A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.
A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.
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DOI：
10.1093/glycob/cwac082
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发表时间：
2023-03-06
期刊：
影响因子：
4.3
通讯作者：
中科院分区：
文献类型：
--
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重组聚糖和糖缀合物的糖工程是一个快速发展的领域。然而，聚糖的生产和开发落后于蛋白质和核酸。生物合成的糖缀合物生产需要细菌细胞内三个关键组分的协调合作：底物蛋白、偶联寡糖基转移酶和聚糖生物合成位点。虽然受体蛋白和寡糖基转移酶是单基因的产物，但聚糖是多基因代谢途径的产物。通常，聚糖生物合成基因座被克隆并从天然生物体整体转移至合适的大肠杆菌菌株。然而，这些途径内的基因表达已经通过天然宿主中的自然选择进行了优化，并且对于不相关生物体中的异源生产来说不太可能是最佳的。近年来，合成生物学通过解构这些途径并自下而上重建它们来解决多基因系统异源表达的挑战。DNA组装方法的使用允许通过在单个步骤中将限定的部分与必需的编码序列组合来方便地组装此类途径。在本研究中，我们将组合组装应用于大肠杆菌中空肠弯曲菌N-糖基化（pgl）途径的异源生物合成。杆菌我们设计了重建的生物合成簇，忠实地复制了C。空肠七糖聚糖。此外，在单轮组合组装和筛选之后，我们鉴定了优于天然未修饰的pgl簇的聚糖和糖缀合物生产的途径克隆。该平台提供了一种灵活的方法，用于优化E.杆菌
Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.
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影响因子：
3.5
作者：
Ihssen, Julian;Kowarik, Michael;Thoeny-Meyer, Linda
通讯作者：
Thoeny-Meyer, Linda
影响因子：
4.7
作者：
Bassalo, Marcelo C.;Garst, Andrew D.;Gill, Ryan T.
通讯作者：
Gill, Ryan T.
影响因子：
3.6
作者：
Linton, D;Dorrell, N;Wren, BW
通讯作者：
Wren, BW
用于提高大肠杆菌糖基化效率的系统代谢工程。
DOI：
10.1016/j.bbrc.2012.02.020
发表时间：
2012-03-16
影响因子：
3.1
作者：
Pandhal J;Desai P;Walpole C;Doroudi L;Malyshev D;Wright PC
通讯作者：
Wright PC
影响因子：
5.8
作者：
Kay EJ;Yates LE;Terra VS;Cuccui J;Wren BW
通讯作者：
Wren BW