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.     

Production of brown tyrosine pigments by the yeast Yarrowia lipolytica

AIMS: To study the mechanism of production of brown pigments from tyrosine in the yeast Yarrowia lipolytica. METHODS AND RESULTS: Pigment formation was followed during growth in tyrosine medium, and the presence of the pigment precursor in the medium was assessed by evaluating pigment formation after removing the cells at different times of incubation. It was observed that the pigment precursor accumulated outside the cells during the exponential phase of growth, but pigment formation only occurred during the stationary phase of growth and resulted from the oxidation of the precursor. Pigment formation was repressed by glucose and L-glutamine, and promoted by lactic acid, L-asparagine and glycine. Spectra of 1H and 13C-NMR revealed that the brown pigment was derived from tyrosine and was a polymer composed of a core of aromatic residues. CONCLUSION: The results indicate that pigments result from the extracellular accumulation and auto-oxidation of an intermediate of tyrosine catabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the mechanism of pigment production from tyrosine in a yeast species.     

Characterization of the Melanogenic System in Vibrio cholerae,ATCC 14035

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine. Other phenolic chemicals related to L-tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami-nases that transforms L-tyrosine into p-hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.     

The biosynthesis of cyanogenic glucosides in higher plants. The (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime as intermediates in the biosynthesis of dhurrin in Sorghum bicolor (L.) Moench

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (M?ller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled.     

Interference of some tryptophan metabolites in the formation of melanin in vitro

Melanin (eumelanin) is commonly produced in mammals starting from tyrosine and/or 3,4-dioxyphenylalanine (DOPA) under the action of tyrosinase. 3-Hydroxyanthranilic acid and 3-hydroxykynurenine are intermediates occurring in the kynurenine pathway of tryptophan catabolism. In this paper, we show that these substances can interfere in melanin formation in vitro when tyrosine or DOPA is oxidized by molecular oxygen under catalysis by tyrosinase. In particular, when 3-hydroxyanthranilic acid is present, a brown and apparently water-soluble pigment is formed, whereas the typical eumelanin granules seem to become more and more rare as the concentration of 3-hydroxyanthranilic acid increases. Also in the presence of the latter, the rate of tyrosine and/or DOPA consumption decreases. A very complicated (13)C-NMR spectrum indicates the high complexity of the reaction. This involves both the true melanin precursor(s) and the tryptophan metabolite, even if with peculiar mechanism and kinetics. When 3-hydroxykynurenine is substituted for 3-hydroxyanthranilic acid the reaction leads to reddish pigments whereas xanthommatins (the typical oxidation products of 3-hydroxykynurenine) are absent. A possible relationship between some dischromic pathologies and tryptophan metabolic disorders is suggested.     

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.     

Production by Bacillis subtilis of brown sporulation-associated pigments

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.     

Serum-induced hypha formation in the dimorphic yeast Yarrowia lipolytica

The dimorphic yeast Yarrowia lipolytica forms true hyphae in a medium containing N-acetylglucosamine. We made a new finding that serum is a very effective inducer of hypha formation of Y. lipolytica: serum induced its hyphal growth very quickly compared to N-acetylglucosamine (4 h vs. 10 h). Osmotic and oxidative stresses (0.2 M NaCl and 20 mM H2O2) inhibited the hypha formation induced by N-acetylglucosamine, but did not suppress the hypha formation triggered by serum. Serum-specific morphological mutants, which formed hyphae in the N-acetylglucosamine medium but not in serum medium, could be isolated. These results suggest that the signal triggered by serum may be transduced through a different pathway, at least in part, from that used for the N-acetylglucosamine signal in Y. lipolytica.     

Methionine catabolism in Saccharomyces cerevisiae

The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol.     

Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species.

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.     

Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.     

The effects of aeration on glucose catabolism in Penicillium expansum.

