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Systematic engineering of transport and transcription to boost alkaline α-amylase production in Bacillus subtilis

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Abstract

In the present work, we used systematic engineering at transport and transcription levels to significantly enhance alkaline α-amylase production in Bacillus subtilis 168M. Signal peptide YwbN’ proved to be optimal. Alkaline α-amylase production was elevated by deleting a putative peptide segment of YwbN’. Insertion of arginine (R) between residues 5 and 6 of YwbN’∆p further increased the protein yield. Enhancing positive charges at sites 4 and 10 and decreasing the hydrophobicity of the H-region of YwbN’∆p were critical for improving alkaline α-amylase production in B. subtilis 168M. PHpaII was the optimal promoter, and deleting − 27T or − 31A from PHpaII enhanced the transcription of the target gene. Using a single-pulse feeding-based fed-batch system, alkaline α-amylase activity of B. subtilis 168M P∆−27T was increased by 250.6-fold, compared with B. subtilis 168M A1.

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Funding

This work was funded by National Natural Science Foundation of China (21406089), Natural Science Foundation of Jiangsu Province (BK20140152, BK20191185), and the Open Project Program of the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, China (KLCCB-KF201802), 111 Project (111-2-06).

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Authors and Affiliations

  1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Haiquan Yang, Yingfang Ma & Yuan Zhao

  2. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Haiquan Yang, Yingfang Ma, Wei Shen & Xianzhong Chen

  3. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Wei Shen & Xianzhong Chen

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  1. Haiquan Yang
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  2. Yingfang Ma
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  3. Yuan Zhao
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  4. Wei Shen
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  5. Xianzhong Chen
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Contributions

H.Y. and X.C. designed the research; H.Y. and Y.M. performed the research; W.S., Y.Z., and X.C. analysed the data; H.Y., Y.M., and X.C. wrote this paper.

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Correspondence to Haiquan Yang or Xianzhong Chen.

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This work follows ethical standards. It contains no experiments with animals performed or human participants.

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Yang, H., Ma, Y., Zhao, Y. et al. Systematic engineering of transport and transcription to boost alkaline α-amylase production in Bacillus subtilis. Appl Microbiol Biotechnol 104, 2973–2985 (2020). https://doi.org/10.1007/s00253-020-10435-z

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