Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete chrysosporium

The lignin-degrading basidiomycete Phanerochaete chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol) via phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol. Study of the conversion of 3,4-dimethoxycinnamyl alcohol to veratrylglycerol and veratryl alcohol showed is to be (a) catalyzed by a secondary metabolic system, (b) markedly suppressed by culture agitation, and (c) strongly inhibited by l-glutamate. The amount of veratryl alcohol synthesized de novo was positively correlated with the O₂ concentration after primary growth. Other work has shown that the cinnamyl alcohol terminal residue in a lignin substructure model compound is degraded via arylglycerol and benzyl alcohol structures in ligninolytic cultures of P. chrysosporium, and that the ligninolytic system exhibits traits (a)-(c) above. Ligninolytic activity is also strongly and positively correlated with O₂ concentration. The results here suggest, therefore, that the actual biosynthetic secondary metabolic product is 3,4-dimethoxycinnamyl alcohol, but that this is degraded by the ligninolytic system to veratryl alcohol via veratrylglycerol. Veratryl alcohol is only slowly metabolized by the fungus, and accumulates.Non-standard abbreviation tlc thin layer chromatography     

Metabolism of non-phenolic β-O-4 lignin model compounds by the white-rot fungus Phlebia radiata

Summary The degradation of three non-phenolic -O-4 diarylpropane lignin model compounds was studied in cultures of the white-rot fungus Phlebia radiata. The degradation pattern of the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) was also compared with that of Phanerochaete chrysosporium under conditions where both fungi were cultivated without agitation in an oxygen atmosphere. Compound I was readily degraded by both fungi, and qualitatively the degradation patterns were quite similar. The product, after C-C bond cleavage, was veratraldehyde (IV) which was almost stoichiometrically reduced to veratryl alcohol (V). However, large amounts of V were detected only in P. chrysosporium cultures. Experiments with the model compound 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (II) showed that in the presence of II, the total amount of veratryl compounds accounted for 15–33 m in standing cultures of Phlebia radiata. The model compound 1-(3,4-dimethoxyphenyl)-2-(4-methoxyphenoxy) propane-1,3-diol (III) was more readily degraded than I and II. The results indicated that, in P. radiata cultures, the acting enzymes were lignin peroxidases and IV reducing enzyme, while laccase was less important. Offprint requests to: A. Hatakka     

Lignin peroxidase production by strains of Phanerochaete chrysosporium grown on glycerol

Summary Lignin peroxidase production by several strains of Phanerochaete chrysosporium was determined during growth on glycerol under conditions of nitrogen sufficiency. Fungal strains which grew poorest on glycerol produced the highest titres of lignin peroxidase whereas enzyme levels were much lower when marginally greater biomass values were recorded. In the case of P. chrysosporium strain INA-12, the nature of the nitrogen source had a pronounced effect on both growth and enzyme production. Highest biomass values were obtained when l-glutamate or l-glutamine served as the major nitrogen source but enzyme synthesis was normally repressed completely. Lignin peroxidase activity in this strain was maximal when the initial pH of the culture medium was adjusted to pH 5.0.     

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (V_max) of the system was 3.86 units (U) mg^–1 protein with arabinoxylan as substrate and the substrate concentration giving half V_max (S_0.5) was 0.52 mg ml^–1. At concentrations of arabinoxylan greater than 15 mg ml^–1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components. Correspondence to: P. Broda     

Biodegradation of Cyanide by a White Rot Fungus, Trametes versicolor

The cyanide degradation abilities of three white rot fungi, Trametes versicolor ATCC 200801, Phanerochaete chrysosporium ME 496 and Pleurotus sajor-caju, were examined. T. versicolor was the most effective with 0.35 g dry cell/100 ml degrading 2 mm KCN (130 mg/l) over 42 h, at 30°C, pH 10.5 with stirring at 150 rpm.     

