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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.

Increase of pectinase activity is especially important in fermentation industry. Understanding of the metabolic mechanisms can find metabolic modulation approach to promote high yield of pectinase. Higher activity of pectinase was detected in DY1 than DY2, two strains of Bacillus licheniformis. GC-MS-based metabolomics identified differential metabolome of DY2 compared with DY1, characterizing the increased TCA cycle and biosynthesis of fatty acids. Elevated activity of pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) showed global elevation of carbon metabolism, which is consistent with the result that lowers glucose in DY2 than DY1. Inhibitors malonate, furfural and triclosan, of PDH, SDH and biosynthesis of fatty acids, promoted pectinase activity, where triclosan increased pectinase activity by 179%. These results indicate that functional metabolomics is an effective approach to understand metabolic mechanisms of fermentation production and provides clues to develop new methods for changing bacterial physiology and production.

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]

• 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]