RESEARCH LETTER
YddG from Escherichia coli promotes export ofaromatic amino acids Vera Doroshenko, Larisa Airich, Maria Vitushkina, Alexandra Kolokolova, Vitaliy Livshits & Sergey Mashko Ajinomoto-Genetika Research Institute, Moscow, Russian Federation
Correspondence: Vera Doroshenko, Ajinomoto-Genetika Research Institute, 1st Dorozhny proezd, Moscow 117545, Russian Federation. Tel.: 17 495 314 1858; fax: 17 495 315 0001; e-mail:
[email protected] Received 28 April 2007; revised 25 July 2007; accepted 27 July 2007. First published online September 2007. DOI:10.1111/j.1574-6968.2007.00894.x Editor: Anthony George Keywords YddG; Escherichia coli ; amino acid efflux; aromatic amino acid exporter.
Abstract The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of L-phenylalanine and aromatic amino acid analogues, DL-p-fluorophenylalanine, DL-o-fluorophenylalanine and DL-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of L-phenylalanine, L-tyrosine or L-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. coli L-phenylalanine-producing strain containing overexpressed yddG accumulated less L-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.
Introduction Over the last decade, investigations have shown that bacteria could expel from cells not only harmful substances (antibiotics, heavy metals and detergents) but also internally formed metabolites such as amino acids and purine derivatives (Eggeling & Sahm, 2003; Zakataeva et al., 2006). Characterization of new potential exporters of amino acids and other commercially important metabolites are currently a rather dynamic field of research. A dozen amino acid efflux pumps were recently shown to augment amino acid production by industrial Escherichia coli and Corynebacterium glutamicum production strains (Burkovski & Kr¨amer, 2002; Zakataeva et al., 2006). The identified E. coli amino acid exporters (RhtA, YdeD or RhtB, RhtC, YfiK, YeaS) belong to two widespread membrane protein families: DMT and RhtB/LysE (M.H. Saier,: www.tcdb.org). The proteins of each family resemble each other in their molecular masses, the topology of the membrane-spanning helices and the presence of a few conserved sequence signatures. The majority of prokaryotic genomes contain up to 10 or more genes encoding putative proteins of this kind. Therefore, new amino acid exporters could be found among paralogues of the known efflux proteins. In the E. coli genome, ten RhtA paralogues have 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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been identified (Livshits et al., 2003b). One of them was the cysteine exporter YdeD (Daßler et al., 2000). The preliminary results showed that another RhtA paralogue, YddG, when overexpressed in E. coli-producing strains, enhanced the L-phenylalanine (Phe) or L-tryptophan (Trp) yield (Livshits et al., 2003a). Earlier, it was shown that YddG from Salmonella enterica sv. Typhimurium, having more than 95% identical amino acids with E. coli YddG, in concert with the outer membrane porin OmpD, is involved in methyl viologen (MV) export (Santiviago et al., 2002). The membrane location of YddG was confirmed using PhoA/ GFP fusion analysis (Rapp et al., 2004). In this study data indicating that YddG is capable of exporting aromatic amino acids in E. coli are presented. However, the resistance of E. coli MG1655 cells to MV might be not dependent on YddG.
Materials and methods Bacterial strains, plasmids and growth conditions The E. coli strains and plasmids used in this study are listed in Table 1. The media used were Luria–Bertani (LB), SOB or M9 minimal medium, with 0.4% glucose as a carbon source FEMS Microbiol Lett 275 (2007) 312–318
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Exporter of aromatic amino acids
Table 1. Escherichia coli strains used in this study Strain or plasmid Strain MG1655 DV033 DV036 DV064 DV157 DV158 DV654 DV666 DV683 DV686 DV688 DV1060 DV1061 DV1062 DV1063 Plasmids pKD46 pMW-int-xis pMDV3-Cm pMH10 pMW118-Cm
Genotype
Source or reference
Wild type MG1655 htrE
