Detection of low level benzene exposure in supermarket wrappers by urinary muconic acid

Women who use the 'hot wire' and 'cool rod' machines to wrap meat in supermarkets are potentially exposed to low levels of benzene and polycyclic aromatic hydrocarbons present in fumes emitted during the thermal decomposition of the plastic used to wrap meat. In order to evaluate whether the benzene metabolite trans, trans-muconic acid (MA) can be used to monitor these low levels, we collected urine samples from supermarket workers, and assayed the urine for MA. Geometric mean after-shift MA levels were highest for subjects who used the 'hot wire' machine, i.e. > 300 ng mg-1 creatinine (Cr). The corresponding levels for subjects who used the 'cool rod' machine were similar to those for subjects who did not use either type of machine, and were much lower. These results indicate that urinary muconic acid has some potential for use in monitoring benzene exposures of less than 1 part per million (ppm). The study detected very high background MA levels (exceeding 2000 ng mg-1 Cr) in some subjects, suggesting that individuals in the general population without occupational exposure to benzene may have urinary MA levels equivalent to exposure to up to 2 ppm benzene in ambient air. However, since non-benzene sources of the metabolite cannot be completely ruled out as partially responsible for these high levels, the public health significance of this finding is not known at the moment.     

Detection of low level benzene exposure in supermarket wrappers by urinary muconic acid

Women who use the 'hot wire' and 'cool rod' machines to wrap meat in supermarkets are potentially exposed to low levels of benzene and polycyclic aromatic hydrocarbons present in fumes emitted during the thermal decomposition of the plastic used to wrap meat. In order to evaluate whether the benzene metabolite trans, trans-muconic acid (MA) can be used to monitor these low levels, we collected urine samples from supermarket workers, and assayed the urine for MA. Geometric mean after-shift MA levels were highest for subjects who used the 'hot wire' machine, i.e. > 300 ng mg-1 creatinine (Cr). The corresponding levels for subjects who used the 'cool rod' machine were similar to those for subjects who did not use either type of machine, and were much lower. These results indicate that urinary muconic acid has some potential for use in monitoring benzene exposures of less than 1 part per million (ppm). The study detected very high background MA levels (exceeding 2000 ng mg-1 Cr) in some subjects, suggesting that individuals in the general population without occupational exposure to benzene may have urinary MA levels equivalent to exposure to up to 2 ppm benzene in ambient air. However, since non-benzene sources of the metabolite cannot be completely ruled out as partially responsible for these high levels, the public health significance of this finding is not known at the moment.     

Chemical structure and biodegradability of halogenated aromatic compounds. Halogenated muconic acids as intermediates.

Substituted muconic acids were prepared from the corresponding catechols by pyrocatechase II from Pseudomonas sp. B13. The stabilities of substituted muconic acids were compared under different pH conditions. 3-Substituted cis, cis-muconic acids cycloisomerized readily in slightly acidic solutions, whereas 2-chloro- and 2-fluoro-cis,cis-muconic acids were stable under these conditions and could be isolated as crystalline compounds. They were isomerized to the cis, trans-form in highly acidic solution (pH 1), particularly when heated to 80 degrees C. Cycloisomerization of 2-chloro-cis,cis-muconic acid in 75% (v/v) H2SO4 yields 4-carboxymethyl-2-chloro-but-2-en-4-olide (4-chloro-2,5-dihydro-5-oxo-3H-furan-2-ylacetic acid). THe cis,cis-configuration of 2-chloromuconic acid was certified by 1H n.m.r. spectroscopy and by enzymic cycloisomerization. Although the cis,cis-configuration of 2-fluoromuconic acid was confirmed by corresponding spectroscopic data, it was not cycloisomerized by crude extracts or cycloisomerase II preparations from Pseudomonas sp. B13.     

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141 mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.     

Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid.

1. An enzyme for the cycloisomerization of 2- and 3-chloro-cis,cis-muconic acid was isolated from 3-chlorobenzoate-grown cells of Pseudomonas sp. B13. It was named muconate cycloisomerase II, because it could it clearly be differentiated by its Km and Vmax. values from an ordinary muconate cycloisomerase, which functioned in benzoate catabolism and exhibited low activity with the chlorinated substrates. 2-Chloro-cis,cis-muconic acid was converted into trans- and 3-chloro-cis,cis--muconic acid into cis-4-carboxymethylenebut-2-en-4-olide together with dehalogenation. 2. An enzyme was isolated from chlorobenzoate-grown cells, which converted the 4-carboxymethylenebut-2-en-4-olides into maleoylacetic acid.     

