Microbial production of O-methylated flavanones from methylated phenylpropanoic acids in engineered Escherichia coli

Journal of Industrial Microbiology & Biotechnology - Methylated flavonoids possess improved bioactivities compared to their unmethylated counterparts. In this study, for the efficient...     

YddG from Escherichia coli promotes export of aromatic amino acids

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. colil-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.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli

Fusion proteins with charged polycationic amino acid tails were constructed for the purpose of simple ion-exchange purification with high purity. A number of positively charged lysine and arginine tails were fused to the C-terminus of cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans and expressed in Escherichia coli. The ionic binding forces provided by the tails allowed the selective recovery of CGTase from recombinant E. coli cell extracts, while CGTase by itself could not bind to the cation exchanger at neutral pH. The type of amino acids used and the length of the tail directly affected the purification factors. Most intracellular proteins of E. coli adsorbed on the cation exchanger could be removed by washing with 400 mM NaCl solution at pH 7.4, suggesting that a fusion partner suitable for purification purpose should be provided with high binding strength and the maintenance of adsorption by washing with NaCl solution. Among the fusion CGTases constructed, the CGTK10ase containing 10 lysine residues provided sufficiently high binding strength to allow purification to its homogeneity through simple ion-exchange chromatography.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Chemotactic response of Escherichia coli to chemically synthesized amino acids.

In Escherichia coli, seven of the commonly occurring amino acids are strong attractants: L-aspartate, L-serine, L-glutamate, L-alanine, L-asparagine, glycine, and L-cysteine, in order of decreasing effectiveness. The chemotactic response to each amino acid attractant is mediated by either methyl-accepting chemotaxis protein I or II, but not by both. Seven of the commonly occurring amino acids are repellents. This work was carried out with chemically synthesized amino acids.     

Properties of ribosomes from erythromycin resistant mutants of Escherichia coli.

Summary We have studied the in vitro properties of ribosomes from several mutants resistant to erythromycin. Mutations in three different genes may confer resistance to erythromycin. Two of them are structural genes for proteins L4 and L22 of the large subunit. The third mutation (in eryC gene) seems to affect mainly the small subunit. The mechanism of action of the antibiotic may involve both subunits.     

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

Isolation of ribosomes from Escherichia coli by chromatography on methyl-albumin-kieselgel.

The present communication shows that ribosomes isolated by differential centrifugation retain their composition and biological activity during subsequent methyl-albumin-kieselgel (MAK)-chromatography. The data obtained were used to develop a novel chromatographic procedure for the isolation of ribosomes from E. coli lysates. It could be demonstrated that purified preparations of active ribosomes can be obtained by MAK-chromatography avoiding ultracentrifugation.     

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.