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.     

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     

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.

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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.

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

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.     

Effect of salicylic acid on protein composition of Tatar buckwheat Fagopyrum tataricum calluses with different ability for morphogenesis

The effect of salicylic acid on the content of soluble proteins and individual polypeptides in Tatar buckwheat Fagopyrum tataricum calluses differing in ability for morphogenesis was studied. Changes in the protein composition of the calluses cultivated in the dark and in the light indicated the higher sensitivity of the non-morphogenic callus. Different response of callus cultures to salicylic acid and conditions of cultivation (light, darkness) is suggested to be associated with the antioxidant defense system, which is, in particular, characterized by the hydrogen peroxide content in the calluses. Salicylic acid increased the H₂O₂ content in non-morphogenic calluses more strongly than in morphogenic calluses, and the difference was more significant for the calluses cultivated in the light.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 390–396.Original Russian Text Copyright © 2005 by Maksyutova, Galeeva, Rumyantseva, Viktorova.     

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.     

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.     

Biosynthesis of phenylalanine,tyrosine, 3-(3-carboxyphenyl)alanine and 3-(3-carboxy-4-hydroxyphenyl)alanine in higher plants: Examples of the transformation possibilities for chorismic acid

¹⁴C-labelled shikimic acid and double labelled shikimic acid tritiated stereospecifically at C-6 are incorporated into 3-(3-carboxyphenyl)alanine, 3-(3-carboxyl-4-hydroxyphenyl)alanine, phenylalanine, and tyrosine in Resda lutea L., Reseda odoratta L., Iris x Hollandica cv. Prof. Blauw, and Iris x hollandica cv. Wedgwood. The experiments with ¹⁴C-labelled shikimic acid confirm that the aromatic carboxyl groups and rings in 3-(3-carboxyphenyl)-alanine and 3-(3-carboxy-4-hydroxyphenyl)alanine derive from the carboxyl group and ring in shikimic acid whereas the experiments with double labelled shikimic acid demonstrate that the pro-6S-hydrogen atom is retained and the pro-6R-hydrogen atom lost in the biosynthesis of 3-(3-carboxyphenyl)alanine, phenylalanine, and tyrosine in the plants used. ³H was located in the ortho-position in the aromatic rings of phenylalanine and tyrosine but in a position para to the alanine side chain of 3-(3-cabroxyphenyl)alanine. No ³H was found in 3-(3-carboxy-4-hydroxyphenyl)alanine. This supports a derivation of the last two compounds from chorismic acidvia isochorismic acid, isoprephenic acid, and 3′-carboxyphenylpyruvic acid and 3′-carboxy-4′-hydroxyphenylpyruvic acid. The ³H/¹⁴ C ratio in 3-(3-carboxyphenyl)alanine was found higher than in the precursor used. This isotope effect must operate by competition between the pathways from isoprephenic acid to 3′-carboxyphenylpyruvic acid and to 3′-carboxy-4′-hydroxyphenylpyruvic acid. The proposed biosynthetic pathways for the two carboxy-substituted amino acids are in agreement with their distribution patterns in the plant kingdom and suggest that they may derive from minor changes of enzymes involved in the general pathways of aromatic biosynthesis.     

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.     

A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo⁵U)² and its methylester, mcmo⁵U. Both became radioactively labelled using [methyl-¹⁴C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo⁵U and, in some cases, (4) mcmo⁵U. The mutant had also become Aro^?, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo⁵U and mcmo⁵U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo⁵U and mcmo⁵U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K₂ or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.     

Shikimate Pathway Activity during Shoot Initiation in Tobacco Callus Cultures

The activity of the shikimic acid pathway during shoot initiation in tobacco (Nicotiana tabacum L. Wisconsin 38) callus was examined. Enhancement of the activities of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase, shikimate kinase, chorismate mutase, and anthranilate synthase was observed during culture of tobacco callus under shootforming conditions in comparison to tissue cultured under non-organforming conditions. Confirmation of these findings was obtained by examining the incorporation of d-[¹⁴C]glucose into quinic and shikimic acids and of [¹⁴C]shikimic acid into tyrosine, phenylalanine, and tryptophan.     

