Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production
Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production
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在酿酒酵母上用两种微型支架蛋白自表面组装纤维素体以生产纤维素乙醇
DOI：
10.1073/pnas.1209856109
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发表时间：
2012-08-14
影响因子：
11.1
通讯作者：
Tan, Tian-Wei
中科院分区：
文献类型：
--
作者：
Fan, Li-Hai;Zhang, Zi-Jian;Tan, Tian-Wei
通过在酿酒酵母的细胞表面展示微纤维素小体，设计了直接将纤维素，特别是微晶纤维素转化为生物乙醇的酵母。纤维素体的构建和细胞表面附着是由两个单独的微咖啡因完成的，以增加显示水平。所有的纤维素酶包括celCCA（内切葡聚糖酶）、celCCE（纤维素生物水解酶）和Ccel_2454 （β-葡萄糖苷酶）都是从纤维素水解梭菌中克隆出来的，确保了纤维素水解和酵母发酵的热相容性。α-因子分泌纤维素酶和一种微咖啡因；因此，对锚定微咖啡因的组装和附着是在细胞外完成的。免疫荧光显微镜、流式细胞分析（FACS）和纤维素乙醇发酵证实了这种复合物在酵母表面的成功展示。对酶-酶协同、酶-邻近协同、纤维素-酶-细胞协同进行了分析，优化了微咖啡因蛋白的锚定长度。将改造后的酿酒酵母用于羧甲基纤维素（CMC）、磷酸膨胀纤维素（PASC）和Avicel的发酵。对微晶纤维素具有显著的水解活性，乙醇效价为1412 mg/L。这表明，同时糖化和发酵结晶纤维素乙醇可以完成酵母，与微纤维素体工程。
Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae. The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (β-glucosidase) were cloned from Clostridium cellulolyticum, ensuring the thermal compatibility between cellulose hydrolysis and yeast fermentation. Cellulases and one of miniscaffoldins were secreted by α-factor; thus, the assembly and attachment to anchoring miniscaffoldin were accomplished extracellularly. Immunofluorescence microscopy, flow cytometric analysis (FACS), and cellulosic ethanol fermentation confirmed the successful display of such complex on the yeast surface. Enzyme–enzyme synergy, enzyme-proximity synergy, and cellulose–enzyme–cell synergy were analyzed, and the length of anchoring miniscaffoldin was optimized. The engineered S. cerevisiae was applied in fermentation of carboxymethyl cellulose (CMC), phosphoric acid-swollen cellulose (PASC), or Avicel. It showed a significant hydrolytic activity toward microcrystalline cellulose, with an ethanol titer of 1,412 mg/L. This indicates that simultaneous saccharification and fermentation of crystalline cellulose to ethanol can be accomplished by the yeast, engineered with minicellulosome.