Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus
Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus
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DOI：
10.1128/aem.64.12.4857-4861.1998
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发表时间：
1998-12
影响因子：
4.4
通讯作者：
T. Murai;M. Ueda;T. Kawaguchi;M. Arai;A. Tanaka
中科院分区：
文献类型：
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作者：
T. Murai;M. Ueda;T. Kawaguchi;M. Arai;A. Tanaka
摘要：由于酿酒酵母缺乏世界上丰富的水解纤维素材料的纤维素酶复合物，因此将两种参与纤维素材料降解为葡萄糖的水解酶基因共固定在其细胞表面，以直接利用纤维素材料，这是我们研究的最终目标之一。将真菌曲霉（aspergillus aculeatus）的fi -羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶基因分别与酵母α-凝集素C端一半（C端320个氨基酸残基）基因融合，导入酿酒酵母（S. cerevisiae）。CMCase和β-葡萄糖苷酶分别通过CMCase天然信号序列的分泌信号序列和米根霉葡萄糖淀淀酶分泌β-葡萄糖苷酶的信号序列传递到细胞表面。这些基因由酿酒酵母的甘油醛-3-磷酸脱氢酶启动子表达。CMCase和β-葡萄糖苷酶活性在细胞颗粒中检测，而在培养上清中检测不到。免疫荧光显微镜证实了CMCase和β-葡萄糖苷酶蛋白在细胞表面的显示。显示这些纤维素酶的细胞可以在纤维素二糖或水溶性纤维素低聚糖作为唯一碳源上生长。薄层色谱法证实了纤维素低聚糖的降解和同化作用。结果表明，加入这些酶的细胞表面工程酵母具有吸收低聚糖的能力。这是酿酒酵母表达异种纤维素酶基因同化纤维素物质的第一步。
ABSTRACT Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungusAspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.