Effects of ozone on organic acids in needles of Norway spruce and Scots pine

Summary The effect of ozone, needle age, and season on the pH of homogenate and acid contents of Scots pine and Norway spruce needles is presented. In addition enzyme activities of cytochrome C-oxidase (cyt. C-ox), phosphoenolpyruvate-carboxylase (PEPC), shikimic acid-dehydrogenase (SHDH) and malate-dehydrogenase (MDH) were measured in Scots pine needles. In freshly sprouted spruce needles the level of quinic acid is high and the pH of the needle homogenate is low. Shikimic acid starts at low levels, increases with increasing needle age and becomes dominant, whereas the quinic acid content decreases. Malic acid has a marked seasonal trend; no trend was found in citric acid. Ozone (200 g/m³) decreased shikimic acid and quinic acid, whereas pH, malic acid and citric acid increased. Ozone (100 g/m³) had a similar effect, except in the current-year spruce needles. In Scots pine needles ozone led to increased enzymatic activities of cyt. C-ox, PEPC and SHDH, and a decrease in the activity of MDH. This effect was more pronounced in summer than in autumn, but the visible damage was greater in autumn. These effects can be found with other stresses and are not specific for ozone.     

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe,Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C^/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D₂O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

光周期对苦荞芽菜生长与品质的影响

苦荞(Fagopyrum tataricum)芽菜是一种新兴健康食品,该研究通过不同光周期(0、4、8、12、16和20 h·d~(-1))处理苦荞芽菜,测定其生物量、叶绿素和主要营养成分的含量以及黄酮合成相关基因的表达水平,以明确不同光周期对苦荞芽菜品质的影响机理。结果表明:(1)随着苦荞芽菜生长时间的增加,芽菜生物量总体呈上升趋势,16 h·d~(-1)光周期时芽菜生长状态最好,鲜重最大。(2)不同光周期对苦荞芽菜各营养成分含量的影响有所差异,可溶性糖含量在芽菜萌发第2天开始逐渐下降,10 d后含量仅为第2天的13.5%～14.5%;花青素含量在芽菜萌发第2天光周期为12 h·d~(-1)时最高(2.16 mg/g),不同光周期处理4～10 d后均显示出降低趋势;芦丁是苦荞芽菜中主要的黄酮类化合物,芦丁含量在不同光周期处理后有所变化且适当的光照有利于其合成和积累,并在处理第4天光周期为16 h·d~(-1)时含量最高(59.60 mg/g)。(3)qRT-PCR分析表明,不同光周期处理的苦荞芽菜中各黄酮合成相关基因的表达量在第2天最高,随生长周期其表达量均不同程度降低,但黄酮醇合成支路关键酶基因FtFLS1和花青素支路基因FtDFR2表达量上升、且显示出强烈的光诱导特征。研究发现,光周期对苦荞芽菜的生物量影响较大,黑暗有利于胚轴伸长,长光周期(16 h·d~(-1))有利于鲜重的增加;在苦荞萌发过程中,苦荞芽菜可溶性糖、花青素和芦丁含量整体呈减少趋势,可溶性糖和花青素含量均在光周期16 h·d~(-1)处理第2天时最高,芦丁含量在光周期为16 h·d~(-1)处理第4天时最高,建议苦荞芽菜在光周期为16 h·d~(-1)生长4 d时采食。     

Interactions of an acid tolerant strain of phosphate solubilizing bacteria with a few acid tolerant crops

