Dataset Information

Improved Expression and Optimization of Trehalose Synthase by Regulation of Pglv in Bacillus subtilis.

ABSTRACT: Trehalose synthase (TreS) converts maltose to trehalose, which has several important functions; therefore, enhancing TreS expression is desirable. Here, a recombinant Bacillus subtilis W800N (?amyE)-Pglv strain was constructed to achieve enhanced expression of TreS. Process optimization strategies were developed to improve the expression level of TreS in B. subtilis W800N (?amyE)-Pglv. Intracellular activity of TreS was induced using 60?g/L of maltose in shake flask culture. The protein activity reached 5211?±?134 U/g at 33?°C and pH 7.0 in Luria-Bertani medium. A fed-batch fermentation strategy was applied in a 30?L fermenter containing 18?L terrific broth to achieve high cell density by replacing glycerol with high maltose syrup as a carbon source and an inducer. After 32?h of fermentation, recombinant B. subtilis W800N (?amyE)-Pglv activity reached 6850?±?287 U/g dry cell weight. Our results demonstrate the efficiency of the Pglv promoter in increasing the expression of TreS in B. subtilis W800N (?amyE)-Pglv.

SUBMITTER: Liu H

PROVIDER: S-EPMC6488592 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Improved Expression and Optimization of Trehalose Synthase by Regulation of Pglv in Bacillus subtilis.

Liu Hongling H Liu Hao H Yang Shaojie S Wang Ruiming R Wang Tengfei T

Scientific reports 20190429 1

Trehalose synthase (TreS) converts maltose to trehalose, which has several important functions; therefore, enhancing TreS expression is desirable. Here, a recombinant Bacillus subtilis W800N (ΔamyE)-Pglv strain was constructed to achieve enhanced expression of TreS. Process optimization strategies were developed to improve the expression level of TreS in B. subtilis W800N (ΔamyE)-Pglv. Intracellular activity of TreS was induced using 60 g/L of maltose in shake flask culture ...[more]

PMID: 31036837

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Project description:BACKGROUND: B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength. RESULTS: Here, site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The transcription level from four mutants was increased. Production of beta-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of beta-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The beta-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated. CONCLUSIONS: In this study, we obtained a mutated promoter Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.

Project description:Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ?gltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ?gltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ?gltA mutant. The ?gltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ?gltA mutant. These results indicate that the ?gltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ?gltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ?gltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCE Bacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

Dataset Information

Improved Expression and Optimization of Trehalose Synthase by Regulation of Pglv in Bacillus subtilis.

Publications

Improved Expression and Optimization of Trehalose Synthase by Regulation of P<sub>glv</sub> in Bacillus subtilis.

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