Improvement Of ?-aminobutyric Acid Biosynthesis Capability Of Lactobacillus

As the major suppressive neurotransmitter in mammalian brain,GABA possesses many physiological functions such as anti-hypertension,anti-anxiety,diuretic and tranquilizer.Owing to these beneficial functions,GABA has a wide range of application in food,pharmaceutical,and chemical industry.Biosynthesis of GABA with lactic acid bacteria is condsiered to be one competitive way for GABA prepareation,so understanding the gene expression profiles of glutamic acid decarboxylase(GAD)system,and improving GABA production performance of lactic acid bacteria through metabolic engineering,protein engineering,and fermentation control methods have important practical significanc for GABA production.In this work,we first analyzed key genes expression profiles of the GAD system in GABA high-yielding strain Lb.brevis CGMCC1306 under different culture condition and probed into effect of inhibiting F1FO-ATPase on the expression of key genes of GAD system.Second,GadB genes were coloned from Lb,plantarum strains with higer GABA-yielding activity under a relatively wide pH range and protein engineering methods were applied to generate one GadB mutant(GadBC378S)with good catalytic activity in close to neutral pH condition.Finally,the mutant enzyme was overexpressed in Lb.brevis CGMCC 1306 to assess its effect on improving GABA-yielding capability of the host strain.The main research results are as follows:(1)The growth rates and expression profiles of key genes(gadA,gadB,and gadC)in Lb.brevis CGMCC 1306 GAD system under different fermentation conditions with addition of DCCD were determined.The growth rates of the bacteria were decreased with the increase of DCCD concentration,and DCCD of high concentration could lead to cell death.However,when culturing Lb.brevis CGMCC 1306 under facultative anaerobic condition at appropriately low DCCD concentration,the expression levels of gadB and gadC were increased(by 25.09%and 37.65%than control group at late logarithmic phase,respectively;by 11.03%and 32.5%respectively than the control group at stationary phase,respectively),and GABA synthesis capability of bacteria was improved.(2)Two Lb.plantarum strains(Lb.plantarum GM1403 and Lb.plantarum PF1409)with higher GABA production were screen using the soluble calcium circle method and target gene screening method.With pET28a(+)as expression vector,the GadB genes from Lb.plantarum GM1403 and Lb.plantarum PF1409 were expressed in BL21(DE3).The optimum temperature and pH for the two corresponding recombinant GAD were identical(40 ? and pH 5.0).When the pH value increased to 5.6,its catalytic activity was about 34.5%of the optimal activity at pH 5.0,which showed a wider favorable pH range than the GADs from other lactic acid bacterium.(3)To improve Lb.plantarum GAD catalytic activity under neutral pH conditions,a C-terminally truncated GadB ?11 mutant was created by semi-rational molecular design method based on the recombinant GAD of Lb.plantarum GM1403.With the help of directed evolution based on random mutation and one high-throughput screening method(based on pH sensitive indicator of cresol red),one mutant C378S which showed 48.86%higher activity than GadB?11 at pH 6 and 103%higher activity at pH 7 was obtained.The optimum pH and temperature for the mutant were 5.0 and 40 ?,respectively.Its Km was 19.03 mM,which was similar to GadB?11.(4)The C378S mutant was expressed in Lb.brevis CGMCC 1306 using pMG36e as expression vector and it improved the GABA procudtion of Lb.brevis CGMCC 1306.After 36 h reaction,the GABA yield reached 38.47 g/L,24%higher than the control group.