Incorporation of Shikimic Acid into Corynecins and Its Regulation

In the biosynthesis of corynecins by Corynebacterium hydrocarboclastus, it appeared that shikimic acid was one of the efficient precursors, where shikimic acid-U-¹⁴C was incorporated into corynecins in the yield of approximately 15%. Analyses of degradation products of labeled corynecins demonstrated that shikimic acid was incorporated specifically into aromatic ring of corynecins.

The incorporation of shikimic acid was inhibited by several aromatic amines such as p-aminophenylserinol-N-propionamide, although the uptake of shikimic acid was not affected, suggesting that biosynthesis of corynecins might be regulated by p-aminophenyl intermediates. Furthermore, p-ammophenylethylalcohol was found to be a potent inhibitor of biosynthesis of corynecins. In contrast, corynecins and other p-nitro-phenyl derivatives, aromatic amino acids and vitamins related to shikimic acid pathway did not inhibit the biosynthesis of corynecins from shikimic acid.     

Transport and utilizing of the biosynthetic intermediate shikimic acid in Escherichia coli

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poort growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced.Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport.Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% contransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not contransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that bot classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (K_m) for shikimate transport.     

The shikimic acid pathway and polyketide biosynthesis

The shikimic acid pathway, ubiquitous in microorganisms and plants, provides precursors for the biosynthesis of primary metabolites such as the aromatic amino acids and folic acid. Several branchpoints from the primary metabolic pathway also provide aromatic and, in some unusual cases, nonaromatic precursors for the biosynthesis of secondary metabolites. We report herein recent progress in the analysis of two unusual branches of the shikimic acid pathway in streptomycetes; the formation of the cyclohexanecarboxylic acid (CHC)-derived moiety of the antifungal agent ansatrienin and the dihydroxycyclohexanecarboxylic acid (DHCHC) starter unit for the biosynthesis of the immunosuppressant ascomycin. A gene for 1-cyclohexenylcarbonyl-CoA reductase, chcA, which plays a role in catalyzing three of the reductive steps leading from shikimic acid to CHC has been characterized from Streptomyces collinus. A cluster of six open reading frames (ORFs) has been identified by sequencing in both directions from chcA and the putative role of these in CHC biosynthesis is discussed. The individual steps involved in the biosynthesis of DHCHC from shikimic acid in Streptomyces hygroscopicus var ascomyceticus has been delineated and shown to be stereochemically and enzymatically distinct from the CHC pathway. A dehydroquinate dehydratase gene (dhq) likely involved in providing shikimic acid for both DHCHC biosynthesis and primary metabolism has been cloned, sequenced and characterized. Received 17 February 1998/ Accepted in revised form 26 April 1998     

Aromatic amino acid biosynthesis in Trichophyton rubrum III. Exogenous studies: Absence of the shikimic acid pathway

Even thoughTrichophyton rubrum is permeable to exogenous shikimic acid, neither shikimic nor quinic acids stimulate the growth of this fungus in a minimal medium deficient in phenylalanine or tyrosine, nor do they serve as substrates for pigmentogenesis in media lacking these amino acids. The respiration of the dermatophyte is unaffected by shikimic or quinic acids and the fungus does not have the capacity to utilize either compound when it is added to the culture medium. Isotope dilution studies with shikimic acid-U-C¹⁴ show that de novo shikimic acid synthesis does not occur. This information supports previous findings that the shikimic acid pathway of aromatic biosynthesis is not involved in the biosynthesis of phenylalanine byTrichophyton rubrum.

---

Zusammenfassung ObwohlT. rubrum fur exogene shikimicsäure durchlässig ist, fördern weder Shikimicsäure noch Quinicsäure das Wachstum dieses Pilzes im Falle eines Mangels von Phenykalanine oder Tyrosine, noch dienen sie als Substanzen für Pigmentgenese in Medien ohne diese Aminosäuren. Die Atmung des Pilzes ist durch Shikimicoder Quinicsäure unbeeinflußt und der Pilz ist unfähig, beide Substanzen zu benützen, wenn sie zum Kulturmedium hinzugefügt werden. Isotope Verdünnungen mit Shikimicsäure-U-C¹⁴ zeigten, daß de novo Shikimicsäure-Synthese nicht erfolgt. Diese Erkenntnis unterstüzt vorherige Befunde, daß Shikimicsäure Richtung der aromatischen Biosynthese in der Biosynthese von Phenylalanine durchT. rubrum nicht begangen wird.

---

---

University of Illinois at the Medical Center Department of Microbiology, Chicago, Illinois 60612     

The cyclohexane moiety of rapamycin is derived from shikimic acid inStreptomyces hygroscopicus

Summary Although the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,¹⁴C-shikimic acid was incorporated into rapamycin to a very high degree.¹³C-Shikimic acid was successfully prepared from 1-[¹³C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that¹³C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.     

