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PubMed2006

A gene cluster for prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomyces cinnamonensis DSM 1042.

Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin-a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA-like monooxygenase gene, and two cloQ-like prenyltransferase genes designated fnq26 and fnq28. Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary-metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S-adenosylhomocysteine (SAH) to S-adenosylmethionine (SAM). Two SAM-dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7-O-methylation and fnq27 for the 6-C-methylation reaction in FNQ I biosynthesis.

Base Sequence↗
PubMed1985

Regulation of purine biosynthesis in G1 phase-arrested mammalian cells.

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30-50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.

Animals↗
PubMed1979

Pyrimidine biosynthesis in normal and transformed cells.

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embryo fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.

Animals↗
PubMedDate not supplied

The cerebro-hepato-renal (Zellweger) syndrome: prenatal detection based on impaired biosynthesis of plasmalogens.

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.

Acyltransferases↗
PubMed1998

Lysophosphatidylcholine inhibits cardiolipin biosynthesis in H9c2 cardiac myoblast cells.

The effect of phospholipase A2 treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with Naja mocambique mocambique phospholipase A2 reduced the pool sizes of phosphatidylcholine and phosphatidylethanolamine compared with controls. The pool sizes of lysophosphatidylcholine and lysophosphatidylethanolamine were elevated, whereas the pool sizes of cardiolipin and other phospholipids were unaffected by phospholipase A2 treatment. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or preincubated in the absence or presence of phospholipase A2. In all experiments, radioactivity incorporated into cardiolipin was reduced in phospholipase A2-treated cells with time compared with controls, indicating attenuated de novo biosynthesis of cardiolipin. The mechanism for the reduction in cardiolipin biosynthesis in phospholipase A2-treated cells was a decrease in the activity of phosphatidic acid:cytidine-5'-triphosphate cytidylyltransferase, the rate-limiting enzyme of cardiolipin biosynthesis, mediated by elevated cellular lysophosphatidylcholine levels. The results suggest that de novo cardiolipin biosynthesis in H9c2 cells may be regulated by the cellular level of lysophosphatidylcholine.

Animals↗
PubMed2000

The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent.

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.

Animals↗
PubMed1994

Influence of androgen on the GnRH-stimulated secretion and biosynthesis of gonadotropins in the pituitary of juvenile female bullfrogs, Rana catesbeiana.

The influence of androgen on pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was investigated in juvenile female bullfrogs. Newly metamorphosed bullfrogs were treated in vivo for 7 days or their pituitaries were treated in vitro for 24 hr with 5 alpha-dihydrotestosterone (DHT). Pituitary sensitivity to GnRH was assessed by incubating glands with 100 ng/ml GnRH. The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was quantified by separate radioimmunoassays. Gth biosynthesis was quantified using immunoprecipitation to measure the incorporation of [35S]methionine. Prior exposure to DHT, in vitro or in vivo, significantly elevated the GnRH-induced secretion of LH and FSH. However, DHT had a differential influence on the two gonadotropins; both the basal and the GnRH-induced secretion of LH was enhanced, whereas only the GnRH-induced secretion of FSH was elevated. DHT did not significantly alter Gth biosynthesis. Treating older frogs (5 months postmetamorphic) in vivo with DHT (for 7 days) combined with a GnRH agonist (GnRHa) for 3 days enhanced the GnRH-stimulated Gth biosynthesis compared to treatment with either DHT or GnRHa alone. Therefore, while DHT may act on the pituitary to enhance gonadotropin secretion in response to GnRH, this action does not result in a concomitant increase in Gth biosynthesis. Juvenile female bullfrogs may require an increase in both GnRH and DHT in order to stimulate Gth biosynthesis.

Androgens↗
PubMedDate not supplied

Genetic disorders of cholesterol biosynthesis in mice and humans.

Over the past few years, the number of identified inborn errors of cholesterol biosynthesis has increased significantly. The first inborn error of cholesterol biosynthesis to be characterized, in the mid 1980s, was mevalonic aciduria. In 1993, Irons et al. ( 1 ) (M. Irons, E. R. Elias, G. Salen, G. S. Tint, and A. K. Batta, Lancet 341:1414, 1993) reported that Smith-Lemli-Opitz syndrome, a classic autosomal recessive malformation syndrome, was due to an inborn error of cholesterol biosynthesis. This was the first inborn error of postsqualene cholesterol biosynthesis to be identified, and subsequently additional inborn errors of postsqualene cholesterol biosynthesis have been characterized to various extent. To date, eight inborn errors of cholesterol metabolism have been described in human patients or in mutant mice. The enzymatic steps impaired in these inborn errors of metabolism include mevolonate kinase (mevalonic aciduria as well as hyperimmunoglobulinemia D and periodic fever syndrome), squalene synthase (Ss-/- mouse), 3beta-hydroxysteroid Delta14-reductase (hydrops-ectopic calcification-moth-eaten skeletal dysplasia), 3beta-hydroxysteroid dehydrogenase (CHILD syndrome, bare patches mouse, and striated mouse), 3beta-hydroxysteroid Delta8,Delta7-isomerase (X-linked dominant chondrodysplasia punctata type 2, CHILD syndrome, and tattered mouse), 3beta-hydroxysteroid Delta24-reductase (desmosterolosis) and 3beta-hydroxysteroid Delta7-reductase (RSH/Smith-Lemli-Opitz syndrome and Dhcr7-/- mouse). Identification of the genetic and biochemical defects which give rise to these syndromes has provided the first step in understanding the pathophysiological processes which underlie these malformation syndromes.

