Journal
MICROBIAL CELL FACTORIES
Volume 17, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s12934-018-1011-y
Keywords
Bacillus subtilis; Pullulanase; Protease deficient; Host construction; Promoter optimization
Categories
Funding
- Science and Technology Planning Project of Guangdong Province [2016A050503016, 2016A010105004]
- Fundamental Research Funds for the Central Universities [2015ZP032]
- Natural Science Foundation of Guangdong Province [2017A030313097]
Ask authors/readers for more resources
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 ATCC605110. ATCC605110 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 P-spovG promoter produced the highest extracellular PUL activity, which achieved 412.9U/mL. Subsequently, sixteen dual-promoter plasmids were constructed and evaluated using this same method. The plasmid containing the dual promoter P-amyL-P-spovG produced the maximum extracellular PUL activity (625.5U/mL) and showed the highest expression level (the dry cell weight of 18.7g/L).ConclusionsTaken together, we constructed an effective B. subtilis expression system by deleting multiple proteases and screening strong promoters. The dual-promoter P-amyL-P-spovG system was found to support superior expression of extracellular proteins in B. subtilis ATCC6051.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available