A malic acid dependent mutant of Schizosaccharomyces malidevorans

The yeast Schizosaccharomyces malidevorans utilizes l-malate when grown on glucose as the carbon source. A mutant of this yeast has been isolated which is dependent on the presence of both l-malate and glucose for growth. The mutant utilizes l-malate as rapidly as the wildtype and the utilization of glucose is greatly reduced. Other TCA cycle intermediates do not relieve the malate dependence.To John Ingraham whose pioneering work with malolactic bucteria made me curious enough about the field of nine microbiology to enter it and whose intense instruction in scientific method has made my continued pursuit of physiological and genetic questions a joy     

Vacuolar localization of 1-sinapolglucose: l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus

The distribution of l-malate, sinapic acid esters and 1-sinapoylglucose: l-malate sinapoyltransferase (SMT) which catalyzes the synthesis of sinapoyl-l-malate were examined in preparations of protoplasts obtained from cotyledons of red radish (Raphanus sativus L. var. sativus). Vacuoles isolated from the protoplasts contained all of the SMT activity, all of the accumulated sinapic acid esters and about 50% of free l-malate present initially in the protoplasts. An esterase activity, acting on 1-sinapoyglucose, was found to be exclusively localized in the cytoplasm and a large proportion was found to be recoverable in a 100 000-g pellet obtained from protoplast lysates. The vacuoles were obtained after lysis of the protoplasts by osmotic shock and purification on a Ficoll gradient. The cytoplasmic contamination of vacuole preparations was found to be about 10%, as judged by enzymatic markers and microscopic inspection. No SMT activity was found in a 100 000-g pellet obtained from vacuole lysates. The results indicate that biosynthesis of sinapoyl-l-malate takes place within the central vacuoles of redradish cotyledons.Abbreviation SMT 1-sinapoylglucose: l-malate sinapol-transferase     

Tissue distribution of phenylpropanoid metabolism in cotyledons of Raphanus sativus L.

The tissue distributions of sinapic acid esters (1-sinapoylglucose, sinapolyl-l-malate, 6,3-disinapoylsucrose), kaempferol glycosides, free malic acid and of the enzyme involved in the synthesis of sinapoyl-l-malate, 1-sinapoylglucose: l-malate sinapoyltransferase (SMT), have been investigated in cotyledons of Raphanus sativus L. seedlings. The kaempferol glycosides were mainly localized in the upper epidermis. The sinapoyl esters were found in all tissues, but differed markedly in their concentrations. While disinapoylsucrose was localized predominantly in the mesophyll, most sinapoylmalate was found in the epidermal layers, as was most SMT activity. Ultraviolet microscopy and microfluorospectrophotometry of isolated epidermal peels indicated that the epidermal sinapoyl esters were restricted to guard cells, guard mother cells and adjacent epidermal cells. Upon excitation by UV light (365 nm) these exhibited strong blue fluorescence with an emission maximum at about 480 nm. Our results indicate a highly tissue-and cell-specific secondary metabolism in Raphanus cotyledons and indicate that the biosynthesis of sinapoylmalate is intimately related to the malic-acid metabolism of the guard cells.Abbreviations HPLC high-performance liquid chromatography - SMT 1-sinapoylglucose: l-malate sinapoyltransferase     

Nitrogen nutrition and the accumulation of free and sinapoyl-bound malic acid in Raphanus sativus cotyledons

Seedlings of red radish (Raphanus sativus L. var. sativus) accumulated high amounts of free malic acid and sinapoylmalate, when grown on nitrate as the sole N-source. In the presence of ammonium (NO ₃ ^– : NH ₄ ⁺ , 1:2) both metabolites failed to accumulate, and the levels of arginine, asparagine, glutamine, histidine, and serine were greatly increased. The extractable activity of 1-sinapoylglucose: l-malate sinapoyltransferase, an enzyme which plays a key role in channelling malic acid into the sinapic-acid metabolism of this plant, was positively correlated with the malic-acid level in cotyledons. The possibility is discussed that free malic acid might be the likely candidate for regulating the activity of 1-sinapoylglucose: l-malate sinapoyltransferase.Abbreviation SMT sinapoylglucose: L-malate sinapoyltransferase     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

