Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing, Germany.
Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
Carbohydr Polym. 2022 Feb 15;278:118951. doi: 10.1016/j.carbpol.2021.118951. Epub 2021 Dec 2.
Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC-MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.
多粘类芽孢杆菌是一种能够产生具有工业应用价值的胞外多糖的微生物。然而，由于聚合物组成的复杂性，很长一段时间以来，对多糖的结构解析一直难以实现。通过使用 CRISPR-Cas9 介导的敲除策略，我们首次在相应的基因簇中单独缺失了所有的单糖基转移酶和 Wzy 聚合酶。由此可见，主要的聚合物组分完全被抑制（或缺失），并可以分离出一种纯的含有岩藻糖的低聚糖，将其命名为 paenan II。通过应用这种组合方法，我们通过 HPLC-MS、GC-MS 和 NMR 确定了这种新型聚合物的单糖组成、序列和连接模式。此外，我们还证明了 PepQ、PepT、PepU 和 PepV 糖基转移酶以及 PepG Wzy 聚合酶的敲除会导致 paenan II 的缺失，这表明这些酶参与了重复单元的组装。