Animals↗
PubMed1998

Clorgyline effect on pineal melatonin biosynthesis in rats with lesioned suprachiasmatic nuclei.

We have reported that clorgyline-induced stimulation of pineal melatonin biosynthesis could be augmented by the exposure to 24h of constant light in young but not in aged rats. Aging is associated with the declined integrity of the suprachiasmatic nuclei (SCN), the major station of the light signal passage from the retina to the pineal gland. The present study aimed to investigate whether SCN integrity is essential for clorgyline effects on pineal melatonin biosynthesis in light-primed rats. Clorgyline (2.5 mg/kg, s.c.) was administered to sham-operated and SCN-lesioned Sprague-Dawley rats kept under regular light/dark cycle or exposed to 24h of constant light. Pineal melatonin and related indoles were evaluated by HPLC-fluorimetric procedure. Clorgyline stimulated pineal melatonin biosynthesis in both SCN-lesioned and sham-operated rats kept under regular light/dark cycle. Exposure to constant light (for 24h) augmented clorgyline-induced stimulation of melatonin biosynthesis in sham-operated rats, but not in SCN-lesioned animals. The obtained results suggest that decline in SCN activity (e.g., age-associated) might contribute to previously reported attenuation of clorgyline-induced stimulation of melatonin biosynthesis in light-primed aged rats.

Animals↗
PubMed2004

Regulation of cephalosporin biosynthesis.

The filamentous fungus Acremonium chrysogenum is the natural producer of the beta-lactam antibiotic cephalosporin C and is as such used worldwide in major biotechnical applications. Albeit its profound industrial importance, there is still a limited understanding about the molecular mechanisms regulating cephalosporin biosynthesis in this fungus. This review focuses on various regulatory levels of cephalosporin biosynthesis. In addition to precursor and antibiotic biosynthesis, molecular genetic characteristics of cephalosporin biosynthesis genes and the knowledge of multiple layers of their regulatory expressional control, as well as the function of activators or repressors on cephalosporin biosynthesis are jointly being surveyed. Furthermore, this review summarizes (i) molecular features, which distinguish strains with different production levels and (ii) examples of molecular engineering approaches to A. chrysogenum.

Acremonium↗
PubMed1993

Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis.

The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 microM). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11 beta-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1% of control [50% inhibitory concentration (IC50) 0.03 microM] while ketoconazole suppressed 11 beta-hydroxylase to only 39% of control activity (IC50 15 microM). Ketoconazole however, most potently blocked the conversion of 17 alpha-hydroxy-progesterone to androstenedione by C17,20-desmolase to about 15% of control activity (IC50 1 microM) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 microM. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17 alpha-hydroxylase (IC50 6-18 microM) and 16 alpha-hydroxylase (IC50 4-8 microM) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11 beta-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.

Adrenal Glands↗
PubMed1992

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart.

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations (< or = 0.1 microM), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine (> or = 0.4 microM) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine. The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine.

Alanine↗
PubMed1996

The effects of lidocaine and hypoxia on phospholipid biosynthesis in the isolated hamster heart.

In this study, the effects of lidocaine and hypoxia on the biosynthesis of phospholipids in the hamster heart were examined. Hamster hearts were perfused with [1,3-3H]glycerol under normal and hypoxic conditions, and in the absence or presence of 0.5 mg/mL lidocaine. After perfusion, the radioactivity incorporated into the various phospholipid fractions was determined. With the exception of phosphatidylcholine, the synthesis of phospholipids was generally stimulated by lidocaine perfusion. In contrast, hypoxia caused a general decrease in phospholipid biosynthesis which was partially restored by lidocaine. ATP and CTP levels were severely reduced under hypoxic conditions, but their levels were not restored by lidocaine treatment. The activities of enzymes for phospholipid synthesis were determined under the various perfusion conditions. The activity of phosphatidic acid phosphatase was elevated by lidocaine and decreased by hypoxic treatment. The activity of CTP:phosphatidic acid cytidylyltransferase was increased under hypoxia, with or without lidocaine. Despite the reduction in phosphatidylcholine biosynthesis, no change in the activity of cytidine diphosphocholine (CDPcholine):diacylglycerol cholinephosphotransferase was detected following lidocaine or hypoxic perfusion. However, enzyme activity was inhibited by the presence of lidocaine in the assay mixture. Our results indicate that the reduction in phospholipid biosynthesis under hypoxic conditions was caused mainly by diminishing high-energy nucleotide levels. The enhancement of phospholipid biosynthesis by lidocaine appeared to be mediated in part by modulation of enzyme activities.