Insensitivity of 5-enolpyruvylshikimic acid-3-phosphate synthase to glyphosate confers resistance to this herbicide in a strain of Aerobacter aerogenes

The broadspectrum herbicide glyphosate (N-[phosphonomethyl]glycine), an inhibitor of the shikimate pathway enzyme 5-enolpyruvyl-shikimic acid-3-phosphate (EPSP)-synthase, inhibits the growth of Aerobacter aerogenes and causes the excretion of shikimic acid-3-phosphate. A strain of A. aerogenes, resistant to inhibition of growth by glyphosate, was isolated and found to have a glyphosate-insensitive EPSP-synthase and to no longer excrete shikimic acid-3-phosphate in the presence of glyphosate. Partial identity of EPSP-synthases from the glyphosate-sensitive and-resistant A. aerogenes strains was demonstrated by immunological procedures.Abbreviation EPSP-synthase 5-enolpyruvylshikimic acid-3-phosphate synthase (EC 2.5.1.19; 3-phosphoshikimate 1-carboxyvinyltransferase)     

Enzymes of Trichophyton rubrum

Jensen, Altschuler &Bard (1957) have surveyed the respiratory enzymes ofTrichophyton mentagrophytes. Enzymes of the hexosediphosphate, hexosemonophosphate, and tricarboxylic acid pathways were found plus a group of miscellaneous enzymes. In view of the close morphological, immunological, and nutritional similarities ofT. mentagrophytes toTrichophyton rubrum, we felt that a study of the enzymology of the latter organism had potential value in forming a basis for the comparison of the two dermatophytes. Our results showed that cell-free extracts ofT. rubrum contain glucokinase, TPN-dependent glucose and fructose dehydrogenases and separate TPN- and DPN-linked glycerol dehydrogenases. These results are in accord with findings of other investigators. ATPase activity was minimal, and shikimic acid kinase, and shikimic and quinic acid dehydrohydrogenases were not detected.     

A transketolase mutant of Corynebacterium glutamicum

A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected. Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 1998     

Zucker,Cyclite und organische Säuren des Cambialsaftes von Pinus silvestris L., Picea Abies Karst. und Abies alba Mill

Summary Samples of cambial sap from each of the three coniferous species Pinus sylvestris L., Picea Abies Karst. and Abies alba Mill. were taken at the time the trees were coming into bud and analysed for low molecular weight carbohydrates, cyclitols and organic acids. They all contained the same free sugars and cyclitols, but in markedly different proportions. Quantitative analyses were carried out for glucose, fructose, sucrose, raffinose, myo-inositol, D-inositol, pinitol, sequoyitol and coniferin.The three main components of the organic acid fractions-quinic acid, shikimic acid and malic acid—were determined quantitatively. The amount of quinic acid greatly exceeded the amount of all the other acids in all three species. ¹⁴C-labelled quinic acid applied to the cut ends of Pinus sylvestris needles was transported to the twig. There was no conversion of quinic acid to shikimic acid over short periods of time.     

4-O-Caffeoylshikimic and 4-O-(p-Coumaroyl)shikimic Acids from the Dwarf Tree Fern,Dicksonia antarctica

Two derivatives of shikimic acid were isolated from croziers of the dwarf tree fern, Dicksonia antarctica, and their structures were elucidated as 4-O-caffeoylshikimic acid and 4-O-(p-coumaroyl)-shikimic acid on the basis of mass spectrometric and NMR spectroscopic evidence.     

3-Dehydroshikimate dehydratase in mung bean cultured cells

Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg²⁺ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase     

Studies on the Biosynthesis of Lignin

We have been further studying on the specific lignification in the gourd fruits as shown in the previous paper. During the process of lignification, the both activities of peroxidase and β-glucosidase were decreased, and so were shikimic acid, while some organic acids including quinic acid were detected by paper chromatography.     

Studies on the production of shikimic acid using the <Emphasis Type="Italic">aroK</Emphasis> knockout strain of <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu^®) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.     

Metabolism of shikimic,quinic, and cyclohexanecarboxylic acids in germfree,conventional, and gnotobiotic rats

By using a new high-pressure liquid chromatography assay, the increase in urinary hipprate following ingestion of shikimic, quinic, and cyclohexanecarboxylic acid was studied to quantitate the extent of aromatization in germfree, gnotobiotic, and converitonal rats. Germfree rats aromatized 2% of a single dose of shikimic acid or quinic acid and 44% of cyclohexanecarboxylic acid. Conventional rats aromatized all three compounds; shikimic (12%), quinic (12%), and cyclohexanecarboxylic acid (61%). A human fecal flora was fed to otherwise germfree rats to determine the degree of association and the resulting effect upon the metabolism of shikimic, quinic, and cyclohexanecarboxylic acids in vivo. Following establishment of the human microflora and subsequent feedings of shikimic or quinic acids, excretion of urinary hippurate was five to seven times greater (10–15% of the dose) than in germfree rats fed the same acids. The results suggest that the intestinal flora is needed to metabolize the shikimic acid to substrate(s) (probably cyclohexanecarboxylic acid). This substrate can then be aromatized by mammalian enzymes.     

