Orthwein Tim, Alford Janette T, Becker Nathalie Sofie, Fink Phillipp, Forchhammer Karl
Interfaculty Institute of Microbiology and Infection Medicine Tübingen, University of Tübingen, Tübingen, Germany.
mBio. 2025 May 14;16(5):e0337824. doi: 10.1128/mbio.03378-24. Epub 2025 Apr 3.
The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been identified as a regulating key point in the carbon storage metabolism of cyanobacteria. Upon nitrogen starvation, the iPGAM is inhibited by the P-interacting regulator PirC, which is released from its interaction partner P due to elevated 2-oxoglutarate levels. analysis of 338 different iPGAMs revealed a deep-rooted distinctive evolution of iPGAMs in cyanobacteria. Remarkably, cyanobacterial iPGAMs possess a unique loop structure and an extended C-terminus. Our mass photometry analysis suggests that iPGAM forms a complex with three individual PirC monomers. Biolayer interferometry revealed that the PirC-iPGAM complex is affected by the unique loop and the C-terminal structural elements of iPGAM. A C-terminally truncated iPGAM enzyme showed loss of control by PirC and twofold increased enzymatic activity compared to the iPGAM-WT (wild type), as demonstrated by enzyme assays. By contrast, deleting the loop structure significantly reduced the activity of this variant. Physiological experiments were carried out with different iPGAM variant strains of , in which these structural elements were deleted. The strain expressing the C-terminally truncated iPGAM showed a similar overproduction of polyhydroxybutyrate as deletion of the iPGAM regulator PirC. However, in contrast to the latter, these strains showed higher overall biomass accumulation, making them a better chassis for the production of polyhydroxybutyrate or other valuable substances than the PirC-deficient mutant.IMPORTANCEThe primordial cyanobacteria were responsible for developing oxygenic photosynthesis early in evolution. In the pathways of fixed carbon allocation, the co-factor-independent phosphoglycerate mutase (iPGAM) plays a crucial role by directing the first CO fixation product, 3-phosphoglycerate, toward central anabolic glycolytic-derived pathways. This work reveals a distinct evolution of iPGAM within oxygenic photosynthetic organisms. We have identified two specific segments in cyanobacterial iPGAMs that affect the control of iPGAM activity through its specific interactor protein PirC. This understanding of iPGAM has allowed us to engineer cyanobacterial strains with altered carbon fluxes. Since cyanobacteria can directly convert CO into valuable products, our results demonstrate a novel approach for developing a chassis for biotechnical use.
不依赖2,3-二磷酸甘油酸的磷酸甘油酸变位酶（iPGAM）已被确定为蓝藻碳储存代谢中的一个关键调控点。在氮饥饿条件下，iPGAM受到与磷相互作用的调节因子PirC的抑制，由于2-酮戊二酸水平升高，PirC从其相互作用伙伴P中释放出来。对338种不同的iPGAM进行分析，揭示了蓝藻中iPGAM的一个根深蒂固的独特进化过程。值得注意的是，蓝藻iPGAM具有独特的环结构和延伸的C末端。我们的质量光度分析表明，iPGAM与三个单独的PirC单体形成复合物。生物层干涉术表明，PirC-iPGAM复合物受到iPGAM独特环和C末端结构元件的影响。如酶活性测定所示，C末端截短的iPGAM酶显示出不受PirC控制，且与野生型iPGAM相比酶活性增加了两倍。相比之下，删除环结构显著降低了该变体的活性。使用删除了这些结构元件的不同iPGAM变体菌株进行了生理实验。表达C末端截短的iPGAM的菌株显示出与删除iPGAM调节因子PirC类似的聚羟基丁酸酯过量产生。然而，与后者不同的是，这些菌株显示出更高的总体生物量积累，使其成为比PirC缺陷型突变体更好的用于生产聚羟基丁酸酯或其他有价值物质的底盘。重要性原始蓝藻在进化早期负责发展有氧光合作用。在固定碳分配途径中，不依赖辅因子的磷酸甘油酸变位酶（iPGAM）通过将第一个CO固定产物3-磷酸甘油酸导向中心合成代谢糖酵解衍生途径发挥关键作用。这项工作揭示了iPGAM在有氧光合生物中的独特进化。我们已经在蓝藻iPGAM中鉴定出两个特定片段，它们通过其特定的相互作用蛋白PirC影响iPGAM活性的控制。对iPGAM的这种理解使我们能够构建具有改变的碳通量的蓝藻菌株。由于蓝藻可以直接将CO转化为有价值的产品，我们的结果展示了一种开发用于生物技术用途的底盘的新方法。
