Yang Peizhou, Wu Yun, Zheng Zhi, Cao Lili, Zhu Xingxing, Mu Dongdong, Jiang Shaotong
College of Food and Biological Engineering, Anhui Key Laboratory of Intensive Processing of Agricultural Products, Hefei University of Technology, Hefei, China.
Front Microbiol. 2018 Oct 10;9:2436. doi: 10.3389/fmicb.2018.02436. eCollection 2018.
The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of expression cassette containing glyceraldehyde-3-phosphate-dehydrogenase gene () promoter in the chromosome. The target insertion site was found to be located in the hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered . By using orange peel as the fermentation substrate, the filter paper, endo-1,4-β-glucanase, exo-1,4-β-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type , respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.
木质纤维素生物乙醇的发展在替代石化能源和农业废弃物的高值利用方面发挥着重要作用。通过将成簇规律间隔短回文重复序列-Cas9方法应用于含有甘油醛-3-磷酸脱氢酶基因（）启动子的表达盒在染色体中的整合，实现了纤维素酶基因的安全稳定表达。通过设计gRNA表达载体，发现目标插入位点位于己糖激酶2中。重组SESTC蛋白在工程菌中表现出约44 kDa的大小。以橙皮为发酵底物，转化体的滤纸酶、内切-1,4-β-葡聚糖酶、外切-1,4-β-葡聚糖酶活性分别为1.06、337.42和1.36 U/mL，分别比野生型高35.3倍、23.03倍和17倍。处理6小时后，获得了约20 g/L的葡萄糖。在厌氧条件下，48小时发酵后最高乙醇浓度达到7.53 g/L，比野生型（0.2 g/L）高37.7倍。工程菌株可为木质纤维素乙醇的开发提供有价值的材料。
