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Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization

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Abstract

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell’s outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL−1. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.

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Funding

This work received financial support from the National Key Research and Development Program of China (2019YFA0706900), the National Natural Science Foundation of China (31730067, 31901633), the 111 Project (No. 111–2-06), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018–03), the Natural Science Foundation of Jiangsu Province (BK20180082), and the Jiangnan University Postdoctoral Science Foundation (5818088201191160).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Kang Zhang, Lingqia Su & Jing Wu

  2. School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Kang Zhang, Lingqia Su & Jing Wu

  3. International Joint Laboratory On Food Safety, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Kang Zhang, Lingqia Su & Jing Wu

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  1. Kang Zhang
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  2. Lingqia Su
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  3. Jing Wu
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Contributions

K Zhang and LQ Su designed the study. K Zhang performed the experiment. K Zhang, LQ Su, and J Wu analyzed the data. K Zhang wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jing Wu.

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Zhang, K., Su, L. & Wu, J. Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization. Appl Biochem Biotechnol 194, 1206–1220 (2022). https://doi.org/10.1007/s12010-021-03617-6

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  • DOI: https://doi.org/10.1007/s12010-021-03617-6

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