Investigation of enzymatic quality and quantity using pyridoxal 5'-phosphate (PLP) regeneration system as a decoy in Escherichia coli.

Pyridoxal 5'-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V).

Cultures of Shewanella putrefaciens grown in medium containing 10mM 1,4-diamino-2-butanone (DBO) as an inhibitor of ornithine decarboxylase and 10mM 1,5-diaminopentane (cadaverine) showed the simultaneous biosynthesis of the macrocyclic dihydroxamic acids: putrebactin (pbH2), avaroferrin (avH2) and bisucaberin (bsH2). The level of DBO did not completely repress the production of endogenous 1,4-diaminobutane (putrescine) as the native diamine substrate of pbH2. The relative concentration of pbH2:avH2:bsH2 was 1:2:1, which correlated with the substrate selection of putrescine:cadaverine in a ratio of 1:1. The macrocycles were characterised using LC-MS as free ligands and as 1:1 complexes with Fe(III) of the form [Fe(pb)]+, [Fe(av)]+ or [Fe(bs)]+, with labile ancillary ligands in six-coordinate complexes displaced during ESI-MS acquisition; or with Mo(VI) of the form [Mo(O)2(pb)], [Mo(O)2(av)] or [Mo(O)2(bs)]. Chromium(V) complexes of the form [CrO(pb)]+ were detected from solutions of Cr(VI) and pbH2 in DMF using X-band EPR spectroscopy. Supplementation of S. putrefaciens medium with DBO and 1,3-diaminopropane, 1,6-diaminohexane or 1,4-diamino-2(Z)-butene (Z-DBE) resulted only in the biosynthesis of pbH2. The work has identified a native system for the simultaneous biosynthesis of a suite of three macrocyclic dihydroxamic acid siderophores and highlights both the utility of precursor-directed biosynthesis for expanding the structural diversity of siderophores, and the breadth of their coordination chemistry.

Polyamine transporters and polyamines increase furfural tolerance during xylose fermentation with ethanologenic Escherichia coli strain LY180.

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.

Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC-MS, LC-MS and 1D NMR techniques.

Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and possesses anticancer and antiviral activities. This species contains a plethora of phytochemicals including terpenoids, saponins, flavonoids, polyamines and polysaccharides. The full complement of bioactive compounds has yet to be elucidated, a step necessary in order to explain its medicinal use. There are over 30 species in the Glycyrrhiza genus world-wide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Here, large scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Glycyrrhiza species chemical composition. UV, MS and NMR spectra of extracted components were connected with NMR, MS, and multivariate analyses data from Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza echinata. Major peaks in (1)H NMR and MS spectra contributing to the discrimination among species were assigned as those of glycyrrhizin, 4-hydroxyphenyl acetic acid, and glycosidic conjugates of liquiritigenin/isoliquiritigenin. Primary metabolites profiling using GC-MS revealed the presence of cadaverine, an amino acid, exclusively found in G. inflata roots. Both LC-MS and NMR were found effective techniques in sample classification based on genetic and or geographical origin as revealed from derived PCA analysis.

The impact of Cu treatment on phenolic and polyamine levels in plant material regenerated from embryos obtained in anther culture of carrot.

