Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5

  • Yao, Zhuang (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Meng, Yu (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Le, Huong Giang (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Lee, Se Jin (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Jeon, Hye Sung (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Yoo, Ji Yeon (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science (BK21 Four), Graduate School, Gyeongsang National University)
  • Received : 2021.03.15
  • Accepted : 2021.05.06
  • Published : 2021.06.28
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Abstract

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Keywords

Acknowledgement

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03030037) and also by a NRF grant funded by the Korea government (MSIT) (NRF-2020R1A2C100826711). Yao Z, Meng Y, Lee SJ, Jeon HS, and Yoo JY were supported by BK21 program, MOE, Republic of Korea. Le HG was supported by full time graduate student scholarship from Gyeongsang National University.

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