The enzymic conversion of hydroxycinnamic acids to p-coumarylquinic and chlorogenic acids in tomato fruits

Two enzymes thought to be involved in the biosynthesis of chlorogenic acid have been separated and purified by ion exchange chromatography and their properties studied. These two enzymes, p-coumarate CoA ligase and hydroxycinnamyl CoA: quinate hydroxycinnamyl transferase, acting together catalyse the conversion of p-coumaric acid to 5′-p-coumarylquinic acid and of caffeic acid to chlorogenic acid. The ligase has a higher affinity for p-coumaric than for caffeic acid and will in addition activate a number of other cinnamic acids such as ferulic, isoferulic and m-coumaric acids but not cinnamic acid. The transferase shows higher activity and affinity with p-coumaryl CoA than caffeyl CoA. It also acts with ferulyl CoA but only very slowly. The enzyme shows high specificity for quinic acid; shikimic acid is esterified at only 2% of the rate with quinic acid and glucose is not a substrate. The transferase activity is reversible and both chlorogenic acid and 5′-p-coumarylquinic acids are cleaved in the presence of CoA to form quinic acid and the corresponding hydroxycinnamyl CoA thioester.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose

Fumaric acid is a dicarboxylic acid used extensively in synthetic resins, food acidulants, and other applications, including oil field fluids and esters. The filamentous fungus Rhizopus oryzae is known for its ability to produce and accumulate high levels of fumaric acid under aerobic conditions. In this work, the overexpression of native fumarase encoded by fumR and its effect on fumaric acid production in R. oryzae were investigated. Three plasmids containing the endogenous fumR gene were constructed and used to transform R. oryzae, and all transformants showed significantly increased fumarase activity during both the seed culture (growth) and fermentation (fumaric acid production) stages. However, fumarase overexpression in R. oryzae yielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase also catalyzed the hydration of fumaric acid to malic acid. The results suggested that the overexpressed fumarase, encoded by fumR, by itself was not responsible for the over-production of fumaric acid in R. oryzae.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species

Caffeic acid derivatives (CADs) are a group of bioactive compounds which are produced in Echinacea species especially Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida. Echinacea is a popular herbal medicine used in the treatment of common cold and it is also a prominent dietary supplement used throughout the world. Caffeic acid, chlorogenic acid (5-O-caffeoylquinic acid), caftaric acid (2-O-caffeoyltartaric acid), cichoric acid (2, 3-O-dicaffeoyltartaric acid), cynarin, and echinacoside are some of the important CADs which have varied pharmacological activities. The concentrations of these bioactive compounds are species specific and also they vary considerably with the cultivated Echinacea species due to geographical location, stage of development, time of harvest, and growth conditions. Due to these reasons, plant cell and organ cultures have become attractive alternative for the production of biomass and caffeic acid derivatives. Adventitious and hairy roots have been induced in E. pupurea and E. angustifolia, and suspension cultures have been established from flask to bioreactor scale for the production of biomass and CADs. Tremendous progress has been made in this area; various bioprocess methods and strategies have been developed for constant high-quality productivity of biomass and secondary products. This review is aimed to discuss biotechnological methods and approaches employed for the sustainable production of CADs.     

Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.     

Bifidobacterium breve with α-Linolenic Acid and Linoleic Acid Alters Fatty Acid Metabolism in the Maternal Separation Model of Irritable Bowel Syndrome

The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (10⁹ microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.     

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-¹⁴C]cresol was recovered predominantly as [¹⁴C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.     

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.     

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml⁻¹ for two of the three test organisms).     

Metabolism of linoleic Acid by barley lipoxygenase and hydroperoxide isomerase

The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9-hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13-epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13-hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source.     

Application of a Sex Pheromone,Pheromone Analogs,and Verticillium lecanii for Management of Heterodera glycines

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Seed lipids of the lythraceae

Fatty acid composition of seed lipids for 20 of the 26 genera in the Lythraceae and seed oil and protein content for nine genera are reported. The percent oil ranges from 2.7 to 34% of total weight and protein from 11.3 to 24.9%. Linoleic acid is the dominant fatty acid in seed lipids of all genera surveyed. Variations in pattern emphasize palmitic or oleic acid or both as second most abundant lipid component. There are three exceptions: in Diplusodon capric acid ranks second in abundance; in Adenaria lauric acid and oleic acid occur in approximately equal amounts as second most abundant fatty acid; in Decodon an unusual trienoic acid, previously reported only from the Compositae, is the main secondary component. Fatty acid composition of seeds in the genera is compared to that of the previously studied lythraceous genus Cuphea. Among all the genera, only Cuphea seed produces large quantities of lauric, capric, or caprylic acids, as well as a diversity of fatty acid patterns. No relationship between oil content or seed weight and habit is apparent in any genus studied, nor are differences in seed morphology reflected in composition of the seed lipids. The fatty acid patterns are judged evolutionarily conservative, with the strong exception of Cuphea, which remains unique in the Lythraceae and among all angiosperms for the diversity of patterns displayed.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.     

Uptake of the β-Lactam Precursor α-Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease

External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (K_m values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.     

Further studies on the biosynthesis of mangiferin in Anemarrhena asphodeloides: hydroxylation of the shikimate-derived ring

The hydroxylation at C-3′ of maclurin, an intermediate in mangiferin biosynthesis, has been studied. Labelled cinnamic acid, p-coumaric acid, caffeic acid, iriflophenone and maclurin were fed to Anemarrhena asphodeloides. Cinnamic acid and p-coumaric acid were better precursors than caffeic acid for mangiferin, and iriflophenone as well as maclurin was effectively incorporated into mangiferin and isomangiferin. These results show that maclurin is biosynthesized via hydroxylation of iriflophenone derived from p-coumarate in this plant.     

The synthesis of folic acid-like compounds by Lactobacillus arabinosus

In the presence of p-aminobenzoic acid (PABA), Lactobacillus arabinosus synthesizes one or more compounds with folic acid (FA)-like activity during growth. The total FA activity formed is proportional to the amount of PABA added, up to 200 mμg./tube. Most of the FA activity is in a bound form which is present chiefly in the cells, and the remainder is present in free form which is mainly in the culture medium. Increasing levels of sulfanilamide in the medium competitively inhibit the utilization of PABA for growth of L. arabinosus and decrease the amount of free and combined FA compounds formed.Equimolecular amounts of p-aminobenzoylglutamic acid and PABA have approximately the same growth-promoting and antisulfanilamide activities for L. arabinosus under the conditions employed, and lead to the synthesis of similar amounts of the FA-like compound by L. arabinosus.Thymidine can replace PABA for growth of L. arabinosus and its activity is inhibited very little by sulfanilamide. Thymine and thymidylic acid are much less effective than thymidine. Crystalline vitamin B_c conjugate and vitamin B₁₂ cannot replace PABA for growth of L. arabinosus and do not augment the antisulfonamide activity of PABA.The growth-promoting and antisulfanilamide activities of synthetic folid acid (pteroylglutamic acid), citrovorum factor, and formylfolic acid preparations for L. arabinosus are much poorer than those of equimolecular amounts of PABA. The possibility that the compound(s) with FA-like activity produced by L. arabinosus may differ from pteroylglutamic acid, citrovorum factor, or formylfolic acid is discussed.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.