Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production

Abstract

Background: L-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, L-ornithine production is based on microbial fermentation, and few microbes are used for producing L-ornithine owing to unsatisfactory production titer.

Results: In this study, Corynebacterium glutamicum S9114, a high glutamate-producing strain, was developed for L-ornithine production by pathway engineering. First, argF was deleted to block L-ornithine to citrulline conversion. To improve L-ornithine production, ncgl1221 encoding glutamate transporter, argR encoding arginine repressor, and putP encoding proline transporter were disrupted. This base strain was further engineered by attenuating oxoglutarate dehydrogenase to increase L-ornithine production. Plasmid-based overexpression of argCJBD operon and lysine/arginine transport protein LysE was tested to strengthen L-ornithine synthesis and transportation. This resulted in efficient L-ornithine production at a titer of 18.4 g/L.

Conclusion: These results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient L-ornithine production and provide new targets for strain development.

Keywords: Corynebacterium glutamicum; L-Ornithine production; Metabolic engineering.

Fig. 1

Metabolic pathway abbreviated drawing for l-ornithine biosynthesis in C. glutamicum. Glc, glucose; G6P, glucose-6-P; PEP, phosphoenolpyruvate; Qxa, oxaloacetate; Mal, malate; Fum, fumarate; Suc, succiate; Suc-CoA, succinyl-coA; Oxo, 2-oxoglutarate; Iso, isocitrate; Cit, citrate; argF, encoding ornithine carbamoyltransferase; odhA, a subunit of ketoglutarate dehydrogenase; argCJBD, an operon involved in arginine synthesis; argR, a repressor for argCJBD; ncgl1221, encoding glutamate transporter; lysE, encoding lysine/arginine transporter; putP, encoding l-proline transporter. The red fonts represent gene deletion, green font means gene overexpression, and blue font means gene attenuation

Fig. 2

Influence of deleting argF on cell growth and l-ornithine production during shake-flask cultivations. a The growth of strain S9114 and Sorn1. b l-Ornithine production. Results of standard deviations present in three individual experiments

Fig. 3

The residual glucose, glutamate and lactate concentration in fermentation broth of Sorn1, Sorn2, Sorn3 and Sorn4. a Glucose and lactate concentration; b l-Glutamate concentration. Results of standard deviations present in three individual experiments

Fig. 4

Effect of ncgl1221 deletion on l-ornithine production and cell growth during shake-flask cultivations. a The growth of Sorn1 and Sorn2 (Sorn1 with ncgl1221 deletion); b l-ornithine curves with temporal change. Results of standard deviations present in three individual experiments

Fig. 5

Effect of argR and putP deletion on l-ornithine production in Sorn2. a The growth of Sorn2, Sorn3 (Sorn2 with argR deletion) and Sorn4 (Sorn3 with putP deletion). b l-Ornithine concentration in fermentation supernatant. Results of standard deviations present in three individual experiments

Fig. 6

Effect of lysE overexpression on l-ornithine production. a The glucose concentration and growth curve of Sorn8 and Sorn11. The solid line represents the glucose concentration curve; the dotted line represents growth curve. b l-Ornithine production curves of Sorn8 and Sorn11. Results of standard deviations present in three individual experiments