Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis

• Main Authors: Chaoyang Zhou, Bin Ye, Shan Cheng, Leizhen Zhao, Yuanxin Liu, Jiandong Jiang, Xin Yan
• Format: Article
• Language: English
• Published: BMC 2019-06-01
• Series: Microbial Cell Factories
• Subjects: Bacillus subtilis; Promoter engineering; Chromosomal integration; Highly expression
• Online Access: http://link.springer.com/article/10.1186/s12934-019-1159-0

Abstract Background Bacillus subtilis is developed to be an attractive expression host to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. Chromosomal integration is a stable expression strategy while the expression level is not ideal compared with plasmid expression. Thus, to meet the requirement of protein overexpression, promoter, as one of the key elements, is important. It is necessary to obtain an ideal promoter for overproduction of foreign proteins from a single copy expression cassette. Results The activity of promoter P ylb was further enhanced by optimizing the − 35, − 10 core region and upstream sequence (UP) by substituting both sequences with consensus sequences. The final engineered promoter exhibited almost 26-fold in β-galactosidase (BgaB) activity and 195-fold in super-folded green fluorescent protein (sfGFP) intensity than that of WT. The two proteins account for 43% and 30% of intracellular proteins, respectively. The promoter was eventually tested by successful extracellular overproduction of Methyl Parathion Hydrolase (MPH) and Chlorothalonil hydrolytic dehalogenase (Chd) to a level of 0.3 g/L (144 U/mL) and 0.27 g/L (4.4 U/mL) on shake-flask culture condition. Conclusions A strong promoter was engineered for efficient chromosomally integrated expression of heterologous proteins.