused by anthropogenic CO2 emissions due to the combustion of fossil fuels. Accordingly, climate change and its consequences are of main public interest, which led to a rethinking and the political commitment for the establishment of a CO2-neutral bio-economy in the next decades. This also requires sustainable, e.g. biotechnological processes based on organic carbon (C) delivered by photosynthetic, i.e. light-driven CO2 fixation. As cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, they receive growing interest as biocatalysts in photo-biotechnological applications nowadays. To rationally engineer cyanobacteria by channeling metabolic fluxes to obtain the maximum yield of a desired chemical product, it is also important to consider native molecular processes that control primary metabolism. Although we are only at the beginning of a full understanding of the regulation of cyanobacterial metabolism, recent research has opened the window into a more comprehensive view on fundamental control principles. For instance, we recently discovered a key control step of central C metabolism. The metabolic flux of newly fixed C is majorly controlled at the phosphoglycerate-mutase (PGAM) reaction, which converts the first CO2 fixation product 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA). This reaction acts as a metabolic valve as C is taken out from the Calvin-Benson cycle and re-directed towards lower glycolysis, i.e. into the reaction sequence from glyceraldehyde 3-phosphate (G3P) to pyruvate. As revealed recently, the catalysis provided by PGAM is controlled by the interaction with the small protein PirC, which itself is under control of the central PII signal transduction protein. As another level of metabolic engineering we aim to target the PirC-PGAM switch as key hub of regulatory engineering. In the frame of the project, we will construct chassis strains of the cyanobacterial model strain Synechocystis sp. PCC 6803 with engineered PGAM valves by using three complementary approaches: tuning PGAM gene expression, tuning PGAM activity by modulating PirC abundance and eliminating the control of PII over PirC. In addition, we will combine our approach with “classical” metabolic engineering strategies, e.g. the introduction and expression of genes encoding (heterologous) pathways, feeding or otherwise supporting reactions or the deletion of competing reactions. The significance of regulatory engineering to direct metabolic flux towards the anticipated product will be demonstrated by two representative chemicals derived either from the Calvin Benson cycle (sucrose) or the TCA cycle (succinate). Our study will provide a proof of concept for the next level of molecular engineering of cyanobacteria. Accordingly, it will be crucial to fully exploit their biocatalytic potential, i.e. for the future design of photosynthesis-driven biotechnological applications.

今天，人们普遍认为，全球变暖是由于化石燃料燃烧造成的人为二氧化碳排放造成的。因此，气候变化及其后果是主要的公共利益，这导致了重新思考和政治承诺，在未来几十年建立一个二氧化碳中性生物经济。这还需要可持续的生物技术工艺，例如基于通过光合作用提供的有机碳（C）的生物技术工艺，即光驱动的CO2固定。由于蓝藻是唯一能进行光合作用的原核生物，它们作为光生物催化剂在光生物技术中的应用越来越受到人们的关注。为了通过引导代谢通量来合理地工程化蓝藻以获得所需化学产品的最大产量，考虑控制初级代谢的天然分子过程也很重要。虽然我们只是在一个充分了解蓝藻代谢的调节的开始，最近的研究已经打开了一个窗口，更全面地了解基本的控制原则。例如，我们最近发现了中央C代谢的关键控制步骤。新固定的C的代谢通量主要由磷酸甘油酸变位酶（PGAM）反应控制，该反应将第一个CO2固定产物3-磷酸甘油酸（3-PGA）转化为2-磷酸甘油酸（2-PGA）。该反应充当代谢阀，因为C从Calvin-Benson循环中取出并重新导向较低的糖酵解，即进入从甘油醛3-磷酸（G3P）到丙酮酸的反应序列。正如最近所揭示的，PGAM提供的催化作用是由与小蛋白PirC的相互作用控制的，PirC本身是在中央PII信号转导蛋白的控制下。作为代谢工程的另一个层面，我们的目标是将PirC-PGAM开关作为调控工程的关键枢纽。在该项目的框架下，我们将通过使用三种互补的方法构建具有工程PGAM阀的蓝藻模式菌株集胞藻属PCC 6803的底盘菌株：调节PGAM基因表达，通过调节PirC丰度来调节PGAM活性，以及消除PII对PirC的控制。此外，我们将联合收割机与"经典"代谢工程策略相结合，例如，引入和表达编码（异源）途径的基因，喂养或以其他方式支持反应或删除竞争反应。将通过来自卡尔文本森循环（蔗糖）或TCA循环（琥珀酸）的两种代表性化学物质证明调控工程对引导代谢通量朝向预期产物的重要性。我们的研究将为下一阶段的蓝藻分子工程提供概念验证。因此，至关重要的是要充分利用其生物催化潜力，即未来设计光合作用驱动的生物技术应用。