The influence of copper sulphate on the regeneration of carrot (Daucus carota L.) androgenic embryos and changes in the levels of phenolic substances and polyamines that might be indicative of the response to oxidative stress were investigated. The cultivation on the regeneration medium supplemented with Cu(2+) at the concentrations 1 and 10 microM for 15 weeks resulted in significant dose-dependent inhibition of the growth and organogenic ability of carrot embryos. The total content of phenolic acids (represented by the sum of all soluble and insoluble fractions) in the Cu(2+)-treated carrot cultures did not change in comparison with the control (0.1 microM Cu(2+)). However, the levels of phenolic acids in the individual fractions showed significant differences. The cultivation in the presence of increased Cu(2+) evoked first of all the rise of free chlorogenic and caffeic acids, and the increase in soluble ester-bound ferulic acid. Marked dose-dependent decline in the amount of ferulic acid incorporated into the cell walls of the Cu(2+)-treated carrot cultures was partly compensated by the increase in the content of p-hydroxybenzoic acid. Decline in the total polyamine contents in the carrot tissues cultivated in the presence of increased Cu(2+) concentrations was observed. The most abundant polyamine, both in a free and PCA-soluble conjugated forms, was putrescine, the least abundant was spermine, which occurred in free form only. While the levels of free polyamines slightly decreased in a dose-dependent manner in the Cu(2+)-treated cultures, those of PCA-soluble conjugates markedly rose (enhancement to 135 and 170% in 1 and 10 microM Cu(2+), respectively, compared with the control). The decline in the total polyamine contents was caused mainly by the decline in the levels of PCA-insoluble conjugates. The decrease observed in this fraction was approximately to 70 and 50% in 1 and 10 microM Cu(2+)-treated cultures, respectively, when compared with the control. The role of phenolic acids and polyamines in preventing Cu(2+)stress in the carrot tissues is discussed.

A rapid transglutaminase assay for high-throughput screening applications.

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca(2+)-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The K(m) of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 muM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

Molecular cloning and characterization of a maize transglutaminase complementary DNA.

Two related complementary DNA clones, TGZ15 and TGZ21, encoding active maize transglutaminase (TGase) have been isolated for the first time in plants by molecular cloning (Patent Pending PCT/ES03/00247). Southern and northern blot analyses indicate that the two cDNAs probably corresponded to two different single-copy genes in the maize genome. Northern blot analyses revealed that the transcript is expressed preferentially in young leaves and differentiated embryogenic maize callus. This expression is dependent on light exposure time. TGase activity of the proteins encoded by clones TGZ15 and TGZ21 was detected in bacterial extracts overexpressing them, using two enzymatic assays. TGase activity was significantly higher than that of the empty-phagemid bacterial extracts. As in other TGases, this activity was inhibited by monodansyl cadaverine (MDC), GTP and the absence of exogenous Ca(2+). Likewise, light-stimulated Ca(2+)-dependent TGase activity was detected in thylakoids and grana of maize chloroplast, which was inhibited by MDC, GTP, DIECA and Diuron.

Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements.

A number of studies show that environmental stress conditions such as drought, high salt, and air pollutants increase polyamine levels in plant cells. However, little is understood about the physiological function of elevated polyamine levels. We report here that polyamines regulate the voltage-dependent inward K(+) channel in the plasma membrane of guard cells and modulate stomatal aperture, a plant "sensor" to environmental changes. All natural polyamines, including spermidine, spermine, cadaverine, and putrescine, strongly inhibited opening and induced closure of stomata. Whole-cell patch-clamp analysis showed that intracellular application of polyamines inhibited the inward K(+) current across the plasma membrane of guard cells. Single-channel recording analysis indicated that polyamine regulation of the K(+) channel requires unknown cytoplasmic factors. In an effort to identify the target channel at the molecular level, we found that spermidine inhibited the inward K(+) current carried by KAT1 channel that was functionally expressed in a plant cell model. These findings suggest that polyamines target KAT1-like inward K(+) channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.

Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine.

Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acetyl-spermidine, compared with those of exported putrescine, were observed to accumulate in the culture medium. A careful analysis of the changes in the intracellular concentration of putrescine relative to the steady-state net rate of putrescine export, as the doubling time of the cultures increased from 16 h to 22 h, indicated that an inverse relationship existed between these two parameters. As the intracellular putrescine concentrations increased, the net rate of putrescine export decreased markedly. Determination of the rate of putrescine uptake indicated that putrescine uptake also decreased significantly as the cultures neared confluency, and at no time during the growth of the culture did the rate of putrescine uptake approximate to the high rate of putrescine efflux. The decrease in the putrescine export rate seen as the cells grew toward confluency was determined to be primarily due to the inhibitory effect of the effluxed putrescine in the medium (Ki = 2 microM), and not to contact inhibition. The data suggested that the efflux of putrescine and cadaverine is not mediated to a significant degree by a process involving simple diffusion.

