De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55. Formation of 3-chloro-anisaldehyde from glucose.

The white-rot fungus Bjerkandera sp. BOS55 produced de-novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi, other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3-chloro-anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 microM veratraldehyde and 50 microM 3-chloro-anisaldehyde. This is the first report of de-novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. Anisaldehyde and 3-chloro-anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium.     

Physiological Role of Chlorinated Aryl Alcohols Biosynthesized De Novo by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H₂O₂ production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.     

Aryl alcohols in the physiology of ligninolytic fungi

Abstract: White-rot fungi have a versatile machinery of enzymes which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. This review will focus on the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H₂0₂. The aryl alcohol/aldehyde couple is well protected against degradation by the fungi's extracellular ligninolytic enzymes and their concentrations in the extracellular fluid are highly regulated by intracellular enzymes.     

Manganese Regulation of Veratryl Alcohol in White Rot Fungi and Its Indirect Effect on Lignin Peroxidase

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.     

Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L-phenylalanine is proposed.     

Significant Biogenesis of Chlorinated Aromatics by Fungi in Natural Environments

Common wood- and forest litter-degrading fungi produce chlorinated anisyl metabolites. These compounds, which are structurally related to xenobiotic chloroaromatics, occur at high concentrations of approximately 75 mg of chlorinated anisyl metabolites kg of wood^-1 or litter^-1 in the environment. The widespread ability among common fungi to produce large amounts of chlorinated aromatic compounds in the environment makes us conclude that these kinds of compounds can no longer be considered to originate mainly from anthropogenic sources.     

Manganese Is Not Required for Biobleaching of Oxygen-Delignified Kraft Pulp by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 extensively delignified and bleached oxygen-delignified eucalyptus kraft pulp handsheets. Biologically mediated brightness gains of up to 14 ISO (International Standards Organization units) were obtained, providing high final brightness values of up to 80% ISO. In nitrogen-limited cultures (2.2 mM N), manganese (Mn) greatly improved manganese-dependent peroxidase (MnP) production. However, the biobleaching was not affected by the Mn nutrient regimen, ranging from 1,000 (mu)M added Mn to below the detection limit of 0.26 (mu)M Mn in EDTA-extracted pulp medium. The lowest Mn concentration tested was at least several orders of magnitude lower than the K(infm) known for MnP. Consequently, it was concluded that Mn is not required for biobleaching in Bjerkandera sp. strain BOS55. Nonetheless, fast protein liquid chromatography profiles indicated that MnP was the predominant oxidative enzyme produced even under culture conditions in the near absence of manganese. High nitrogen (22 mM N) and exogenous veratryl alcohol (2 mM) repressed biobleaching in Mn-deficient but not in Mn-sufficient culture medium. No correlation was observed between the titers of extracellular peroxidases and the biobleaching. However, the decolorization rate of the polyaromatic dye Poly R-478 was moderately correlated to the biobleaching under a wide range of Mn and N nutrient regimens.     

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.     

Hydrogen Peroxide Production as a Limiting Factor in Xenobiotic Compound Oxidation by Nitrogen-Sufficient Cultures of Bjerkandera sp. Strain BOS55 Overproducing Peroxidases

The overproduction of ligninolytic peroxidase by the N-deregulated white rot fungus Bjerkandera sp. strain BOS55 under nitrogen-sufficient conditions had no noteworthy effect on the oxidation of anthracene or the decolorization of the polymeric aromatic dye Poly R-478 in 6-day-old cultures. Only when the endogenous production of H(inf2)O(inf2) was increased by the addition of extra oxygen and glucose could a 2.5-fold increase in the anthracene oxidation rate and a 6-fold increase in the Poly R-478 decolorization rate be observed in high-N cultures with 10- to 35-fold higher peroxidase activities than N-limited cultures. Further increase of the H(inf2)O(inf2) generation rate in high-N cultures with glucose oxidase led to an additional 3.5-fold increase in the anthracene oxidation rate (350 mg liter(sup-1) day(sup-1)) and a 10-fold increase in the Poly R-478 decolorization rate. These results indicate that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H(inf2)O(inf2). The absence of Mn, which decreased the manganese peroxidase titers and increased the lignin peroxidase titers, was associated with up to 95% improvements in the anthracene oxidation rate. The simultaneous presence of Mn and veratryl alcohol was observed to have a synergistic negative effect on the oxidation of anthracene and the decolorization of Poly R-478.     

