Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum.

The content of alpha-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into alpha-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of alpha-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing alpha-aminoadipic acid accumulation. The repression of alpha-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of alpha-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts alpha-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.     

Biosynthesis and stereochemical configuration of N5-(1-carboxyethyl)ornithine. An unusual amino acid produced by Streptococcus lactis

In a recent communication (Thompson, J., Curtis, M. A., and Miller, S.P.F. (1986) J. Bacteriol. 167, 522-529) we described the purification and characterization of N5-(1-carboxyethyl)ornithine from cells of Streptococcus lactis 133. This unusual amino acid has not previously been found in nature. Radiotracer experiments presented here reveal that exogenous [14C]ornithine serves as the precursor for biosynthesis of [14C]arginine, [14C]N5-(1-carboxyethyl)ornithine, and [14C]N5-acetylornithine by cells of S. lactis K1 during growth in a defined medium lacking arginine. In the absence of both arginine and ornithine, cells of S. lactis K1 can also generate intracellular [14C]N5-(1-carboxyethyl)ornithine from exogenous [14C]glutamic acid. Previously we showed that the properties of N5-(1-carboxyethyl)ornithine prepared from S. lactis were identical to one of the two diastereomers [2S, 7S) or (2S, 7R] present in a synthetic preparation of (2S, 7RS)-N5-(1-carboxyethyl)ornithine. The two diastereomers have now been unambiguously synthesized by an Abderhalden-Haase condensation between (2S)-N2-t-butoxycarbonyl-ornithine and the chiral (2S)-, and (2R)-bromopropionates. By 13C-NMR spectroscopy it has been established that the preparation from S. lactis is exclusively (2S, 7S)-N5-(1-carboxyethyl)ornithine. has been demonstrated in a cell-free extract of S. lactis 133. The requirements for ornithine, pyruvic acid, and NAD(P)H suggest that biosynthesis of N5-(1-carboxyethyl)ornithine occurs via a reductive condensation mechanism. A general survey revealed that N5-(1-carboxyethyl)ornithine was produced only by certain strains of Group N streptococci. These findings may indicate a plasmid locus for the gene(s) encoding the enzyme(s) for N5-(1-carboxyethyl)ornithine biosynthesis.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Saccharomyces

Hydroxylysine acts as a growth inhibitor of Saccharomyces for a certain period of time. The inhibition is concentration-dependent and is reversed by a small amount of lysine in the medium. After the growth-inhibitory period, the wild-type cells are able to grow rapidly even in the presence of hydroxylysine. Both lysine auxotrophs and wild-type cells are unable to utilize hydroxylysine in place of lysine. Hydroxylysine, mimicking lysine, controls the biosynthesis of lysine and thereby limits the availability of biosynthetic lysine to the cells. Hydroxylysine affects the biosynthesis of lysine at a number of enzymatic steps. Accumulation of homocitric acid, the first intermediate of lysine biosynthesis, in the mutant strains 19B and A B9 is reduced significantly in the presence of hydroxylysine. Hydroxylysine, like lysine, exerts a significant inhibition in vitro on the homocitric acid-synthesizing activity. Enzymes following the alpha-aminoadipic acid step respond in a noncoordinate fashion to hydroxylysine. Level of the enzyme saccharopine reductase, but not of alpha-aminoadipic acid reductase or saccharopine dehydrogenase, is reduced significantly. These regulatory effects of hydroxylysine are similar to those observed for lysine.     

Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum

Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.     

乙型脑炎患者脑脊液和血浆氨基酸代谢的变化

本文采用气相色谱法对５７例乙型脑炎患者极期和２１例恢复期患者的脑脊液（ＣＳＦ）和血浆１４种游离氨基酸浓度进行分析。结果表明,乙脑极期ＣＳＦ游离氨基酸浓度除苏氨酸、丝氨酸值低于正常值外,其它氨基酸值均增高,其中谷氨酸、鸟氨酸、赖氨酸、缬氨酸、脯氨酸值上升明显（ａｌｌＰ＜０．０１）,至乙脑恢复期不同幅度下降。乙脑极期血浆游离脯氨酸、赖氨酸、苯丙氨酸、酪氨酸、鸟氨酸、谷氨酸浓度高于正常值,而其它８种氨基酸值均下降明显,至乙脑恢复期,多数氨基酸恢复至接近正常水平。实验提示,乙脑病毒能明显影响血浆和ＣＳＦ中氨基酸的含量,而二者具有代谢差异。     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

黄独微型块茎低温离体保存的GC/MS代谢组学分析

GC/MS检测方法采用初步探明黄独低温离体保存微型块茎的差异代谢物。与黄独微型块茎25℃离体保存相比较,黄独微型块茎4℃离体保存的差异性代谢物有丙氨酸（Alanine）、儿茶素（Catechin）、N,N-双（2-羟乙基）甲胺（N,N-Di-（2-Hydroxyethyl）-methanamine）、水杨酸（Salicylic acid）、柠檬酸（Citric acid）和山梨糖（Sorbose）等。在黄独微型块茎4℃离体保存中,丙氨酸（Alanine）参与氰基氨基酸代谢;儿茶素（Catechin）参与次生代谢产物生物合成、黄酮类化合物的生物合成和苯丙素的生物合成;水杨酸（Salicylic acid）参与多环芳烃降解、微生物在不同环境中的代谢、植物激素信号转导、次生代谢产物生物合成、二恶英降解、苯丙氨酸代谢、芳烃降解、植物激素生物合成、铁载体组非核糖体肽合成和苯丙素的生物合成等。柠檬酸（Citric acid）参与来自鸟氨酸、赖氨酸和烟酸的生物碱生物合成、组氨酸和嘌呤的生物碱生物合成、微生物在不同环境中的代谢、植物次生代谢产物的生物合成、2-氧代羧酸代谢、萜类和类固醇的生物合成、原核生物固碳途径、次生代谢产物生物合成、来自莽草酸途径的生物碱生物合成、来自萜类化合物和聚酮的生物碱生物合成、柠檬酸循环（TCA循环）、植物激素生物合成、乙醛酸和二羧酸代谢、双组分系统、苯丙素的生物合成以及来自鸟氨酸,赖氨酸和烟酸的生物碱生物合成等。黄独低温离体保存微型块茎差异代谢物的初步发现为进一步了解其低温离体保存的分子机制奠定了基础,也为低温离体保存黄独微型块茎的破除休眠以及其后续萌发提供了理论依据。     

