Enhancing cellulase biosynthesis of Bacillus subtilis Z2 by regulating intracellular NADH level

IF 4.1 2区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES iScience Pub Date : 2025-05-16 Epub Date: 2025-04-03 DOI:10.1016/j.isci.2025.112341

Shuai Liu , Yi Li , Lin Quan , Hai-Xia Liu , Yang Luo , Yong-Zhong Wang

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Abstract

Optimizing cellulase biosynthesis in Bacillus subtilis 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 B. subtilis Z2. Employing a modular engineering strategy with four distinct modules, we directed metabolic flux to enhance NAD⁺ biosynthesis and NADH regeneration. Key genes (ycel, nadV, nadM, mdh, and sucB) were identified, and their co-expression in B. subtilis 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 LaCl₃ treatment and spcF gene deletion, unequivocally established the calcium signaling pathway’s role in regulating cellulase gene overexpression in response to elevated intracellular NAD(H/⁺) levels.

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通过调节胞内NADH水平促进枯草芽孢杆菌Z2纤维素酶的合成

优化枯草芽孢杆菌的纤维素酶合成对提高木质纤维素生物质转化的酶产率至关重要。然而，胞内NAD（H/+）水平与纤维素酶生产之间的调控机制仍不明确。在本研究中，我们系统筛选了枯草芽孢杆菌Z2中与NAD+生物合成和NADH再生相关的13个基因。采用四个不同模块的模块化工程策略，我们指导代谢通量来增强NAD+生物合成和NADH再生。鉴定出关键基因（ycel、nadV、nadM、mdh等），它们在枯草芽孢杆菌Z2中的共表达导致细胞内NADH水平增加13.09倍，滤纸水解酶（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]）活性分别增加2.24倍和2.04倍。实验验证，包括拮抗剂LaCl3治疗和spcF基因缺失，明确确立了钙信号通路在细胞内NAD（H/+）水平升高时调节纤维素酶基因过表达的作用。

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来源期刊

iScience Multidisciplinary-Multidisciplinary

CiteScore

7.20

自引率

1.70%

发文量

1972

审稿时长

6 weeks

期刊介绍： Science has many big remaining questions. To address them, we will need to work collaboratively and across disciplines. The goal of iScience is to help fuel that type of interdisciplinary thinking. iScience is a new open-access journal from Cell Press that provides a platform for original research in the life, physical, and earth sciences. The primary criterion for publication in iScience is a significant contribution to a relevant field combined with robust results and underlying methodology. The advances appearing in iScience include both fundamental and applied investigations across this interdisciplinary range of topic areas. To support transparency in scientific investigation, we are happy to consider replication studies and papers that describe negative results. We know you want your work to be published quickly and to be widely visible within your community and beyond. With the strong international reputation of Cell Press behind it, publication in iScience will help your work garner the attention and recognition it merits. Like all Cell Press journals, iScience prioritizes rapid publication. Our editorial team pays special attention to high-quality author service and to efficient, clear-cut decisions based on the information available within the manuscript. iScience taps into the expertise across Cell Press journals and selected partners to inform our editorial decisions and help publish your science in a timely and seamless way.