Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

Fungal metabolism and detoxification of fluoranthene.

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate.

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Fungal metabolism of acenaphthene by Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized within 72 h of incubation approximately 64% of the [1,8-14C]acenaphthene added. The radioactive metabolites were extracted with ethyl acetate and separated by thin-layer chromatography and reversed-phase high-performance liquid chromatography. Seven metabolites were identified by 1H nuclear magnetic resonance, UV, and mass spectral techniques as 6-hydroxyacenaphthenone (24.8%), 1,2-acenaphthenedione (19.9%), trans-1,2-dihydroxyacenaphthene (10.3%), 1,5-dihydroxyacenaphthene (2.7%), 1-acenaphthenol (2.4%), 1-acenaphthenone (2.1%), and cis-1,2-dihydroxyacenaphthene (1.8%). Parallel experiments with rat liver microsomes indicated that the major metabolite formed from acenaphthene by rat liver microsomes was 1-acenaphthenone. The fungal metabolism of acenaphthene was similar to bacterial and mammalian metabolism, since the primary site of enzymatic attack was on the two carbons of the five-member ring.     

Fungal metabolism of tert-butylphenyl diphenyl phosphate

The fungal metabolism of tert-butylphenyl diphenyl phosphate (BPDP) was studied. Cunninghamella elegans was incubated with BPDP for 7 days, and the metabolites formed were separated by thin-layer, gas-liquid, or high-pressure liquid chromatography and identified by 1H nuclear magnetic resonance and mass spectral techniques. C. elegans metabolized BPDP predominantly at the tert-butyl moiety to form the carboxylic acid 4-(2-carboxy-2-propyl)triphenyl phosphate. In addition, 4-hydroxy-4'-(2-carboxy-2-propyl)triphenyl phosphate, triphenyl phosphate, diphenyl phosphate, 4-(2-carboxy-2-propyl)diphenyl phosphate, 2-(4-hydroxyphenyl)-2-methyl propionic acid, and phenol were detected. Similar metabolites were found in the 28 fungal cultures which were examined for their ability to metabolize BPDP. Experiments with [14C]BPDP indicated that C. elegans metabolized 70% of the BPDP after 7 days and that the ratio of organic-soluble metabolites to water-soluble metabolites was 8:2. The results indicate that fungi preferentially oxidize BPDP at the alkyl side chain and at the aromatic rings to form hydroxylated derivatives. The trace levels of mono- and diaryl metabolites and the low level of phosphotriesterase activity measured in C. elegans indicate that phosphatase cleavage is a minor pathway for fungal metabolism of BPDP.     

红球菌R04联苯/多氯联苯代谢相关调控蛋白RHOGL007659的生理功能

【目的】研究红球菌(Rhodococcus sp.)R04调控蛋白RHOGL007659的生理功能及其缺陷菌株的代谢特性,初步探究红球菌R04降解联苯的调控机制。【方法】通过基因同源重组敲除红球菌R04联苯代谢相关基因RHOGL007659。比较红球菌R04(野生型)和缺陷型菌株R04Δ7659(基因RHOGL007659缺陷型的R04)在不同碳源培养下的生长情况,HPLC分析R04和R04Δ7659转化联苯的能力。提取R04和R04Δ7659的总RNA,实时荧光定量PCR检测联苯降解关键基因的转录表达。纯化Bph B(联苯降解脱氢酶)和Bph D(联苯降解水解酶),制备多克隆抗体。Western blot分析Bph B和Bph D蛋白在R04和R04Δ7659中的表达水平。【结果】获得了RHOGL007659基因的缺陷型菌株R04Δ7659,与R04相比,R04Δ7659在联苯培养条件下的生物量趋近于零。HPLC分析表明,RHOGL007659基因的缺失使红球菌R04丧失转化联苯的能力。实时荧光定量PCR结果表明,在联苯培养条件下,缺失RHOGL007659后的R04,其联苯降解关键基因均有不同程度的下调表达。Western blot分析显示RHOGL007659缺失后,联苯降解关键酶Bph B和Bph D表达量均降低,这与实时荧光定量PCR结果相一致。【结论】RHOGL007659是红球菌R04联苯降解关键基因簇的调控蛋白,该蛋白对红球菌R04代谢联苯过程具有正调控作用。     

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850.

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.     

The effect of an NADPH-regenerating system on biphenyl metabolism in isolated rat hepatocytes.

Biphenyl 4-hydroxylation was studied in isolated rat hepatocytes. It was found that there was in inter-relationship between 4-hydroxylase activity and glucuronidase activity, removal of 4-hydroxybiphenyl by conjugation being necessary to stimulate a second phase of hydroxylation. Addition of an NADPH-regenerating system resulted in an initial depression of both processes, but later their activities were enhanced. This action could not be explained by the presence of non-viable cells.     

Fungal metabolism and detoxification of the nitropolycyclic aromatic hydrocarbon 1-nitropyrene.

