Functional Reconstitution of Cellulose Synthase in Escherichia coli
Functional Reconstitution of Cellulose Synthase in Escherichia coli
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DOI：
10.1021/bm501217g
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发表时间：
2014-11-01
影响因子：
6.2
通讯作者：
Sugiyama, Junji
中科院分区：
文献类型：
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作者：
Imai, Tomoya;Sun, Shi-jing;Sugiyama, Junji
纤维素是一种高分子量的β 1 -> 4-d-葡聚糖多糖，广泛分布于自然界，从植物细胞壁到细菌的胞外多糖。纤维素合酶与细胞膜中的其它辅助亚基一起促进纤维素聚合物链成纤维状组装成微纤维。编码纤维素合酶催化亚基的基因是cesA，并且已经在许多纤维素生产生物中鉴定。然而，很少有研究表明重组CesA蛋白合成纤维素聚合物，但CesA蛋白合成纤维素微纤维的机制尚不清楚。在这里，我们表明，纤维素合成活性在大肠杆菌中成功地重建通过表达的细菌纤维素合成酶复合物的木葡糖酸醋杆菌：CesA和CesB（原BcsA和BcsB，分别）。然而，只有当CesA和CesB与双胍基环化酶（DGC）共表达时才能检测到纤维素合酶活性，所述双胍基环化酶（DGC）合成环状-二-GMP（c-di-GMP），其反过来激活细菌中的纤维素合成活性。电子显微镜直接观察发现E.大肠杆菌细胞，其通过纤维素酶处理去除。这种纤维结构不太可能是纤维素I的天然晶体形式，因为它是通过比以往描述的更温和的化学处理转化为纤维素II的。因此，我们可以得出结论，这种细纤维是一种前所未有的纤维素结构。尽管重组酶不能合成纤维素的天然结构，但本研究中描述的名为“CESEC（纤维素合成E。Coli））"，代表了纤维素合成酶的功能分析和播种新纳米材料的有用工具。
Cellulose is a high molecular weight polysaccharide of beta 1 -> 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.