Physiology Engineering Strategy To Improve Gamma-aminobutyrate Production In Lactic Acid Bacteria

γ-aminobutyric acid(GABA)as a natural non-protein amino acid,has several signification physiological functions and is widely used as a pharmaceutical,nutraceutical,and as a precursor for synthesizing materials for industrial use.The GABA production by using Lactic acid bacteria as bacterial cell factories has been a focus of research.Since understanding the role and regulation of GAD system is a prerequisite for exerting the expected characteristics in acid resistance and GABA production,it is very necessary to gain a deeper insight into the glutamate decarboxylase-dependent system in the physiological role of LAB.To address this issue,in the present work,we focused our attention on a high GABA-producing strain Lactobacillus brevis CGMCC1306,with the aim to identify the genes that could concur to explain its growth ability in acid conditions and its GABA production.Subsequently,in order to improve the production of GABA from MSG,the engineered strains of Lb.brevis were designed based on a physiology-oriented engineering strategy.Furthermore,the production of GABA by the recombinant strains were investigated.The main results obtained are as follows:(1)In Lb.brevis CGMCC1306,the glutamate decarboxylase(GAD)system comprises a transcriptional regulator(GadR),a glutamate/γ-aminobutyrate(GABA)antiporter(GadC)and two glutamate decarboxylases(GadA and GadB),which catalyzes the conversion of L-glutamate to GABA and CO2 in a proton-consuming reaction contributing to the pH homeostasis.Molecular analysis revealed that the gadB is co-transcribed in tandem with the upstream gadC gene,and the expression of gadCB is greatly upregulated in response to low ambient pH when cells enter late exponential growth phase.In contrast,the gadA gene is located separately from the other gad genes,and its expression was consistently lower and was not induced by mild acid treatment.Analysis of deletion mutations in the gad genes of Lb.brevis demonstrated the decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild type>△gadA>△gadB>△gadC>△gadAB,indicating that the GAD activity mainly endowed by GadB is an indispensable step in the GadCB complex mediated acid resistance to this organism.(2)The gadA,gadB,gadC,gadCB and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e,and strain Lb.brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities.Subsequently,a physiology-oriented engineering strategy was adopted to construct an FlFo-ATPase deficient strain NRA6 with higher GAD activity.As expected,strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42%GABA conversion rate within 48 h in GYP fermentation medium,which is 1.22-fold higher than that obtained by wild type strain in the same condition.This demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency,and contributes to research into the development of the physiology-oriented engineering.(3)An engineered GABA high-producing strain Lb.brevis LpGadB△C11-LbGadC was constructed by co-expressing a C-terminally truncated GadB mutant and GadC,which is active in expanded pH range.In fed-batch fermentations,temperature,pH and initial glutamate concentration were fixed as 35℃,5.2 and 75 g/L.The experiments were performed in the 5-L fermentor under following conditions:medium volume 2 L,inoculum size 10%(v/v),agitation speed 100 rpm,and fermentation time 72 h.200 ml aliquots of MSG(140 g per aliquot)were supplemented into the bioreactor at 24 h,36 h and 48 h,respectively.Under the selected fermentation conditions,GABA was rapidly produced at the first 48 h,and concentration of GABA reached 104.38 g/L at 72 h.Similarity,the recombinant Lc.lactis NZ9000/pNZ8148-LpgadB△11-LcgadC was also constructed,and under the optimal fermentation conditions,GABA was rapidly produced at the first 36 h,and the maximum concentration of GABA reached 32.16 g/L with a 87.92%conversion rate at 48 h.This is the highest yield ever reported of GABA production by Lc.lactis strains.(4)The controlled expression of the lytic genes lytH and lytA,which encode the holin and lysin proteins of the lactococcal bacteriophage rlt,respectively,was accomplished by application of a nisin-inducible expression system.Production of holin and lysin proteins lead to lysis of the host cells.Similarity,another controlled expression of the enlA,which encode the N-acetylmuraminidase of Enterococcus faecalis LMG 2333 has also been accomplished.And based on these "platfonn strains",the production efficiency of GABA has been promoted during fermentation by mixed strains of Lc.lactics NZ9000/pNZ81Z8-/cgadB and pNZ8148-enlA-PpepN-LcgadB,with a final concentration of 37.64 g/L at 36 h.Similarly,the concentration of GABA reached 126.85 g/L at 48 h when the fermentation process was performed with the mixed cultures of Lc.lactics NZ9000/pNZ8148-enlA-PpepN-LcgadB and Lb.brevis LpGadB△C11-LbGadC.