Polyacrylamide-disc gel electrophoresis and quantitative enzyme assays showed that the pathways of glucose catabolism and secondary metabolism in Penicillium expansum were dependent on the degree of aeration of the cultures. The isoenzyme patterns and specific activities of aldolase and succinate dehydrogenase indicated that glycolysis and the tricarboxylic acid cycle operated under conditions of both limited and efficient aeration (i.e. in cultures grown statically or on an orbital shaker). At high levels of aeration the growth rate was faster and synthesis of extracellular pectolytic enzymes was enhanced, whilst the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase showed that the pentose-phosphate shunt was important in glucose catabolism during the trophophase of growth. In contrast, under conditions of low aeration this latter pathway was virtually undetectable, growth was slower, pectolytic enzyme production low and large concentrations of secondary metabolites (6-methylsalicylic acid, patulin and citrinin) accumulated.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Copper extrusion after accumulation during growth of copper-tolerant yeast Yarrowia lipolytica

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.     

Biosynthesis of phytoquinones. Homogentisic acid: a precursor of plastoquinones, tocopherols and α-tocopherolquinone in higher plants, green algae and blue–green algae

1. By means of (14)C tracer experiments and isotope competition experiments the roles of d-tyrosine, p-hydroxyphenylpyruvic acid, p-hydroxyphenylacetic acid, phenylacetic acid, homogentisic acid and homoarbutin (2-methylquinol 4-beta-d-glucoside) in the biosynthesis of plastoquinones, tocopherols and alpha-tocopherolquinone by maize shoots was investigated. It was established that d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid can all be utilized for this purpose, whereas p-hydroxyphenylacetic acid, phenylacetic acid and homoarbutin cannot. Studies on the mode of incorporation of d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid showed that their nuclear carbon atoms and the side-chain carbon atom adjacent to the nucleus give rise (as a C(6)-C(1) unit) to the p-benzoquinone rings and nuclear methyl groups (one in each case) of plastoquinone-9 and alpha-tocopherolquinone and the aromatic nuclei and nuclear methyl groups (one in each case) of gamma-tocopherol and alpha-tocopherol. 2. By using [(14)C]-homogentisic acid it has been shown that homogentisic acid is also a precursor of plastoquinone, tocopherols and alpha-tocopherolquinone in the higher plants Lactuca sativa and Rumex sanguineus, the green algae Chlorella pyrenoidosa and Euglena gracilis and the blue-green alga Anacystis nidulans.     

Control of Lysine Biosynthesis in Yeast by a Feedback Mechanism

Homocitric acid (β-hydroxy-β-carboxyadipic acid; HC) is accumulated by a lysine-requiring yeast mutant when grown in a chemically defined medium, supplemented with limited amounts of lysine. A study of the formation of HC in relation to the depletion of lysine from the growth medium indicates that HC accumulated only when the concentration of lysine was low. The enzymatic formation of HC from α-ketoglutarate plus acetyl-coenzyme A in cell-free extracts of the same organism was also inhibited by lysine. The inhibitory effect of lysine on the formation of HC in both whole cells and cell-free extracts is indicative of the functional existence of a feedback control mechanism in the pathway for lysine biosynthesis in yeast.     

Modeling lipid accumulation and degradation in Yarrowia lipolytica cultivated on industrial fats

A modeling approach was used to quantify the kinetic behavior of a Yarrowia lipolytica strain capable of producing significant lipid amounts when cultivated on industrial fats. Biomass and cellular lipid evolution were successfully simulated, while the optimized parameter values were similar to those experimentally measured. The maximum specific formation rate of fat-free biomass seemed unaffected by the substrate fatty acid composition. On the contrary, the maximum concentration of lipid accumulated inside the yeast cell, as well as the maximum specific accumulation rate of cellular lipids, was favored in high stearic acid content media. The microorganism presented the tendency to degrade its accumulated lipids, although remarkable substrate fat amounts remained unconsummated in the culture medium. This degradation slowly occurred in the yeast cell as the specific rate of the intracellular carbon pool (storage lipid consumption) was significantly lower compared with that of the extracellular carbon pool (substrate fat). However, the fat-free biomass yield on storage lipids (g of fat-free biomass formed per g of storage lipids consumed) was higher than the one on the substrate (g of fat-free biomass formed per g of medium fat consumed).     

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid.