Microbial biotransformation: a tool for drug designing

For centuries microbial biotransformation has proved to be an imperative tool in alleviating the production of various chemicals used in food, pharmaceutical, agrochemical and other industries. In the field of pharmaceutical research and development, biotransformation studies have been extensively applied to investigate the metabolism of compounds (leads, lead candidates, etc.) using animal models. The microbial biotransformation phenomenon is then commonly employed in comparing metabolic pathways of drugs and scaling up the metabolites of interest discovered in these animal models for further pharmacological and toxicological evaluation. Microorganisms can conveniently afford drugs difficult obtained via synthesis. The plethora of reported microbial biotransformations along with its added benefits has already invoked further research in bioconversion of novel and structurally complex drugs. This review alternatively discusses the prospect of microbial biotransformation studies as a significant element ameliorating drug discovery and design in terms of cost-effectiveness, environment protection and greater structural diversity as compared to animal models used to study metabolism. To explicate the microbial biotransformation paradigm in drug designing 3 main areas in this aspect have been analyzed: 1—lead expansion: obtaining pharmacologically improved metabolites from bioactive molecules; 2—biosynthesis of precursors/intermediates involved in the production of bioactive molecules; 3—resolution of racemic mixture to obtain enantiomers possessing different pharmacological profiles.     

Biotransformation of drugs by microbial cultures for predicting mammalian drug metabolism

This review discusses the microbial transformation studies of drugs, correlating them with the corresponding metabolism (biotransformation) in animal systems. Approaches are provided for development of microbial models for mammalian metabolism. Emphasis is placed on the potential of microorganisms to mimic mammalian metabolism and provide ways for structural elucidation and toxicological and pharmacological studies of metabolites. Microorganisms can provide difficult-to-synthesize drugs and assist in identifying metabolic pathways of drugs.     

Cunninghamella--a microbial model for drug metabolism studies--a review

Drug metabolism studies constitute an important and necessary step in the evaluation of drug efficacy and safety. In vivo drug metabolism studies suffer from many disadvantages. Hence there is a rise in validation of in vitro microbial models. This review describes the transformation studies of drugs by the fungus, Cunninghamella and correlating them with the metabolism/biotransformation in animal systems and providing technical methods to develop microbial models. Emphasis is laid on the potential of Cunninghamella fungus to mimic mammalian drug biotransformations and to use as in vitro model for drug metabolism studies and for further toxicological and pharmacological studies of metabolites.     

A commentary on methodological aspects of hydrolysable tannins metabolism in ruminant: a perspective view

Although, the application of tannic acid (TA), gallic acid (GA), natural hydrolysable tannins (HT)-rich ingredients, and HT-rich feeds in ruminant feeding have been explored in order to modify or manipulate microbial activities of digestive tract of animals, the interaction between HT and gastrointestinal microbiota and the fate of HT metabolites (GA, ellagic acid, pyrogallol, resorcinol, phloroglucinol, catechol and urolithin) derived from gastrointestinal microbial HT metabolism in the animal as a whole and animal products are missing. Incomplete biotransformation of HT and TA to GA, pyrogallol, resorcinol, phloroglucinol and other phenolic metabolites is a prevalent phenomenon discovered by researchers who examine the fate of HT metabolites in ruminant. While the rest of fellow researchers do not even examine the fate of HT metabolites and assume the complete biotransformation and fermentation of HT metabolites to volatile fatty acids (VFA). Only three studies have successfully identified the complete biotransformation and fermentation of HT metabolites to VFA in ruminant. The HT metabolites, mostly pyrogallol, produced through incomplete biotransformation of HT have adverse effects on gastrointestinal microbiota and host animal. Lack of awareness regarding the metabolism of HT metabolites and its consequences would compromise ruminant gastrointestinal microbiota, animal welfare, our environment and the power of research papers’ findings. In this perspective paper, I will bring to attention a new angle on the biotransformation and fermentation of HT metabolites in gastrointestinal tract, the role of gastrointestinal microbiota and deficiency of current approach in isolating tannin-degrading bacteria from rumen. Also, suggestions for better monitoring and understanding HT metabolisms in ruminant are presented.     

