Our solution to overcome this problem was to express Lal under the control of a stationary phase-specific promoter in order to separate the production period from the growth period. The resulting strain JKYPQ3/pPE167 produced >100 mM Ala-Gln extracellularly, in a 5-L jar fed-batch cultivation in a glucose–ammonia salt medium. As an alternative solution to overcome the growth inhibition, we focused on a dipeptide efflux system. Because LysE, a basic amino acid exporter of Corynebacterium glutamicum, was found (Vrljic et al., 1996), amino acid exporter proteins have been extensively
characterized in C. glutamicum and Escherichia coli from scientific and practical interests (Eggeling & Sahm, 2003; Marin & Krämer, 2007). It
has already been shown that these exporter proteins are useful for increasing productivity in amino acid fermentations (Simic et al., 2002). However, dipeptide exporter proteins have not been identified for any Palbociclib mw bacteria so far. Our objectives in this study were (1) to identify the genes relevant to dipeptide efflux from predicted multidrug-efflux transporter genes learn more of E. coli and (2) to examine their role in dipeptides production. The E. coli strains and plasmids used in this study are listed in Table 1. Strain DH5α was used as a host for cloning. We previously reported that whenever the pepD gene was disrupted, the strain became an l-proline auxotroph (Tabata & Hashimoto, 2007). The result of comparative genomic hybridization indicated that pepD was encoded on F′ plasmid as proAB in JM101 (unpublished data). From these reasons, we concluded that disruption of pepD was accompanied by a loss of F′ plasmid. Strain Δpeps was obtained from strain JKYP7 by disrupting pepA as described previously (Tabata & Hashimoto, 2007). Gene disruption was performed based on the phage lambda Red recombinase system (Datsenko & Wanner, 2000). Strain Δpeps was used to select the genes conferring resistance to growth
inhibitory dipeptides. Luria–Bertani (LB) medium and M9 minimal medium (Sambrook & Russell, 2001) was used for general cultivation. l-Proline was added to M9 minimal medium at 0.2 g L−1 under all conditions. Solid plates were prepared by the addition of Bacto agar (Difco) to 1.6%. If necessary, 100 mg L−1 kanamycin and/or 25 mg L−1 chloramphenicol was Atezolizumab added. For dipeptide resistance assay, dipeptides were added at 0.2 mM. Serial 10-fold dilutions of cells were plated and cultivated at 30 °C for 2 days. Test tube cultivations for dipeptide production were carried out as described previously (Tabata & Hashimoto, 2007). For l-alanyl-l-branched chain amino acids (Ala-BCAA) production, branched chain amino acid was added at 0.02% to test tube (TT) medium. DNA manipulations were basically performed according to the method of Sambrook & Russell. The primers used in this study are listed in Table 1.