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导致的人为二氧化碳排放造成的。因此，气候变化及其后果关系到公众的主要利益，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合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受到越来越多的关注。为了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是重要的。虽然我们对蓝藻新陈代谢调节的全面了解才刚刚开始，但最近的研究已经打开了一扇窗，使我们能够更全面地了解基本控制原理。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的代谢通量主要由磷酸甘油-变位酶(PGAM)反应控制，该反应将第一个二氧化碳固定产物3-磷酸甘油酸酯(3-PGA)转化为2-磷酸甘油酸酯(2-PGA)。当C从Calvin-Benson循环中取出并重定向到低级糖酵解，即从3-磷酸甘油醛(G3P)到丙酮酸的反应序列时，该反应起到代谢阀的作用。最近发现，PGAM的催化作用是由与小蛋白Pirc的相互作用控制的，而Pirc本身受中央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Pirc-PGAM开关作为调节工程的关键枢纽。在该项目的框架内，我们将构建蓝藻模式菌株聚球藻的底盘菌株。通过三种互补的方法：调节PGAM基因的表达，通过调节Pirc丰度来调节PGAM的活性，以及消除PII对Pirc的控制。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、喂养或以其他方式支持反应或删除竞争反应。来自Calvin Benson循环(蔗糖)或TCA循环(琥珀酸)的两种代表性化学物质将证明调控工程将代谢流量引向预期产品的重要性。我们的研究将为蓝藻下一步的分子工程提供概念验证。因此，充分开发它们的生物催化潜力将是至关重要的，即对于未来光合作用驱动的生物技术应用的设计。

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.