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.