Overexpression of a Thermostable α-Amylase through Genome Integration in <i>Bacillus subtilis</i>
Yifan Yang,
Xiaoping Fu,
Xingya Zhao,
Jianyong Xu,
Yihan Liu,
Hongchen Zheng,
Wenqin Bai,
Hui Song
Affiliations
Yifan Yang
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
Xiaoping Fu
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Xingya Zhao
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Jianyong Xu
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Yihan Liu
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
Hongchen Zheng
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Wenqin Bai
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
Hui Song
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702.
