Figure 2: GABA transaminase activity was introduced into A. tumefaciens.

(A) Amino acid sequence comparison of GABA transaminase from E. coli K12 (gabTSCA772438), P. syringae pv. tomato DC3000 (gabT1: gabT1-PSPTO0259, gabT2: gabT2-PSPTO0301), P. aeruginosa PAO1 (gabT-PA0266). “ClustalW2” (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and “Jalview” (http://www.jalview.org) were used for calculation of amino acid multiple sequence alignment and display the alignment result, respectively. The residues were coloured according to their physicochemical properties as follows; Aliphatic/Hydrophobic, Aromatic, Positive, Negative, Hydrophilic, Conformationally Special and Cystein. Arrowheads indicate 3 of the 12 invariant amino acid residues among 16 aminotransferases with highly homologous peptides. The red box indicates a conserved motif (Ser [or Thr]-X-X-Lys) in the pyridoxalphosphate-binding peptide of aspartate aminotransferase (AAT) and histidinol-phosphate transaminases. The blue box indicates near identity between the homologous peptides of the histidinol-phosphate transaminases from E. coli K12 and Saccharomyces cerevisiae. (B) Construction of a plasmid for the expression of GABA transaminase (gabT) in A. tumefaciens. HindIII and XbaI fragments (ca. 1.6 kb) containing the GABA transaminase gene from E. coli K12 were ligated into the HindIII and XbaI sites of the broad-host-range plasmid pBBR1MCS-5, resulting in pBBRgabT. The expression of the GABA transaminase gene gabT was under the control of the lac promoter. MCS: multiple cloning site. (C) Detection of GABA activity in A. tumefaciens. Glutamic acid accumulation in the reaction buffer was measured according to the method of Akihiro et al.⁴⁸. The open and closed circles indicate A. tumefaciens GV2260 (pBBRgabT, pIG121-Hm) and A. tumefaciens GV2260 (pBBR1MCS-5, pIG121-Hm), respectively. Bars represent the standard deviation (n = 3).