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Antibiotic-free production of sucrose isomerase in Bacillus subtilis by genome integration

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Abstract

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose to form isomaltulose, a valuable functional sugar widely used in the food industry. However, the lack of safe and efficient heterologous expression systems hinders SIase production and application. In this study, we achieved antibiotic-free SIase expression in Bacillus subtilis through genome integration. Using CRISPR/Cas9 system, SIase expression cassettes were integrated into various genomic loci, including amyE and ctc, both individually and in combination, resulting in single-copy and muti-copy integration strains. Engineered strains with a maltose-inducible promoter effectively expressed and secreted SIase. Notably, multi-copy strain exhibited enhanced SIase production, achieving 4.4 U/mL extracellular activity in shake flask cultivations. Furthermore, crude enzyme solution from engineered strain transformed high concentrations sucrose into high yields of isomaltulose, reaching a maximum yield of 94.6%. These findings demonstrate antibiotic-free SIase production in B. subtilis via genome integration, laying the foundation for its industrial production and application.

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Data availability

The data that support the findings of the present study are available from the corresponding author on reasonable request.

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Funding

This research was funded by Scientific Research Fund of Liaoning Provincial Education Department (Grant Number LJKFZ20220213), Dalian Polytechnic University College Student Innovation and Entrepreneurship Training Program Project (Grant Number 202310152083), National Natural Science Foundation of China (Grant Number 32072160), and Key Research Project of the Education Department of Liaoning Province (Grant Number LJKZZ20220061).

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Investigation, data curation, writing-original draft, Mingyu Li and Ming Xu; Investigation, data curation, Xinrui Bai, Xiang Wan, and Meng Zhao; Investigation, data curation, supervision, conceptualization, writing-review and editing, funding acquisition, Conggang Wang; writing-review and editing, Xianzhen Li, Xiaoyi Chen, and Fan Yang. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Conggang Wang or Fan Yang.

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The authors have no relevant financial or non-financial interests to disclose.

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This study does not contain any experiments involving human or animal subjects.

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The authors agreed to publish this paper in Biotechnology Letters.

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Li, M., Xu, M., Bai, X. et al. Antibiotic-free production of sucrose isomerase in Bacillus subtilis by genome integration. Biotechnol Lett (2024). https://doi.org/10.1007/s10529-024-03501-3

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  • DOI: https://doi.org/10.1007/s10529-024-03501-3

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