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Microb Cell Fact. 2010 Jul 10;9:55. doi: 10.1186/1475-2859-9-55.

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

Author information

1
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.

PMID:
20618987
PMCID:
PMC2908567
DOI:
10.1186/1475-2859-9-55
[Indexed for MEDLINE]
Free PMC Article

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