GABA Synthesis And Regulation Of Streptococcus Thermophilus

Streptococcus thermophilus,the main starter for the production of yogurt and hard cheese,utilizes lactose and casein of milk to produce a variety of active metabolites,giving products special texture,flavor and nutrition.Gamma-aminobutyric acid(GABA),a non-protein amino acid,has been reported to produce lots of probiotic functions,such as analgesic,anti-anxiety,sleep-promoting and anti-inflammatory.Many strains of S.thermophilus are able to synthesize GABA,while the regulation of GABA synthesis remains unclear.In addition,S.thermophilus is often subjected to stress such as shortage of available nitrogen source and low pH during the growth and fermentation,while the mechanism responding to the above environmental stresses is unknown.To improve the GABA synthesis ability of S.thermophilus,this thesis determined the gadBC gene cluster and the characteristics of GABA synthesis in shortage of available nitrogen source and acid stress.The regulators were screened by Pull-down assays,Mass spectrometry and EMSA.The role and mechanism of the cell-envelope protease PrtS in promoting GABA synthesis were explored by intracellular proteomics of S.thermophilus,providing a theoretical guide for GABA-enriched yogurt making.The specifically markable achievements are as follows:1 Genetic composition and functional analysis of GABA synthesis gene cluster in S.thermophilusBased on the genomic sequence of S.thermophilus retrieved from the GenBank database,GABA synthesis gene cluster of S.thermophilus was composed of the glutamate decarboxylase(GAD)encoding gene gadB,the glutamate-GABA transporter encoding gene gadC and two constitutive promoters,PgadB and PgadC.However,the gadR gene encoding for the transcriptional regulator GadR was lacking.The transposable element IS was flanking the gene cluster,indicated that the GABA synthesis ability of S.thermophilus was derived from horizontal gene transfer.The above unique genetic composition was different from that of reported lactic acid bacteria.Furthermore,the presence of above gene cluster and GABA synthesis ability of 41 S.thermophilus strains stored in our laboratory were measured.The results showed that 37 strains carried the conserved gadBC gene cluster and 35 strains had the ability to synthesize GABA among them.In order to determine the function of gadBC gene cluster,the transcription levels of gadB and gadC genes of S.thermophilus SDMCC050242 were analyzed to transcribe normally in different growth stages.Comparing the amino acid sequences of GAD in the above 37 strains,it was found that the mutation of amino acid N81S led to the inactivation of the enzyme,while the mutations of amino acid sites S147P,S385G,N433T and R452H had no effect on GABA synthesis ability.Moreover.the recombinant strains expressing different gadBC gene clusters showed the same GABA synthesis ability.Therefore,the GABA synthesis of S.thermophilus was constitutive expression,and GABA synthesis ability was regulated by environmental stress or other regulatory factors.2 Identification of GABA synthesis regulators in S.thermophilusMilk is an oligotrophic substrate with few free amino acids.For rapidly growing in milk,S.thermophilus converted carbon metabolic skeleton to amino acid synthetic skeleton regulated by the global regulator CodY.However,it is unclear whether the GABA synthesis ability of S.thermophilus is regulated by CodY.In order to find out the regulation mechanism of GABA synthesis in S.thermophilus,two proteins binding to the gadB gene promoter PgadB of strain SDMCC050242 were screened by DNA Pull-down assays and identified by LC-MS/MS as CodY,the global transcription regulator involved in nitrogen metabolism,and GlnR,the repressed transcriptional regulator of glutamine synthase,respectively.Further analysis showed that the deletion of codY gene downregulated gadB and gadC gene transcription levels,and decreased GABA production,indicating that CodY promoted GABA synthesis.Electrophoretic mobility shift assay(EMSA)results confirmed the direct binding of CodY to the promoters PgadB and PgadC,therefore,CodY played a positive regulatory role.Similarly,overexpression of glnR gene downregulated gadB and gadC gene transcription levels,resulting in decreased GABA production,showing a negative regulatory effect.The above results suggested that regulators of nitrogen metabolism in S.thermophilus were involved in GABA synthesis,CodY played a positive regulatory role and GlnR played a negative regulatory role.3 The promotion of cell-envelope protease PrtS on GABA biosynthesis in S.thermophilusCell-envelope protease PrtS hydrolyzed casein to free amino acids and oligopeptides,thus providing available