To enhance the transcriptional strength of the inducible promoter P_malA and achieve efficient extracellular expression of target proteins, in this study, error-prone PCR was employed to construct a promoter mutant library based on a previously established maltose-inducible expression system (MATE expression system). Using green fluorescent protein (GFP) as a reporter protein, a high-expression mutant designated as M3, harboring a single-base substitution in the-35 region, was identified. The mutant promoter M3 was then utilized as a regulatory element to construct a recombinant expression vector for glucose-6-phosphate isomerase (phospho-glucose isomerase, Pgi), which was fused at its N-terminus to the Sec-type signal peptide AmyS to facilitate secretion. To further optimize inducible expression, various carbon sources were evaluated, and the optimal condition was applied in a 3-L bioreactor for high-density fermentation, yielding a maximum Pgi titer of 7.26 g/L. Functional assays confirmed that the catalytic activity of Pgi expressed in this system was comparable to that of the control produced in Escherichia coli, indicating that enhanced expression did not compromise protein functionality. This study establishes an optimized and efficient MATEbased expression system, offering a feasible strategy for high-level expression of key enzymes in metabolic engineering and supporting the application of Bacillus subtilis in the development of industrial enzyme preparations.