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Chemically inducible chromosome evolution of Corynebacterium glutamicum for producing 4-hydroxyisoleucine

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Abstract

4-Hydroxyisoleucine (4-HIL) holds potential value in the treatment of diabetes. It can be produced by expressing the exogenous isoleucine dioxygenase gene ido in L-isoleucine (Ile) producing Corynebacterium glutamicum strains. But the stable expression of ido on plasmids relies on the usage of antibiotics. To make the harboring of ido independent of plasmid, this study developed a chromosome-engineered strain for synthesizing 4-HIL directly from glucose. First, the ido-cat-ido expressing cassette was inserted into the chromosome of C. glutamicum, and the copy number of ido was increased through chemically inducible chromosome evolution (CIChE). After successive rounds of CIChE by increasing chloramphenicol concentration, 7 copies of ido were integrated in the chromosome of C. glutamicum SE04, and the 4-HIL production reached 20.3 ± 4.99 g/L, 3.5-fold higher than the initial strain SC12 harboring two-copies of ido. To cease further homologous recombination, recA was deleted in CIChE strains, but cell growth and 4-HIL production were damaged. Notably, the stability of chromosomally inserted genes in the evolved strain SE04 was confirmed. Ultimately, the evolved C. glutamicum SE04 strain produced 30.3 g/L of 4-HIL in a 2-L bioreactor. This study established a plasmid-free strain of C. glutamicum for 4-HIL production, offering new insights into utilizing multi-copy integration methods for producing other valuable biochemical substances in C. glutamicum.

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Authors and Affiliations

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

    Weiqing Chen, Rui Chen & Feng Shi

  2. State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, 214122, China

    Weiqing Chen, Rui Chen & Feng Shi

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  1. Weiqing Chen
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  2. Rui Chen
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Contributions

FS conceived and designed research. WC and RC conducted experiments. WC and FS analyzed data. FS, WC and RC wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Feng Shi.

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Chen, W., Chen, R. & Shi, F. Chemically inducible chromosome evolution of Corynebacterium glutamicum for producing 4-hydroxyisoleucine. Syst Microbiol and Biomanuf 5, 1252–1260 (2025). https://doi.org/10.1007/s43393-025-00350-7

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