Optimization Of Bioprocess For High Production Of ?-aminobutyric Acid By Lactobacillus Brevis CD0817 And A Preliminary Study Of GadA Traceless Knockout

Gamma-aminobutyric acid(GABA)is a kind of small molecule amino acid with many probiotic functions,which has been widely used in food,medicine and feed industries and has a broad application prospect.Currently,due to the low efficiency of GABA industrial production,the convenience of fermentation method and the recognized safety of lactic acid bacteria,the lactic acid bacteria fermentation method has already become the developing trend of high efficient GABA production.In this paper,the single-factor optimization strategy was firstly adopted to strengthen the GABA fermentation process parameters of Lactobacillus brevis CD0817 through shaking flask experiment.Then,according to the optimized parameters,a small test transformation process was established in a 10 L fermenter.In order to further explore the mechanism of high GABA production,a recombinant plasmid for gad A knockout was constructed based on suicide plasmid,and a traceless knockout technology of lactic acid bacteria was discussed.The main results of this paper are as follows:(1)Initial medium and culture conditions.Single factor flask shaking test was used to screen the key factors and their optimal levels.The seed medium was formulated as(g/L):glucose 10,yeast extract FM408 35,sodium glutamate 28,Mn SO4 0.05,Tween80 1,p H 5.0.The fermentation medium formula was(g/L):glucose 5,yeast extract FM408 35,Mn SO₄ 0.05,tween-80 1,L-glutamic acid 650.The results showed that glucose,yeast extract,Mn SO₄,tween-80 and culture temperature were the key factors affecting the production of GABA by Lactobacillus brevis CD0817.(2)The effect of the above fermentation process was verified in a 10-L fermenter.The seed culture was 10%(v/v)inoculated into 3 L fermentation system at 30?with50 rpm for fermentation.The yield of GABA reached 321.88±6.73 g/L 48 h after inoculation,which was 27.73%higher than that of the original fermentation method,and the substrate conversion rate was 99.80%.(3)The recombinant knockout plasmid p K18?Cm R?up&down of Lactobacillus brevis CD0817 glutamate decarboxylase gad A based on suicide plasmid p K18mob Sac B was constructed.The recombinant plasmid contains the fusion fragment of the upstream and downstream homologous sequences of gad A(u&d),which was obtained by two-step fusion PCR,for homologous recombination with Lactobacillus brevis CD0817 genome.Chloramphenicol resistance gene(Cm R)was used as a positive screening marker;The Sac B gene was used to induce a second homologous recombination and negative screening.Theoretically,markerless deletion of gad A can be achieved after double cross over with genomic DNA.The transformants by electric shock transformation could be obtained on selective medium,while aimed fragments were not detected both by PCR amplification and sequencing.In order to complete the traceless knockout,more comprehensive optimization and exploration are needed.