Analysis Of The Process And The Metabolites In The Fermentation Of The Recombinant Corynebacterium Glutamicim

L-arginine,one kind of semi-dispensable amino acids,is a intermediate metabolite in the urea cycle,and has important physiologic function in organism.Therefore,L-arginine has numerous applications in food,pharmaceuticals and feed and L-arginine demand is constantly increasing.Coynebacterium glutamicim is the main arginine producing strain.Its genome has been sequenced.what’s more,the vector system of Coynebacterium glutamicim and gene knockout system has been construted.So wo can use the skill of metabolic engineering to breed,Preliminary studies was found that orignal strain had no the accumulaton of L-arginine.When the mutation of the key enzyme arg BH268 E which is resistant to feedback inhibition by L-arginine was overexpressed in 14067,the L-arginie synthesis was largely enhanced.But the yield is greatly influenced by oxgen supply level,oxygen supply is the key to the fermentation.So the method of analysis of amino acid in fermentation broth was established to study the effect of different oxygen supply level on L-arginine production.The metabolic flux distribution under different oxygen supply level was used to explain the effect of arginine systhsis mechnism to determine the appropriate oxygen supply conditions.Meanwhile,the recombinant strain would accumulate a lot of citriline under the sufficent oxygen supply.So we over expressed the argBH268 E and argGH on the original strain ATCC 14067 to enhance the arginine biosysthetic pathway,and increased the arginine production.The effect of over expression of argBH268 E and arg GH was analysised by metabolic flux distribution.Firstly,the method of analysis of amino acid in fermentation broth was established to lay the foundation for metabolic flux analysis.The method used pre-column derivatization with2,4-dintrofluorobenzene(DNFB).The amino acids were separated in a Phenomenex Gemini NX C18 column(4.6mm×250mm,5μm)at 30℃.The mobile phase was a mixture of phase A:50mM sodium acetate buffer(pH 6.8)and phase B: 1:1(v/v)acetonitrile/water through the gradient elution.Detection was performed using a UV detector at 360 nm.The method showed a good reproducibility and sensitivity.Secondly,the effect of different oxygen supply level on L-arginine production was studied.By analysising the fermentation charictorsics under the different oxygen supply level,we found that high oxgen supply was in favour of arginine production and oxygen shortage would seriously inhibit the synthesis of L-arginine.Further analysising the metabolic flux distribution in fermentation under different oxygen supply level,we found that TCA cycle,the flux to oxaloacetae(OAA)from the Phosphoenolpyruvate(PEP)and the flux to pathwayof glutamate from α-ketoglutarate node were enhanced on the high oxygen supply.So the arginine synthesis was enhanced.L-arginine production of fed feimentation under high oxygen increased 86% because of extending fermentation time.Sometimes,the citriline,arginine metabolic intermediates,were found to have a lot of accumlation in fermentation process.Finally,we over-expressed arg BH267 E and argGH on ATCC14067 to enhance arginine biosynthetic pathway,and arginine produciton was increased.An expression vector Peftuarg BH268E-arg GH which carries arg BH268 E and argGH gene constructed had been construted and was transformed to ATCC14067.Analysising the result of fed fermentation of recombinant strains 14067-Peftu-arg BH268E-argGH in 3L jar,we found arginine production reached 10.75g/L,which was 130% higher than single expression of argBH268 E.Citrulline production significantly reduced to 0.37g/L,which was only 14% of single expression arg BH268 E.This indicated that expression of argBH268 Eand argGH enhanced the conversion from citrulline to arginine,and increased arginine biosynthesis metabolic flux.Analysising the metabolic flux distribution in fed fermentation,we found the expression of argBH268 E and arg GH enhanced the flux to arginine biosynthesis from α-ketoglutarate node and reduced the flux to citrulline.Sometime,the enhancement of the flux to OAA from PEP made up for the deficiency of OAA.Because of this,arginine production would be great improved.