Rational metabolic engineering of Corynebacterium glutamicum to create a producer of L-valine.

L-Valine is one of the nine amino acids that cannot be synthesized de novo by higher organisms and must come from food. This amino acid not only serves as a building block for proteins, but also regulates protein and energy metabolism and participates in neurotransmission. L-Valine is used in the food and pharmaceutical industries, medicine and cosmetics, but primarily as an animal feed additive. Adding L-valine to feed, alone or mixed with other essential amino acids, allows for feeds with lower crude protein content, increases the quality and quantity of pig meat and broiler chicken meat, as well as improves reproductive functions of farm animals. Despite the fact that the market for L-valine is constantly growing, this amino acid is not yet produced in our country. In modern conditions, the creation of strains-producers and organization of L-valine production are especially relevant for Russia. One of the basic microorganisms most commonly used for the creation of amino acid producers, along with Escherichia coli, is the soil bacterium Corynebacterium glutamicum. This review is devoted to the analysis of the main strategies for the development of L- valine producers based on C. glutamicum. Various aspects of L-valine biosynthesis in C. glutamicum are reviewed: process biochemistry, stoichiometry and regulation, enzymes and their corresponding genes, export and import systems, and the relationship of L-valine biosynthesis with central cell metabolism. Key genetic elements for the creation of C. glutamicum-based strains-producers are identified. The use of metabolic engineering to enhance L-valine biosynthesis reactions and to reduce the formation of byproducts is described. The prospects for improving strains in terms of their productivity and technological characteristics are shown. The information presented in the review can be used in the production of producers of other amino acids with a branched side chain, namely L-leucine and L-isoleucine, as well as D-pantothenate.

[Nucleotide sequence and structural analysis of cryptic plasmid pBL90 from Brevibacterium lactofermentum].

The nucleotide sequence of cryptic plasmid (designated as pBL90) detected in the cells of Brevibacterium lactofermentum DSM 1412 was determined. The length of plasmid DNA is 67826 bp. Comparison of the nucleotide sequence of pBL90 with known plasmid sequences showed no long regions of significant homology. Computer analysis of the plasmid DNA revealed 29 open reading frames (ORFs). The amino acid sequences of 15 ORFs (approximately 25% of plasmid length) have a high (>70%) level of identity to proteins from different plasmids of Corynebacterium representatives, including replicative proteins. Unusual in pBL90 is the presence of replicative genes from two different families and types of replication.

[Cloning and analysis of a new aliphatic amidase gene from Rhodococcus erythropolis TA37].

A new aliphatic amidase gene (ami), having a level of similarity with the nearest homologs of no more than 77%, was identified in the Rhodococcus erythropolis TA37 strain, which is able to hydrolyze a wide range of amides. The amidase gene was cloned within a 3.7 kb chromosomal locus, which also contains putative acetyl-CoA ligase and ABC-type transportergenes. The structure of this locus in the R. erythropolis TA37 strain differs from the structure of loci in other Rhodococcus strains. The amidase gene is expressed in Escherichia coli cells. It was demonstrated that amidase (generated in the recombinant strain) efficiently hydrolyzes acetamide (aliphatic anmide) and does not use 4'-nitroacetanilide (N-substituted amide) as a substrate. Insertional inactivation of the amidase gene in the R. erythropolis TA37 strain results in a considerable decrease (by at least 6-7 times) in basal amidase activity, indicating functional amidase activity in the R. erythropolis TA37 strain.

A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

Aliphatic amidase from Rhodococcus rhodochrous M8 is related to the nitrilase/cyanide hydratase family.

A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous.

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.

A study of the surface-active properties of the Mg2+-activated ATPase from cytoplasmic membranes of Streptococcus faecalis.

The surface activity and enzymic properties of the factor F1, the catalytic moiety of Streptococcus faecalis H+-ATPase, has been studied at the air-water and phospholipid-water interfaces. F1 does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F1 molecule at the air-water interface have been estimated. In the presence of Mg2+, base area is S = 1.8 . 10(4) A2, height h = 27 A. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F1 subunits should all lie in the same plane. The ATPase activity of F1 at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution. Adsorption of F1 at the interface shifts the isoelectric point of tiscussed relative to the proton-active transport mechanism.