Molecular breeding of a Brevibacterium lactofermentum l-phenylalanine producer using a cloned prephenate dehydratase gene

Summary The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermentum, AJ11957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPH11 and pPH14 complemented a phenylalanine auxotroph of B. lactofermentum, A-15, provided the transformant with the desensitized enzyme and caused an increased level of the enzyme compared to that of a wild strain. Plasmid pPH14 was introduced into l-phenylalanine producers genetically induced from B. lactofermentum; MF358 and FP-1 excreting l-tyrosine and anthranilate, respectively, as by-products. Both transformants predominantly accumulated l-phenylalanine at the expense of by-product formation. Co-existence of pPH14 and pTAR16, a recombinant plasmid expressing desensitized 3-deoxy-d-arabino-hepturosonate-7-phosphate synthase had a marked effect on further improvement in l-phenylalanine productivity, accompanied by an increase in the corresponding enzyme activity. The parent, MF358, accumulating 5.5 g/l l-phenylalanine, 6.8 g/l l-tyrosine and 0.3 g/l anthranilate turned into a potent l-phenylalanine producer producing 18.2 g/l l-phenylalanine and 1.0 g/l l-tyrosine by-product. Offprint requests to: Hisao Ito     

Depression of by-product formation during l-isoleucine production by a living-cell reaction process

Summary Two unnatural and unwanted amino acids, norvaline (Nva) and O-ethylhomoserine (O-EH) are formed as by-products in l-isoleucine production by Brevibacterium flavum AB-07 using a new process named the living cell reaction process. Nva formation was depressed by using a leucine auxotrophic mutant (AB-07-Leu-2) derived from strain AB-07. It was found that Nva formation was closely related to leucine biosynthesis. O-EH formation was repressed by addition of l-methionine to the reaction mixture. However, the homoserine-O-acetyltransferase of AB-07-Leu-2 was not subject to either inhibition or repression by addition of l-methionine. Furthermore, the O-EH-forming enzyme, which converts O-acetylhomoserine to O-EH, was speculated to be repressed by l-methionine. Offprint requests to: H. Yukawa     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Cell Selective Conditional Null Mutations of Serine Racemase Demonstrate a Predominate Localization in Cortical Glutamatergic Neurons

d-Serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-d-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of d-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and d-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in d-serine levels as quantified by HPLC, whereas d-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain d-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral–CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where d-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and d-serine is required for optimal activation of post-synaptic NMDA receptors.     

Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect d-hydantoinase-producing microorganisms by direct colony hybridization

Pseudomonas putida strain DSM 84 produces N-carbamyl-d-amino acids from the corresponding d-5-monosubstituted hydantoins. The sequence of the d-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect d-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed d-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganims from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated d-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Clues from a halophilic methanogen about aromatic amino acid biosynthesis in archaebacteria

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP⁺ (preferred) or NAD⁺ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.Abbreviation DAHP 3-deoxy-d-arabino-heptulosonate-7-phosphate     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K₂HPO₄ (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases. Received: 20 November 1997 / Accepted: 28 February 1997     

Molecular cloning, expression, and characterization of a thermostable glutamate racemase from a hyperthermophilic bacterium, Aquifex pyrophilus

A gene encoding glutamate racemase has been cloned from Aquifex pyrophilus, a hyperthermophilic bacterium, and expressed in Escherichia coli. The A. pyrophilus glutamate racemase is composed of 254 amino acids and shows high homology with glutamate racemase from Escherichia coli, Bacillus subtilis, or Lactobacillus brevis. This racemase converts l- or d-glutamate to d- or l-glutamate, respectively, but not other amino acids such as alanine, aspartate, and glutamine. The cloned gene was expressed and the protein was purified to homogeneity. The A. pyrophilus racemase is present as a dimer but it oligomerizes as the concentration of salt is increased. The K _m and k_cat values of the overexpressed A. pyrophilus glutamate racemase for the racemization of l-glutamate to the d-form and the conversion of d-glutamate to the l-form were measured as 1.8 ± 0.4 mM and 0.79 ± 0.06 s⁻¹ or 0.50 ± 0.07 mM and 0.25 ± 0.01 s⁻¹, respectively. Complete inactivation of the racemase activity by treatment with cysteine-modifying reagents suggests that cysteine residues may be important for activity. The protein shows strong thermostability in the presence of phosphate ion, and it retains more than 50% of its activity after incubation at 85°C for 90 min. Received: September 11, 1998 / Accepted: January 12, 1999     

Isolation and characterization of a bacterium possessing L-phenylalanine dehydrogenase activity

The new enzyme phenylalanine dehydrogenase [L-phenylalanine: NAD⁺-oxidoreductase (deaminating)] was detected in the crude extract of a strain of Brevibacterium spec. The bacterium was isolated from a soil sample by enrichment with phenylalanine. This strain was the only one containing phenylalanine dehydrogenase out of 173 tested strains, among them 22 of the genus Brevibacterium, 74 strains from soil samples and 77 strains from a culture collection belonging to several genera. The enzyme is involved in the degradation of phenylalanine and could be induced by addition of L-, D-, D,l-phenylalanine or L-histidine, the optimum inducer concentration of phenylalanine being 1%.The reaction mechanism of a reductive amination was confirmed by demonstrating the close coupling between NADH-consumption and phenylalanine production; ammonia could not be replaced by L-glutamate or L-aspartate as amino donor. The -keto acid of L-tyrosine was converted too, while the corresponding compound of histidine was inactive. The optimum pH value for reductive amination in the crude extract was 8.5 and for oxidative desamination 10.5.     

