Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Biotechnol Biofuels. 2014 Jan 14;7(1):8. doi: 10.1186/1754-6834-7-8.
The recombinant yeast strains displaying the heterologous cellulolytic enzymes on the cell surface using the glycosylphosphatidylinositol (GPI) anchoring system are considered promising biocatalysts for direct conversion of lignocellulosic materials to ethanol. However, the cellulolytic activities of the conventional cellulase-displaying yeast strains are insufficient for the hydrolysis of cellulose. In this study, we constructed novel gene cassettes for the efficient cellulose utilization by cellulase-displaying yeast strains.
The novel gene cassettes for the cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reeseii endoglucanase II (EGII) were constructed using the promoter and the GPI anchoring region derived from Saccharomyces cerevisiae SED1. The gene cassettes were integrated into the S. cerevisiae genome, then the β-glucosidase activity of these recombinant strains was evaluated. We revealed that simultaneous utilization of the SED1 promoter and Sed1 anchoring domain in a gene cassette enabled highly-efficient enzyme integration into the cell wall. The β-glucosidase activity of recombinant yeast cells transduced with the novel gene cassette was 8.4-fold higher than that of a conventional strain. The novel EGII-displaying strain also achieved 106-fold higher hydrolysis activity against the water-insoluble cellulose than a conventional strain. Furthermore, direct ethanol production from hydrothermally processed rice straw was improved by the display of T. reeseii EGII using the novel gene cassette.
We have developed novel gene cassettes for the efficient cell-surface display of exo- and endo-type cellulolytic enzymes. The results suggest that this gene cassette has the wide applicability for cell-surface display and that cellulase-displaying yeasts have significant potential for cost-effective bioethanol production from lignocellulosic biomass.
利用糖基磷脂酰肌醇（GPI）锚定系统在酵母细胞表面展示异源纤维素酶的重组酵母菌株被认为是将木质纤维素材料直接转化为乙醇的有前途的生物催化剂。然而，传统的纤维素酶展示酵母菌株的纤维素酶活性不足以水解纤维素。在本研究中，我们构建了新型基因盒，以提高纤维素酶展示酵母菌株的纤维素利用效率。
利用来自酿酒酵母 SED1 的启动子和 GPI 锚定区构建了新型的曲霉β-葡萄糖苷酶（BGL1）和里氏木霉内切葡聚糖酶 II（EGII）的细胞表面展示基因盒。将基因盒整合到酿酒酵母基因组中，然后评估这些重组菌株的β-葡萄糖苷酶活性。我们揭示了在基因盒中同时利用 SED1 启动子和 Sed1 锚定结构域可以高效地将酶整合到细胞壁中。转导新型基因盒的重组酵母细胞的β-葡萄糖苷酶活性比传统菌株高 8.4 倍。新型 EGII 展示菌株对水不溶性纤维素的水解活性也比传统菌株高 106 倍。此外，使用新型基因盒展示里氏木霉 EGII 可提高水热处理后的水稻秸秆的直接乙醇产量。
我们开发了新型的基因盒，用于高效地在酵母细胞表面展示外切型和内切型纤维素酶。结果表明，该基因盒具有广泛的适用性，用于细胞表面展示，并且纤维素酶展示酵母具有从木质纤维素生物质中生产具有成本效益的生物乙醇的巨大潜力。
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