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 different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towards l-leucine 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.
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Acknowledgements
All the authors are thankful for the financial support of the National Natural Science Foundation of China (31671840) and National Key R&D Program of China (2018YFA0901700).
Supporting information
Supplementary Table 1—Bacterial strains used in this study.
Supplementary Table 2—Plasmids used in this study.
Supplementary Table 3—Primers used in this study.
Supplementary Table 4—Concentration of l-glutamate in strains.
Supplementary Figure 1—Chromosomal replacement of promoter in C. glutamicum ATCC14067 via the RecET-Cre/loxP system.
Supplementary Figure 2—Flask fermentation of WT, WT-pEC-T18-Peftu-leuA, WT-pEC-T18-Peftu-leuA_13032 and WT-pEC-T18-Peftu-leuAr_13032, and production of l-leucine and the major by-products l-valine and l-isoleucine.
Supplementary Figure 3. Flask fermentation of WT, WT-pEC-XK99E-Peftu-ilvBNC, WT-pEC-XK99E-Peftu-ilvBNrC, WT-pEC-XK99E-Peftu-ilvBNrC (E251K) and WT-pEC-XK99E-Peftu-ilvBNC_CP, and production of l-leucine and the major by-products l-valine and l-isoleucine.
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Luo, G., Zhao, N., Jiang, S. et al. Application of RecET-Cre/loxP system in Corynebacterium glutamicum ATCC14067 for l-leucine production. Biotechnol Lett 43, 297–306 (2021). https://doi.org/10.1007/s10529-020-03000-1
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DOI: https://doi.org/10.1007/s10529-020-03000-1