Limits...
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.

Show MeSH
RT-PCR analysis of the mutated promoters (A) and the production of β-Gal driven by these promoters (B, C and D). (A) Real-time PCR analysis of the transcription amount from pLJ-7, pJRINM1, pJRINM2, pJRINM3 and pJRINM4, respectively (16 s rDNA used as the control). (B) The production of β-Gal when cultured in LB. 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. (C) The production of β-Gal when cultured in LB supplemented with 5% maltose. 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; (D) SDS-PAGE analysis of β-Gal from the recombinants in B. subtilis 1A747 after 24 h cultured on a 12% SDS-polyacrylamide gel. β-Gal is indicated by the arrow. Molecular mass marker indicates (top to bottom): 116, 66, 45, 35 and 25 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908567&req=5

Figure 1: RT-PCR analysis of the mutated promoters (A) and the production of β-Gal driven by these promoters (B, C and D). (A) Real-time PCR analysis of the transcription amount from pLJ-7, pJRINM1, pJRINM2, pJRINM3 and pJRINM4, respectively (16 s rDNA used as the control). (B) The production of β-Gal when cultured in LB. 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. (C) The production of β-Gal when cultured in LB supplemented with 5% maltose. 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; (D) SDS-PAGE analysis of β-Gal from the recombinants in B. subtilis 1A747 after 24 h cultured on a 12% SDS-polyacrylamide gel. β-Gal is indicated by the arrow. Molecular mass marker indicates (top to bottom): 116, 66, 45, 35 and 25 kDa.

Mentions: To examine the expression efficiency of the obtained four mutants, they were sub-cloned and engineered with synthetic ribosome binding site. The resultant recombinants pJRINM1, pJRINM2, pJRINM3 and pJRINM4, in which the bgaB was under the control of four mutants respectively, were transformed into B. subtilis 1A747 to investigate the expression of β-Gal. Real-time PCR assay (Figure 1A) showed that compared with the pLJ-7, the transcription amount from the pJRINM1, pJRINM2 and pJRINM3 were increased in different degrees, in which the mutant pJRINM1 is obviously prior to the pLJ-7. This suggested the site-directed mutagenesis of the Pglv promoter is efficient in these three mutants.


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

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

RT-PCR analysis of the mutated promoters (A) and the production of β-Gal driven by these promoters (B, C and D). (A) Real-time PCR analysis of the transcription amount from pLJ-7, pJRINM1, pJRINM2, pJRINM3 and pJRINM4, respectively (16 s rDNA used as the control). (B) The production of β-Gal when cultured in LB. 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. (C) The production of β-Gal when cultured in LB supplemented with 5% maltose. 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; (D) SDS-PAGE analysis of β-Gal from the recombinants in B. subtilis 1A747 after 24 h cultured on a 12% SDS-polyacrylamide gel. β-Gal is indicated by the arrow. Molecular mass marker indicates (top to bottom): 116, 66, 45, 35 and 25 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: RT-PCR analysis of the mutated promoters (A) and the production of β-Gal driven by these promoters (B, C and D). (A) Real-time PCR analysis of the transcription amount from pLJ-7, pJRINM1, pJRINM2, pJRINM3 and pJRINM4, respectively (16 s rDNA used as the control). (B) The production of β-Gal when cultured in LB. 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. (C) The production of β-Gal when cultured in LB supplemented with 5% maltose. 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; (D) SDS-PAGE analysis of β-Gal from the recombinants in B. subtilis 1A747 after 24 h cultured on a 12% SDS-polyacrylamide gel. β-Gal is indicated by the arrow. Molecular mass marker indicates (top to bottom): 116, 66, 45, 35 and 25 kDa.
Mentions: To examine the expression efficiency of the obtained four mutants, they were sub-cloned and engineered with synthetic ribosome binding site. The resultant recombinants pJRINM1, pJRINM2, pJRINM3 and pJRINM4, in which the bgaB was under the control of four mutants respectively, were transformed into B. subtilis 1A747 to investigate the expression of β-Gal. Real-time PCR assay (Figure 1A) showed that compared with the pLJ-7, the transcription amount from the pJRINM1, pJRINM2 and pJRINM3 were increased in different degrees, in which the mutant pJRINM1 is obviously prior to the pLJ-7. This suggested the site-directed mutagenesis of the Pglv promoter is efficient in these three mutants.

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