Biosorption of tributyltin and other organotin compounds by cyanobacteria and microalgae

Biosorption of triorganotin compounds by the cyanobacteria Synechocystis PCC 6803 and Plectonema boryanum and the microalga Chlorella emersonii, incubated in 2-(N-morpholino)ethanesulphonic acid (MES) buffer, pH 5.5, in the presence of 0.5 mm organotin (supplied as chlorides), increased with molecular mass of the organotins, the order being triphenyltin > tributylin (Bu₃SnCl) > tripropyltin >- trimethyltin >- triethylin. In the butylin series, monobutyltin biosorption was lowest, although levels of dibutyltin uptake were greater than for Bu₃SnCl. Cyanobacterial Bu₃SnCl biosorption was complete in 5 min with no subsequent accumulation. In contrast, a second phase of uptake in C. emersonii resulted in an approximate 2.4-fold increase in cellular Bu₃SnCl between 5 min and 2 h. The external pH had a marked influence on biosorption of Bu₃SnCl by Synechocystis PCC 6803 and P. boryanum, with maximal uptake at pH 5.5 and 6.5, respectively. Effects of pH were less evident in C. emersonii. In all the organisms examined, no inhibition of Bu₃SnCl biosorption was observed between 0.05 and 50 mm NaCl. However, an increase in the external NaCl concentration from 50 to 500 mm resulted in an approximate 55–65% reduction in Bu₃SnCl uptake. Biosorption increased at increasing Bu₃SnCl concentrations (0.25–3.0 mm). Saturation of Bu₃SnCl biosorption at the higher concentrations was most evident in the cyanobacteria, although uptake levels were greater in these organisms at <- 2 mm Bu₃SnCl. Theoretical maximum biosorption levels at complete cell saturation, derived from reciprocal Langmuir plots, were approximately 565, 525 and 1050 nmol Bu₃SnCl mg^–1 dry weight, for Synechocystis PCC 6803, P. boryanum and C. emersonii, respectively. Correspondence to: G. M. Gadd     

Decoloration of azo dyes by three whiterot fungi: influence of carbon source

Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high nitrogen medium with glucose, could decolorize two azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). When starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12mM NH₄Cl, whether or not veratric acid was present. In medium containing 1.2mM NH₄Cl and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2mM NH₄Cl, decoloration by the local isolate was enhanced but not that by the other strains.The authors are with the Department of Microbiology, Soochow University, Shih Lin, Taipei, Taiwan     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday     

Oxalate production by Basidiomycetes,including the white-rot species Coriolus versicolor and Phanerochaete chrysosporium

Oxalate was found to accumulate in liquid culture media from the growth of the white-rot basidiomycetes Coriolus versicolor, Heterobasidion annosum, Pleurotus florida and Phanerochaete chrysosporium. Whereas little oxalate accumulated during active growth, millimolar concentrations of oxalate were detected in culture media during the stationary phase. The basidiomycete Agaricus bisporus, the cultivated mushroom, also accumulated oxalate in its culture medium in the stationary phase. In comparison, the brown-rot fungi Amyloporia xantha, Coniophora marmorata, C. puteana and Poria vaporaria accumulated oxalate in the primary metabolic phase and throughout growth up to 35 days. Oxalate accumulation (0.04–10.0 mm) in white-rot cultures did not lower the pH of the medium during growth, whereas in brown-rot cultures oxalate (2.0–20.0 mm) reduced the media pH during growth. Cultures of Agaricus bisporus, C. puteana and Coriolus versicolor grown on solid media containing high levels of calcium (50 or 100 mm calcium chloride) produced calcium oxalate crystals to varying extents on the surface of the hyphae. Correspondence to: C. S. Evans     

Degradation of chloroanilines in soil slurry by specialized organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichloroaniline was examined as single compounds as well as a mixture in soil slurries. At 30°C the degradation of chloroanilines by indigenous soil populations in soil slurries was observed when soil slurry was freshly contaminated or precontaminated to allow binding of chloroanilines to the soil matrix. Within 6 weeks, 3-chloro- and 3,4-dichloroaniline (each 2 mm) were degraded more rapidly (about 50% chloride elimination) than 4-chloro- and 2-chloroaniline, due to stronger adsorption of 4-chloroaniline and greater resistance of 2-chloroaniline. The addition of various supplements such as buffer, mineral salts and acetate only slightly influenced the degradation of chloroanilines by the indigenous soil populations. The mineralization was drastically enhanced when laboratory-selected chloroaniline-degraders (8·10⁶ cells/g) such as Pseudomonas acidovorans strain BN3.1 were supplemented to the soil slurries so that complete elimination of chloride from the chloroanilines occurred within 10 days. Correspondence to: F. R. Brunsbach     

