Applied Biochemistry and Biotechnology, Vol.191, No.3, 955-967, 2020
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
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 (SNK118 Delta argR Delta argF Delta ncgl2228/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.