Biogenic amines in silage. 2. The dynamics of the formation of biogenic amines in silage.

When analysing a series of laboratory silages made from orchardgrass, red clover and oats, the fluctuating dynamics of biogenic amines were observed. For levels of putrescine and cadaverine, a rapid exponential increase culminating approximately 30-50 days after ensiling is typical. A small decrease, reaching the minimum at approximately the 100th day, is sometimes followed by a second increase in amine concentrations achieving its maximum approximately 200-230 days after ensiling. Irregular curves of the dynamics probably originate in the simultaneous decarboxylation and deamination reactions along with other relevant amine degradation processes. The dynamics of other biogenic amines-spermidine, spermine and histamine are more difficult to predict. The changes in histamine levels resembled those in the diamines. Some 200 days after ensiling, considerable increases in this toxic amine were observed. The dynamics of some quality criteria, especially the degree of proteolysis, were in many cases similar to those of the amines.

Competition of homologous substrates, putrescine and cadaverine, in the reaction catalyzed by pea diamine oxidase.

The determination of diamine oxidase activity with the ninhydrin reagent was used for monitoring of simultaneous oxidation of two homologous substrates, putrescine and cadaverine, which give different colour products (519 and 417 nm). We measured the reaction rates of oxidation of both substrates in different proportion and compared them with the total reaction rate determined by the guaiacol method. The substrates show competition with inhibition constants of putrescine against cadaverine of 0.14 mmol.l-1 and cadaverine against putrescine of 6.4 mumol.l-1.

Conversion of orally administered 2-n.pentylaminoacetamide into glycinamide and glycine in the rat brain.

Male Sprague Dawley albino rats were treated orally with 2-n.pentylaminoacetamide (10 to 100 mg/kg b.wt). This oral administration provoked a dose-related and time-dependent accumulation of glycinamide in forebrain, cerebellum, and medulla, and to increased levels of glycine in the three brain areas, and of serine in medulla. In kidney, liver and plasma, the accumulation of glycinamide was lower and there was no increase in glycine and serine levels. With a dose of 100 mg/kg b.wt, 28% of the drug were eliminated unchanged and 16% as glycinamide, in urines collected for 24 h. In all tissues examined, 2-n.pentylaminoacetamide and glycinamide levels peaked at 1 h and were nil again after 24 h, the ratio of 2-n.pentylaminoacetamide over glycinamide decreasing more rapidly in brain than in kidney and liver. Contrasting with the effects of 2-n.pentylaminoacetamide, the oral administration of glycinamide (66 mg/b.wt) led, 2 hours later, to similar low rises of glycinamide in plasma and brain. In another control experiment, the intraperitoneal injection of a large dose of glycine (450 mg/kg b.wt) provoked, 30 min later, modest rises of glycine levels in the central nervous system that merely reflected a contamination by plasma glycine.

Cadaverine in the rat brain: regional distribution and acylation of [14C]cadaverine in vivo and uptake in vitro.

The regional distribution and acylation of intraventricularly injected [14C]cadaverine was studied in the rat brain over a 48-h period. The concentrations of labeled cadaverine and its acyl derivatives, N-monoacetylcadaverine and N-monopropionylcadaverine, were determined in the telencephalon, striatum, hypothalamus, midbrain, cerebellum, and medulla-pons by TLC of their 5-dimethylamino-1-naphthalenesulfonyl derivatives, followed by liquid scintillation spectrometry. The apparent passage of radioactivity from the ventricular space into brain tissue was slow, with the concentrations reaching a peak at 24 h after injection. The percentage of radioactivity in the acyl forms of cadaverine, however, was maximal 4 h after injection, with the propionyl form predominating. The telencephalon, striatum, and hypothalamus contained the highest concentrations of radioactivity, in all three forms, at all elapsed times. A high-affinity uptake mechanism for cadaverine was demonstrated in slices of these tissues. This process was completely inhibited by equimolar concentrations of unlabeled putrescine.