The use of various substrates and substrate concentrations by a Hyphomicrobium sp. isolated from soil: Effect on growth rate and growth yield

The content of Hyphomicrobium sp. was estimated from a clay loam soil using the most probable number technique with methanol as the sole carbon source. The method enumerated Hyphomicrobia as 0.2% of the total bacteria determined by acridine orange direct counts. Hyphomicrobium sp. was not able to use C-C compounds such as glucose or acetate for growth. Maximal growth yield and growth rate were obtained when the concentration of methanol was in the range of 0.5–5 mg C/liter. Substrate affinity measurements revealed K_s values of 0.8 m and 5.8 m when the methanol concentration was 0.5–2.5 m and 5–200 m, respectively. Hyphomicrobium sp. had the ability to assimilate volatile organic compounds from air for growth. A growth yield of 0.7 mg/liter cell carbon was obtained in a mineral medium that contained no additions of organic compounds but had been stored for 4 weeks in flasks, allowing volatile compounds from the air to dissolve in the medium. When air was pumped into the culture during cultivating, the growth yield was proportional to the flow rate of air into the culture. Correspondence to: Kari Aa     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Metabolism of 4,4′-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142

Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4′-dichlorobiphenyl (4,4′-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4′-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4′-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4′-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4′-DCB was elucidated by the identification of metabolites upon addition of 4,4′-DCB and its metabolic intermediates. 4,4′-DCB was initially metabolized to 2-hydroxy-4,4′-DCB and 3-hydroxy-4,4′-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4′-DCB to 3-hydroxy-4,4′-DCB and 4-hydroxy-3,4′-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4′-DCB was transformed to 3-methoxy-4,4′-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4′-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4′-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.     

Comparison of two lignin-degrading fungi:Phlebia radiata andPhanerochaete chrysosporium

Summary The ligninolytic enzymes ofPhlebia radiata were produced in static conditions earlier developed forPhanerochaete chrysosporium. The production pattern of lignin peroxidases resembled that ofP. chrysosporium. The extracellular proteins ofPhlebia radiata were separated by isoelectric focusing. Four proteins with acidic isoelectric points (4.15) were detected by peroxidase staining. The peroxidases ofP. radiata reacted with antibodies produced against a peroxidase ofPhanerochaete chrysosporium and vice versa. Thus the lignin peroxidases of the two fungi have major similarities despite slight differences in their isoelectric points and molecular weights. Veratryl alcohol was produced by both fungi and degraded to veratraldehyde, two lactones and a quinone by the ligninolytic cultures.     

Determination of a catalytic tyrosine in <Emphasis Type="Italic">Trametes cervina</Emphasis> lignin peroxidase with chemical modification techniques

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine¹⁸¹ at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan¹⁷¹ have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine¹⁸¹. These results strongly suggest that tyrosine¹⁸¹ is at the catalytic site in T. cervina LiP.     

Formation and partial characterization of glucose-2-oxidase,a H2O2 producing enzyme in Phanerochaete chrysosporium

Summary A sugar oxidizing enzyme which produces H₂O₂ during glucose starvation in the white-rot fungus Phanerochaete chrysosporium has been purified from mycelial extracts and somewhat characterized. Enzyme purity was confirmed by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis, both techniques revealing a homogeneous protein. The enzyme is active over a broad pH range with maximum activity at pH 7.5. Of several sugars tested, glucose was the preferred substrate although -d-gluconolactone and d-xylose were also oxidized at significant rates (at 60% and 37%, respectively, of the rate observed with glucose). K _m-values for glucose and xylose are 1.03 and 20 mM respectively and the glucose oxidation product was idenitified as d-arabino-2-hexosulose. The possible importance of glucose-2-oxidase in lignin degradation is discussed.     