AMINO ACID AND UREA UPTAKE IN TEN SPECIES OF CHLOROPHYTA1

The Uptake of radioartively-labelled mixed amino acids, arginine, lysine, leucine, glutamic acid and urea was examined in six species of Volvocales and four species of Chlarococcales grown in nitrate-containing medium. Nonradioactive amino acids in excess were used to estimate specificity of amino and carriers in selected cases. All ten species possess salurable (hence, carrier-mediated) systems for uptake of both arginine and urea. In all Volvacales and one Chlorococcales, the arginine-speciftc carrier (which also transported lysine with lower efficiency) was the only amino acid carrier detected. Three species of Chlororoccales appear to possess a separate carrier for lysine and two of these appear to possess at least one additional carrier that is involved in uptake of non-basic amino acids.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.     

arg-13可能参与鸟氨酸在粗糙脉孢霉线粒体的过膜转运(英文)

arg-13为精氨酸代谢途径里的一个渗露型突变。经研究发展了该突变的严格选择方法。该法省略了基本培养基的氮源而加上相似浓度的鸟氨酸与赖氨酸。此法在严紧山梨糖/葡萄糖条件下能强烈抑制arg-13突变株生长,但在斑点试验条件下允许arg-13突变株生长。由于鸟氨酸是通过线粒体合成和由细胞质至线粒体的过膜转运而积累,我们构建了arg-4,arg-13双突变株,其中arg-4阻断了线粒体鸟氨酸合成。在斑点试验条件下,arg-4,arg-13双突变株能利用鸟氨酸作为唯一氮源与精氨酸合成前体,但受赖氨酸与刀豆氨酸强烈抑制。具正常鸟氨酸转运功能的arg-4单突变株在鸟氨酸基本培养基的生长只受微弱的赖氨酸抑制。已有报道arg-13为嘧啶合成代谢途径里pyr-3(CPSACT~ )突变的部分抑制基因,序列分析表明arg-13编码一线粒体转运酶。本文数据提示arg-13在线粒体鸟氨酸过膜转运过程中起主要作用。arg-13突变株仍携带一定的线粒体鸟氨酸转运功能并受碱性氨基酸赖氨酸、刀豆氨酸抑制,可能为另一线粒体碱性氨基酸转运酶介导。     

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.     

arg—13可能参与鸟氨酸在粗糙脉孢霉线粒体的过膜转运

arg-13 is a leaky mutation involved in arginine metabolism. A tight selection is developed using similar amount of lysine and ornithine replacing other nitrogen source in minimal medium. This selection strongly inhibits the growth of arg-13 under stringent sorbose/glucose condition but allows arg-13 to grow under spot test conditions. As ornithine is build up through mitochondrial ornithine biosynthesis and transport from cytoplasm to mitochondria, arg-13 is combined in genetic crosses with arg-4 which blocks mitochondrial ornithine synthesis. Under spot test conditions, double mutant arg-4, arg-13 is able to use ornithine as sole nitrogen source and arginine biosynthesis precursor, but subject to strong lysine and canavanine inhibition. While the usage of ornithine in arg-4 single mutant with intact ornithine transport function is only slightly inhibited by lysine. All available data suggest arg-13 plays a major role in mitochondrial ornithine transport. The strain carrying the mutation at the arg-13 locus allows inefficient mitochondrial ornithine trafficking, possibly mediated by another distinct basic amino acid carrier.     

Distribution of amino acids in bran, embryo and milled endosperm and shifts in storage protein subunits of in vitro-selected and lysine-enhanced mutant and wild type rice

Experiments were designed to test whether an enhanced lysine phenotype from in vitro selections was expressed in milled rice, Oryza sativa L. Analysis of the endosperm tissue after removal of the aleurone and outer layers proved the grains maintained enhanced lysine after milling. Mutant endosperm had 15% greater lysine than the control and the enhanced lysine was correlated with reduced glutamic acid in two different mutants. Glutelins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed increases in the protein quantity of the 33-kDa alpha and 21-kDa beta subunits.     

Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms.

The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences.     

Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis.

Targeted gene insertion methodology was used to study the effect of perturbing alpha-aminoadipic acid precursor flux on the overall production rate of beta-lactam biosynthesis in Streptomyces clavuligerus. A high-copy-number plasmid containing the lysine epsilon-aminotransferase gene (lat) was constructed and used to transform S. clavuligerus. The resulting recombinant strain (LHM100) contained an additional complete copy of lat located adjacent to the corresponding wild-type gene in the chromosome. Biological activity and production levels of beta-lactam antibiotics were two to five times greater than in wild-type S. clavuligerus. Although levels of lysine epsilon-aminotransferase were elevated fourfold in LHM100, the level of ACV synthetase, whose gene is located just downstream of lat, remained unchanged. These data strongly support the notion that direct perturbation of alpha-aminoadipic acid precursor flux resulted in increased antibiotic production. This strategy represents a successful application of metabolic engineering based on theoretical predictions of precursor flux in a secondary metabolic pathway.