SupplementaryTable 1 Strains and plasmids used in this study

Strain or Plasmid / Description / Source
Strains
CICC 10055 / Soil-isolated Bacillus megaterium strain, capable of glutamic-acid production / CICC , China
DH5α / Escherichia coli derivative; Competent cells for general cloning / Takara
BL21 (DE3) / Escherichia coliderivative; Competent cells for protein expression / Novagen
Plasmids
pET21b / Bacterial expression vector; contains T7 promoter and 6×His tag; AmpR / Novagen
pET21b-Bmgad / pET21b derivative; contains the gad gene from B. megaterium CICC 10055 / This study
pET21b-BmgadD38K / pET21b-Bmgad derivative; contains themutant gad gene at the residue D38 / This study
pET21b-BmgadD92A / pET21b-Bmgad derivative; contains themutant gad gene at the residue D92 / This study
pET21b-BmgadE179K / pET21b-Bmgad derivative; contains themutant gad gene at the residue E179 / This study
pET21b-BmgadD231R / pET21b-Bmgad derivative; contains themutant gad gene at the residue D231 / This study
pET21b-BmgadE294R / pET21b-Bmgad derivative; contains themutant gad gene at the residue E294 / This study
pET21b-BmgadH467A / pET21b-Bmgad derivative; contains themutant gad gene at the residue H467 / This study
pET21b-Bmgad∆466-467 / pET21b-Bmgad derivative; contains themutant gad gene lacking the residues H466 and H467 / This study

SupplementaryTable 2 Primers used in this study

Primers / Nucleotide sequence (5'-3')
BmGad-F-NdeI / AGTCCATATGCCTCAATGGCATCCGCATCGT
BmGad-R-HindIII / GTACAAGCTTATGATGAAATCCATTGTCGT
D38K-F / CCAAGACTGCGTATCGGTAAACAAGGTATGCTTCCGGAAAC
D38K-R / GTTTCCGGAAGCATACCTTGTTTACCGATACGCAGTCTTGG
D92A-F / TGATAAAAATATGATCGATAAAGCTGAGTATCCGCAGACAG
D92A-R / CTGTCTGCGGATACTCAGCTTTATCGATCATATTTTTATCA
E179K-F / TCGCAAACTATTGGGACGTAAAGCCTCGTTATGTGAATATT
E179K-R / AATATTCACATAACGAGGCTTTACGTCCCAATAGTTTGCGA
D231R-F / GCTGCTATCGCAAAAGCATTACGTGAGTTACAGGAAAAAAC
D231R-R / GTTTTTTCCTGTAACTCACGTAATGCTTTTGCGATAGCAGC
E294R-F / GATTTGGAGACGAAAAGAGGACTTGCCTGAAGATCTTATT
E294R-R / TTCAGGCAAGTCCTCTTTTCGTCTCCAAATCACCCATCCCA
H467A-F / AAATACGACAATGGATTTCATGCTAAGCTTGCGGCCGCACT
H467A-R / AGTGCGGCCGCAAGCTTAGCATGAAATCCATTGTCGTATTT
Δ466-467-F / ACAATGGATTTAAGCTTGCGGCCGCACTCGAGCACCACCAC
Δ466-467-R / TCGAGTGCGGCCGCAAGCTTAAATCCATTGTCGTATTTCGT

The restriction sites are underlined, and mutation positions are in boldface.

Supplementary Figure 1 Enzymatic transformation of glutamate to GABA at near-neutral pH. The enzymatic transformation of purified BmGad was calculated at pH 6.0. The reaction mixturecontaining 50 g monosodium glutamate/l, 0.2 mM PLP, and 0.3 mg/mlpurified enzyme. The GABA concentration was determined by the method describe above.The data are presented as means ± standard deviation (sd) from three independent experiments.

Supplementary Fig. 2. Determination of kinetic parameters for the recombinant BmGAD. The Michaelis-Menten plot was shown, and a reciprocal plot was shown as an inset in each panel.The data are derived from duplicate assays in three independent experiments, and the standard deviations are less than 10% of the mean values.

Supplementary Figure 3Multiple alignments of Bacillus megaterium GAD with other reported GADs. Identical residues are highlighted in black, and three conserved domains are shown. Asterisksindicate the point-mutation sites. Ec_GADB, Escherichia coli GADB; Lb_GADB1, Lactobacillus brevis lb85 GADB1; Ph_GAD, Pyrococcus horikoshii GAD; Bm_GAD; Bacillus megaterium CICC 10055 GAD.

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