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Pectinases are enzymes that catalyze pectin degradation. There is a global demand for pectinases because of their wide utility and catalytic efficiency. Optimization of the fermentation process to increase the pectolytic enzyme activity is generally practiced to lower process costs, but whether temperature influences the metabolome, enhancing pectinase activity, is not known. Here, we developed a metabolomics approach to explore it. The activity of P-DY2 pectinase produced by Bacillus licheniformis DY2 was higher in cells grown at 30°C than those grown at 37°C. Differential metabolome analysis revealed fluctuating tricarboxylic acid (TCA) cycle at 30°C. Consistently, the transcripts of TCA cycle genes and activities of pyruvate dehydrogenase and α-Ketoglutaric dehydrogenase were lower at 30°C than 37°C. Furthermore, inhibition of pyruvate dehydrogenase and succinate dehydrogenase enhanced the activity of P-DY2, supporting the conclusion that the inactivated pyruvate metabolism and TCA cycle were required for pectinase activity, and that P-DY2 was TCA cycle-independent. Collectively, these findings indicated that fermentation temperature affected P-DY2 activity by metabolic modulation, with an inactivated TCA cycle as a characteristic feature of high P-DY2 activity. More importantly, the present study highlights an approach of promoting pectinase activity through metabolic modulation by using metabolic pathway inhibitors. [ABSTRACT FROM AUTHOR]

• Pectinase activity varied in DY2 during 48 h submerged fermentation. • Metabolic shift is related to the fermentation period. • Reduced fatty acid biosynthesis is believed to be required for pectinase yield. • Verified experiments were carried out by adding key enzymes' inhibitors. Fermentation conditions affect pectinase production. Among these conditions, fermentation period is one of the important parameters to be optimized for saving the process costs. However, information regarding whether metabolic regulation plays a role is not available. Here, a Bacillus licheniformis strain DY2 was incubated in submerged fermentation for pectinase production for 48 h. The pectinase activity was assayed every 4 h from 8th h of fermentation. The pectinase activity was low at 8 h, middle at 28 h, and peak at 44 h. Consistently, metabolomics analysis showed that the metabolic shift is related to the fermentation period. The metabolomes exhibited almost of differential metabolites in the enriched metabolic pathways, which were decreased at 28 h and 44 h compared to 8 h in a time-dependent manner. Furthermore, reduced palmitic acid and stearic acid in a time-dependent manner were identified as crucial biomarkers at 48 h. These findings in our work reveal that the reduced fatty acid biosynthesis is required for pectinase yield, which was further demonstrated by addition of inhibitors furfural and triclosan to inhibit pyruvate dehydrogenase and fatty acid biosynthesis, respectively. Our work reveals a potential possibility to elevate pectinase yield through metabolomics modulation. [ABSTRACT FROM AUTHOR]