Study On Breeding And Metabolic Regulation Of L-Citruliine Production Strains

As a kind of non-protein amino acids and an intermediate metabolite of urea cycle,L-citrulline plays an important role in physiological function of human.In the nature,some microorganisms can produce arginine deiminase converting L-arginine to L-citrulline,which is known as the enzymatic production of L-citrulline.This method works in the mild condition,has higher conversion rate,and also environment friendly,would have a good prospect of application in the mass industrial production.This study not only independently screened high L-citrulline yield strains from watermelon greenhouse soil,but also explored the influences of various factors on production of L-citrulline in the process of fermentation and conversion.Forty-five L-citrulline producing strains were screened from Hangzhou watermelon greenhouse soil through preliminary screening（using arginine dihydrolase selective medium）and secondary screening（determing the concentration of citrulline by HPLC）.The strain 15 had the highest ability of converting L-arginine to L-citrulline and the highest concentration of L-citrulline.The strain was identified as Aeromonas by morphology observation,physiological and biochemical characteristic and molecular biological identification,and was named as Aeromonas sp.YQ.Bred the mutant Aeromonas sp.YQ-89 through the ultraviolet mutagensis.And its L-citrulline-producing capacity still good and stable after five generationsSeveral factors affecting on the production of L-citrulline were studied in the process of fermentation and conversion.The optimum conditions are as follow:2.5 g/L sucrose as the carbon source,15 g/L yeast extract and 10 g/L peptone as nitrogen source,concentration of inducer of L-arginine is 20 g/L,concentration of Co²⁺is 10^-7mol/L,inoculation volume is 3%,p H of medium is 7,medium volume is 100m L/250 m L,180 r/min,37℃.0.252 g biomass（dry weight）after ferment ing 20 h in this conditions,can converted 160 g/L substrate of L-arginine to 93.5 g/L concentration of L-citrulline in the 60 m L transformation system containing 0.4 mol/L acetate buffer（p H=6）after 4 h conversion.The highest concentration of L-citrulline reached to 113.04 g/L,and conversion rate was 70.24%.In order to accumulate L-citrulline to high concentration without being degraded,we used Red homologous recombination technology to knock out the gene of ornithine carbamoyl transferase which can metabolize L-citrulline.The gene of kana was successfully integrated into the genome of Aeromonas sp.YQ-89 which has been given kanamycin resisitance,but not able to replace the original ornithine carbamyl transferase gene that still existed,which might be due to the defects of the targeting of homologous arm designed and lead to failure.The following studies can design more specific homologous arm to knock out the gene of ornithine carbamyl transferase.