Kakacek Baily E, Liang Jiabao, Dickerson Kyle A, Gardner Jeffrey G
Department of Biological Sciences, University of Maryland - Baltimore County, Baltimore, Maryland, USA.
Appl Environ Microbiol. 2025 Sep 4:e0081825. doi: 10.1128/aem.00818-25.
Microbial deconstruction of plant polysaccharides is important for environmental nutrient cycling, and bacteria proficient at this process have extensive suites of polysaccharide-specific enzymes. In the gram-negative saprophyte , genome annotation suggests that 17 genes are predicted to encode Carbohydrate-Active enZymes (CAZymes) with roles in cellulose degradation; however, previous work suggested that only a subset of these genes is essential. Building upon that work, here, we identify the required and minimally sufficient set of enzymes for complete degradation of cellulose using a combination of transcriptomics, gene deletion analysis, heterologous expression studies, and metabolite analysis. We identified six CAZyme-encoding genes required for cellulose deconstruction in , which are , , , , , and . These genes encode for a β-glucosidase, an endoglucanase, a cellobiohydrolase, a lytic polysaccharide mono-oxygenase, and two carbohydrate-binding proteins, respectively. These CAZyme-encoding genes are essential for growth using insoluble cellulose by and sufficient for using soluble cellulose when heterologously expressed in . Moreover, during growth using insoluble cellulose, we detected no cellodextrins in the medium, which suggested that cello-oligosaccharide uptake is highly efficient. RNA-seq analysis corroborates these results as we observed several genes significantly upregulated during growth using cellulose that encode TonB-dependent and ABC transporters. Our revised model of cellulose utilization by suggests a greater importance for the Cbp2D and Cbp2E proteins than previously thought and that rapid cellodextrin uptake by is a mechanism to maximize the energetic return on investment for the production and secretion of CAZymes.
Bacterial contributions to fixed carbon turnover in the soil are increasingly being shown as vital to global nutrient cycling. Additionally, the discovery and characterization of bacterial polysaccharide deconstruction enzymes are fundamental for industrial and biomedical applications, such as the production of renewable fuels, sustainable detergents, and nutritional supplements. Our analysis of cellulose deconstruction is significant because it provides a roadmap for analyzing a suite of enzymes that may be functionally redundant and identifies those that are truly essential. Specifically, our results indicate that the functions of the Cbp2D and Cbp2E proteins, previously thought to have only a supporting or accessory role, in fact play a more direct part in cellulose deconstruction in . Furthermore, our results suggest that a strategy where recovery of soluble oligosaccharides is a priority to maximize the energetic economics for polysaccharide degradation in .
植物多糖的微生物解构对于环境养分循环很重要，擅长此过程的细菌拥有大量多糖特异性酶。在革兰氏阴性腐生菌中，基因组注释表明有17个基因预计编码参与纤维素降解的碳水化合物活性酶（CAZymes）；然而，先前的研究表明这些基因中只有一部分是必需的。在此基础上，我们结合转录组学、基因缺失分析、异源表达研究和代谢物分析，确定了完全降解纤维素所需的最少且足够的酶集。我们确定了腐生菌中纤维素解构所需的6个编码CAZyme的基因，分别是 、 、 、 、 和 。这些基因分别编码一种β-葡萄糖苷酶、一种内切葡聚糖酶、一种纤维二糖水解酶、一种裂解多糖单加氧酶和两种碳水化合物结合蛋白。这些编码CAZyme的基因对于腐生菌利用不溶性纤维素生长至关重要，并且在异源表达于 时足以利用可溶性纤维素。此外，在腐生菌利用不溶性纤维素生长期间，我们在培养基中未检测到纤维糊精，这表明纤维寡糖的摄取效率很高。RNA测序分析证实了这些结果，因为我们观察到在利用纤维素生长期间有几个编码依赖TonB的转运蛋白和ABC转运蛋白的基因显著上调。我们修正后的腐生菌纤维素利用模型表明，Cbp2D和Cbp2E蛋白比以前认为的更重要，并且腐生菌对纤维糊精的快速摄取是一种机制，可使CAZyme生产和分泌的能量投资回报最大化。
细菌对土壤中固定碳周转的贡献越来越被证明对全球养分循环至关重要。此外，细菌多糖解构酶的发现和表征对于工业和生物医学应用至关重要，例如可再生燃料、可持续洗涤剂和营养补充剂的生产。我们对腐生菌纤维素解构的分析具有重要意义，因为它为分析一组可能功能冗余的酶提供了路线图，并确定了那些真正必不可少的酶。具体而言，我们的结果表明，以前认为仅具有支持或辅助作用的Cbp2D和Cbp2E蛋白的功能，实际上在腐生菌的纤维素解构中发挥了更直接的作用。此外，我们的结果表明，一种将可溶性寡糖的回收作为优先事项的策略，以最大限度地提高腐生菌中多糖降解的能量经济性。
