Improvement Of L-ornithine Production By Attenuation Of ArgF In Engineered Corynebacterium Glutamicum

L-Ornithine,a non-essential amino acid,owns lots of important physiological functions and had explored enormous applications in food,chemical,and pharmaceutical industries.In Corynebacterium glutamicum(C.glu)argF is an important gene that controlling L-ornithine catabolism.Accompanied with arginine auxotrophy and growth deficiency,deletion of argF increased L-ornithine production from 0.61 g/L(CO)to 4.27 g/L(CO-1).Here growth deficiency brought by arginine auxotrophy relieved by extra arginine addition but L-ornithine producing decreased owning to arginine causes feedback inhibition of N-acetyl-l-glutamate kinase activity.Thus other genetic engineering methods should be taken to attenuate argF expression and to avoid the disadvantages brought by directly deletion of argF gene.Firstly,methods of attenuating argF gene expression on translation level had been taken.Such as:using an SsrA-mediated peptide tagging to construct unstable ArgF variants;ribosome binding site substitution and start codon replacement.But no expected L-ornithine production had detected.Then methods on transcriptional level had been applied.Transcription terminators play key roles in controlling gene expression and in regulating natural genetic systems.We cloned a strong terminator named rrnB and inserted it before argF sequence.After 48 h of incubation terminator method showed its talent on improving L-ornithine production and relieving growth disturbance.Specifically L-ornithine production on CO-6 are 5.53 g/L with OD600= 13.42.To further evaluate the application possibility of the terminator method,we chose 17 terminators with a terminator strength ranged from 382.13 to 10.94 and individually inserted them before argF creating strains CO-7-CO-23.Terminator method shows commonly ability on improving L-ornithine production.The max L-ornithine production had achieved 6.1 g/L on strain CO-9 with terminator T4.Transcription analysis showed that by introducing terminators with different terminator strength we can accurately down-regulate gene expression and improve the transcriptional level of upstream genes simultaneously.Thus terminator can act as promising technology on metabolic engineering to precise control gene expression.