Effect of TatAdCd translocases overexpression on the secretion of lipase in Bacillus subtilis

Abstract:

[Objective] This study focuses on the influence of overexpression of TatAdCd translocases in Tat pathway on the secretion of LipA in Bacillus subtilis. [Methods] The promoter and mRNA leader region of the operon tatAD-CD were replaced by the tandem promoter and mRNA leader region of cdd gene and this modified gene was inserted into sacB gene. The expression level of the tatAD-CD operon was characterized by qRT-PCR. We transformed the plasmid pHP13L which can express lip gene of Bacillus subtilis excessively into the recombinant strain. The influence of overexpression of TatAdCd translocases on the secretion of LipA was assessed by SDS-PAGE and measuring the enzyme activity of the recombinant in fermentation broth. [Results] The modified tatAD-CD operon was expressed successfully. Compared with the control strain, the relative transcription level was improved by 185 times. The enzyme activity of recombinant in fermentation broth was increased by 40% due to overexpression of TatAdCd translocases. [Conclusion] The tandem promoter and mRNA leader region of the cdd gene can increase the expression level of the target gene effectively. LipA can be secreted to extracellular through Sec pathway and Tat pathway in Bacillus subtilis. The overexpression of tatAD-CD operon can promote the secretion of LipA.

Key words:Bacillus subtilis, Lipase fermentation, Tat secretion pathway, tatAD-CD operon, Gene expression