Crawford Conor J, Reintjes Greta, Solanki Vipul, Ricardo Manuel G, Harder Jens, Amann Rudolf, Hehemann Jan-Hendrik, Seeberger Peter H
Max Planck Institute for Colloids and Interfaces, Potsdam 14476, Germany.
Faculty of Biology/Chemistry, University of Bremen, Bremen 28359, Germany.
J Am Chem Soc. 2025 Jul 23;147(29):25799-25805. doi: 10.1021/jacs.5c07546. Epub 2025 Jul 8.
Glycans shape microbiomes in the ocean and the gut, driving key steps in the global carbon cycle and human health. Yet, our ability to track microbial glycan turnover across microbiomes is limited, as identifying active degraders without prior genomic knowledge remains a key challenge. Here, we introduce an activity-based fluorescence resonance energy transfer (FRET) probe that enables direct visualization and quantification of glycan metabolism in complex microbial communities. As a proof of concept, we investigated α-mannan degradation, a prominent polysaccharide in algal blooms. Using automated glycan assembly, we synthesized a mannan hexasaccharide bearing a fluorescein-rhodamine FRET pair. The probe was validated using a recombinantly expressed -α-mannanase (GH76) from sp. Hel_I_6. It was shown to function in cell lysates, pure cultures, and complex microbiomes (via plate assays and microscopy). This probe enabled spatiotemporal visualization of in situ α-mannan turnover in a marine microbiome. Glycan FRET probes are versatile tools for tracking glycan degradation across biological scales from single enzymes to microbiomes.
聚糖塑造了海洋和肠道中的微生物群落，推动着全球碳循环和人类健康的关键步骤。然而，我们追踪微生物群落中聚糖周转的能力有限，因为在没有先验基因组知识的情况下识别活性降解者仍然是一个关键挑战。在这里，我们引入了一种基于活性的荧光共振能量转移（FRET）探针，该探针能够直接可视化和量化复杂微生物群落中的聚糖代谢。作为概念验证，我们研究了α-甘露聚糖的降解，α-甘露聚糖是藻华中的一种主要多糖。通过自动聚糖组装，我们合成了一种带有荧光素-罗丹明FRET对的甘露聚糖六糖。该探针使用来自Hel_I_6菌的重组表达α-甘露聚糖酶（GH76）进行了验证。结果表明，它在细胞裂解物、纯培养物和复杂微生物群落中均能发挥作用（通过平板试验和显微镜观察）。该探针能够对海洋微生物群落中原位α-甘露聚糖的周转进行时空可视化。聚糖FRET探针是用于追踪从单一酶到微生物群落等生物尺度上聚糖降解的通用工具。
