State Key Laboratory of Heavy Oil Processing & Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, China.
J Basic Microbiol. 2013 Apr;53(4):381-9. doi: 10.1002/jobm.201100602. Epub 2012 Aug 23.
In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production.
在这项研究中，通过在细胞表面展示活性纤维素酶，对酿酒酵母进行了基因工程改造，使其具有利用纤维素生产生物乙醇的能力。从芽孢杆菌 C-9 中克隆了内切 1,4-β-葡聚糖酶基因 egX，并通过将 egX 与 aga2 融合，将其表达产物 EGX 纤维素酶展示在酿酒酵母的细胞表面，aga2 编码酿酒酵母细胞壁蛋白 α-凝集素的结合亚基。为了获得高基因拷贝数和稳定性，通过将融合 aga2-egX 基因整合到酿酒酵母染色体的 rDNA 区域，实现了多拷贝整合。为了实现高效表达和表面展示效率，aga2-egX 基因在强启动子的控制下表达。通过羧甲基纤维素酶活性测定和免疫荧光显微镜观察，验证了具有酶活性的纤维素酶融合蛋白在酿酒酵母细胞表面的存在。这项工作提出了一种很有前途的策略，可通过基因工程改造酵母，使其有效地发酵纤维素材料生产生物乙醇。