Production of catechols and muconic acids from various aromatics by the styrene-degraderRhodococcus rhodochrous NCIMB 13259

Summary Intact cells ofRhodococcus rhodochrous NCIMB 13259 which had been grown in the presence of styrene were used to produce metabolic intermediates from other aromatic substrates in the presence of 3-fluorocatechol as an inhibitor of catechol oxygenase. Toluene and ethylbenzene gave 3-methylcatechol and 3-ethylcatechol respectively. The enol form of 3-acetylcatechol was produced from acetophenone and 1-phenylethanol. Benzoic acid was produced from cinnamic acid. This organism cannot metabolise muconic acids, and methylmuconic acid and ethylmuconic acid accumulated under appropriate conditions. 2-Fluoromuconic acid was also produced from the added 3-fluorocatechol. This organism provides a convenient tool for producing catechols and muconic acids from a variety of substrates without need for gentic manipulation.     

Biotechnological production of muconic acid: current status and future prospects

Muconic acid (MA), a high value-added bio-product with reactive dicarboxylic groups and conjugated double bonds, has garnered increasing interest owing to its potential applications in the manufacture of new functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals. At the very least, MA can be used to produce commercially important bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid. Recently, great progress has been made in the development of biotechnological routes for MA production. This present review provides a comprehensive and systematic overview of recent advances and challenges in biotechnological production of MA. Various biological methods are summarized and compared, and their constraints and possible solutions are also described. Finally, the future prospects are discussed with respect to the current state, challenges, and trends in this field, and the guidelines to develop high-performance microbial cell factories are also proposed for the MA production by systems metabolic engineering.     

Substrate interactions during aerobic biodegradation of benzene.

This study dealt with the interactions with benzene degradation of the following aromatic compounds in a mixed substrate: toluene, o-xylene, naphthalene, 1,4-dimethylnaphthalene, phenanthrene, and pyrrole. The experiment was performed as a factorial experiment with simple batch cultures. The effect of two different types of inocula was tested. One type of inoculum was grown on a mixture of aromatic hydrocarbons; the other was grown on a mixture of aromatic hydrocarbons and nitrogen-, sulfur-, and oxygen-containing aromatic compounds (NSO compounds), similar to some of the compounds identified in creosote waste. The culture grown on the aromatic hydrocarbons and NSO compounds was much less efficient in degrading benzene than the culture grown on only aromatic hydrocarbons. The experiments indicated that toluene- and o-xylene-degrading bacteria are also able to degrade benzene, whereas naphthalene-, 1,,4-dimethylnaphthalene-, and phenanthrene-degrading bacteria have no or very little benzene-degrading ability. Surprisingly, the stimulating effect of toluene and o-xylene was true only if the two compounds were present alone. In combination an antagonistic effect was observed, i.e., the combined effect was smaller than the sum from each of the compounds. The reason for this behavior has not been identified. Pyrrole strongly inhibited benzene degradation even at concentrations of about 100 to 200 micrograms/liter. Future studies will investigate the generality of these findings.     

The metabolism of higher chlorinated benzene isomers.

The metabolism of the isomeric tri- and tetrachlorobenzene isomers, penta- and hexachlorobenzene was investigated in the rabbit. The major urinary metabolites of the isomeric tri- and tetrachlorobenzenes were identified as the corresponding tri- and tetrachlorophenols whose structures were confirmed by chromatographic analyses. The genesis of the formation of metabolites is discussed in terms of their possible arene oxide intermediates in which the NIH shift of a chlorine atom is observed in the oxidation of many of the isomers. Pentachlorobenzene is metabolized to give both pentachlorophenol and a dechlorination-hydroxylation product which was identified as 2,3,4,5-tetrachlorophenol. The hexachlorobenzene substrate did not give any phenolic metabolites.     

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity,number of granulopoietic stem cells and frequency of micronuclei in mice

The effects of benzene and benzene metabolites (hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.