Liu Zhuo, Inokuma Kentaro, Ho Shih-Hsin, Haan Riaan den, Hasunuma Tomohisa, van Zyl Willem H, Kondo Akihiko
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501 Japan.
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, 657-8501 Japan.
Biotechnol Biofuels. 2015 Sep 26;8:162. doi: 10.1186/s13068-015-0344-6. eCollection 2015.
Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed.
In this study, heterologous endoglucanase (EG) and cellobiohydrolase I (CBHI) were produced in a β-glucosidase displaying S. cerevisiae strain using cell-surface display, secretion, or a combined strategy. Strains EG-D-CBHI-D and EG-S-CBHI-S (with both enzymes displayed on the cell surface or with both enzymes secreted to the surrounding medium) showed higher ethanol production (2.9 and 2.6 g/L from 10 g/L phosphoric acid swollen cellulose, respectively), than strains EG-D-CBHI-S and EG-S-CBHI-D (with EG displayed on cell surface and CBHI secreted, or vice versa). After 3-cycle repeated-batch fermentation, the cellulose degradation ability of strain EG-D-CBHI-D remained 60 % of the 1st batch, at a level that was 1.7-fold higher than that of strain EG-S-CBHI-S.
This work demonstrated that placing EG and CBHI in the same space (on the cell surface or in the medium) was favorable for amorphous cellulose-based ethanol fermentation. In addition, the cellulolytic yeast strain that produced enzymes by the cell-surface display strategy performed better in cell-recycle batch fermentation compared to strains producing enzymes via the secretion strategy.
对酿酒酵母进行工程改造以生产异源纤维素酶被认为是从木质纤维素生产生物乙醇的一种有前景的策略。纤维素酶的生产通常通过两种策略之一来实现：将酶展示在细胞表面或分泌到培养基中。然而，据我们所知，尚未在酵母菌株中采用这两种策略的组合。
在本研究中，使用细胞表面展示、分泌或组合策略，在展示β-葡萄糖苷酶的酿酒酵母菌株中生产异源内切葡聚糖酶（EG）和纤维二糖水解酶I（CBHI）。菌株EG-D-CBHI-D和EG-S-CBHI-S（两种酶都展示在细胞表面或两种酶都分泌到周围培养基中）显示出比菌株EG-D-CBHI-S和EG-S-CBHI-D（EG展示在细胞表面而CBHI分泌，或反之亦然）更高的乙醇产量（分别从10 g/L磷酸膨胀纤维素中产生2.9 g/L和2.6 g/L）。经过3轮重复分批发酵后，菌株EG-D-CBHI-D的纤维素降解能力保持在第一批的60%，比菌株EG-S-CBHI-S高1.7倍。
这项工作表明，将EG和CBHI置于同一空间（细胞表面或培养基中）有利于基于无定形纤维素的乙醇发酵。此外，与通过分泌策略生产酶的菌株相比，通过细胞表面展示策略生产酶的纤维素分解酵母菌株在细胞循环分批发酵中表现更好。
