YjeH Is a Novel Exporter of l -Methionine and Branched-Chain Amino Acids in Escherichia coli

Abstract

c Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of Lmethionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of L-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical "5 ؉ 5" transmembrane ␣-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of L-methionine export by the overexpression of yjeH in an L-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering. Membrane transport mediates the exchange of materials and energy with the surroundings to facilitate cell viability (1). Amino acids play central roles both as the building blocks of proteins and as intermediates in metabolism. Amino acid transport processes, including uptake and efflux, exist widely in bacteria. Amino acids in the surrounding environment can be imported into cells directly to participate in protein synthesis or carbon and nitrogen metabolism without spending energy for anabolism (2). The export process, however, is essential to maintain the intracellular amino acid pool and has significant applications for amino acid overproduction.Amino acid transport processes are mostly transporter mediated in prokaryotes. In Escherichia coli, the L-methionine uptake system MetNIQ is a typical primary transporter which utilizes the energy of ATP binding and hydrolysis to transport substrates (3), while the amino acid/polyamine/organocation (APC) superfamily is one of the largest superfamilies with nearly 250 secondary transporters. These transporters vary in length from 350 to 850 amino acid residues (4). Many amino acids are transported through APC carriers that function as solute-cation symporters and solute-solute antiporters, such as the lysine importer LysP in E. coli (5) and the aromatic amino ...

A Novel L-Serine Exporter and Its Positive Regulator in Corynebacterium glutamicum

“…The recombinant plasmids were constructed as follows: the genes thrE and serE were amplified, digested, and ligated to the pDXW-10 plasmid that was digested with Hind III/ Bgl II. The plasmid harboring the fusion protein, SerE-EGFP (enhanced green fluorescent protein) was constructed by using the method reported in a previous study (16). To confirm the role of NCgl0581 on NCgl0580 expression, the fragment consisting of intergenic region of NCgl0581 and NCgl0580 and EGFP with or without NCgl0581 was ligated to the plasmid pDXW-11 by Clon Express MultiS One Step Cloning Kit (Vazyme, Nanjing, China).…”

“…Amino acid excretion was initiated by adding 2 mM Ser-Ser (another dipeptide). HPLC was used to determine the concentrations of amino acids (16).…”

“…It has been reported that homologs similar to the exporters in E. coli might fulfil a comparable function in C. glutamicum (14, 16, 17). Accordingly, we hypothesized that the homolog to eamA ( L -serine exporter in E. coli ) (1) might be involved in L -serine export in C. glutamicum .…”

High yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum

“…In E. coli, Mundhada et alfound that intracellular L-serine accumulation was toxic to the engineered strain modified to produce L-serine, and that following overexpression of eamA (which encodes L-cysteine exporter in E. coli), the engineered strain exhibited increased tolerance toward L-serine with higher L-serine productivity [2]. Therefore, L-serine exporter in C. glutamicum could be a potential target for strain optimization to further improve L-serine production.It has been reported that homologs similar to the exporters in E. coli might fulfil a comparable function in C. glutamicum [17,19,20]. Accordingly, we hypothesized that the homolog to eamA (Lserine exporter in E. coli) might be involved in L-serine export in C. glutamicum.…”

“…In brief, pre-incubated cells (in seed medium) were washed once with CGXⅡ minimal medium [41], inoculated into CGXⅡ minimal medium with 2 mM Ser-Ser (other dipeptide), and incubated for 2 h at 30 o C. Then, the cells were harvested, washed once with cold CGXⅡ minimal medium, and resuspended in CGXⅡ minimal medium. Amino acid excretion was initiated by adding 2 mM Ser-Ser (another dipeptide).HPLC was used to determine the concentrations of amino acids [19].…”