Enigmatic Gratuitous Induction of the Covalent Flavoprotein Vanillyl-Alcohol Oxidase in Penicillium simplicissimum

When Penicillium simplicissimum is grown on veratryl alcohol, anisyl alcohol, or 4-(methoxymethyl)phenol, an intracellular covalent flavin-containing vanillyl-alcohol oxidase is induced. The induction is highest (up to 5% of total protein) during the growth phase. In addition to vanillyl-alcohol oxidase, an intracellular catalase-peroxidase is induced. Induction of vanillyl-alcohol oxidase in P. simplicissimum is prevented by the addition of isoeugenol to veratryl alcohol-containing media, but growth is unaffected. The inhibitory effect of isoeugenol on induction is not observed when anisyl alcohol or 4-(methoxymethyl)phenol is used as the growth substrate. Based on the induction experiments and the degradation pathways for veratryl and anisyl alcohol, we propose that induction of vanillyl-alcohol oxidase is superfluous when P. simplicissimum is grown on these aromatic alcohols. However, the enzyme plays an essential role in the degradation of the methyl ether of p-cresol, 4-(methoxymethyl)phenol.     

Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus pleurotus pulmonarius

It has recently been reported that Pleurotus pulmonarius secretes a versatile peroxidase that oxidizes Mn2+, as well as different phenolic and nonphenolic aromatic compounds; this enzyme has also been detected in other Pleurotus species and in Bjerkandera species. During culture production of the enzyme, the activity of the main peak was as high as 1,000 U/liter (measured on the basis of the Mn3+-tartrate formation) but this peak was very ephemeral due to enzyme instability (up to 80% of the activity was lost within 15 h). In culture filtrates inactivation was even faster; all peroxidase activity was lost within a few hours. Using different inhibitor compounds, we found that proteases were not responsible for the decrease in peroxidase activity. Peroxidase instability coincided with an increase in the H2O2 concentration, which reached 200 μM when filtrates were incubated for several hours. It also coincided with the onset of biosynthesis of anisylic compounds and a decrease in the pH of the culture. Anisyl alcohol is the natural substrate of the enzyme aryl-alcohol oxidase, the main source of extracellular H2O2 in Pleurotus cultures, and addition of anisyl alcohol to filtrates containing stable peroxidase activity resulted in rapid inactivation. A decrease in the culture pH could also dramatically affect the stability of the P. pulmonarius peroxidase, as shown by using pH values ranging from 6 to 3.25, which resulted in an increase in the level of inactivation by 10 μM H2O2 from 5 to 80% after 1 h. Moreover, stabilization of the enzyme was observed after addition of catalase, Mn2+, or some phenols or after dialysis of the culture filtrate. We concluded that extracellular H2O2 produced by the fungus during oxidation of aromatic metabolites is responsible for inactivation of the peroxidase and that the enzyme can protect itself in the presence of different reducing substrates.     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).     

Ubiquinone biosynthesis in Escherichia coli K-12. Accumulation of an octaprenol, farnesylfarnesylgeraniol, by a multiple aromatic auxotroph

Cell extracts of a multiple aromatic auxotroph of Escherichia coli K-12, strain AB2830, grown in the absence of precursors of the quinone rings of the ubiquinone and menaquinone molecules, converted 4-hydroxy[U-¹⁴C]benzoate into a mixture of 3-octaprenyl-4-hydroxybenzoate and 2-octaprenylphenol. An octaprenol, farnesylfarnesylgeraniol, was isolated from such cell extracts and characterized by n.m.r. and mass spectroscopy. Neither the octaprenol, nor polyprenylation of 4-hydroxy[U-¹⁴C]benzoate, could be detected in cell extracts of strain AB2830 grown in the presence of 0.1mm-4-hydroxybenzoate. It was concluded that, in the biosynthesis of ubiquinone, the polyprenyl side chain is added to 4-hydroxybenzoate as a C₄₀ unit, the active form of which is converted by cell extracts into farnesylfarnesylgeraniol. The multiple aromatic auxotroph, when grown in the absence of 4-hydroxybenzoate but in the presence of 4-aminobenzoate, converted the latter compound into 3-octaprenyl-4-aminobenzoate. This compound was isolated from whole cells and characterized by n.m.r. and mass spectroscopy.     

