Expression, purification and crystallization of N-acetyl-(R)-ß-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme.

N-Acetyl-(R)-ß-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-ß-phenylalanine to produce enantiopure (R)-ß-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-ß-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure-function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-ß-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P41212, with unit-cell parameters a = b = 112.70-112.97, c = 341.50-343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-ß-phenylalanine acylases were clarified to be dimeric in solution.

Purification and characterization of enantioselective N-acetyl-ß-Phe acylases from Burkholderia sp. AJ110349.

For the production of enantiopure ß-amino acids, enantioselective resolution of N-acyl ß-amino acids using acylases, especially those recognizing N-acetyl-ß-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-ß-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-ß-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-ß-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-ß-Phe (>99% ee) and (S)-ß-Phe (>99% ee) were obtained from the racemic substrate.

Glutamate is excreted across the cytoplasmic membrane through the NCgl1221 channel of Corynebacterium glutamicum by passive diffusion.

The NCgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (Glu) overproduction by Corynebacterium glutamicum, but direct evidence of Glu excretion through this channel has not yet been provided. In this study, by electrophysiological methods, we found direct evidence of Glu excretion through this channel by passive diffusion. We found that the introduction into Phe-producing Escherichia coli of mutant NCgl1221 genes that induce Glu overproduction by C. glutamicum improved productivity. This suggests a low-substrate preference of this channel, indicates its potential as a versatile exporter, and more broadly, indicates the potential of exporter engineering.

The protein encoded by NCgl1221 in Corynebacterium glutamicum functions as a mechanosensitive channel.

The function of the NCgl1221-encoded protein of Corynebacterium glutamicum was analyzed using Bacillus subtilis as host because a method for preparing the giant provacuole required for electrophysiological studies has been established. Expression of NCgl1221 in a strain deficient in mscL and ykuT, both of which encode mechanosensitive channels, resulted in an 8.9-fold higher cell survival rate upon osmotic downshock than the control. Electrophysiological investigation showed that the giant provacuole prepared from this strain, expressing NCgl1221, exhibited significantly higher pressure-dependent conductance than the control. These findings show that the NCgl1221-encoded protein functions as a mechanosensitive channel.

Changes in enzyme activities at the pyruvate node in glutamate-overproducing Corynebacterium glutamicum.

Glutamate is industrially produced by fermentation using Corynebacterium glutamicum. The key factor for efficient glutamate production by this microorganism has been considered to be a metabolic change at the 2-oxoglutarate dehydrogenase (ODH) branch point caused by a decrease in ODH activity under glutamate-overproducing conditions. However, this change would be insufficient because the ODH branch is merely the final branch in the glutamate biosynthetic pathway, and efficient glutamate production requires a balanced supply of acetyl-CoA and oxaloacetate (OAA), which are condensed to form a precursor of glutamate, namely, citrate. Therefore, there must be another (other) change(s) in metabolic flux. In this study, we demonstrated that a decrease in pyruvate dehydrogenase (PDH) activity catalyzes the conversion of pyruvate to acetyl-CoA. It is speculated that carbon flux from pyruvate to acetyl-CoA decreases under glutamate-overproducing conditions. Furthermore, an increase in pyruvate carboxylase (PC) activity, which catalyzes the reaction of pyruvate to OAA, is evident under glutamate-overproducing conditions, except under biotin-limited condition, which may lead to an increase in carbon flux from pyruvate to OAA. These data suggest that a novel metabolic change occurs at the pyruvate node, leading to a high yield of glutamate through adequate partitioning of the carbon flux.

Crystallization and preliminary crystallographic analysis of DtsR1, a carboxyltransferase subunit of acetyl-CoA carboxylase from Corynebacterium glutamicum.

DtsR1, a carboxyltransferase subunit of acetyl-CoA carboxylase derived from Corynebacterium glutamicum, was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 6000 as a precipitant. The crystal belongs to the trigonal system with space group R32 and contains three subunits in the asymmetric unit. A molecular-replacement solution was found using the structure of transcarboxylase 12S from Propionibacterium shermanii as a search model.

Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum.

L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.

Temperature-sensitive cloning vector for Corynebacterium glutamicum.

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.

Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum.

Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects. In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.

Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity.

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.

Purification and characterization of malate dehydrogenase from Corynebacterium glutamicum.

The malate dehydrogenase (MDH) (EC 1.1.1.37) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 8) matched the sequence (residues 2 to 9) of the MDH from C. glutamicum (GenBank accession no. CAC83073). The molecular mass of the native enzyme was 130 kDa. The protein was a homotetramer, with a 33-kDa subunit molecular mass. The enzyme was almost equally active both for NADU and NADPH as coenzyme on the bases of the k(cat) values at pH 6.5 which is the optimum pH for the both coenzymes. Plotting of the logarithms of the 1/Km, k(cat), and k(cat)/K(m) values with respect to oxalacetate against pH lead to speculation that imidazolium is possibly a functional group in the active center of the enzyme. Citrate activated the enzyme in the oxidation of malate to oxalacetate and inhibited it in the reverse reaction.

Corynebacterium efficiens sp. nov., a glutamic-acid-producing species from soil and vegetables.

Three glutamic-acid-producing coryneform strains were isolated from soil and vegetable samples. Chemotaxonomic investigations indicated that these strains belonged to the genus Corynebacterium. Phylogenetic studies, based on 16S rDNA analysis, demonstrated that the three strains formed a distinct cluster within the genus Corynebacterium and that their nearest relatives were Corynebacterium glutamicum and Corynebacterium callunae, also known as glutamic-acid-producing species. The data from 16S rDNA sequence and DNA-DNA relatedness studies clearly indicated that the three isolates represented a new species within the genus Corynebacterium. All of the isolates could grow at 45 degrees C and produced acid from dextrin; these were the most significant characteristics differentiating the three isolates from their neighbours. On the basis of the data presented here, it is proposed that the three glutamic-acid-producing isolates together be classified as Corynebacterium efficiens sp. nov., the type strain of which is YS-314T (= AJ 12310T = JCM 11189T = DSM 44549T).