Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis
Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis
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DOI：
10.1101/2020.03.23.001982
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发表时间：
2020-03
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影响因子：
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通讯作者：
Ayushi Agrawal;C. K. Bandi;Tucker Burgin;Y. Woo;H. Mayes;Shishir P. S. Chundawat
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文献类型：
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作者：
Ayushi Agrawal;C. K. Bandi;Tucker Burgin;Y. Woo;H. Mayes;Shishir P. S. Chundawat
用于化学酶促合成定制寡糖的碳水化合物活性酶如糖苷酶的工程化受到缺乏合适的基于定向进化的蛋白质工程化方法的限制。目前还没有超高通量筛选方法可用于使用叠氮基糖作为底物的进化的糖合酶的快速和高灵敏度的基于单细胞的筛选。在这里，我们报告了一种基于荧光的方法，采用点击化学的选择性检测糖基叠氮化物（相对于游离无机叠氮化物），促进超高通量的单细胞在体内测定的糖合酶活性。这一发现导致了定向进化方法的发展，用于筛选和分选用于合成所需岩藻糖基化寡糖的糖合酶突变体。我们的筛选技术促进了大肠杆菌中表达的糖合酶变体（>106个突变体）的大型文库的快速荧光激活细胞分选。大肠杆菌中鉴定了几种新的突变体，这些突变体具有增加的β-岩藻糖基-叠氮化物活化的供体糖对所需受体糖的活性，证明了这种方法的更广泛的适用性。
Engineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.