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO₃ (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Alicyclic acid metabolism in plants V. Biosynthesis of alicyclic acids from 14C-labeled glucose and erythrose in some plant tissues

Etiolated seedlings of Phaseolus mungo were fed with ¹⁴C-glucoseand the incorporation of ¹⁴C into shikimic and quinic acidswas determined. The incorporation of ¹⁴C into shikimic acidwas enhanced when non-labeled shikimic, quinic or 5-dehydroquinicacid was not significantly affected by these alicyclic acids.To examine whether the difference in biosynthetic patterns betweenshikimic and quinic acids is common in higher plants, flowersand leaves of several plants were fed with ¹⁴C-glucose or ¹⁴C-erythroseand the effciencies of these labeled sugars as precursors ofshikimic and quinic acids were compared. In seven of eight plantsamples, erythrose was superior to glucose as the precursorof shikimic acid, while there was no great difference in theefficiency of either sugar as the precursor of quinic acid.The possibility that the biosynthetic mechanism for quinic aciddiffers from that for shikimic acid is discussed. (Received September 12, 1973; )     

Changes of shikimate pathway in glyphosate tolerant alfalfa cell lines with reduced embryogenic ability

Glyphosate tolerant cell lines were selected from highly embryogenic cell suspension culture ofMedicago sativa L. Resistant cell lines showed significant reduction of embryogenic ability and during long-term culture in the presence of glyphosate gradual loss of this ability was observed. After glyphosate treatment the increased activity of 5-enolpyruvylshikimate-3-phosphate synthase in tolerant cell lines overcame the block in aromatic amino acid synthesis which was observed in control cell lines. Glyphosate caused marked increase in the content of shikimic acid in both control and tolerant cell lines but the accumulation of shikimic acid was considerably lower in tolerant calli. Significant qualitative and quantitative differences were found in the content of individual phenolic acids. The considerable decrease in the amount of cinnamic acid derivates and broader spectrum of hydroxybenzoic acids suggest in tolerant cell lines the activation of alternative pathway not regulated by phenylalanine ammonia lyase. The possible role of altered pool of phenolic acids on the embryogenic ability is discussed.     

Pigment and isozyme variation in aspen shoots regenerated from callus culture

Pigment as well as isozyme variations were observed among aspen (Populus tremuloides Michx.) plants regenerated from callus cultures. Out of more than 600 plantlets, two chimeric plants (one with green base and two albino shoots and the other with an albino shoot) were produced. Callus derived from albino shoots produced albino as well as chimeric plants when transferred to shoot inducing medium. Isozyme patterns of 119 plants were examined by starch gel electrophoresis. Thirty plants showed variation in shikimic dehydrogenase isozyme and 41 in isocitric dehydrogenase. Variation was also observed in malate dehydrogenase and phosphoglucose isomerase. No variation was seen in 6-phosphogluconate dehydrogenase. Pigment variation was not associated with any isozyme changes.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - GD Gresshoff & Doy medium - WPM woody plant medium - SKD shikimic dehydrogenase - IDH isocitric dehydrogenase - MDH malate dehydrogenase - PGI phosphoglucose isomerase - 6-PGD 6-phosphogluconate dehydrogenase     

Zinc Accumulation and Tolerance in Solanum nigrum are Plant Growth Dependent

Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulator, was studied during pre- and post-flowering stages of development. The plants, when challenged with Zn concentrations lethal to plantlets, showed an increase in tolerance from pre-flowering to post-flowering, which was accompanied by a reduction of Zn translocation to the aerial plant parts. Treatment with Zn induced a differential response in organic acids according to the plant organ and developmental stage. In the roots, where Zn concentrations were similar in pre- and post-flowering plants, a general decrease in organic acid in pre-flowering roots contrasted with the increase observed in post-flowering plants. In the stems, Zn induced a generalized increase in organic acids at both growth stages while in the leaves, a slight increase in malic and shikimic was observed in pre-flowering plants and only shikimic acid levels were significantly increased in post-flowering plants. This work shows that Zn accumulation and tolerance in S. nigrum vary during plant development – an observation that may be important to improve the efficiency of phytoremediation approaches. Furthermore, the data suggest the involvement of specific organic acids in this response.     

Aromatic amino acid biosynthesis in Trichophyton rubrum

Summary WhenTrichophyton rubrum is grown in a minimal medium containing glucose, the carbon skeleton of fungal phenylalanine and tyrosine is derived from the glucose carbon. Tracer experiments with variously labeled glucose-C¹⁴ indicate that phenylalanine synthesis is linked to glycolysis, but suggest that the pentose phosphate pathway is not involved. These findings suggest that aromatic amino acid biosynthesis may not be linked to the shikimic acid pathway inT. rubrum.