Metabolic characterisation of a novel vanillylmandelate-degrading bacterium

A newly isolated gram-negative bacterium, possibly Brevundimonas diminuta, utilised d,l-vanillylmandelate (d,l-VMA) as a sole carbon and energy source. The organism converted d,l-VMA to vanillylglyoxylate using a soluble NAD-dependent dehydrogenase specific for d-VMA and a dye-linked, membrane-associated l-VMA dehydrogenase. Vanillylglyoxylate was further metabolised by decarboxylation, dehydrogenation and demethylation to protocatechuate. A 4,5-dioxygenase cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. Partially purified d-VMA dehydrogenase exhibited optimal activity at 30° C and pH 9.5 and had an apparent K _m for d-VMA of 470 μM. Although induced by several substituted mandelates, the enzyme had a narrow substrate specificity range with virtually no activity towards d-mandelate. Such properties render the enzyme of potential use in both diagnostic and biosynthetic applications. Received: 23 January 1996 / Accepted: 9 April 1996     

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ

l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml⁻¹ cell mass incubated at 40°C for 60 min. The combination of CuSO₄ and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml⁻¹, respectively. The activated charcoal 2 mg ml⁻¹ was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg⁻¹ of tyrosinase. Kinetic studies showed significant values of Y _p/s (0.994), Q _s (0.500) and q _s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.     

Mid- and late-Holocene vegetation and fire history at Biviere di Gela, a coastal lake in southern Sicily, Italy

The vegetation and fire history of few coastal sites has been investigated in the Mediterranean region so far. We present the first paleoecological reconstruction from coastal Sicily, the largest island in the Mediterranean Sea. We analysed pollen and charcoal in the sediments of Biviere di Gela, a lake (lagoon) on the south coast of Sicily. Our data suggest that the area became afforested after a marine transgression at ca. 7200 cal b.p. (5250 b.c.). Build-up of forest and shrublands took ca. 200–300 years, mainly with the deciduous trees Quercus, Ostrya and Fraxinus. Juniperus expanded ca. 6900 cal b.p. (4950 b.c.), but declined again 6600 cal b.p. (4650 b.c.). Afterwards, evergreen trees (Q. ilex-type and Olea) became dominant in the forest and Pistacia shrublands were established. Forest and shrubland reached a maximum ca. 7000–5000 cal b.p. (5050–3050 b.c.); subsequently forest declined in response to human impact, which was probably exacerbated by a general trend towards a more arid climate. During the Neolithic, fire was used to open the landscape, significantly reducing several arboreal taxa (Q. ilex, Fraxinus, Juniperus) and promoting herbs and shrubs (Achillea, Cichorioideae, Brassicaceae, Ephedra). Final forest disruption occurred around 2600 cal b.p. (650 b.c.) with the onset of the historically documented Greek colonization. We conclude that the open maquis and garrigue vegetation of today is primarily the consequence of intensive land-use over millennia. Under natural or near-natural conditions arboreal taxa such as Q. ilex, Olea and Pistacia would be far more important than they are today, even under the hot and rather dry coastal conditions of southern Sicily.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Characterization of a recombinant thermostable D-lyxose isomerase from Dictyoglomus turgidum that produces D-lyxose from D-xylulose

A putative d-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for d-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by gel-filtration chromatography. The activity of the enzyme was highest for d-lyxose, suggesting that it is a d-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co²⁺, with a half-life of 108 min, K _m of 39 mM, and k _cat of 3,570 1/min. The enzyme is the most thermostable d-lyxose isomerase among those characterized to date. It converted 500 g d-xylulose/l to 380 g d-lyxose/l after 2 h. This is the highest concentration and productivity of d-lyxose reported thus far.     

Vegetation history of the SE section of the Zagros Mountains during the last five millennia; a pollen record from the Maharlou Lake, Fars Province, Iran

A pollen diagram was derived from a 150 cm core taken from the shallow hypersaline Lake Maharlou in the south-eastern part of the Zagros Mountains, SW Iran. The pollen record shows that Quercus brantii woodland and Pistacia–Amygdalus scrub dominated the area during the late Holocene. The record starts at around 5700 cal b.p. with a dry period during which both Pistacia–Amygdalus scrub and Quercus brantii woodland were at their minimum extent. This period was followed by the expansion of Pistacia–Amygdalus scrub in the area and the spread of Quercus brantii woodlands at higher altitudes. An important occupation phase, characterized by the appearance of several cultivated tree species such as Juglans, Olea, Vitis and Platanus, started at ca. 4300 cal b.p., coinciding with the onset of the Bronze Age civilization of Jiroft in Central Iran. Human activities become very clear after 3700 cal b.p. Around 2700 cal b.p., extensive stands of Pistacia–Amygdalus scrub became profoundly degraded, presumably under strong human pressure coinciding with the beginning of the Persian Empires. The maximum expansion of the Quercus brantii woodland occurred about 2100 to 1700 cal b.p. This woodland remained relatively stable until the end of the diagram at 400 cal b.p.     

Overexpression of a thermostable lipase gene from Pseudomonas fluorescens in Escherichia coli

Summary A thermostable lipase gene from Pseudomonas fluorescens SIK W1 was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl--d-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 m urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95°C and the increment of temperature to reduce heating times by 90% (z _d value) was 76°C.Offprint requests to: J. S. Rhee     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation

An open reading frame encoding a putative bi-functional β-d-xylosidase/α-l-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual β-d-xylosidases and α-l-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75°C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like d-glucose and l-arabinose.