Biotransformation of Meloxicam by <Emphasis Type="Italic">Cunninghamella blakesleeana</Emphasis>: Significance of Carbon and Nitrogen Source

Influence of carbon and nitrogen source, on biotransformation of meloxicam was studied by employing Cunninghamella blakesleeana NCIM 687 with an aim to achieve maximum transformation of meloxicam and in search of new metabolites. The transformation was confirmed by HPLC and based on LC–MS–MS data and previous reports the metabolites were predicted as 5-hydroxymethyl meloxicam, 5-carboxy meloxicam and a novel metabolite. The quantification of metabolites was performed using HPLC peak areas. The results obtained indicate that glucose as carbon source, ammonium nitrate as nitrogen source, were found to be optimum for maximum transformation of meloxicam. The study suggests the significance of these factors in biotransformation of meloxicam using microbial cultures. The fermentation was scaled up to 1 l level.     

Transformation of Schizandrin into Gomisin A by Use of Microbial O-Demethylation and Chemical Reactions

The transformation of schizandrin (I) into gomisin A (II) was accomplished by use of a combination of biotransformation and chemical reactions. The biotransformation, microbial O-demethylation of I by Cuntiinghamella echinulata var. elegans (ATCC 9245) produced two novel metabolites [3-norschizandrin (IV) and 2-norschizandrin (VI)] and two known metabolites [gomisin T (III) and 13-norschizandrin (V)]. Among those metabolites, compound III was derived to II by the O-demethylation with a Lewis acid in the presence of an acid scavenger, followed by methylenation.     

8-氧化咖啡因和嘧啶类生物碱在普洱熟茶中的存在

应用柱层析分离技术,从普洱熟茶中首次分离到8-氧化咖啡因,嘧啶类生物碱(胸腺嘧啶脱氧核苷、胸腺嘧啶和尿嘧啶) ,黄酮类配糖体(黄杞甙) ,以及简单酚类化合物(1 ,2 ,4-苯三酚、1 ,3-苯二酚和4-甲基-1 ,2-二苯酚)。由于普洱熟茶是由大叶茶经微生物后发酵生产的, 8-氧化咖啡因显然是茶叶中的咖啡因在微生物作用下形成的转化产物。胸腺嘧啶脱氧核苷亦可能是茶叶中的嘧啶类生物碱与微生物中的核苷类化合物在后发酵过程中缩合形成的。二者均为新发现的普洱熟茶的特征性成分。     

Pathways of reductive 2,4‐dinitroanisole (DNAN) biotransformation in sludge

As the use of the insensitive munition compound 2,4‐dinitroanisole (DNAN) increases, releases to the environment may pose a threat to local ecosystems. Little is known about the environmental fate of DNAN and the conversions caused by microbial activity. We studied DNAN biotransformation rates in sludge under aerobic, microaerophilic, and anaerobic conditions, detected biotransformation products, and elucidated their chemical structures. The biotransformation of DNAN was most rapid under anaerobic conditions with H₂ as a cosubstrate. The results showed that the ortho nitro group in DNAN is regioselectively reduced to yield 2‐methoxy‐5‐nitroaniline (MENA), and then the para nitro group is reduced to give 2,4‐diaminoanisole (DAAN). Both MENA and DAAN were identified as important metabolites in all redox conditions. Azo and hydrazine dimer derivatives formed from the coupling of DNAN reduction products in anaerobic conditions. Secondary pathways included acetylation and methylation of amine moieties, as well as the stepwise O‐demethylation and dehydroxylation of methoxy groups. Seven unique metabolites were identified which enabled elucidation of biotransformation pathways. The results taken as a whole suggest that reductive biotransformation is an important fate of DNAN leading to the formation of aromatic amines as well as azo and hydrazine dimeric metabolites. Biotechnol. Bioeng. 2013; 110: 1595–1604. © 2012 Wiley Periodicals, Inc.     

