Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue.

A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase.     

Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate

Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni . During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase ( aspA ) mutant (unable to utilize any amino acid except serine) and a Cj0762 c ( aspB ) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA -dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.     

Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants]

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.     

Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168.

The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonucleoside-requiring mutants of Bacillus subtilis.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 degrees C but not at 25 degrees C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.     

Saccharomyces cerevisiae porphobilinogenase: some physical and kinetic properties

1. Properties of porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were studied in a wild strain, D273-10B and a mutant, B231, of Saccharomyces cerevisiae. 2. A well-defined maximum of enzyme activity was observed for the mutant strain after 20 hr of growth; whilst the activity in the wild strain did not vary significantly during growth. 3. Neither PBG consumption nor uroporphyrinogen formation were modified by the presence of air either in the wild or in the mutant strain. 4. In both the wild and mutant strains uroporphyrinogen formation increased linearly with both protein concentration and incubation time. 5. The addition of a mixture of sodium and magnesium salts to the assay system inhibited the enzyme activity of both strains by 50% without modifying the isomer composition. 6. The same optimum pH (7.4) and mol. wt (50,000 +/- 5000) was found for the enzyme from both strains. 7. The enzyme from both the wild and mutant strains shows Michaelis-Menten kinetics when isolated from cells at either the exponential or the stationary phases of growth. Accumulation of porphyrins and delta-aminolevulinic acid occurring during the exponential phase in the mutant strain, did not modify the kinetics.     

Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.

We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.     

A non-NadB type L-aspartate dehydrogenase from Ralstonia eutropha strain JMP134: molecular characterization and physiological functions

We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.     

Aspartate transcarbamylase synthesis ceases prior to inactivation of the enzyme in Bacillus subtilis.

Aspartate transcarbamylase is synthesized during exponential growth of Bacillus subtilis and is inactivated when the cells enter the stationary phase. This work is a study of the regulation of aspartate transcarbamylase synthesis during growth and the stationary phase. Using specific immunoprecipitation of aspartate transcarbamylase from extracts of cells pulse-labeled with tritiated leucine, we showed that the synthesis of the enzyme decreased very rapidly at the end of exponential growth and was barely detectable during inactivation of the enzyme. Synthesis of most cell proteins continued during this time. When the cells ceased growing because of pyrimidine starvation of a uracil auxotroph, however, synthesis and inactivation occurred simultaneously. Measurement of pools of pyrimidine nucleotides and guanosine tetra- and pentaphosphate demonstrated that failure to synthesize aspartate transcarbamylase in the stationary phase was not explained by simple repression by these compounds. The cessation of aspartate transcarbamylase synthesis may reflect the shutting off of a "vegetative gene" as part of the program of differential gene expression during sporulation. However, aspartate transcarbamylase synthesis decreased normally at the end of exponential growth at the nonpermissive temperature in a mutant strain that is temperature-sensitive in sporulation and RNA polymerase function. Cessation of aspartate transcarbamylase synthesis appeared to be normal in three other temperature-sensitive RNA polymerase mutants and in several classes of spo0 mutants.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Malate Dehydrogenase Mutants in Escherichia coli K-12

Mutants devoid of malate dehydrogenase activity have been isolated in Escherichia coli K-12. They do not possess detectable malate dehydrogenase when grown aerobically or anaerobically on glucose as sole carbon source. All mutants revert spontaneously; a few partial revertants have been found with a malate dehydrogenase exhibiting altered electrophoretic mobility. Therefore, only one such enzyme appears to exist in the strains examined. No evidence could be obtained for the presence of a malate dehydrogenase not linked to nicotinamide adenine dinucleotide. Mutants deficient in both malate dehydrogenase and phosphoenol pyruvate carboxylase activities will grow anaerobically on minimal glucose plus succinate medium; also, malate dehydrogenase mutants do not require succinate for anaerobic growth on glucose. The anaerobic pathway oxaloacetate to succinate or succinate to aspartate appears to be accomplished by aspartase. Malate dehydrogenase is coded for by a locus somewhere relatively near the histidine operon, i.e., a different chromosomal location than that known for other citric acid cycle enzymes.     

Determination of aspartase activity in dairy Propionibacterium strains

Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix. Here a method for aspartase activity was established and a number of dairy propionibacteria belonging to P. freudenreichii ssp. shermanii and freudenreichii were screened for this enzyme activity. A wide range of aspartase activity could be found in PAB isolates originating from cheese. The majority, i.e. 70% of the 100 isolates tested, showed very low levels of aspartate activity.