Rapid Method for Detection and Quantitation of Hydroxylated Aromatic Intermediates Produced by Microorganisms

Tetrazotized o-dianisidine was used for quantitative measurements of the amount of 1-naphthol formed from naphthalene by fungi in liquid cultures. The reagent was also used to detect the accumulation of phenols and cis-dihydrodiols in fungal and bacterial colonies on solid media.     

代谢工程在芳香化合物生物合成研究中的应用

生物技术和代谢工程的发展促进了生物合成研究。概述了近年来利用微生物莽草酸途径进行芳香化合物生物合成研究的现况、代谢工程在提高天然芳香化合物产量和扩大合成非天然产生的芳香化合物范围的应用的进展 ,特别是整体代谢工程对提高第二代工程菌产量的作用。指出了生物合成法是生产氨基酸及其它生物小分子如奎尼酸、维生素和抗生素等的未来趋势 ,在工业化生产中有着广阔的应用前景。     

显色培养基在几种食源性致病菌快速检测中的应用

用显色培养基鉴定微生物是一种新的微生物快速检测技术,该技术以生化反应为基础,通过在培养基中加入细菌特异性酶的显色底物直接根据菌落颜色对菌种作出鉴定。常见食源性致病菌检测中,李斯特菌显色培养基（BCM^TM Listeria mormcytogenes,Rapid’LMONO agar．CHROMagar^TM Listeria）、大肠杆菌显色培养基（CHROMagar^TM Ecoli）、沙门氏菌显色培养基（Rambach agar）、金黄色葡萄球菌显色培养基（CHROMagar Staphylococcus aureus）等已被广泛应用于食品、医药和环境监测等领域,极大提高了微生物检测的效率。但是,显色培养基也存在一些假阳（阴）性等问题,其设计尚待优化。     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

微生物代谢尼古丁的研究及其应用

尼古丁是烟叶中最主要的生物碱,也是影响烟叶品质的重要因素,其含量过高不但影响吸食的安全性,而且对环境也会造成危害.有些微生物能在烟叶醇化过程中代谢尼古丁,降低尼古丁的含量,而不影响烟叶原有的品质,并降低其对人类健康的危害.因此,尼古丁降解微生物有着非常好的应用潜力.本文综述了国内外尼古丁代谢微生物的种类、代谢途径、酶学研究、遗传研究以及相关应用研究的进展情况,展示了微生物方法和技术在处理烤烟尼古丁中的应用前景.     

Degradation of Phenolic Compounds and Ring Cleavage of Catechol by Phanerochaete chrysosporium

POL-88, a mutant of the white-rot fungus Phanerochaete chrysosporium, was selected for diminished phenol-oxidizing enzyme activity. A wide variety of phenolic compounds were degraded by ligninolytic cultures of this mutant. With several o-diphenolic substrates, degradation intermediates were produced that had UV spectra consistent with muconic acids. Extensive spectrophotometric and polarographic assays failed to detect classical ring-cleaving dioxygenases in cell homogenates or in extracts from ligninolytic cultures. Even so, a sensitive carrier-trapping assay showed that intact cultures degraded [U-C]catechol to [C]muconic acid, establishing the presence of a system capable of 1,2-intradiol fission. Significant accumulation of [C]muconic acid into carrier occurred only when evolution of CO(2) from [C]catechol was inhibited by treating cultures with excess nutrient nitrogen (e.g., l-glutamic acid) or with cycloheximide. l-Glutamic acid is known from past work to repress the ligninolytic system in P. chrysosporium and to mimic the effect of cycloheximide. The results here indicate, therefore, that the enzyme system responsible for degrading ring-cleavage products to CO(2) turns over faster than does the system responsible for ring cleavage.     

Formation of Catechol Compounds from Phenylalanine and Tyrosine with Isolated Nerve Endings

THERE is much evidence that catecholamines may act as synaptic transmitters in the mammalian brain¹. Enzymatic activities necessary for the synthesis of catecholamines have been located in central neurones¹ and it is generally believed that tyrosine hydroxylase² is the rate limiting enzyme in brain as well as peripheral tissues containing catecholamines³. While it is clear that tyrosine can serve as a precursor of catecholamine synthesis in the brain^{1, 3, 4}, the significance of phenylalanine is problematic. It was believed that the mammalian brain is devoid of enzymatic activity necessary to convert phenylalanine to tyrosine^{6, 7}, while liver is known to be rich in the enzyme phenylalanine hydroxylase⁸. The earlier attempts to demonstrate hydroxylation of phenylalanine in brain tissue may have been unsuccessful due to methodological problems⁹. Recent evidence suggests that tyrosine hydroxylase prepared from peripheral sympathetically innervated tissues or from brain can hydroxylate either phenylalanine or tyrosine⁹. Initially, the rate of hydroxylation of phenylalanine by tyrosine hydroxylase was thought to be as little as 5% that of tyrosine⁹. It has been found recently, however, that structural variations in the pteridine cofactor present in the incubation mixture lead to striking changes in the ability of partially purified tyrosine hydroxylase from bovine adrenal medulla to hydroxylate phenylalanine¹⁰. Thus, tetrahydrobiopterin allowed the hydroxylation of phenylalanine to proceed at least as rapidly as that of tyrosine or faster¹⁰. As the structure of the endogenous pteridine cofactor of tyrosine hydroxylase is not known, it is possible that synthesis of catecholamines from phenylalanine as well as tyrosine could occur in intact neuronal tissues. Evidence has been presented that after the injection of large quantities of ¹⁴C-phenylalanine into the lateral ventricle of the rat brain, small amounts of labelled tyrosine and traces of newly synthesized catecholamines were detected in brain tissues, giving qualitative evidence that catecholamines may be synthesized in brain from phenylalanine in vivo¹¹.     

