Trettel Daniel S, Hoang Y, Vecchiarelli Anthony G, Gonzalez-Esquer Cesar R
Los Alamos National Laboratory, Bioscience Division, Microbial and Biome Sciences group, Los Alamos, NM, USA.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 USA.
bioRxiv. 2024 Oct 8:2024.10.08.617227. doi: 10.1101/2024.10.08.617227.
Carboxysomes are polyhedral protein organelles that microorganisms use to facilitate carbon dioxide assimilation. They are composed of a modular protein shell which envelops an enzymatic core mainly comprised of physically coupled Rubisco and carbonic anhydrase. While the modular construction principles of carboxysomes make them attractive targets as customizable metabolic platforms, their size and complexity can be a hinderance. In this work, we design and validate a plasmid set - the pXpressome toolkit - in which α-carboxysomes are robustly expressed and remain intact and functional after purification. We tested this toolkit by introducing mutations which influence carboxysome structure and performance. We find that deletion of vertex-capping genes results in formation of larger carboxysomes while deletion of facet forming genes produces smaller particles, suggesting that adjusting the ratio of these proteins can rationally affect morphology. Through a series of fluorescently labeled constructs, we observe this toolkit leads to more uniform expression and better cell health than previously published carboxysome expression systems. Overall, the pXpressome toolkit facilitates the study and redesign of carboxysomes with robust performance and improved phenotype uniformity. The pXpressome toolkit will support efforts to remodel carboxysomes for enhanced carbon fixation or serve as a platform for other nanoencapsulation goals.
羧酶体是多面体蛋白质细胞器，微生物利用它们来促进二氧化碳同化。它们由模块化蛋白质外壳组成，该外壳包裹着一个酶核心，主要由物理耦合的核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）和碳酸酐酶组成。虽然羧酶体的模块化构建原则使其成为有吸引力的可定制代谢平台目标，但它们的大小和复杂性可能是一个障碍。在这项工作中，我们设计并验证了一组质粒——pXpressome工具包，其中α-羧酶体能够被稳健表达，并且在纯化后保持完整和功能。我们通过引入影响羧酶体结构和性能的突变来测试这个工具包。我们发现，顶点封闭基因的缺失会导致形成更大的羧酶体，而小面形成基因的缺失会产生更小的颗粒，这表明调整这些蛋白质的比例可以合理地影响形态。通过一系列荧光标记构建体，我们观察到这个工具包比以前发表的羧酶体表达系统能带来更均匀的表达和更好的细胞健康状况。总体而言，pXpressome工具包有助于对羧酶体进行研究和重新设计，使其具有强大的性能和改善的表型均匀性。pXpressome工具包将支持为增强碳固定而对羧酶体进行重塑的努力，或作为实现其他纳米封装目标的平台。
