Xiao Yan, Dong Sheng, Liu Ya-Jun, You Chun, Feng Yingang, Cui Qiu
CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Shandong Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China; Shandong Energy Institute, Qingdao, China; Qingdao New Energy Shandong Laboratory, Qingdao, China; Dalian National Laboratory for Clean Energy, Qingdao, China; University of Chinese Academy of Sciences, Beijing, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Int J Biol Macromol. 2023 Oct 1;250:126226. doi: 10.1016/j.ijbiomac.2023.126226. Epub 2023 Aug 7.
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery.
嗜热细菌热纤梭菌能有效地将多糖降解为寡糖。在热纤梭菌中，β-1,4-连接的纤维寡糖的代谢由三种酶启动，即纤维糊精磷酸化酶（Cdp）、纤维二糖磷酸化酶（Cbp）和β-葡萄糖苷酶A（BglA）。相比之下，对于含有其他连接方式的寡糖是如何被利用的，人们却知之甚少。在本研究中，我们发现BglA水解β-1,3-二糖层二糖的活性远高于其水解β-1,4-二糖纤维二糖的活性。通过晶体结构测定和分子对接分析了底物特异性的结构基础。分别对BglA和Cbp进行基因缺失，并对细胞提取物进行酶分析，结果表明BglA是负责层二糖代谢的关键酶。此外，BglA的缺失会抑制Cbp的表达，而Cbp的缺失会上调BglA的表达，这表明BglA和Cbp存在交叉调节，且BglA对纤维二糖代谢也至关重要。这些见解为深入了解热纤梭菌的代谢和调控奠定了基础，并强调了木质纤维素生物炼制中含有β-1,3-连接糖苷键的多糖的降解和利用的重要性。