Phosphate solubilizing bacteria (PSB) were isolated from sixty soil samples of various soil classes and cropping histories in Himalayan regions of Uttar Pradesh, India by enrichment culture techniques. Phosphate solubilization and acid tolerance of each strain was estimated. A strain (PAS-2) isolated froma pasture and waste land of pH 4.8, organic matter 2.6% available N 265kg ha^-1, available P₂O₅(Bray's II) 2.3kg ha^-1 and available K₂O 353 kg ha^-1 had the highest P-solubilization (45 µg P per mL per day) and also highest acid tolerance rating 42. The strain was identified as Bacillus sp. Seed inoculation of this bacterial strain resulted in significant increases in grain and vegetative yield of fingermillet (Elosine coracana), maize (Zea mays), amaranth (Amaranthus hypochondriacus), buckwheat (Fagopyrium esculentum), frenchbean (Phaseolus vulgaris) with or without added P sources. The significant grain yield (quintol ha^-1) with phosphate and seed inoculation ranged from 33.85 in maize, 26.33 in frenchbean, 22.41 in buckwheat, 20.71 in amaranth and 19.19 in fingermillet as compared to controls. The highest response was observed with frenchbean followed by fingermillet, buckwheat, amaranth and maize. Phosphate use efficiency was highest in frenchbean followed by maize and lowest and almost at par in buckwheat, amaranth and fingermillet. Available phosphate was also highest in frenchbean cultivated plot followed by amaranth, fingermillet, buckwheat and maize. The MPN count of phosphate solubilizing bacteria were also influenced by seed inoculation of strain PAS-2. Frenchbean exerted greaterrhizosphere effect followed by pseudocereals and cereals. Likewise, phosphate nutrition of crops were also improved through seed inoculation irrespective of added P sources. The study thus demonstrated that selection of efficient strain of PSB from acid soil and its seed inoculation in selected crop genotype is beneficial in boosting up crop yield in low productive hill soil. Seed inoculation also created greater rhizosphere effect over uninoculation which improved P-nutrition of crops and also available soil P.     

Application of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> (Chlorophyceae) as supplement and/or an alternative medium for the in vitro cultivation of <Emphasis Type="Italic">Schomburgkia crispa</Emphasis> (Orchidaceae)

Schomburgkia crispa Lindley (Orchidaceae) is an epiphytic species found in gallery forests and dry vegetation in the Brazilian Cerrado. It is typically unable to germinate or exhibits low germination because of dependency on mycorrhizal associations. In vitro cultivation techniques have helped circumvent difficulties involved in propagation from seeds. Alternative media and organic biostimulant substances that reduce costs and promote satisfactory in vitro growth are constantly sought. This study evaluated in vitro multiplication and rooting of S. crispa in a modified culture medium containing extract of the microalga Chlorella sorokiniana. We analyzed supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium. The extracts were added to WPM instead of distilled water. The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively. Both growth regulators were tested at 0, 2.5, and 5.0 mg L^?1. During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L^?1 BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L^?1 IBA supplemented with microalgae extract stimulated in vitro rooting. Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone. Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa.     

Search for the wild ancestor of buckwheat III. The wild ancestor of cultivated common buckwheat, and of tatary buckwheat

By surveying wild Fagopyrum species and their distribution in southern China and the Himalayan hills, I arrived at the conclusion that the newly discovered subspecies F. esculentum ssp. ancestralis Ohnishi is the wild ancestor of cultivated common buckwheat, while previously known wild tatary buckwheat,F. tataricum ssp. potanini Batalin is the wild ancestor of tatary buckwheat. Their original birthplace is revealed to be northwestern corner of Yunnan province for common buckwheat judging from the distribution of wild ancestor, and to be the northwest part of Sichuan province for tatary buckwheat judging from allozyme variability in wild tatary buckwheat. F. cymosum is not the ancestor of cultivated buckwheat; it is only distantly related to cultivated buckwheat, in morphology, isozymes and cpDNA. Several genetic, ecological and taxonomic categories which should be taken into consideration in examining the origin of buckwheat were discussed. Key Words: Fagopyrum esculentum ssp. ancestralis; Fagopyrum tataricum ssp. potanini; southern China theory of origin of buckwheat. Contribution from Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University. No. 78.     

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.