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Development of a Bacillus subtilis expression system using the improved Pglv promoter.

Ming YM, Wei ZW, Lin CY, Sheng GY - Microb. Cell Fact. (2010)

Bottom Line: Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity.The beta-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system.Thus, we provided a valuable expression system in B. subtilis.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China. yangjinxin@vip.163.com

ABSTRACT

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.

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Characterization of the mutated promoters. The production of β-Gal from B. subtilis 1A747 harboring mutants supplemented with 5% maltose plus 5% glucose. The black square, white triangle, white circle, black diamond and white diamond represents the production of β-Gal from B. subtilis 1A747 harboring pJRINM1, pJRINM2, pJRINM3, pLJ-7 and pJRINM4, respectively; (B) Reduced sugar concentration of culture of B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose; (C) Glucose actual concentration and β-Gal activity detection from B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose plus 5% glucose. The black triangle represents the β-Gal production. The white triangle represents the glucose actual concentration.
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Figure 2: Characterization of the mutated promoters. The production of β-Gal from B. subtilis 1A747 harboring mutants supplemented with 5% maltose plus 5% glucose. The black square, white triangle, white circle, black diamond and white diamond represents the production of β-Gal from B. subtilis 1A747 harboring pJRINM1, pJRINM2, pJRINM3, pLJ-7 and pJRINM4, respectively; (B) Reduced sugar concentration of culture of B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose; (C) Glucose actual concentration and β-Gal activity detection from B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose plus 5% glucose. The black triangle represents the β-Gal production. The white triangle represents the glucose actual concentration.

Mentions: To examine the effect of mutant on alleviating repression caused by glucose, the four recombinants were cultured in LB medium supplemented with 5% maltose and 5% glucose, and the growth curves (Additional file 3) and β-Gal production (Figure 2A) was measured, respectively. The β-Gal production from wildtype promoter was only 1/17 and 1/5 of that in medium without glucose supplement at 24 h and 30 h, respectively. Whereas the β-Gal production from pJRINM1 was 1/4 and 1/3 of that in medium without glucose supplement at 24 h and 30 h, respectively. While, the β-Gal production from pJRINM2, pJRINM3 and pJRINM4 was 1/6, 1/6 and 1/4 of that in medium without glucose supplement at 24 h, respectively. Thus, these mutants alleviated the repression caused by glucose, especially pJRINM1.


Development of a Bacillus subtilis expression system using the improved Pglv promoter.

Ming YM, Wei ZW, Lin CY, Sheng GY - Microb. Cell Fact. (2010)

Characterization of the mutated promoters. The production of β-Gal from B. subtilis 1A747 harboring mutants supplemented with 5% maltose plus 5% glucose. The black square, white triangle, white circle, black diamond and white diamond represents the production of β-Gal from B. subtilis 1A747 harboring pJRINM1, pJRINM2, pJRINM3, pLJ-7 and pJRINM4, respectively; (B) Reduced sugar concentration of culture of B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose; (C) Glucose actual concentration and β-Gal activity detection from B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose plus 5% glucose. The black triangle represents the β-Gal production. The white triangle represents the glucose actual concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908567&req=5

Figure 2: Characterization of the mutated promoters. The production of β-Gal from B. subtilis 1A747 harboring mutants supplemented with 5% maltose plus 5% glucose. The black square, white triangle, white circle, black diamond and white diamond represents the production of β-Gal from B. subtilis 1A747 harboring pJRINM1, pJRINM2, pJRINM3, pLJ-7 and pJRINM4, respectively; (B) Reduced sugar concentration of culture of B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose; (C) Glucose actual concentration and β-Gal activity detection from B. subtilis 1A747 harboring pJRINM1 in LB supplemented with 5% maltose plus 5% glucose. The black triangle represents the β-Gal production. The white triangle represents the glucose actual concentration.
Mentions: To examine the effect of mutant on alleviating repression caused by glucose, the four recombinants were cultured in LB medium supplemented with 5% maltose and 5% glucose, and the growth curves (Additional file 3) and β-Gal production (Figure 2A) was measured, respectively. The β-Gal production from wildtype promoter was only 1/17 and 1/5 of that in medium without glucose supplement at 24 h and 30 h, respectively. Whereas the β-Gal production from pJRINM1 was 1/4 and 1/3 of that in medium without glucose supplement at 24 h and 30 h, respectively. While, the β-Gal production from pJRINM2, pJRINM3 and pJRINM4 was 1/6, 1/6 and 1/4 of that in medium without glucose supplement at 24 h, respectively. Thus, these mutants alleviated the repression caused by glucose, especially pJRINM1.

Bottom Line: Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity.The beta-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system.Thus, we provided a valuable expression system in B. subtilis.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China. yangjinxin@vip.163.com

ABSTRACT

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.

Show MeSH