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Microbial pectinases are important sources due to the ease of production and unique physicochemical properties. Here, DY2, a strain of Bacillus licheniformis, was identified from 14 strains of bacteria as a pectinase-producing bacterium with good application potential. Optimized carbon sources of submerged fermentation led to the identification of glucose as an ideal carbon source for activity and production of P-DY2, the pectinase produced by DY2. GC-MS based metabolomics was used to explore metabolic mechanisms mediated by glucose, showing the frustrated TCA cycle is necessary to elevate the activity and production of P-DY2. Decreased activity of α-ketoglutaric dehydrogenase and succinate dehydrogenase of DY2 in glucose-treated samples supports the conclusion that P-DY2 production is the TCA cycle-independent. These results reveal a metabolic mechanism of high-activity pectinase mediated by exogenous glucose. These findings highlight the way to understand metabolic mechanisms and promote pectinase yield through metabolomics approach and metabolic modulation, respectively. [ABSTRACT FROM AUTHOR]