CAZyChip: Dynamic assessment of exploration of glycoside hydrolases in microbial diversity ecosystems.. Bacteria

Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides since they produce a vast variety of glycoside hydrolases. The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharide or glycans. This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares and microarrays were directly synthetized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, previously characterized by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota. The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems. Overall design: 26 plasmidic GH were tested once time, 23 fosmids were tested in triplicate

微生物是参与环境碳循环与植物多糖降解的酶类储备库，因其可产生种类繁多的糖苷水解酶（glycoside hydrolases）。本研究开发的CAZy芯片（CAZyChip）可实现这些糖苷水解酶的转录组水平快速表征，并能鉴定作用于多糖或聚糖水解的酶类。这款DNA生物芯片包含了CAZy数据库（CAZy database）中收录的55,220个细菌糖苷水解酶的特征序列。探针通过两款软件设计完成，微阵列则采用原位喷墨技术（in situ ink-jet technology）直接合成。CAZy芯片的特异性与重复性通过对重组大肠杆菌（E. coli）菌株中提取的已知糖苷水解酶RNA进行杂交实验得到验证，这些菌株此前已通过功能宏基因组学方法完成表征。研究团队还利用瘤胃来源的微生物群，在生物工艺条件下对糖苷水解酶库进行了分析。CAZy芯片可作为一款操作便捷的工具，用于对多种糖苷水解酶的表达谱进行分析。该芯片可用于研究功能多样性的时间动态变化，从而助力从各类生态系统中筛选出新型高效的酶促转化候选酶。实验整体设计：26个质粒携带的糖苷水解酶样本单次检测，23个黏粒（fosmids）样本进行三次重复检测。