Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco’s CO2 fixation, yet hornwort Rubisco remains poorly characterized. Here we assembled the hornwort Anthoceros agrestis Rubisco (AaRubisco) using the Arabidopsis thaliana SynBio expression system and observed the formation of stalled intermediates, prompting us to develop a new SynBio system with A. agrestis cognate chaperones. We successfully assembled AaRubisco and Rubisco from three other hornwort species. Unlike A. thaliana Rubisco, AaRubisco assembly is not dependent on RbcX or Raf2. Kinetic characterization reveals that hornwort Rubiscos exhibit a range of catalytic rates (3–10 s−1), but with similar affinity (∼30 μM) and specificity (∼70) for CO2. These results suggest that hornwort Rubiscos do not comply with the long-held canonical catalytic trade-off observed in other land plants, providing experimental support that Rubisco kinetics may be phylogenetically constrained. Unexpectedly, we observed a 50% increase in AaRubisco catalytic rates when RbcX was removed from our SynBio system, without any reduction in specificity. Structural biology, biochemistry, and proteomic analysis suggest that subtle differences in Rubisco large-subunit interactions, when RbcX is absent during biogenesis, increases the accessibility of active sites and catalytic turnover rate. Collectively, this study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discoveries will contribute to developing new approaches for engineering Rubisco with superior kinetics.

中文翻译：

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合成生物学系统揭示的金缕梅 Rubiscos 的独特生物发生和动力学


金獨草是唯一使用类焦烯来优化 Rubisco 的 CO2 固定的陆生植物，但金缕梅 Rubisco 的特征仍然很差。在这里，我们使用拟南芥 SynBio 表达系统组装了金缕草 Anthoceros agrestis Rubisco （AaRubisco），并观察到停滞中间体的形成，促使我们开发了一种具有 A. agrestis 同源伴侣的新 SynBio 系统。我们成功地组装了来自其他三种金缕梅的 AaRubisco 和 Rubisco。与 A. thaliana Rubisco 不同，AaRubisco 组装不依赖于 RbcX 或 Raf2。动力学表征表明，金缕梅 Rubiscos 表现出一系列催化速率 （3–10 s-1），但对 CO2 具有相似的亲和力 （∼30 μM） 和特异性 （∼70）。这些结果表明，金缕梅 Rubiscos 不符合在其他陆地植物中观察到的长期规范催化权衡，为 Rubisco 动力学可能受到系统发育限制提供了实验支持。出乎意料的是，当从我们的 SynBio 系统中删除 RbcX 时，我们观察到 AaRubisco 催化速率增加了 50%，而特异性没有任何降低。结构生物学、生物化学和蛋白质组学分析表明，当生物发生过程中不存在 RbcX 时，Rubisco 大亚基相互作用的细微差异会增加活性位点的可及性和催化周转率。总的来说，这项研究揭示了一个以前未知的 Rubisco 动力学参数空间，并提供了一个 SynBio 底盘来扩展对其他 Rubisco 动力学的研究。我们的发现将有助于开发具有卓越动力学的 Rubisco 工程新方法。