Rerouting the plant phenylpropanoid pathway by expression of a novel bacterial enoyl-CoA hydratase/lyase enzyme function.

The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin). Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol. HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas beta-1,3-glucanase was suppressed. This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools.

Physicochemical characteristics of onion (Allium cepa L.) tissues.

The structure and mechanical properties of onions are important factors affecting their textural quality. The onion bulb consists of several layers of pigmented, papery scales surrounding fleshy storage scales that comprise an upper epidermis, an intermediate parenchyma tissue, and a lower epidermis. The purpose of this study was to examine the chemical composition of cell walls from the papery scales and outer fleshy scales of onion (Allium cepa L. cv. Sturon) in relation to their mechanical properties. Cell-wall material (CWM) was prepared from the component tissues and analyzed for its carbohydrate and phenolic composition. The CWMs were rich in uronic acid and glucose, with smaller quantities of arabinose, galactose, and xylose. In the fleshy scales, the lower epidermis contained relatively more galactose-rich pectic polysaccharides, whereas the upper epidermis and the papery scales contained virtually no galactose. Analysis of mechanical properties showed that the order of strength of the tissues was papery scales > fleshy scales, which were in the order lower epidermis > upper epidermis > intermediate parenchyma. The upper epidermis of fleshy scales was stronger in the vertical than the horizontal direction, and both orientations showed negligible notch sensitivity. Cyclohexane-trans-1,2-diaminetetraacetate-induced vortex-induced cell separation of the intermediate layer of fleshy scales indicated that calcium cross-linking may play an important role in cell-cell adhesion. A small but significant amount of ferulic acid was found in the walls, predominantly in the thick cuticle of the lower epidermis of fleshy scales. Alkali-labile wall-bound flavonoids were also detected.

4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL)--An enzyme of phenylpropanoid chain cleavage from Pseudomonas.

The enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), which catalyzes a hydration and two-carbon cleavage step in the degradation of 4-hydroxycinnamic acids, has been purified and characterized from Pseudomonas fluorescens strain AN103. The enzyme is a homodimer and is active with three closely related substrates, 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA (Km values: 5.2, 1.6, and 2.4 microM, respectively), but not with cinnamoyl-CoA or with sinapinoyl-CoA. The abundance of the enzyme reflects a low catalytic center activity (2.3 molecules s-1 at 30 degrees C; 4-coumaroyl-CoA as substrate).

Post-translational modification of nisin. The involvement of NisB in the dehydration process.

The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.

Isolation and characterisation of the acetyl-CoA carboxylase gene from Aspergillus nidulans.

The putative gene encoding acetyl-CoA carboxylase, accA, has been isolated from Aspergillus nidulans. This single-copy gene has an open reading frame (ORF) of 6864 bp and contains two small introns near the 5'-end. A short ORF upstream of the ATG start codon has been identified in this gene by RT-PCR. Based on sequence homology to acetyl-CoA carboxylases from other organisms, putative biotin-, ATP-, HCO3-- and acetyl-CoA- binding sites have been assigned. Northern data and ACC enzyme-activity measurements from A. nidulans suggested that expression of accA was higher in media containing nitrate than ammonia as a sole nitrogen source. Deletion of accA in A. nidulans was unsuccessful. The failure of A. nidulans to grow in the presence of the ACC-specific inhibitor, soraphen A, supplemented with C16-18 fatty acids suggested that ACC is an essential enzyme.

Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester.

A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens. Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin. The gene was isolated by a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression in Escherichia coli under the control of a T7 RNA polymerase promoter. The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.

Tropane alkaloid production in transformed root cultures of brugmansia Candida.

Transformed root cultures of BRUGMANSIA CANDIDA were established by infection with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones with different growth index and tropane alkaloid pattern and content were obtained and two were examined in depth. The alkaloid content and pattern changed during the time course. At 21 days of culture clone 1 revealed an alkaloid spectrum dominated by 3alpha-acetoxytropane (about 50% of the total alkaloid) and hyoscyamine (about 25%), with a ratio of hyoscyamine to scopolamine of 11.2. In clone 40 this ratio was 1.5 and the content of 3alpha-acetoxytropane was low (2%). The maximum concentrations of hyoscyamine were obtained at 3 weeks of culture, and were 700 and 500 microg/g FW in clone 1 and 40, respectively.

Alternative metabolic fates of hygrine in transformed root cultures of Nicandra physaloides.

Alkaloid production by several Agrobacterium rhizogenes-transformed root lines of Nicandra physaloides was studied. Early in the culture cycle all lines contained predominantly hygrine, which has previously been reported to be the major alkaloid in roots of this plant. A number of other hygrine-derived alkaloids were identified, and these generally increased in importance as the cultures matured. Some lines were found to differ quite markedly in the relative importance of the different hygrine-derived alkaloids. The possibility is raised that alkaloid biosynthesis in N. physaloides root cultures may be a good model system in which to study metabolite partitioning in branched metabolic pathways.

Strategies for the genetic manipulation of alkaloid-producing pathways in plants.

Increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. In the pathways forming indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. Such knowledge contributes the basis upon which it has become feasible to design a strategy by which the flux in these pathways may be enhanced at the genomic level. This review presents a summary of the state-of-the-art pertaining to these pathways and discusses the strategy to be adopted for a molecular approach to their manipulation, together with some of the pitfalls that may arise when trying to alter their natural regulation.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 1. The kinetics of alkaloid production and the influence of feeding intermediate metabolites.

The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 2. On the relative contributions of L-arginine and L-ornithine to the formation of the tropane ring.

The relative contributions made by the L-arginine/agmatine/N-carbamoylputrescine/putrescine and the L-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, DL-α-difluoromethylarginine or DL-α-difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of DL-α-difluoromethylornithine.

Abiotic factors elicit sesquiterpenoid phytoalexin production but not alkaloid production in transformed root cultures of Datura stramonium.

The treatment of root cultures of Datura stramonium with copper and cadmium salts at external concentrations of approximately 1mM has been found to induce the rapid accumulation of high levels of sesquiterpenoid defensive compounds, notably lubimin and 3-hydroxylubimin. These compounds were undetectable in unelicited cultures. No net change was seen in the alkaloid content of the system following treatment with Cu(2+) or Cd(2+), the tropane alkaloid titre apparently being insensitive to elicitation. However, a considerable rapid and, in some instances, reversible release of alkaloid was observed. This resulted in the appearance of up to 50-75% of the total alkaloid in the medium after 40-60 h. Subsequently, in cultures treated with Cu(2+) ions, though not in cultures treated with Cd(2+) ions, this alkaloid was re-absorbed. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors.

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

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.

Alkaloid production by transformed root cultures of Catharanthus roseus.

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05µg/g dry weight.

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.

Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures.

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes.

'Hairy root' cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of 'in vitro' plant tissue culture for the production of valuable plant secondary products is identified and confirmed.

Apparent free space and cell volume estimation: A non-destructive method for assessing the growth and membrane integrity/viability of immobilised plant cells.

Pre-existing methods for measuring cell or organelle volume based on the selective permeability of biological membranes have been modified to make them suitable for determining the intracellular volume of immoblised cells. When a freely permeable substance (e.g. tritiated water) and an impermeable substance ((14)C labelled mannitol is often suitable) are mixed with an immobilised cell culture, the two substances are diluted to different degrees. The extent of the difference allows the total intracellular volume of intact cells to be calculated. This volume is shown to be a useful parameter for assessing cell growth. The application of the method to follow membrane integrity and cell viability is also discussed.

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity.

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5-6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.