In this case, it is expected that the enhancement of efflux of intracellular dipeptides improves growth deficiency of strain Δpeps. Overexpression of bcr, norE, ydeE and yeeO partially PLX4032 cost restored the growth defect (Fig. 2b). This observation suggested that intracellular accumulation of dipeptides inhibited cell growth and that dipeptide transporter candidates excreted intracellular dipeptides into the medium.
We assumed that transformants overexpressing dipeptide transporter candidates excreted considerable amounts of Ala-Gln into the medium and had decreased intracellular Ala-Gln levels. Strain Δpeps overexpressing each of the dipeptide transporter candidate was cultivated in the medium supplemented with 50 mM Ala-Gln, and after that the intracellular Ala-Gln levels were compared. The intracellular Ala-Gln levels of strain Δpeps overexpressing each of dipeptide transporter candidates were reduced to between 2% and 83% of strain Δpeps harboring the vector only (Fig. 3). This result suggested PD0332991 concentration that these genes might be involved in Ala-Gln export into the medium. The most drastic reduction was observed with the strain overexpressing ydeE. In order to confirm whether the four multidrug-efflux transporter genes selected by dipeptides resistance is involved in Ala-Gln production
in E. coli, each plasmid expressing a dipeptide transporter candidate or the control vector pSTV28 was introduced into strain JKYPQ3 harboring pPE167, which carries the gene (lal) coding for Lal and the gene (ald) coding for Ald from B. subtilis under the control of uspA promoter. The transformed cells were grown in TT medium, and the amount of Ala-Gln was analyzed. Strain JKYPQ3/pPE167 harboring pSydeE did not grow in TT medium (data not shown). This result suggested that the excessive Resveratrol expression of ydeE affected the growth of Ala-Gln-producing strain. Therefore, ydeE and its native promoter were cloned into the reverse direction
of lac promoter of pSTV28 in order to reduce ydeE expression. As shown in Fig. 4a, strain JKYPQ3/pPE167 overexpressing bcr, norE, ydeE or yeeO showed a 1.4–3.0-fold increase in Ala-Gln production. As previously shown (Tabata et al., 2005), Lal accepts branched-chain amino acids as C-terminal residues and forms Ala-BCAA. The effects of overexpression of dipeptide transporter candidate genes on l-alanyl-l-valine (Ala-Val), l-alanyl-l-leucine (Ala-Leu) and l-alanyl-l-isoleucine (Ala-Ile) production were examined. Each plasmid expressing a dipeptide transporter candidate or the control vector pSTV28 was introduced into strain JKYP9 harboring pPE167. The transformed cells were grown in TT medium supplemented with the substrate l-branched chain amino acids, and the amounts of Ala-BCAA were analyzed. In these production systems, l-alanine was fermented from glucose and l-branched chain amino acids were imported from the medium and these two amino acids were ligated by Lal. As shown in Fig.