An evolvant ofEscherichia coli that employs thel-fucose pathway also for growth onl-galactose andd-arabinose

Summary l-Galactose,d-arabinose, andl-fucose form six-membered rings with identical stereoconfigurations. However, onlyl-fucose can serve as the sole carbon and energy source of wild-typeEscherichia coli K-12. A mutant that can grow onl-galactose andd-arabinose was isolated by alternate selection on the two sugars. Thel-fucose pathway became inducible by all three sugars. Transduction into the mutant of the wild-type fuc⁺ region containing both the regulatory and structural genes abolished the novel growth abilities onl-galactose andd-arabinose, whereas transduction into the mutant of a fuc deletion abolished the growth abilities on all three sugars. Introduction of the wild-type fucR⁺ (which encodes the activator protein for the fuc regulon) on a multicopy plasmid depressed the growth abilities of the mutant onl-galactose andd-arabinose, but not onl-fucose. The results suggest that the effector specificity of the activator protein in the mutant was broadened. It is proposed that an adaptive response of an activator-controlled system is more likely than that of a repressor-controlled system to achieve fixation in a population, because the first variant to emerge in response to a novel metabolic demand has a good chance of having an altered specificity of regulation. Such a change entails little or no metabolic liability during the absence of the novel substrate. In contrast, the first variant of a negatively controlled system to emerge has an overwhelming chance of being the result of a random mutation that destroys repressor function. Although negatively controlled systems can be more opportunistic in exploiting new conditions than positively controlled systems, an adaptive change is less likely to become fixed because of the cost associated with gratuitous constitutive gene expression in the absence of the substrate.     

Kinetic mechanism of NADP-malic enzyme from maize leaves

The kinetic mechanism of NADP-dependent malic enzyme purified from maize leaves was studied in the physiological direction. Product inhibition and substrate analogues studies with 3 aminopyridine dinucleotide phosphate and tartrate indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism. NADP is the leading substrate followed by l-malate and the products are released in the order of CO₂, pyruvate and NADPH. The enzyme also catalyzes a slow, magnesium-dependent decarboxylation of oxaloacetate and reduction of pyruvate and oxaloacetate in the presence of NADPH to produce l-lactate and l-malate, respectively.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

Adenine nucleotides,substrate utilization and dinitrogen fixation in Rhodobacter capsulatus

Rhodobacter capsulatus strain 37b4 was grown diazotrophically in phototrophic chemostat culture with 30 mM of d,l-malate and 2 mM of ammonium. Illumination was varied at constant dilution rate (D) and vice versa, respectively. When D was raised from 0.035 to 0.165 h^-1 at 30 klx, the steady state cell protein level as well as malate consumption decreased. d-malate was utilized only at D=0.035 h^-1. Specific cellular activities of nitrogenase, as determined by acetylene reduction as well as by dinitrogen (N₂) fixation, increased and approached constancy at D>0.075 h^-1. Specific ATP contents of cells increased with increasing D, while specific ADP and AMP contents exhibited no significant variations. Consequently, energy charge values as well as molar ratios of ATP/ADP (T/D) increased. Raising illumination from 6 to 30 klx at D=0.075 h^-1 resulted in an increase of the steady state protein level as well as of l-malate consumption. d-malate was not utilized under these conditions. Specific nitrogenase activity of cells increased at the lower and levelled off at the higher illuminations. Specific ATP contents of cells stayed constant but specific ADP contents increased with increasing illumination. The energy charge did not vary significantly, while the T/C ratio decreased between 6 and 18 klx and stayed constant at the higher illuminations. The results do not reveal any relationship between nitrogenase activity and the cellular levels or relative proportions of different adenine nucleotides. However, when steady state amounts of fixed N₂ were plotted versus steady state T/D ratios, an inverse proportion became apparent, irrespective of the growth conditions employed. On the other hand, specific nitrogenase activity increased linearly when the rate of malate consumption increased. The results suggest that under steady state conditions the T/D ratio reflects the amount of ATP required to keep the amount of fixed N₂ at a given level, while the rate at which nitrogenase functions depends on the rate at which the carbon and electron source, malate, is utilized by the organisms.     

Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus

The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000 but were distinguished by their different affinities to the two substrates, l-threonine and l-serine.Abbreviations SDH l-serine dehydratase - TDH l-threonine dehydratase     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

Molecular analysis of the phosphoenolpyruvate-dependent l-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure

We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent l-sorbose: phosphotransferase system (PTS). The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found. The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14000 for SorF, 19000 for SorB, 25000 for SorA and 27000 for SorM. SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIA^Sor and EIIB^Sor domains of the l-sorbose PTS. SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops. They correspond to domains EIIC^Sor and EIID^Sor. The four proteins of the l-sorbose PTS resemble closely (27%–60%) the four subunits of a d-fructose PTS (EIIA^Lev, EIIB^Lev, EIIC^Lev, and EIID^Lev) from Bacillus subtilis and the three subunits of the d-mannose PTS (EIIA,B^Man, EIIC^Man, and EIID^Man) from Escherichia coli K-12. The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membranebound proteins. A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate-immunoreactive neurons in the rat enteric nervous system

l-Aspartate (l-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of l-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered l-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many l-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that l-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some l-Asp-immunoreactive neurons might function as interneurons. These results suggest that l-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric l-Asp-immunoreactive neurons provides strong support for the proposal that l-Asp is a neuromodulator in the rat ENS.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Fermentation of 2-methoxyethanol by Acetobacterium malicum sp. nov. and Pelobacter venetianus

Anaerobic bacteria degrading 2-methoxyethanol were enriched from freshwater sediments, and three strains were isolated in pure culture. Two of them were Grampositive non-spore-forming rods and grew strictly anaerobically by acetogenic fermentation. Optimal growth occurred at 30°C, initial pH 7.5–8.0. 2-Methoxyethanol and 2-ethoxyethanol were fermented to acetate and corresponding alcohols. Hydrogen plus carbon dioxide, formate, acetoin, l-malate, lactate, pyruvate, fructose, and methoxyl groups of 3,4,5-trimethoxybenzoate and 3,4,5-trimethoxycinnamate were fermented to acetate. 1,2-Propanediol was fermented to acetate, propionate, and propanol. Strain MuME1 was described as a new species, Actetobacterium malicum. It had a DNA base composition of 44.1 mol% guanine plus cytosine. The third strain, which was identified as Pelobacter venetianus, fermented 2-methoxyethanol to methanol, ethanol, and acetate.     

Introduction of a stress-responsive gene, yggG, enhances the yield of l-phenylalanine with decreased acetic acid production in a recombinant Escherichia coli

A stress-responsive gene, yggG, was introduced into an l-phenylalanine producer, Escherichia coli AJ12741. In shake-flask culture, the yggG-containing recombinant strain (named AJ12741/pHYGG) produced 6.4 g l-phenylalanine l⁻¹ at the end of culture and its yield on glucose was 0.16 g l-phenylalanine g glucose⁻¹. These values are much higher than those of the original AJ12741 strain (3.7 g l-phenylalanine l⁻¹ and 0.09 g l-phenylalanine g glucose⁻¹, respectively). On the other hand, AJ12741/pHYGG strain produced only 4.5 g acetic acid l⁻¹ and its yield on glucose was about a half of that of the AJ12741 culture. Analysis of gene expression revealed that in late growth phase, the expression levels of genes involved in acetic acid production (pta, ackA, and poxB) were relatively low in AJ12741/pHYGG cells. In particular, the level of poxB expression in AJ12741/pHYGG strains was one-seventh of that of the original strain. These results suggest that the formation of a bottleneck for acetic acid production brings about a metabolic flow favorable to l-phenylalanine synthesis in the recombinant strain over-expressing the yggG gene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.