Preparation of Metabolites of Spironolactone by Microbial Oxygenation

It has been postulated that 3-(3-oxo-7alpha-methylthio-4-androsten-17alpha-yl)propionic acid gamma-lactone (I) is a key intermediate in the metabolism of the anti-aldosterone agent, spironolactone (3-[3-oxo-7alpha-acetylthio-17beta-hydroxy-4-androsten-17alpha-yl] propionic acid gamma-lactone). In the present study it was found that microbial oxygenation of I by Chaetomium cochloides QM 624 gave three metabolites which retained sulfur in their molecules and were found to be identical to the human metabolites of spironolactone.     

Microbial Transformation of Terpenoids. I. Indentification of Metabolites Produced by a Pseudomonad from Citronellal and Citral

A bacterium capable of utilizing citronellal or citral as the sole source of carbon and energy has been isolated from soil by the enrichment culture technique. It metabolizes citronellal to citronellic acid (65%), citronellol (0.6%), dihydrocitronellol (0.6%), menthol (0.75%), and 3,7-dimethyl-1,7-octane diol (1.7%). The metabolites of citral were geranic acid (62%), 6-methyl-5-heptanoic acid (0.5%), 3-methyl-2-butenoic acid (1%), and 1-hydroxy-3, 7-dimethyl-6-octen-2-one (0.75%).     

Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

Recently, high-throughput screening (HTS) has become the mainstream technique for drug discovery. Compounds that are synthesized by combinatorial chemistry might be more suitable than natural products to apply to HTS, because the purification procedure is a drawback of using natural products. Nevertheless, natural products remain an extremely important source of drugs. To overcome the demerits of natural products, we are constructing the RIKEN Natural Products Depository (NPDepo) that is focused primarily on microbial metabolites. In this review, I describe (i) engineering pathways for biosynthetic gene clusters of microbial metabolites, (ii) construction of fraction libraries of microbial metabolites, and (iii) the development of a new screening system using a chemical array and a protein library produced by GLORIA.     

微生物代谢产物库研究进展

微生物代谢物具有极大的化学结构多样性和复杂性,建立微生物代谢物库对发现新药有重要意义。对几种重要的微生物代谢物库及建库方法作一综述。     

Microbial Metabolites with Insecticidal Properties

A screen of fungi for insecticidal activity revealed the ability of Aspergillus versicolor to make versimide, methyl-alpha-(methylsuccinimido)acrylate, a novel contact insecticide. The larvicidal activities of Alternaria tenuis and Fusarium lateritium were found to be due to tenuazonic acid and diacetoxyscirpenol, respectively. Thiolutin, cycloheximide, rubratoxin, patulin, trichothecin, an actinomycin, and scirpene-producing fungi also had insecticidal activity.     

内生放线菌对鬼臼毒素的微生物转化

调查桃儿七根茎内生放线菌对鬼臼毒素的微生物转化,以期获得一些鬼臼毒素的结构类似物或衍生物。利用表面消毒法分离内生放线菌;采用薄层层析和高效液相色谱(HPLC)方法筛选转化鬼臼毒素的内生放线菌;利用硅胶柱层析和制备HPLC分离纯化生物转化产物;应用波谱技术解析转化产物的化学结构;通过形态学、生理生化特征和16S rRNA基因序列分析对内生放线菌进行初步鉴定。从桃儿七根茎中分离出20株内生放线菌,经筛选发现其中1株放线菌能转化鬼臼毒素,其产物为4’-去甲基表鬼臼毒素。初步鉴定该内生放线菌为Streptomyces sp.。内生放线菌Streptomyces sp.能对鬼臼毒素进行去甲基和异构化修饰,推测其可能具有O-去甲基化酶和异构化酶。     

Microbial Transformation of Quercetin by Bacillus cereus

Biotransformation of quercetin was examined with a number of bacterial cultures. In the presence of a bacterial culture (Bacillus cereus), quercetin was transformed into two crystalline products, identified as protocatechuic acid and quercetin-3-glucoside (isoquercitrin).     

Microbial Transformation of Sterols

The ability of accumulating androsta-1, 4-diene-3, 17-dione (ADD) in the digestion of cholesterol by Arthrobacter simplex IAM 1660 was examined with 167 compounds and (i) chelating agents, (ii) Ni²⁺, Co²⁺, Hg²⁺, As³⁺, Sb³⁺, Bi³⁺, Cd²⁺, , and ions, and (iii) redox dyes were found effective for ADD accumulation. Ionic state of the chelating agents was unfavorable for ADD accumulation but inactive ethylaenediamine tetraacetic acid could be turned effective with aid of surface active agents and penicilline. Lipophilic structure of the chelating agents was required probably for its penetration through the cell membrane. The target process of the ADD accumulating agents was supposed as 9α-hydroxylation and their possible mechanism of inhibiting 9α-hydroxylation is discussed.     

Microbial Transformation of Evonine

Arthrobacter citreus ATCC 11624 cleaved selectively only acetyl groups in the polyester alkaloid evonine, whilst the other two hydroxyl groups, esterified with dicarboxylic evonic acid, were retained in the isolated metabolite. Pentadeacetylevonine was identified as the sole product of this microbial transformation, which proceeds quantitatively within 4 days.     

Microbial Transformation of Flavonoids

The ability of a number of fungal spores, and in particular of resting vegetative mycelia, to transform naringin and naringenin was studied. In general, only hydrolytic cleavage of the sugar moieties of naringin to produce prunin and naringenin was observed. Two cultures, Penicillium charlesii and Helminthosporium sativum, also produced two unidentified flavonoid compounds but in very low yields. No transformation of aglycone was detected, although the compound was metabolized by some cultures when supplied as the glycoside prunin. A fluorodensitometric method was developed for the quantitative analysis of flavonoid compounds.     

