CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced l-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase

Abstract

Here, Corynebacterium glutamicum SNK118 was metabolically engineered for l-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of l-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for l-ornithine overproduction. In fed-batch fermentation, l-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of l-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote l-ornithine production by C. glutamicum strains.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (31601463), National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-07), National Key R&D Program (2018YFA0901700), Top-notch Academic Programs Project of Jiangsu Higher Education Institutions, and the Program of Introducing Talents of Discipline to Universities (111-2-06).

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Dong, J., Kan, B., Liu, H. et al. CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced l-Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase. Appl Biochem Biotechnol 191, 955–967 (2020). https://doi.org/10.1007/s12010-020-03231-y

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Keywords

  • Corynebacterium glutamicum
  • l-ornithine
  • CRISPR-Cpf1
  • NADPH pool
  • Glyceraldehyde-3-phosphate dehydrogenase