Nicholas J. Walton

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida × D. aurea hybrid.

Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ℴ flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.

Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica

Abstract Cadaverine (1–10 mM) stimulated the production of anabasine by hairy root cultures of Nicotiana rustica transformed with Agrobacterium rhizogenes . In control cultures, nicotine accounted for at least 70–80% of the total alkaloid produced, whereas in cultures supplemented with 5 mM cadaverine about two-thirds of the alkaloid was anabasine and nicotine production was markedly diminished. Putrescine and agmatine caused some stimulation of alkaloid production, but the ratio of nicotine to anabasine was essentially unaffected. Lysine caused no substantial increase in anabasine formation.

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna.

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine δ-Nmethyltransferase and δ-N-methylornithine decafboxylase were undetectable, indicating that δ-N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-14C]ornithine, l-[U-14C]arginine, [U-14C]agmaine and [1,4-14C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl-α-difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl-α-difluoromethylornithine. Together with the demonstration that label was incorporated from [U-14C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-14C] ornithine and l-[U-14C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.

Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave Km values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and Ki values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a Ki of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.

The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures of Nicotiana hesperis

Unlabelled cadaverine did not diminish the incorporation into anabasine of 14C from L-[U-14C] lysine supplied to hairy root cultures of Nicotiana nesperis, despite causing a stimulation of anabasine production. The finding is discussed in the context of previous observations indicating that free cadaverine is not an intermediate in the biosynthesis of anabasine from lysine.

Diamine oxidation and alkaloid production in transformed root cultures of Nicotiana tabacum

Abstract Cadaverine, putrescine and N -methylputrescine were all oxidized by extracts of transformed roots of Nicotiana tabacum , the apparent K m values being, respectively, 2.75, 0.35 and 0.08 mM and the relative maximal oxidation rates, 100, 48 and 34%. For the same substrates, partially purified pea seedling diamine oxidase (DAO, EC 1.4.3.6) gave apparent K m values of 0.026, 0.040 and 0.046 mM, with relative maximal activities of 100, 65 and 65% respectively. For the Nicotiana extract, with equimolar substrate concentrations (1 and 0.1 mM), N -methylputrescine inhibited the oxidation of both cadaverine and putrescine, whereas the converse inhibitions did not occur. N -Methylputrescine (1 mM) caused only a modest stimulation of nicotine and nornicotine production when fed to transformed root cultures of N. tabacum , which was inhibited by equimolar cadaverine. Conversely, N -methylputrescine diminished the stimulation of anabasine production by cadaverine.

Tryptophan decarboxylase strictosidine synthase and alkaloid production by Cinchona ledgeriana suspension cultures

Abstract The activities of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) have been compared with the production of quinoline alkaloids in light-grown and dark-grown suspension cultures of Cinchona ledgeriana transformed with Agrobacterium tumefaciens . Enzyme activities and alkaloid production were both substantially greater in the dark-grown cultures than in the light-grown cultures. Under the conditions of assay, SS was in all cases at least 80 times more active than TDC and did not vary very substantially over a 22-day culture period. In contrast, TDC activity in the dark-grown cells rose rapidly to a maximum after 13 days and then declined. Total alkaloid production was largely growth-related, but the cellular alkaloid content declined sharply (on account of release into the medium) within the first 4 days after subculture, prior to the rise in TDC activity. TDC activity over the 22-day period was only 2-to 3-fold greater than that required to account for alkaloid production and is therefore potentially rate-limiting under in vivo conditions.

Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase

BackgroundThe aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells).ResultsA genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells.Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells. Numerous genes encoding transporters for carbohydrates and organic alcohols and acids were down-regulated in old cells only.ConclusionNetwork analysis revealed very different transcriptional activities during the lag period in old and young cells. Rejuvenation seems to take place during exponential growth by replicative dilution of old cellular components.

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.

Effects of illumination and glycollate oxidation in promoting glyoxylate decarboxylation by pea leaf protoplasts

During glycollate oxidation, glyoxylate was decarboxylated by pea leaf protoplasts. The characteristics of the reaction were similar to those of the reaction in leaf extracts (Walton, 1982, Planta 155, 218–224). Glyoxylate decarboxylation was not promoted by illumination of the protoplasts.