Abstract
l-Ornithine, a non-essential amino acid, has enormous industrial applications in food, pharmaceutical, and chemical industries. Currently, l-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high l-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. l-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-l-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable l-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved l-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of l-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of l-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.
Highlights
Introduction lOrnithine, a non-essential amino acid, plays an important role in urea cycle (Jiang et al 2013); has various applications in the treatment of diseases such as liver diseases, gyrate atrophy, and cancers in humans; and is capable of improving athletic performance (Zajac et al 2010)
Results l‐Ornithine accumulation and cell growth deficiency led by deletion of argF ArgF, encoding ornithine carbamoyl transferase (OTC), plays a critical role in the biodegradation of l-ornithine
When l-arginine was added up to a concentration of 5 g/L, the l-ornithine production of strain CO-1 dropped from 4.27 to 0.61 g/L. These results suggested that deletion of argF in the engineered strain CO-1 led to growth deficiency, which was relieved by l-arginine supplementation, but addition of l-arginine into the fermentation broth inhibited l-ornithine production in this strain
Summary
Introduction lOrnithine, a non-essential amino acid, plays an important role in urea cycle (Jiang et al 2013); has various applications in the treatment of diseases such as liver diseases, gyrate atrophy, and cancers in humans; and is capable of improving athletic performance (Zajac et al 2010). Due to its numerous applications, l-ornithine high-titer production has become an important task. Due to the problems of high cost, complicated operation, and environmental harm, l-ornithine production by chemical. To attenuate the expression of target genes, numerous strategies such as RBS modification, translational start codon exchange, promoter replacement, and RNA interference have been carried out and widely applied in the past decades (Man et al 2016; Shen et al 2017). Promoter replacement and RNA interference were useful strategies for regulation of the transcription process. Transcription is a process that includes recruitment of RNA polymerase (RNAP) to a promoter, synthesis of mRNA, and dissociation of RNAP at a terminator sequence. The terminator is an important component of the transcription process, which is known to be crucial for protein expression (Nakamura et al 2015)
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