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

今天，人们普遍认为全球变暖是由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，这导致了对未来几十年建立二氧化碳中性生物经济的重新思考和政治承诺。这也需要可持续的，例如基于光合作用提供的有机碳(C)的生物技术过程，即光驱动的二氧化碳固定。由于蓝藻是唯一进行氧光合作用的原核生物，它们作为生物催化剂在光生物技术中的应用越来越受到关注。通过引导代谢通量来合理地改造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。虽然我们只是在开始充分了解蓝藻代谢的调节，最近的研究已经打开了一扇窗，进入一个更全面的基本控制原理的观点。例如，我们最近发现了中枢C代谢的一个关键控制步骤。新固定C的代谢通量主要控制在磷酸甘油酸-变异酶（PGAM）反应中，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）。当C从Calvin-Benson循环中取出并重新定向到较低的糖酵解，即进入从甘油醛3-磷酸（G3P）到丙酮酸的反应序列时，该反应就像一个代谢阀。最近发现，PGAM提供的催化作用是通过与小蛋白PirC的相互作用来控制的，而小蛋白PirC本身受中心PII信号转导蛋白的控制。作为代谢工程的另一个层次，我们的目标是将pgc - pgam开关作为调控工程的关键枢纽。在本项目框架内，我们将采用三种互补的方法：调节PGAM基因表达、通过调节PirC丰度调节PGAM活性和消除PII对PirC的控制，构建带有工程PGAM阀的蓝藻模型菌株PCC 6803的基壳菌株。此外，我们将把我们的方法与“经典”代谢工程策略结合起来，例如引入和表达编码（异源）途径的基因，喂养或其他支持反应或删除竞争反应。调控工程对代谢通量导向预期产物的重要性将通过两种来自卡尔文本森循环（蔗糖）或TCA循环（琥珀酸盐）的代表性化学物质来证明。我们的研究将为蓝藻分子工程的下一阶段提供概念证明。因此，充分利用它们的生物催化潜力，即未来设计光合作用驱动的生物技术应用将是至关重要的。

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.