nitrogen source for the growth of S.thermophilus.However,a little strains of S.thermophilus possessed prtS gene(PrtS+strain).In order to study the role of PrtS in GABA biosynthesis of S.thermophilus,the prtS gene of PrtS+strain SDMCC050242 was inactivated by homologous recombination.The GABA production of mutant strain SDMCC050242ΔprtS decreased by 79%,and then increased with the addition of PrtS enzyme,indicating that PrtS promoted GABA synthesis in S.thermophilus.Comparing the intracellular proteome between strains SDMCC050242 and SDMCC050242ΔprtS,it was found that the presence of PrtS increased the abundance of peptidases and oligopeptide transporters,while decreased the abundance of enzymes involved in amino acid synthases such as methionine,alanine and cysteine,indicating that PrtS provided amounts of available nitrogen source for growth to inhibit amino acid synthesis.The transcription levels of the relative genes and codY gene determined by RT-PCR further confirmed above results,indicating that PrtS provided available nitrogen source for growth and up-regulated codY gene transcription to synthesize GABA.4 The promotion of methionine on GABA biosynthesis in S.thermophilusS.thermophilus and Lactobacillus delbrueckii were the main starters for yogurt.Due to the long-term utilization of the nitrogen source provided by the protease system of L.delbrueckii,some genes involved in the amino acid synthesis of S.thermophilus were lost or pseudogenic,resulting in different amino acid nutrient deficiency types.In order to further analyze the difference of amino acid synthesis ability between S.thermophilus SDMCC050242 and SDMCC050242ΔprtS in milk,the extracellular free amino acid levels were determined.The results showed that the contents of methionine,alanine,cysteine and GABA in strain SDMCC050242 were higher than that in mutant strain SDMCC050242ΔprtS,indicating that the PrtS provided enough amino acids for growth.Exogenous addition of above different amino acids to milk,it was found that only methionine restored the GABA synthesis ability of the mutant strain SDMCC050242ΔprtS.Therefore,methionine was the key regulator of GABA synthesis promotion by PrtS and its content directly affected the GABA production by upregulating codY gene transcription.At the same time,the promotion of methionine on GABA synthesis was generally applicable to PrtS-wild type strains of S.thermophilus.5 GABA synthesis in response to acid stress in S.thermophilusPrtS promoted the growth and acidification of S.thermophilus.In order to investigate the mechanism of GABA synthesis in respond to acid stress,two strains of S.thermophilus with different GABA production,SDMCC050242 and SDMCC050243,were chosen to determine the growth and metabolism.The results showed that the higher transcription level of lacZ and ldh genes and their corresponding enzyme activities in the exponential growth phase,the higher GABA production.In addition,the activity of the promoter PgadB increased with the decrease of pH,indicating that acid production promoted the transcription of the gadB gene,thereby synthesizing GABA.Meanwhile,higher GABA production improved the survival of the strain under acid stress,confirming the protective effect of GABA synthesis on bacterial cells.Milk experiments further confirmed that GABA synthesis responded to acid stress caused by the itself fast-acidification to protect bacterial cells,which was the adaptive self-regulation mechanism of S.thermophilus.6 Genetic composition and functional analysis of GAD system in Enterococcus aviumE.avium,a common lactic acid bacterium colonized in the intestines of human and other animals,could synthesis GABA to endow fermented food with probiotic functions.However,the genetic composition and functions of GAD system in E.avium are still unclear.Here,a GABA-producing E.avium SDMCC050406 was isolated from feces.Prediction of sequence showed that the strain contained a complete gene cluster composed of three gadB genes(gadB1,gadB2 and gadB3)together with gadC and gadR genes for GABA synthesis.In order to analyze the role of the three gadB genes in GABA synthesis,the transcription levels and corresponding enzyme activities of the above three genes were determined.The results showed that gadB3 gene had the highest transcription level,and GAD3 encoded by gadB3 gene had the highest enzymatic activity and substrate affinity.Inactivation of gadB3 gene showed that the GABA production of the mutant strain SDMCC050406ΔgadB3 was significantly reduced,accompanying by a decrease in the acid resistance.The above results indicated that gadB3 gene played an important role in GABA biosynthesis in E.avium,enriching the genetic diversity of GABA synthesis in lactic acid bacteria.