Biosynthetic origin of [R-(Z)]-4-amino-3-chloro-2-pentenedioic acid in Streptomyces viridogenes

The biosynthesis of the chlorinated amino acid [R-(Z)]-4-amino-3-chloro-2-pentenedioic acid (ACPA) was investigated. Feeding studies with Streptomyces viridogenes were conducted in resting cells. Substantial incorporation from [(15)N]- and [(13)C]-enriched glutamate and proline indicated that the biosynthetic origin of ACPA is one of these amino acids. Experiments with deuterated glutamate and proline imply that chlorination does not occur via a radical mechanism, but rather suggest that a FADH(2)-dependent halogenase is involved.     

Manganese-lignin peroxidase hybrid from Bjerkandera adusta oxidizes polycyclic aromatic hydrocarbons more actively in the absence of manganese

We studied polycyclic aromatic hydrocarbon (PAH) oxidation using whole cells and purified manganese-lignin peroxidase (MnLiP) from Bjerkandera adusta UAMH 8258. Although the metabolism of PAHs by B. adusta has been previously demonstrated, less than 5% mineralization of 14C-labelled PAHs occurred in this study over a 40-day period. Oxidation of PAHs was examined by a purified MnLiP hybrid isoenzyme in the presence and absence of manganous ions. The rate of PAH oxidation was decreased by the presence of Mn. The substrates were anthracene and its methyl derivatives, pyrene and benzo[a]pyrene, PAHs with ionization potentials of 7.43 eV or lower. The PAH metabolites of the Mn-independent reaction were identified as the corresponding quinones. The pH optimum of the Mn-independent oxidation was generally about 4, while for the Mn-dependent reaction it was 3. The kinetic constants for the Mn-independent oxidation of 2-methylanthracene at pH 4 were determined, and the values we obtained were a kcat of 145/min, KM,app of 23.8 mmol/L for the aromatic substrate, and KM,app of 0.2 mmol/L for hydrogen peroxide. This is the first report of PAH oxidation by a MnLiP hybrid isoenzyme from white rot fungi.     

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.     

Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus.

A variety of simple aromatic compounds were identified in liquid cultures of the basidiomycetes Pleurotus cornucopiae, P. eryngii, P. floridanus, P. pulmonarius, P. ostreatus, and P. sajor-caju by using gas chromatography-mass spectrometry. Such compounds were detected in fungal cultures on lignin- and straw-containing media, but it was found that they were also produced in the absence of aromatic precursors. Anisylic and hydroxybenzylic compounds (such as alcohols, aldehydes, and acids) were identified, p-anisaldehyde being the most characteristic extracellular metabolite synthesized by these ligninolytic fungi. Small amounts of 3-chloro-p-anisaldehyde were also detected in several species. It is postulated that the balance between the more-or-less-oxidized aromatic compounds can be explained in terms of the activity of fungal enzymes, including aryl-alcohol oxidase and dehydrogenase. The former enzyme shows high affinity for p-anisyl alcohol, which is oxidized to p-anisaldehyde with production of H2O2. The aryl-alcohol dehydrogenase was detected only in the mycelium, where it reduces aromatic aldehydes in the presence of NADPH. Both enzymes could be involved in the redox cycling of these aromatic compounds, providing H2O2 to ligninolytic peroxidases.     

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.     

Expression of soluble versatile peroxidase of Bjerkandera adusta in Escherichia coli

Versatile peroxidase from white rot fungus Bjerkandera adusta was over-expressed in a soluble form in Escherichia coli. In the constructed enzyme model based on the selected gene from B. adusta, the active sites for oxidation of Mn(2+) ions and for oxidation of aromatic substrates were identified, both characteristic for versatile peroxidase. For over-expression of the recombinant enzyme different host strains, media formulations, growth temperatures, and fusion partners were tested. With the bacterial strain BL21(DE3)pLysS cultivated at 25 degrees C in auto-induction medium and presence of heme, a soluble peroxidase with incorporated heme and activity against different substrates was obtained. By exploiting an appropriate expression system and providing suitable culture conditions, the recombinant fungal peroxidases in soluble form can be produced in bacteria.