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Metabolic engineering of Escherichia coli for efficient ectoine production

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Abstract

Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry, cosmetics, and biomedicine. In this study, rational engineering strategies have been implemented in Escherichia coli to efficiently produce ectoine. First, the synthetic pathway of ectoine was constructed in E. coli MG1655 by introducing an artificial thermal switch system harboring the ectABC cluster from Halomonas elongate, and the resulting strain produced 1.95 g/L ectoine. Second, crr encoding the glucose-specific enzyme II domain A of phosphotransferase system and iclR encoding the glyoxylate shunt transcriptional repressor were deleted in E. coli for enhancing the oxaloacetate supply, leading to the increasement of the ectoine titer to 9.09 g/L. Third, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was removed from the genome to weaken the competitive pathway; simultaneously, an endogenous feedback-resistant lysC was overexpressed to complement the enzymatic activity deficiency of the aspartate kinase, leading to 30.36% increase of ectoine titer. Next, the expression of phosphoenolpyruvate carboxylase was modulated with varying gradient strength promoters to accelerate the biosynthesis efficiency of ectoine. Finally, aspDH encoding aspartate dehydrogenase from Pseudomonas aeruginosa PAO1 was overexpressed to further improve the biosynthesis of ectoine. The final strain MWZ003/pFT28-ectABC-EclysC*-aspDH-ppc3 produced 30.37 g/L ectoine after 36-h fed-batch fermentation with a yield of 0.132 g/g glucose and a productivity of 0.844 g/(L h).

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Availability of data and material

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. In addition, the GeneBank accession number of the 2433-bp codon-optimized ectABC gene cluster is MW316739.

Code availability

Not applicable.

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Funding

This study was supported by the National Key R&D Program of China (2018YFA0900300), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-10), the Key Technology Project of Inner Mongolia Autonomous Region in China (2019GG302), and the Collaborative Innovation Center of Jiangsu Modern Industrial Fermentation.

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Author notes

  1. Shuyan Zhang and Yu Fang contributed equally to this work.

Authors and Affiliations

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

    Shuyan Zhang, Yu Fang, Lifei Zhu, Hedan Li, Zhen Wang, Ying Li & Xiaoyuan Wang

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

    Shuyan Zhang, Yu Fang, Lifei Zhu, Hedan Li, Zhen Wang, Ying Li & Xiaoyuan Wang

  3. International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China

    Xiaoyuan Wang

Authors
  1. Shuyan Zhang
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Contributions

SZ, YF and XW conceived and designed the research. SZ, YF, LZ, HL, ZW, and YL conducted the experiments. SZ, YF and XW analyzed the data and wrote the manuscript. All the authors read and approved the manuscript.

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Correspondence to Xiaoyuan Wang.

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On behalf of all the authors, the corresponding author states that there is no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Zhang, S., Fang, Y., Zhu, L. et al. Metabolic engineering of Escherichia coli for efficient ectoine production. Syst Microbiol and Biomanuf 1, 444–458 (2021). https://doi.org/10.1007/s43393-021-00031-1

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  • DOI: https://doi.org/10.1007/s43393-021-00031-1

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