Nitropolycyclic aromatic hydrocarbons are ubiquitous environmental pollutants, many of which are potent mutagens in bacterial and mammalian cells and carcinogenic to rodents. In this study, we investigated the fungal metabolism of 1-nitropyrene and determined the mutagenic activity of the metabolites toward Salmonella typhimurium TA98, TA98NR, and TA100. Cunninghamella elegans metabolized 1-nitropyrene to form glucoside conjugates of 6-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene. The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by application of UV absorption, 1H-nuclear magnetic resonance, and mass spectroscopy. Mutagenicity assays performed on samples extracted from incubations of C. elegans with 1-nitropyrene indicated that mutagenic activity decreased with time. Consistent with the loss in mutagenic activity, the glucoside conjugates of 6- and 8-hydroxy-1-nitropyrene were nonmutagenic in the Salmonella reversion assay. The results indicate that the fungus C. elegans metabolizes 1-nitropyrene to detoxified products.     

Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons

The mutagenic activity of ethyl acetate extracts of culture medium from Cunninghamella elegans incubated 72 h with various polycyclic aromatic hydrocarbons (PAHs) was evaluated in the Salmonella typhimurium reversion assay. All of the PAH extracts were assayed in tester strains TA98 and TA100 both with and without metabolic activation using a liver fraction from Aroclor 1254-treated rats. None of the extracts from fungal incubations with the mutagenic PAHs, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene and benz[a]anthracene, as well as the non-mutagenic PAHs, naphthalene, phenanthrene and anthracene, displayed any appreciable mutagenic activity. In addition, time course experiments indicated that the rate of decrease in mutagenic activity in the extracts from cultures incubated with benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene was coincident with the rate of increase in total metabolism. The results demonstrated the ability of the fungus C. elegans to detoxify known carcinogens and mutagens and suggests that this organism may play an important role in the metabolism and inactivation of PAHs in the environment.Abbreviations hplc high performance liquid chromatography - tlc thin layer chromatography - PAH polycyclic aromatic hydrocarbon     

Microbial metabolism of alkylbenzene sulphonates Fungal metabolism of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate

A study was made of the biodegradation of 1-phenylundecane-p-sulphonate and 1-phenyldodecane-p-sulphonate byCladosporium resinae (CMI 88968) which was capable of growth on a number of such alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of detergent homologues were metabolised by the fungus. The alkyl side-chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidised by a β-oxidation pathway. Three enzymes of the β-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and β-hydroxyacyl-CoA dehydrogenase were identified in detergent-grown cell-free extracts of the fungus. The sulphonate moiety was released as sulphate by a desulphonating enzyme. The combined results of continuous sampling programmes monitored by both TLC and sulphate appearance in the growth medium indicated that desulphonation of the aromatic moiety was an early event in the overall biodegradation of synthetic detergent homologues. The presence of 1-phenylvalerate, 1-phenylpropionate, benzoate,p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on 1-phenylundecane-p-sulphonate was indicated by GLC analysis. Cells grown on 1-phenyldodecane-p-sulphonate were shown to contain 1-phenylhexanoate, 1-phenylbutyrate, phenylacetate,p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. The aromatic nuclei remaining after alkyl side-chain biodegradation were further metabolised by an oxidation sequence involving an “ortho-cleavage” pathway. An overall metabolic pathway for the biodegradation of alkylbenzene sulphonates byCladosporium resinae is proposed.     

Fungal secondary metabolism - from biochemistry to genomics

Much of natural product chemistry concerns a group of compounds known as secondary metabolites. These low-molecular-weight metabolites often have potent physiological activities. Digitalis, morphine and quinine are plant secondary metabolites, whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal secondary metabolites. Although chemically diverse, all secondary metabolites are produced by a few common biosynthetic pathways, often in conjunction with morphological development. Recent advances in molecular biology, bioinformatics and comparative genomics have revealed that the genes encoding specific fungal secondary metabolites are clustered and often located near telomeres. In this review, we address some important questions, including which evolutionary pressures led to gene clustering, why closely related species produce different profiles of secondary metabolites, and whether fungal genomics will accelerate the discovery of new pharmacologically active natural products.     

Fungal metabolism of (-)-deprenyl and pargyline

Deprenyl and pargyline were readily metabolized by Cunninghamella echinulata ATCC 9244 and the products obtained were identified by gas-liquid chromatography and mass spectrometry. Deprenyl was completely metabolized to four products; amphetamine, N-methylamphetamine, 1-phenyl 2-propanone oxime and N-acetylamphetamine. Pargyline metabolism extracts contained substrate and five products; N-propargylbenzylamine, N-hydroxy-N-propargylbenzylamine, N-methylbenzylamine, N-acetylbenzylamine and benzylamine. Substrate concentration influenced the relative amounts of metabolites recovered. C. echinulata is an excellent organism for use as a model of mammalian metabolism.     

Flux control analysis for biphenyl metabolism by Rhodococcus pyridinovorans R04

The flux control coefficients of the four enzymes involved in the upper pathway of biphenyl degradation were determined from transient metabolite concentrations. The first enzyme was indicated as the major rate-limiting step of the pathway with a control coefficient of 0.48. The flux control coefficients of the other three enzymes were 0.03, 0.23 and 0.27, respectively. This is the first experimental evidence of the control step in the pathway of biphenyl degradation using metabolic control analysis.