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

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