L-ornithine is an important component of the urea cycle and is a non-essential amino acid. It is known to be beneficial in the treatment of liver diseases. The growth hormone levels in the serum after heavy exercise are elevated by L-ornithine supplementation. The metabolic engineering of microorganisms founded as a way to produce L-ornithine. Engineered E.coli could synthesize 13.2mg of L-ornithine per gram cell dry weight accompanied by addition of glutamate in the culture medium. The 3 gene knockout strain C.glutamicum ATCC 13032 could increase the L-ornithine content in the cell by 16.49mg/gram dry cell weight and there was 179.14mg/L of L-ornithine increase in the culture medium.
In this study, speE gene was knocked out from the C.glutamicum ATCC 13032 strain deficient in argF and proB genes. The resultant mutant is made to grow in an adaptive environment to evolve metabolically and improve the production of L-ornithine in it.

Results of the study
The spermidine synthase gene speE engineered in C.glutamicum could over-express L-ornithine compared to the wild type species. The mutant of C.glutamicum to argF and proB was used to delete speE gene to result in APE mutant of C.glutamicum. The deletion of speE could elevate the production of L-ornithine and the APE mutant strain generated 11.3+/-0.3g/L of L-ornithine. This value was higher than the L-ornithine levels (10.2+/-0.2g/L) generated by argFproB mutant of C.glutamicum. Therefore, APE mutant strain of C.glutamicum was used as the initial strain for adaptive evolution.

Adaptive evolution
Cornybacterium glutamicum ΔAPE was allowed for adaptive evolution by growing them in fermentation medium added with glucose and L-ornithine. The organism will grow without any blockages. After adaptive evolution, the clone C.glutamicum ΔAPE6937 was used for further investigation. L-ornithine was produced by C.glutamicum ΔAPE6937 strain as 13.6+-0.5g/L. This was 20 percent greater than the L-ornithine produced by the parent strain C.glutamicum ΔAPE (11.3+/-0.3g/L).

Evolved strain - Characterization
The L-ornithine biosynthesis enzymes present in the evolved strain of C.glutamicum ΔAPE6937 are pgi (glucose-6 phosphate isomerase), gap (glyceraldehyde-3-phosphate dehydrogenase), gdh (glutamate dehydrogenase), argB (acetyl glutamate kinase), pfkA (6-phospho fructokinase), pyk (pyruvate kinase), gltA (citrate synthase), argJ (ornithine acetyl transferase/N-acetyl glutamate synthase) and pyc (pyruvate carboxylase). All these genes present in the evolved strain of C.glutamicum ΔAPE6937 were upregulated. Analysis of these genes did not result in any identification of mutations compared to the parent strain.

Genetic modification in the evolved C.glutamicum
The argB gene expression was found to act as a regulator of L-ornithine biosynthesis in the evolved strain. Over-expression of argB gene taken from C.glutamicum or E.coli was done in the evolved strain. The concentration of L-ornithine in C.glutamicum increased with the over-expression of argB gene than the over-expression of argB gene in E.coli.

The arg operon associated with the regulation of L-ornithine biosynthesis pathway was regulated by ArgR or arginine repressor. The removal of argR gene is another method of increasing the arg operon expression. The removal of argR gene of evolved strain C.glutamicum ΔAPE6937 will result in the C.glutamicum ΔAPE6937R42 strain. The strain C.glutamicum ΔAPE6937R42 could synthesize 17.3+/-0.4g/L of L-ornithine which was observed as 27 percent greater than that of C.glutamicum ΔAPE6937.
The transcription of the genes that are involved in the L-ornithine biosynthesis in C.glutamicum ΔAPE 6937R42 were analyzed using qRT-PCR. This data was compared with that obtained from C.glutamicum ΔAPE. The upregulation of pgi, argB, argJ, pfkA genes was done by deleting argR gene. The transcription levels of pgi, argB, pfkA, and argJ genes in C.glutamicum ΔAPE6937R42 were 5.6, 5, 9.4 and 16.6 times greater than that of the C.glutamicum ΔAPE strain. The transcripts of the same genes are about 3.7, 3.5, 2.3 and 4 times higher than the transcripts of the parent strain of C.glutamicum ΔAPE.

The L-ornithine biosynthesis pathway is known to utilize NADPH and these reactions are catalyzed by NADP dependent isocitrate dehydrogenase, NADP dependent -acetyl gamma glutamyl reductase and NADP dependent glutamate dehydrogenase. The process of analysis of the relevance of NADPH in L-ornithine biosynthesis was carried out by deleting argR gene of the strain C.glutamicum ΔAPE to obtain C.glutamicum ΔAPER strain. Later, the NADPH levels of both the strains were measured. The NADPH and L-ornithine production was higher in the C.glutamicum ΔAPE6937R42 strain than in C.glutamicum ΔAPER strain.

To understand the influence of NADPH effectively, the transcript levels of genes associated with the NADPH synthesis in both the strains ΔAPER and ΔAPE6937R42 of C.glutamicum were compared. The genes related to NADPH synthesis called zwf, icd and gnd of C.glutamicum ΔAPE 6937R42 were upregulated by 3.8 times, 2.7 and 2.5 times respectively. The ppnk gene was also upregulated by 1.8 times. The concentration of NADPH in C.glutamicum ΔAPER was enhanced by the over-expression of zwf, icd and gnd genes. The production of L-ornithine was enhanced by the over-expression of ppnK gene. The increased NADPH level due to the increased transcriptonal levels of ppnK gene improved the production of L-ornithine.

Ling Yan Jiang, Shang-Guang Chen,Yuan-Yuan Zhang and Jian-Zhong Liu. Metabolic evolution of Cornybacterium glutamicum for increased production of L-ornithine. BMC Biotechnology 2013, 13:47.

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