Synthesis Of GABA Catalyzed By Whole-cell Of Lactobacillus Brevis And Construction Of DAR-PCR System

Gamma-aminobutyric acid（GABA）is a non-protein amino acid widely present in the central system of mammals and has physiological functions such as anti-epileptic,blood pressure lowering and diuretic effects.Lactic acid bacteria（LAB）can utilize endogenous glutamic acid decarboxylase（GAD）to catalyze the decarboxylation of L-glutamic acid（L-Glu）to produce GABA,and it is an important strain for microbial production of GABA.The whole-cell transformation method of microorganisms utilizes cells as catalysts to exert the efficacy of endogenous glutamate decarboxylase to catalyze the synthesis of GABA.Compared with traditional fermentation,the composition of the reaction liquid is simple,which is conducive to the separation and purification of productsIn this work,the fermentation system of L.brevis CD0817 was optimized,in order to obtain cells with the high number and high activity.Secondly,the influence of key factors such as reaction temperature on GABA production and conversion rate was investigated,and the whole-cell catalytic process parameters were determined.In order to explore the mechanism of high GABA production of strains at the molecular level,we established differential annealing-mediated racket PCR（DAR-PCR）,that was used to obtain the unknown flank sequence of known DNA.The main results of this paper are as follows:（1）We optimized the culture time,substrate selection,and carbon source concentration that affect the growth and high decarboxylation activity of L.brevis CD0817 during fermentation.The main ingredients（g/L）are:glucose,20;yeast extract,35;Tween-80,1;Mn SO₄·H₂O,0.05;L-Glu,300;L.brevis CD0817 cells with high decarboxylation activity can be obtained by culturing at 30°C for 16 h;the GABA concentration of fermentation broth is 204.2 g/L;（2）The single factor method was used to explore the effects of temperature,cell concentration,and recycling rate on the catalytic process of cells with high decarboxylation activity.The optimal transformation conditions were determined by GABA yield and transformation rate,and the whole-cell catalytic production process of GABA was established:transformation temperature,45℃;wet cell concentration,25 g/L;transformed for 12 h;L-Glu conversion rate was 91.1%;GABA concentration was 94.0 g/L;（3）We use the basic purification process to obtain white GABA products after the conversion solution is concentrated,ethanol precipitated,and dried;the GABA recovery rate is 89.4%;the product purity is 97.1%;（4）A new tool,DAR-PCR,has been established for the efficient determination of unknown DNA.This method was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and rice.The key to DAR-PCR is the use of primer-mediated intra-strand annealing（ISA）.An ISA primer consists of a 5′root homologous to the known sequence and a heterologous 3′bud.In the single low-stringency cycle,the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer（SSP）1 site,thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer.In the subsequent more stringent cycles,its complementary strand is accumulated,owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1.The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root.A loop-back extension subsequent to ISA occurs,creating a racket-like DNA with the known region positioned at both ends of the unknown sequence.This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1.More than one clear band appeared in all the secondary PCR reactions.The longest DNA fragments obtained in each walking experiment ranged from 1.5 to 5.0 kb.Sequencing data demonstrated that all the dominant bands were target products,verifying the high specificity of the current method.