Aromatic and Volatile Acid Intermediates Observed during Anaerobic Metabolism of Lignin-Derived Oligomers

Anaerobic enrichment cultures acclimated for 2 years to use a ¹⁴C-labeled, lignin-derived substrate with a molecular weight of 600 as a sole source of carbon were characterized by capillary and packed column gas chromatography. After acclimation, several of the active methanogenic consortia were inhibited with 2-bromoethanesulfonic acid, which suppressed methane formation and enhanced accumulation of a series of metabolic intermediates. Volatile fatty acids levels in 2-bromoethanesulfonic acid-amended cultures were 10 times greater than those in the uninhibited, methane-forming consortia with acetate as the predominant component. Furthermore, in the 2-bromoethanesulfonic acid-amended consortia, almost half of the original substrate carbon was metabolized to 10 monoaromatic compounds, with the most appreciable quantities accumulated as cinnamic, benzoic, caffeic, vanillic, and ferulic acids. 2-Bromoethanesulfonic acid seemed to effectively block CH₄ formation in the anaerobic food chain, resulting in the observed buildup of volatile fatty acids and monoaromatic intermediates. Neither fatty acids nor aromatic compounds were detected in the oligolignol substrate before its metabolism, suggesting that these anaerobic consortia have the ability to mediate the cleavage of the β-aryl-ether bond, the most common intermonomeric linkage in lignin, with the subsequent release of the observed constituent aromatic monomers.     

Biosynthesis of cis,cis-Muconic Acid and Its Aromatic Precursors,Catechol and Protocatechuic Acid,from Renewable Feedstocks by Saccharomyces cerevisiae

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.     

Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by Rhodococcus Species

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-¹⁴C]VC was degraded by cell suspensions, with the production of greater than 66% ¹⁴CO₂ and 20% ¹⁴C-aqueous phase products and incorporation of 10% of the ¹⁴C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-¹⁴C]TCE to ¹⁴CO₂, with approximately 40% appearing in ¹⁴C-aqueous phase products and another 10% of ¹⁴C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Asymmetric Biosynthesis of Key Aromatic Intermediates in the Synthesis of an Endothelin Receptor Antagonist

The feasibility of five potential biocatalytic routes were investigated for the chiral synthesis of key intermediates of an experimental endothelin receptor antagonist. Two asymmetric bioreductions of a ketoester and a chlorinated ketone to their corresponding chiral alcohol yielded very encouraging leads. Pichia delftensis (strain MY 1569) and Rhodotorula piliminae (ATCC 32762) were found to respectively bioreduce the esterified ketone and chlorinated substrate to their corresponding (S) alcohol with enantiomeric excesses > 98% and > 99% respectively. When scaled up in laboratory bioreactors (23-liter scale), both processes produced the desired (S) alcohol intermediate with elevated yield, about 88% and 97% for the ketoester and chloroketone respectively. Investigative chemical syntheses employing the (S) ester alcohol showed that unfavorable racemization occurred during the subsequent synthetic steps. However, the use of the (S) chloroalcohol as chiral synthon for the production of the endothelium receptor antagonist was successfully demonstrated at a preparative scale.     

Use of Aromatic Compounds for Growth and Isolation of Zoogloea

Nine Zoogloea strains, were examined for their ability to utilize 35 aromatic compounds. Benzoate, m-toluate, and p-toluate, as well as phenol, o-cresol, m-cresol, and p-cresol, were utilized by eight strains. These strains exhibited meta cleavage of catechol and of methyl-substituted catechols. With the exception of L-tyrosine, none of the aromatic compounds tested supported growth of Z. ramigera ATCC 19623. A medium containing sodium m-toluate was used to isolate 37 zoogloea-forming bacteria from various polluted environments. The isolates were identified as strains of Zoogloea.     

Degradation of Monochlorinated and Nonchlorinated Aromatic Compounds under Iron-Reducing Conditions

The capacity for Fe(sup3+) to serve as an electron acceptor in the microbial degradation of monochlorinated and nonchlorinated aromatic compounds was investigated in anoxic sediment enrichments. The substrates tested included phenol, benzoate, aniline, their respective monochlorinated isomers, o-, m-, and p-cresol, and all six dimethylphenol isomers. Phenol and 2-, 3-, and 4-chlorophenol were utilized by anaerobic microorganisms, with the concomitant reduction of Fe(sup3+) to Fe(sup2+). The amount of Fe(sup2+) produced in the enrichments was 89 to 138% of that expected for the stoichiometric degradation of these substrates to CO(inf2), suggesting complete mineralization at the expense of Fe reduction. Under Fe-reducing conditions, there was initial loss of benzoate and 3-chlorobenzoate but not of 2- or 4-chlorobenzoate. In addition, there was initial microbial utilization of aniline but not of the chloroaniline isomers. There was also initial loss of o-, m-, and p-cresol in our enrichments. None of the dimethylphenol isomers, however, was degraded within 300 days. Furthermore, we tested the capacity of an Fe-reducing, benzoate-grown culture of Geobacter metallireducens GS-15 to utilize monochlorinated benzoates and phenols. G. metallireducens was able to degrade benzoate and phenol but none of their chlorinated isomers, suggesting that the degradation of chlorophenols in our sediment enrichments may be due to novel Fe-reducing organisms that have yet to be isolated.