Abstract
BackgroundBacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Extracellular expression of pullulanase from recombinant B. subtilis is still limited due to the issues on promoters of B. subtilis expression system. This study was undertaken to develop a new, high-level expression system in B. subtilis ATCC6051.ResultsTo further optimize B. subtilis ATCC6051 as a expression host, eight extracellular proteases (aprE, nprE, nprB, epr, mpr, bpr, vpr and wprA), the sigma factor F (spoIIAC) and a surfactin (srfAC) were deleted, yielding the mutant B. subtilis ATCC6051∆10. ATCC6051∆10 showed rapid growth and produced much more extracellular protein compared to the widetype strain ATCC6051, due to the inactivation of multiple proteases. Using this mutant as the host, eleven plasmids equipped with single promoters were constructed for recombinant expression of pullulanase (PUL) from Bacillus naganoensis. The plasmid containing the PspovG promoter produced the highest extracellular PUL activity, which achieved 412.9 U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter PamyL–PspovG produced the maximum extracellular PUL activity (625.5 U/mL) and showed the highest expression level (the dry cell weight of 18.7 g/L).ConclusionsTaken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter PamyL–PspovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051.
Highlights
Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry
We found that using this defective strain to Alkaline β-mannanase l-Asparaginase β-Glucanase Alkaline α-amylase Pullulanase α-Acetolactate decarboxylase Catalase Nattokinase Penicillin G acylase Penicillin G acylase Staphylokinase Xylanase Xylanase Pullulanase Nattokinase Endoxylanase α-Amylase d-Mannose isomerase (MIase)
Eight extracellular proteases were selected as the targets for deletion, because previous studies have demonstrated that the use of proteasedeficient host strains could prevent protein degradation and reduce the secretory stress
Summary
Bacillus subtilis has been widely used as a host for heterologous protein expression in food industry. B. subtilis ATCC6051 is an alternative expression host for the production of industrial enzymes, and exhibits favorable growth properties compared to B. subtilis 168. Bacillus species are regarded as promising host strains with numerous advantages including: non-toxicity, convenience for gene modification and high yields of target proteins, fast growth rate and low Recombinant expression is an important method to facilitate the production of target proteins. Many genetic strategies have been developed to improve the production of recombinant proteins, such as the use of proteasedeficient host strains to prevent degradation [5], the deletion of extracellular protein genes to reduce secretion. B. subtilis ATCC6051 is an alternative expression host for production of industrial enzymes, which exhibits favorable growth properties as compared to the lab strain 168 [14]. Efficient expression vectors have been developed with different promoters like the Pr2 promoter of the sigW gene [17] and the pBL9 promoter of the glvA gene [18], the dualpromoter PgsiB–PHpaII system [19] and the P HpaII–PamyQ′ system [20], etc
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