Metabolic engineering of Corynebacterium glutamicum for the production of L-ornithine and putrescine

Abstract

L-ornithine is a non-essential amino acid which is important precursor of urea cycle. It can be used many applications. Many natural and engineered microorganisms for the L-ornithine production were reported. Among them, Corynebacterium glutamicum was employed for the production of L-ornithine under aerobic condition. For the production of L-ornithine, the proB gene was deleted to block the branching competitive pathway and the argF gene was deleted to block the conversion of L-ornithine into L-citrulline. In addition, the argR gene was also deleted to eliminate regulatory repressor of the L-arginine operon. The resulting strain was able to produce 230 mg/L of L-ornithine in flask culture. This strain was further engineered by plasmid-born (pEK0) overexpression of the argC, argJ, argB, argD genes under the native promoter, which resulted in the production of 7.19 g/L of L-ornithine. Additionally, the start codon of pgi and zwf genes was changed to GTG and ATG, respectively. To en-hance NADPH pool, the native promoter of tkt operon was replaced with the strong sod promoter. The final strain (YW04 (pSY223)) was able to produce 51.5 g/L of L-ornithine in 40 h by fed-batch fermentation with the overall yield and productiviy of 0.12 g/g glucose and 1.28 g/L/h.

Putrescine (1,4-diaminobutane) is four carbon linear chain with two amino groups. It has been used to produce nylon-4,6 because of its high melting point and mechanical strength. In this study, C. glutamicum was metabolically engineered for the production of putrescine. Similar strategy was employed to produce putrescine using the base strain under aerobic condition. Therefore, we further deleted the speE gene encoding spermidine synthase to block the conversion of putrescine. We further deleted monoamine oxidase gene (cgl0223) which could convert putrescine into 4-Aminobyryraldehyde. However, there was no accumulation of putrescine because C. glutamicum hasn’t ornithine decarboxylase(speC) which convert L-ornithine into putrescine. Thus, this strain was further engineered by plasmid-born (pEK0) overexpression of the speC gene under the eftu promoter, which resulted in the production of 36.1 g/L of putrescine in fed-batch fermentation. This study demonstrates the possibility for the industrial production of L-ornithine and putrescine in C. glutamicum.