Animals↗
PubMed1995

Biosynthesis and distribution of insect-molting hormones in plants--a review.

Insect-molting hormones, phytoecdysteroids, have been reported to occur in over 100 plant families. Plants, unlike insects, are capable of the biosynthesis of ecdysteroids from mevalonic acid, and in several cases the biosynthesis of phytoecdysteroids was also demonstrated to proceed via sterols. Spinacia oleracea (spinach) biosynthesizes polypodine B and 20-hydroxyecdysone, which is the predominant insect-molting hormone found in plant species. The onset of ecdysteroid production in spinach requires the appropriate ontogenetic development within the plant, which is related to leaf development. In spinach, lathosterol is the biosynthetic precursor to ecdysone and 20-hydroxyecdysone. Phosphorylated ecdysteroid intermediates, particularly ecdysone-3-phosphate, are required during biosynthesis. Polyphosphorylated forms of ecdysteroids are putative regulatory components of the pathway. During spinach development, the 20-hydroxyecdysone is transported from the sites of biosynthesis to the apical regions. An analysis of the physiological data available suggests that different species may synthesize ecdysteroids in various organs and distribute these ecdysteroids to other sites. Annual plants appear to concentrate ecdysteroids in the apical regions, including flowers and seeds. Perennial plants may recycle their ecdysteroids between their deciduous and their perennial organs over the growing season. Further investigations of ecdysteroid biosynthesis and physiology within plants will be required before an acceptable system can be designed to test phytoecdysteroid effectiveness in vivo against insect herbivory.

Ecdysteroids↗
PubMed1989

Effects of arachidonic acid concentration on prostaglandin biosynthesis and feasibility of semibatch processes.

The reaction characteristics of prostaglandin E2 biosynthesis by PGH-synthase and PGE2 isomerase and the substrate dependency of this biosynthesis were studied. The activity of PG-synthases was blocked by the inhibitory action of one or more byproducts, probably resulting from the action of PGH-synthase. This inhibitory action then appeared to be partly reversible, indicating that the substrate and the inhibitor compete for the catalytic sites. According to these findings, the feasibility of a successful semibatch biosynthesis was investigated. A combination of the substrate concentration reducing procedure and the semibatch process resulted in an about 3.5-fold higher increase in the total amount of PGE2 formed in comparison with the batch results obtained at the substrate concentration of 1.0 mg/cm3. Since the cost of enzyme is a governing factor in this biosynthesis, development of semibatch biosynthesis of PGE2 becomes a matter of economic importance.

Animals↗
PubMed1997

Prospects for the bioengineering of isoprenoid biosynthesis.

Over the last decade, our understanding of isoprenoid biosynthesis has progressed to the stage where specific strategies for the bioengineering of essential oil production can be considered. This review provides a current overview of the enzymology and regulation of essential oil isoprenoid biosynthesis. The reaction mechanisms of the synthases which produce many of the basic isoprenoid skeletons are described in detail. Coverage is also provided of the regulation of isoprenoid biosynthesis, including the roles played by tissue and subcellular compartmentation, and by partitioning of intermediates between different branches of isoprenoid metabolism. This provides necessary context for rationally targeting specific enzymes of metabolic pathways for bioengineering essential oil production. Wherever possible, emphasis is placed on research specific to essential oil isoprenoid biosynthesis, although relevant work related to other isoprenoids is also considered when it can provide useful insights. Finally, building upon this understanding of essential oil isoprenoid biosynthesis, several approaches to the bioengineering of isoprenoid metabolism are considered.

Dimethylallyltranstransferase↗
PubMed2003

Isoprenoid biosynthesis in hereditary periodic fever syndromes and inflammation.

Mevalonate kinase (MK) is an essential enzyme in the isoprenoid biosynthesis pathway which produces numerous biomolecules (isoprenoids) involved in a variety of cellular processes. The indispensability of MK and isoprenoid biosynthesis for human health is demonstrated by the identification of its deficiency as the biochemical and molecular cause of the inherited autoinflammatory disorders mevalonic aciduria and hyperimmunoglobulinemia D and periodic fever syndrome. Since the discovery of the genetic defect, considerable progress has been made in understanding the molecular, biochemical and immunological basis of MK deficiency. Important questions such as which specific protein(s) and/or signaling pathway(s) are affected, however, remain unanswered. Resolving the complete pathophysiology of this disorder is a major challenge, but eventually will give insight into the in vivo role of MK and isoprenoid biosynthesis in inflammation and fever. This may open novel options for antiinflammatory therapies in general. Here, we give a general introduction on isoprenoid biosynthesis, the regulation thereof and deficiencies therein. We review the molecular, biochemical and immunological aspects of MK deficiency and discuss the relations between isoprenoid biosynthesis and inflammation. Finally, we compare MK deficiency with other autoinflammatory syndromes.

Cholesterol↗
PubMed2005

(-)-Menthol biosynthesis and molecular genetics.

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

Genetic Engineering↗