Liu Shuai, Li Yi, Quan Lin, Liu Hai-Xia, Luo Yang, Wang Yong-Zhong
Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, China.
Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing 400030, China.
iScience. 2025 Apr 3;28(5):112341. doi: 10.1016/j.isci.2025.112341. eCollection 2025 May 16.
Optimizing cellulase biosynthesis in is crucial for enhancing enzymatic yield in lignocellulosic biomass conversion. However, the regulatory mechanisms linking intracellular NAD(H/) levels to cellulase production remain elusive. In this study, we systematically screened 13 genes associated with NAD biosynthesis and NADH regeneration in Z2. Employing a modular engineering strategy with four distinct modules, we directed metabolic flux to enhance NAD biosynthesis and NADH regeneration. Key genes (, , , , and ) were identified, and their co-expression in Z2 resulted in a 13.09-fold increase in intracellular NADH levels and a consequential 2.24- and 2.04-fold enhancement in the filter paper-hydrolyzing (FPase [representing total cellulase]) activity and carboxymethylcellulose (CMCase [representing endoglucanase]) activity, respectively. Experimental validations, including antagonist LaCl treatment and gene deletion, unequivocally established the calcium signaling pathway's role in regulating cellulase gene overexpression in response to elevated intracellular NAD(H/) levels.
优化里氏木霉中的纤维素酶生物合成对于提高木质纤维素生物质转化中的酶产量至关重要。然而，将细胞内NAD(H/)水平与纤维素酶生产联系起来的调控机制仍然不清楚。在本研究中，我们系统地筛选了里氏木霉Z2中与NAD生物合成和NADH再生相关的13个基因。采用具有四个不同模块的模块化工程策略，我们引导代谢通量以增强NAD生物合成和NADH再生。鉴定出关键基因（、、、和），它们在里氏木霉Z2中的共表达导致细胞内NADH水平增加13.09倍，相应地，滤纸水解（FPase [代表总纤维素酶]）活性和羧甲基纤维素（CMCase [代表内切葡聚糖酶]）活性分别提高2.24倍和2.04倍。包括拮抗剂LaCl处理和基因缺失在内的实验验证明确证实了钙信号通路在响应细胞内NAD(H/)水平升高时调节纤维素酶基因过表达中的作用。
