Sasaki Yusuke, Takagi Toshiyuki, Motone Keisuke, Kuroda Kouichi, Ueda Mitsuyoshi
Graduate School of Advanced Integrated Studies in Human Survivability, Kyoto University, Sakyo-ku, Kyoto, 606-8306, Japan.
Div. of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Biotechnol Prog. 2017 Jul;33(4):1068-1076. doi: 10.1002/btpr.2478. Epub 2017 Apr 26.
Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d-xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC-displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co ) especially enhanced the activity by 46-fold. Co also contributed to d-xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0- and 2.7-fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC-displaying yeast showed the faster d-xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co-culture system. A xylan-degrading yeast strain was established by displaying two kinds of xylanases; endo-1,4-β-xylanase (Xyn11B) from Saccharophagus degradans, and β-xylosidase (XlnD) from Aspergillus niger. The yeast co-culture system enabled fine-tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)-displaying yeast and XylC-displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10 : 1.39 × 10 : 2.78 × 10 molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co-culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068-1076, 2017.
来自嗜纤维梭菌的木糖异构酶（XylC）能够同时对木质纤维素生物质的主要成分——D-木糖进行异构化和发酵，是一种极具吸引力的候选酶。在本研究中，我们在先前构建的展示XylC的酿酒酵母菌株中优化了特定的金属阳离子。我们研究了每种金属阳离子对展示XylC的酿酒酵母催化功能的影响。结果表明，二价钴离子（Co²⁺）尤其能使活性提高46倍。Co²⁺ 还有助于D-木糖发酵，分别使乙醇产量和木糖消耗率提高了6.0倍和2.7倍。在混合糖存在的情况下，展示了细胞外木糖异构化系统的实用性。展示XylC的酵母比在细胞内产生木糖异构酶（XI）的酵母摄取D-木糖的速度更快。此外，通过独特的酵母共培养系统进行了直接木聚糖糖化和发酵。通过展示两种木聚糖酶构建了一株木聚糖降解酵母菌株，即来自食糖噬纤维菌的内切-1,4-β-木聚糖酶（Xyn11B）和来自黑曲霉的β-木糖苷酶（XlnD）。酵母共培养系统能够通过调整展示木聚糖酶（Xyn11B和XlnD）的酵母与展示XylC的酵母的接种比例，对展示酶的初始比例（Xyn11B:XlnD:XylC）进行微调。当以1:1:2（1.39×10⁶:1.39×10⁶:2.78×10⁶ 个分子）的比例接种这些酶时，从木聚糖中产生了6.0 g/L的乙醇。因此，本研究中开发的辅因子优化和酵母共培养系统可以拓展从木质纤维素生物质生产生物燃料的前景。© 2017美国化学工程师学会生物技术进展，33:1068 - 1076，2017。
