Application of RecET-Cre/ loxP system in Corynebacterium glutamicum ATCC14067 for l -leucine production
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ORIGINAL RESEARCH PAPER
Application of RecET-Cre/loxP system in Corynebacterium glutamicum ATCC14067 for L-leucine production Guangjuan Luo
. Nannan Zhao . Shibo Jiang . Suiping Zheng
Received: 26 November 2019 / Accepted: 3 September 2020 Ó Springer Nature B.V. 2020
Abstract Objective To explore the RecET-Cre/loxP system for chromosomal replacement of promoter and its application on enhancement L-leucine production in Corynebacterium glutamicum (C. glutamicum) ATCC14067. Results The RecET-Cre/loxP system was used to achieve the chromosomal replacement of promoter in C. glutamicum ATCC14067 to adjust the metabolic flux involving the L-leucine synthetic pathway. First, leuAr_13032 from C. glutamicum ATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing
Guangjuan Luo and Nannan Zhao have contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-03000-1) contains supplementary material, which is available to authorized users. G. Luo N. Zhao S. Jiang S. Zheng (&) Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China e-mail: [email protected] G. Luo N. Zhao S. Jiang S. Zheng Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towards Lleucine synthesis. The promoters of pck, odx and pyk2 were overexpressed under the strong promoter Peftu or Psod to improve the supply of pyruvate. Besides, the promoter PilvBNC was employed to dynamically control the transcription level of icd due to its attenuation mechanism by responding to the concentration of L-leucine. The final engineered strain produced 14.05 g L-leucine/L in flask cultivation. Conclusion The RecET-Cre/loxP system is effective for gene manipulation in C. glutamicum ATCC14067. Besides, the results demonstrate the potential of C. glutamicum ATCC14067 for L-leucine production and provide new targets and strategies for strain development. Keywords RecET-Cre/loxP system Corynebacterium glutamicum L-Leucine Dynamic regulation
Introduction The RecET-Cre/loxP system is a powerful method for markerless gene deletion in C. glutamicum ATCC14067 (Huang et al. 2017a, b). The system is composed of two parts: RecE and RecT (RecET)
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protein, an exonuclease-recombinase pairs, and a designed self-excisable linear double-strand DNA (dsDNA) cassette generated by two-steps PCR. The RecET-Cre/loxP system is high-efficient and timesaving, thus, expanding its application ranges in gene manipulation is attractive. L-leucine is an essential amino acid, which is not
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