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

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.