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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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Map of the promoter probe vector pLJ-2. ORI+, ORI- and rep represent the single-strand replication origin, the double strand origin and replication protein in B. subtilis, respectively. ColEI, bgaB and Cm represent E. coli ColEI replicon, chloramphenicol-resistance marker and coding gene of β-Gal. The unique restriction sites are marked on the outside of the map.
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Figure 5: Map of the promoter probe vector pLJ-2. ORI+, ORI- and rep represent the single-strand replication origin, the double strand origin and replication protein in B. subtilis, respectively. ColEI, bgaB and Cm represent E. coli ColEI replicon, chloramphenicol-resistance marker and coding gene of β-Gal. The unique restriction sites are marked on the outside of the map.

Mentions: Using primer pair bga-up/bga-down, the bgaB coding for thermostable β-Gal was polymerase chain reaction (PCR) amplified from plasmid pDL. The obtained 2.0 kb fragment was digested with EcoRI and SacI, and cloned into pGJ103 digested with the same enzymes, resulting in pLJ-2 (Figure 5).


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

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

Map of the promoter probe vector pLJ-2. ORI+, ORI- and rep represent the single-strand replication origin, the double strand origin and replication protein in B. subtilis, respectively. ColEI, bgaB and Cm represent E. coli ColEI replicon, chloramphenicol-resistance marker and coding gene of β-Gal. The unique restriction sites are marked on the outside of the map.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Map of the promoter probe vector pLJ-2. ORI+, ORI- and rep represent the single-strand replication origin, the double strand origin and replication protein in B. subtilis, respectively. ColEI, bgaB and Cm represent E. coli ColEI replicon, chloramphenicol-resistance marker and coding gene of β-Gal. The unique restriction sites are marked on the outside of the map.
Mentions: Using primer pair bga-up/bga-down, the bgaB coding for thermostable β-Gal was polymerase chain reaction (PCR) amplified from plasmid pDL. The obtained 2.0 kb fragment was digested with EcoRI and SacI, and cloned into pGJ103 digested with the same enzymes, resulting in pLJ-2 (Figure 5).

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