Cloning And Expression Of Glutamate Decarboxylase Gene From Streptococcus Thermophilus Y2

y-Aminobutyric acid (GABA) is a natural non-protein amino acid, which exists in animals, plants, and microorganisms. GABA is a major neurotransmitter of inhibition in the mamalian central nervous system. Because of its important physiological function, GABA is applied widely in medicine, food, chemical industry and agriculture. Systhesis of GABA by microorganisms has many advantages over other methods, and has attracted great attention.This paper presents investigations on glutamat decarboxylase gene (gadB) from Streptococcus thermophilus Y2, including cloning and sequence analysis of gadB gene, construction of E. coli expression vectors, construction of Bacillus subtilis expression vectors, heterologous expression of gadB gene.1. By comparison of several glutamate decarboxylase sequences in GenBank, two highly conserved amino acid regions were found. On the basis of CODEHOP analysis, two degenerate primers were designed and used to amplify a 400 bp fragment with genomic DNA of Strepcoccus thermophilus Y2 as the template. The fragment was inserted into pMD19-T vector and sequenced. Blastx analysis indicated that it had homology with glutamate decarboxylases. Flanking sequences of the fragment were amplified by SiteFinding PCR. The PCR products were sequenced and assembly of them generated a fragment of 2036 bp. The sequence was deposited in GenBank under accession number DQ871217. A 1380 bp ORF was inside the sequence and did not show homology with any other genes. Its encoded protein had high similarity with several bacterial glutamate decarboxylases. SignalP software suggested no signal peptide existing in the ORF. Its deduced protein consisted of 459 amino acids, with a calculated molecular weight of 52595.18 kDa. The N-terminal amino acid sequence was NH2-Met-Asn-Glu-Lys-Leu-Phe-Arg-Glu-Ile-, consistent with that of native GAD purified from S. thermophilus Y2. The ORF was designated gadB. Phylogenetic tree of bacterial GAD generated by ClustalX 1.83 and MEGA 4 indicated that GAD of S. thermophilus Y2 showed highest similarity with that of Fusobacterium nucleatum subsp. polymorphum ATCC 10953, and had great difference with that of Streptococcus pneumoniae type 3. The upstream sequence of gadB gene was homologous to bacterial transposases. The downstream sequence of gadB gene encoded a putative protein similar to bacterial glutamate/GABA antiporters, so it was designated gadC. The order of gadB, gadC in S. thermophilus Y2 genome was similar to that of E. coli and Shigella flexneri, but different from that of Lactococcus lactis and Lactobacillus brevis.2. Two primers Gad-f1, Gad-r1 were used to amplify gadB gene. The PCR product was digested with NcoⅠand EcoRⅠ, inserted into E. coli vector pET-DsbA. The resaulting vector, pET-gadB, was introduced into E. coli BL21 (DE3) pLysS compeptent cells. Recombinant E. coli cells were induced with IPTG and gadB gene was expressed under control of T7 promoter. SDS-PAGE analysis indicated that E. coli cells carrying pET-gadB expressed a recombinant protein with a molecular weight of 47 kDa, consistent with that of purified native GAD, close to the calculated molecular weight of 52.6 kDa. Recombinant strain culture exhibited GAD activity of 45.4 mU/ml,2.4 fold that of untransformed E. coli strain, 2.1 fold that of S. thermophilus Y2. For fusion expression purpose, gadB gene was amplified with primers Gad-f2, Gad-r2, then digested with BglⅡ, ligated with BamHI-linealized pET-DsbA. The resaulting vector pET-DsbA-gadB was introduced into E. coli BL21 (DE3) pLysS competent cells. Recombinant strain culture exhibited GAD activity of 60.2 mU/ml,3.2 fold that of untransformed E. coli strain,2.8 fold that of S. thermophilus Y2. The optimal induction time was 2 h, with IPTG opimal concentration of 0.4 mM, induction temperature of 30℃. After centrifugation and sonication, E. coli cell lysate was loaded onto a nickel-charged NTA agarose column and fusion protein DsbA-GAD was purified. SDS-PAGE analysis indicated a clear single band corresponding to 82 kDa, consistent with the calculated molecular weight of 77 kDa. The specific activity of purified recombinant GAD was 2.8 U/(mg protein). The optimal pH was 4.2. The optimal temperature was 52℃.3. To remove P59 promoter and cat::lacZa region, pHB201 was digested with HindⅢand ClaⅠ, and ligated with two complementary oligonucleotides, which recoveried the deleted replication origin of pUC. The resulting plasmid was designated pHB-hc. P43 promoter, degQ gene, promoter and signal peptide sequence of levansucrase gene (sacB) from Bacillus subtilis 168 were amplified and ligated into a fragment with E. coli trpA terminator by SOE-PCR. The fragment TtrpA-degQ-P43-PsacB-SPsacB was digested with ClaⅠand XhoⅠ, inserted into pHB-hc, generated a vector pHPQ. gadB gene was amplified and digested with XhoⅠ, inserted into pHPQ and generated pHPQ-gadB. P43 promoter, degQ gene, degU gene, E. coli trpA terminator were assembled by SOE-PCR. Then the resulting fragment TtrpA-degU-degQ-P43 was digested ClaⅠand BglⅡ, inserted into pHPQ-gadB, generated pHUQ-gadB. Promoter of sacB, signal peptide sequence of nprB gene, gadB gene were assembled by SOE-PCR. The resulting fragment PsacB-SPnprB-gadB was digested with BglⅡand XhoⅠ, inserted into pHUQ-gadB, generated pUQSN-gadB. B. subtilis WB800 competent cells were transformed with pHPQ-gadB, pHUQ-gadB, pUQSN-gadB respectively. Recombinant cells were induced with 2% sucrose, and after centrifugation, supernatant was subjected to GAD activity assay. Recombinant strain carrying pHPQ-gadB did not exhibited GAD activity, and pHUQ-gadB 2.6 mU/ml, pUQSN-gadB 3.4 mu/ml. SDS-PAGE indicated that recombinant strain carrying pHUQ-gadB or pUQSN-gadB expressed a recombinant protein of 47 kDa, similar to the calculated molecular weight of native GAD,52.6 kDa, while pHPQ-gadB did not expressed that protein.