Vitaliy A. Livshits

The novel transmembrane Escherichia coli proteins involved in the amino acid efflux

A novel gene of Escherichia coli, rhtB, has been characterized. Amplification of this gene provides resistance to homoserine and homoserine lactone. Another E. coli gene, rhtC, provides resistance to threonine. The homologues of RhtB are widely distributed among various eubacteria and archaea, from one to 12 copies of family members that differ in their primary structure were found in the genomes. Most of them are genes that encode hypothetical transmembrane proteins. Experimental data that indicate participation of the rhtB product in the excretion of homoserine have been obtained.

Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli.

The rhtA gene known as the ybiF ORF in the genome of Escherichia coli was identified as a new gene involved in threonine and homoserine efflux. This gene encodes a highly hydrophobic membrane protein that contains 10 predicted transmembrane segments. The rhtA23 mutation, which is an A-for-G substitution at position -1 in relation to the ATG start codon, increases the expression level of the rhtA gene. The overexpression of rhtA gene results in resistance to inhibitory concentrations of homoserine, threonine and a variety of other amino acids and amino acid analogues, reduced threonine and homoserine accumulation in resistant cells and increased production of threonine, homoserine, lysine and proline by the respective producing strains. The RhtA protein belongs to a vast family of transporters. The genome of E. coli contains at least 10 paralogues of RhtA. Phylogenetic analysis indicates that a common ancestor of living organisms contained several RhtA homologues.

A new family of amino-acid-efflux proteins.

We thank T. P. Turova and R. L. Tatusov for the SEQEDIT manual alignment software, and D. G. Naumoff and M. S. Gelfand for reading the manuscript and for discussions.

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl‐l‐leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for “leucine export”) was induced by leucine, l‐α‐amino‐n‐butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.

A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3′ end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.

Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens : purification, characterization, and application to increase purine nucleoside production

Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-α-d-ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC 2.7.6.1). Class I PRSs are widespread in bacteria and mammals, are highly conserved among different organisms, and are negatively regulated by two end products of purine biosynthesis, adenosine 5′-diphosphate (ADP) and guanosine 5′-diphosphate (GDP). The D52H, N114S, and L129I mutations in the human PRS isozyme I (PRS1) have been reported to cause uric acid overproduction and gout due to allosteric deregulation and enzyme superactivity. In this study, to find feedback-resistant Bacillus amyloliquefaciens PRS, the influence of the D58H, N120S, and L135I mutations (corresponding to the D52H, N114S, and L129I mutations in PRS1, respectively) on PRS enzymatic properties has been studied. Recombinant histidine-tagged wild-type PRS and three mutant PRSs were expressed in Escherichia coli, purified, and characterized. The N120S and L135I mutations were found to release the enzyme from ADP and GDP inhibition and significantly increase its sensitivity to inorganic phosphate (Pi) activation. In contrast, PRS with the D58H mutation exhibited nearly identical sensitivity to ADP and GDP as the wild-type protein and had a notably greater Pi requirement for activation. The N120S and L135I mutations improved B. amyloliquefaciens and Bacillus subtilis purine nucleoside-producing strains.

Metabolic engineering of Escherichia coli for L-tryptophan production

The review summarizes the main approaches applied during the creation of L-tryptophan producing strains based on Escherichia coli for the industrial production of this amino acid. In addition, some prospects for the further improvement of tryptophan producers to increase their productivity and improve their technological characteristics based on systems metabolic engineering approaches are outlined in the review. These approaches can be used to obtain the producers of other aromatic amino acids and tryptophan precursors or derivatives.

Metabolic engineering of Escherichia coli for the production of phenylalanine and related compounds

In this review, the metabolic engineering approaches including those used by the authors in creating phenylalanine producers based on Escherichia coli were systematized. Optimization of the amino acid biosynthesis was conducted in order to obtain significant quantities of phenylalanine and to ensure the availability of its direct precursors in cell metabolism—erythrose 4-phosphate and phosphoenolpyruvic acid. The possibility of altering global regulation mechanisms was investigated for a full reorientation of the metabolism to phenylalanine synthesis with the use of Csr (carbon storage regulator). The identification of the aromatic amino acids exporter YddG is associated with the use of the phenylalanine producer as a test-system. Novel approaches to phenylalanine producer construction (use of the “leaky” allele tyrA—ssrA, promoters of the phosphate regulon), as well as new methods of obtaining producers of similar amino acid tyrosine, were discussed. Examples of the synthesis of useful aromatic compounds from phenylalanine or its precursor, phenylpyruvic acid, with E. coli as the ecipient for foreign gene expression were examined.