Microbial Transformation of Sterols

The linear double stranded DNA plasmid pGKLl encodes the yeast killer toxin complex (Gunge et al., 1981) of which the killing mechanism is not understood. We isolated and characterized eight mutants in Saccharomyces cerevisiae that were insensitive to both the intracellularly expressed 28-kDa killer subunit and the native killer toxin complex. These mutations (iki1 through iki5) were all recessive, and classified into five complementation groups. The iki2 mutation was mapped to a position near the centromere on chromosome XIII. We developed a novel screening system to isolate the DNA fragments complementing the iki mutations from a Sccharomyces gene library, and isolated three DNA fragments that complement the ikil, iki3, and iki4 mutations, respectively.     

Microbial Transformation of Antibiotics

Microbial transformation of the nucleoside analogue antibiotic showdomycin was performed using some Streptomyces species. Both the growing culture and the resting cells of Streptomyces sp. No. 383 arrested the antibacterial activity of showdomycin. The inactivated showdomycin was isolated from the reaction mixture by carbon chromatography and was identified with an isoshowdomycin sample which has been chemically derived from showdomycin. It is conjectured that the conversion of showdomycin to isoshowdomycin results from isomerization by an enzyme of Streptomyces sp. No. 383.     

Microbial Transformation of Sterols

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.

Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. The degradation pathway of cholesterol, for example, was shown as follows:

Other sterols such as campesterol, β-sitosterol, stigmasterol and 7-dehydrocholesterol were degraded by the same sequence. The pathway exemplified in cholesterol is considered to be the general degradation pathway of sterols by their decomposing microorganisms.

It was further demonstrated that ADD thus formed from sterols was converted into 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione.     

Microbial Transformation of Evonine

Arthrobacter citreus ATCC 11624 cleaved selectively only acetyl groups in the polyester alkaloid evonine, whilst the other two hydroxyl groups, esterified with dicarboxylic evonic acid, were retained in the isolated metabolite. Pentadeacetylevonine was identified as the sole product of this microbial transformation, which proceeds quantitatively within 4 days.     

微生物代谢产物在心血管疾病中的应用

心血管疾病严重威胁着人类健康,研发防治心血管疾病的有效药物一直是广大研究者研究的焦点。微生物的多种代谢产物在预防和治疗心血管疾病方面得到广泛应用,从微生物代谢产物中寻找预防和治疗心血管疾病的药物是比较传统而且简单的方法,文章就微生物代谢产物在心血管疾病方面的应用进行了阐述。     

多孔板-MTT比色法评价植物和微生物代谢产物的抗真菌活性

多孔板-MTF比色法测定植物和微生物代谢产物对真菌抑制活性的步骤为:在多孔板的每孔中依次加入浓度为105孢子/mL的供试真菌孢子悬液90μL,不同浓度的药液10 μL.25℃暗培养48 h,然后每孔中加入8mg/mL的MTT溶液10μL,继续培养10 h后,离心去上清,加入DMS0 150 μL,振荡30 min,离心后上清液在510nm测定吸光值.采用上述条件测定了白屈菜红碱对稻瘟病菌和西瓜枯萎病菌的MIC值分别为80和1.5μg/mL,IC50值分别为21.99和0.78 μg/mL;Diepoxinζ对稻瘟病菌的MIC和IC50值分别为200和96.21 μg/mL.多孔板-MTT比色法为快速有效地筛选和评价植物和微生物抗真菌活性成分创造了条件.     

RETRACTED ARTICLE: Anti-Inflammatory Effects of Chronic Aspirin on Brain Arachidonic Acid Metabolites

Pro-inflammatory and anti-inflammatory mediators derived from arachidonic acid (AA) modulate peripheral inflammation and its resolution. Aspirin (ASA) is a unique non-steroidal anti-inflammatory drug, which switches AA metabolism from prostaglandin E₂ (PGE₂) and thromboxane B₂ (TXB₂) to lipoxin A₄ (LXA₄) and 15-epi-LXA₄. However, it is unknown whether chronic therapeutic doses of ASA are anti-inflammatory in the brain. We hypothesized that ASA would dampen increases in brain concentrations of AA metabolites in a rat model of neuroinflammation, produced by a 6-day intracerebroventricular infusion of bacterial lipopolysaccharide (LPS). In rats infused with LPS (0.5 ng/h) and given ASA-free water to drink, concentrations in high-energy microwaved brain of PGE₂, TXB₂ and leukotriene B₄ (LTB₄) were elevated. In rats infused with artificial cerebrospinal fluid, 6 weeks of treatment with a low (10 mg/kg/day) or high (100 mg/kg/day) ASA dose in drinking water decreased brain PGE₂, but increased LTB₄, LXA₄ and 15-epi-LXA₄ concentrations. Both doses attenuated the LPS effects on PGE₂, and TXB₂. The increments in LXA₄ and 15-epi-LXA₄ caused by high-dose ASA were significantly greater in LPS-infused rats. The ability of ASA to increase anti-inflammatory LXA₄ and 15-epi-LXA₄ and reduce pro-inflammatory PGE₂ and TXB₂ suggests considering aspirin further for treating clinical neuroinflammation.     

Microbial Anaerobic Demethylation and Dechlorination of Chlorinated Hydroquinone Metabolites Synthesized by Basidiomycete Fungi

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.