Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides.
Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides.
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GM1 神经节苷脂短程化学酶全合成的工艺工程和糖基转移酶改进。
DOI：
10.1002/chem.202300005
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Chen,Xi
中科院分区：
文献类型：
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作者：
Yu,Hai;Zhang,Libo;Yang,Xiaohong;Bai,Yuanyuan;Chen,Xi
GM 1是哺乳动物细胞特别是神经系统细胞中重要的神经节苷脂，需要大规模合成以探索其治疗潜力。生物催化生产是实现这一目的的一个有前途的平台。我们在本文中报告了工艺工程和糖基转移酶改进策略的发展，以推进GM 1的化学酶全合成。首先，开发了一种从市售Garner醛化学合成乳糖基鞘氨醇的新的短路线。其次，提高了空肠弯曲菌β1- 4 GalNAcT（CjCgtA）和β1-3-半乳糖基转移酶（CjCgtB）在大肠杆菌中的可溶性表达。通过与N-末端麦芽糖结合蛋白（MBP）融合来提高大肠杆菌的酶稳定性。第三，通过开发多步一锅多酶（MSOPME）策略，在不分离中间体鞘氨醇的情况下，并通过向后续酶促糖基化步骤中添加去污剂胆酸钠，对从乳糖基鞘氨醇酶促合成GM 1鞘氨醇的工艺进行了设计。将所需的脂肪酰基链安装到GM 1鞘糖醇导致目标GM 1神经节苷脂的形成。糖基转移酶的改进与化学和酶促工艺工程的组合代表了在短路线和高效率地通过全化学酶促合成获得含有不同唾液酸形式的GM 1神经节苷脂方面的显著进展。
Large‐scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases includingCampylobacter jejuniβ1–4GalNAcT (CjCgtA) and β1–3‐galactosyltransferase (CjCgtB) were improved on their soluble expression inE. coliand enzyme stability by fusing with an N‐terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep one‐pot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.