The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium

Summary Two important lignin-degrading fungi with existing or potential applications in the production of food, feed and/or fiber products from wood are Lentinus edodes (Berk.; Sing.=Lentinula edodes [Pegler]) and Phanerochaete chrysosporium (Burds). This study discusses their relative ability to degrade lignin and the factors controlling their ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂). Ligninolytic activity in P. chrysosporium is known to develop after the fungus ceases vegetative growth, and to require both O₂ and an exogenous carbon source such as glucose. It has an extracellular ligninase in high titer which is assayed by the oxidation of veratryl alcohol to veratraldehyde. Here, P. chrysosporium was found to have a high capacity for lignin degradation (it was not easily saturated with lignin). Certain inorganic elements, including Fe²⁺, Ca²⁺ and Mo⁶⁺, were found to stimulate its ligninolytic activity. Calcium addition was required, with 40 ppm Ca²⁺ giving the highest activity. As in P. chrysosporium, ligninolytic activity in L. edodes was found to require both O₂ and an exogenous carbon source. However, in contrast to P. chrysosporium, L. edodes was only moderately ligninolytic, had a lower capacity for lignin degradation (was more easily saturated with lignin), and showed maximal activity only during the vegetative growth period. Also in contrast to P. chrysosporium, ligninolytic activity in L. edodes was not stimulated by Ca²⁺. Instead, manganese was required, with 10 ppm Mn²⁺ giving optimal activity. An extracellular ligninase capable of oxidizing veratryl alcohol to veratraldehyde was not detected in L. edodes.     

Fermentation of 12% (w/v) Glucose to 1.2 m Lactate by Escherichia coli Strain SZ194 using Mineral Salts Medium

A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1 m d-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mm betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of d-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth. Revisions requested 17 January 2006; Revisions received 7 February 2006     

Effect of cadmium on the ligninolytic activity ofStereum hirsutum andPhanerochœte chrysosporium

An inhibitory effect of cadmium on the growth and ligninolytic activity of the wood-rotting basidiomycetesPhanerochœte chrysosporium, Pleurotus ostreatus, Pycnoporus cinnabarinus andStereum hirsutum was observed. Delayed decolorization of the polymeric dye Poly R-478 was observed in samples with 0.10 mmol/L Cd. Addition of 0.25 mmol/L Cd to the cultivation medium strongly reduced the activity of both Mn-dependent and Mn-independent peroxidases ofStereum hirsutum, while the activity of laccase was not affected to a similar extent. The maximum of MnP activity in these samples was found during the exponential phase of growth whereas control samples showed the highest activity after the onset of the stationary phase (days 15–21). Cadmium at concentrations higher than 0.50 mmol/L significantly inhibited the activity of all enzymes tested in bothS. hirsutum andP. chrysosporium.     

Isolation and characterization of substituted 4-hydroxy-cyclohex-2-enones as metabolites of 3,4-dimethoxybenzyl alcohol and its methyl ether in ligninolytic cultures of Phanerochaete chrysosporium

The metabolism of quinones formed in the enzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) (Ia) and its methyl ether Ib in ligninolytic cultures of Phanerochaete chrysosporium was studied. A metabolite of 2-hydroxymethyl-5-methoxy-2,5-cyclohexadiene-1,4-dione (IIa, formed from Ia by oxidation) was isolated and identified as cis-4-hydroxy-6-hydroxymethyl-3-methoxy-cyclohex-2-en-one (IVa), formally the reduced hydroquinone IIIa. The formation of IVa was also observed when both veratryl alcohol Ia or 2,5-dihydroxy-4-methoxybenzyl alcohol (IIIa), the hydroquinone of IIa, were used as substrates. Analogously, cis-4-hydroxy-3-methoxy-6-methoxymethyl-cyclohex-2-en-one (IVc) was isolated and identified as a metabolite from either 3,4-dimethoxybenzyl methyl ether (Ib) or from its oxidation product 5-methoxy-2-methoxymethyl-2,5-cyclohexadiene-1,4-dione (IIb) as well as from the corresponding hydroquinone 2,5-dihydroxy-4-methoxybenzyl methyl ether (IIIc). The physiological role of these unprecedented conversions is discussed. Correspondence to: H. E. Schoemaker