Small bugs, big business: the economic power of the microbe

The versatility of microbial biosynthesis is enormous. The most industrially important primary metabolites are the amino acids, nucleotides, vitamins, solvents, and organic acids. Millions of tons of amino acids are produced each year with a total multibillion dollar market. Many synthetic vitamin production processes are being replaced by microbial fermentations. In addition to the multiple reaction sequences of fermentations, microorganisms are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine chemical industry in the production of single-isomer intermediates. Microbially produced secondary metabolites are extremely important to our health and nutrition. As a group, they have tremendous economic importance. The antibiotic market amounts to almost 30 billion dollars and includes about 160 antibiotics and derivatives such as the beta-lactam peptide antibiotics, the macrolide polyketide erythromycin, tetracyclines, aminoglycosides and others. Other important pharmaceutical products produced by microrganisms are hypocholesterolemic agents, enzyme inhibitors, immunosuppressants and antitumor compounds, some having markets of over 1 billion dollars per year. Agriculturally important secondary metabolites include coccidiostats, animal growth promotants, antihelmintics and biopesticides. The modern biotechnology industry has made a major impact in the business world, biopharmaceuticals (recombinant protein drugs, vaccines and monoclonal antibodies) having a market of 15 billion dollars. Recombinant DNA technology has also produced a revolution in agriculture and has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as means of increasing titers and yields of microbial procresses and in discovery of new drugs. Today, microbiology is a major participant in global industry. The best is yet to come as microbes move into the environmental and energy sectors.     

Diamondoids are not forever: microbial biotransformation of diamondoid carboxylic acids

Oil sands process-affected waters (OSPW) contain persistent, toxic naphthenic acids (NAs), including the abundant yet little-studied diamondoid carboxylic acids. Therefore, we investigated the aerobic microbial biotransformation of two of the most abundant, chronically toxic and environmentally relevant diamondoid carboxylic acids: adamantane-1-carboxylic acid (A1CA) and 3-ethyl adamantane carboxylic acid (3EA). We inoculated into minimal salts media with diamondoid carboxylic acids as sole carbon and energy source two samples: (i) a surface water sample (designated TPW) collected from a test pit from the Mildred Lake Settling Basin and (ii) a water sample (designated 2 m) collected at a water depth of 2 m from a tailings pond. By day 33, in TPW enrichments, 71% of A1CA and 50% of 3EA was transformed, with 50% reduction in EC₂₀ toxicity. Similar results were found for 2 m enrichments. Biotransformation of A1CA and 3EA resulted in the production of two metabolites, tentatively identified as 2-hydroxyadamantane-1-carboxylic acid and 3-ethyladamantane-2-ol respectively. Accumulation of both metabolites was less than the loss of the parent compound, indicating that they would have continued to be transformed beyond 33 days and not accumulate as dead-end metabolites. There were shifts in bacterial community composition during biotransformation, with Pseudomonas species, especially P. stutzeri, dominating enrichments irrespective of the diamondoid carboxylic acid. In conclusion, we demonstrated the microbial biotransformation of two diamondoid carboxylic acids, which has potential application for their removal and detoxification from vast OSPW that are a major environmental threat.     

Androstenedione production by biotransformation of phytosterols

Androstenedione is a key intermediate of microbial steroid metabolism. It belongs to the 17-keto steroid family and is used as starting material for the preparation of different steroids. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis. In this review, various methods of biotransformation of sterols to androstenedione are surveyed. It begins with the history and current research status in this field. The existing methods of chemical and biochemical synthesis are examined. Various issues related to these methods and how researchers have addressed them is presented. Among these, the low solubility of sterols in aqueous systems is a critical problem since it limits the product yield. The main content of this review focuses on new methods of biotransformation that are being investigated. Recent biotechnological advances in this field are presented. The review ends with a note on future perspectives.     

Antimicrobial agents

In the microbial transformation of 2-chloro-4-nitrophenol and its 2-aminotridecane compound salt, 2-chloro-4-nitrophenol was found to be degraded to four metabolites only on using strains resistant to 2-chloro-4-nitrophenol. Sensitive strains lacked this property. In the case of the 2-amino-tridecane compound salt, the first reaction produced by strains resistant to 2-chloro-4-nitrophenol was dissociation of the compound salt into the two initial components, followed by degradation of 2-chloro-4-nitrophenol. Microbial degradation resulted in diminished antifungal activity. The other antimicrobi-ally active component of the compound salt, i.e. 2-aminotridecane, was not affected by biotransformation and kept its original activity.     

Enzymatic biotransformation of terpenes as bioactive agents

The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.     

Relative role of eukaryotic and prokaryotic microorganisms in phenanthrene transformation in coastal sediments

THE RELATIVE ROLE OF EUKARYOTIC VERSUS PROKARYOTIC MICROORGANISMS IN PHENANTHRENE TRANSFORMATION WAS MEASURED IN SLURRIES OF COASTAL SEDIMENT BY TWO DIFFERENT APPROACHES: detection of marker metabolites and use of selective inhibitors on phenanthrene biotransformation. Phenanthrene biotransformation was measured by polar metabolite formation and CO(2) evolution from [9-C]phenanthrene. Radiolabeled metabolites were tentatively identified by high-performance liquid chromatography (HPLC) separation combined with UV/visible spectral analysis of HPLC peaks and comparison to authentic standards. Both yeasts and bacteria transformed phenanthrene in slurries of coastal sediment. Two products of phenanthrene oxidation by fungi, phenanthrene trans-3,4-dihydrodiol and 3-phenanthrol, were produced in yeast-inoculated sterile sediment. However, only products of phenanthrene oxidation typical of bacterial transformation, 1-hydroxy-2-naphthoic acid and phenanthrene cis-3,4-dihydrodiol, were isolated from slurries of coastal sediment with natural microbial populations. Phenanthrene trans-dihydrodiols or other products of fungal oxidation of phenanthrene were not detected in the slurry containing a natural microbial population. A predominant role for bacterial transformation of phenanthrene was also suggested from selective inhibitor experiments. Addition of streptomycin to slurries, at a concentration which suppressed bacterial viable counts and rates of [methyl-H]thymidine uptake, completely inhibited phenanthrene transformation. Treatment with colchicine, at a concentration which suppressed yeast viable counts, depressed phenanthrene transformation by 40%, and this was likely due to nontarget inhibition of bacterial activity. The relative contribution of eukaryotic microorganisms to phenanthrene transformation in inoculated sterile sediment was estimated to be less than 3% of the total activity. We conclude that the predominant degraders of phenanthrene in muddy coastal sediments are bacteria and not eukaryotic microorganisms.     

Biotechnological applications of microbial bioconversions

Modern research has focused on the microbial transformation of a huge variety of organic compounds to obtain compounds of therapeutic and/or industrial interest. Microbial transformation is a useful tool for producing new compounds, as a consequence of the variety of reactions for natural products. This article describes the production of many important compounds by biotransformation. Emphasis is placed on reporting the metabolites that may be of special interest to the pharmaceutical and biotechnological industries, as well as the practical aspects of this work in the field of microbial transformations.     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Biotransformation of albendazole by Cunninghamella blakesleeana: effect of carbon and nitrogen source

A filamentous fungus Cunninghamella blakesleeana was screened for its ability to biotransform the anthelmintic drug albendazole. The fungus produced three metabolites in the presence of the carbon and nitrogen sources studied. The transformation was identified by HPLC and the structures of the transformation products were assigned by LC-MS-MS analysis and on the basis of previous reports. The results indicated that the fungus metabolized albendazole into albendazole sulfoxide (M1), albendazole sulfone (M2) and an N-methyl metabolite of albendazole sulfoxide (M3). The effect of carbon and nitrogen source on the biotransformation of albendazole was significant. Among the carbon and nitrogen sources studied, fructose and urea respectively produced maximum extent of biotransformation in terms of substrate depletion. Among the carbon sources studied, maltose produced a higher percentage of M1 whereas M2 and M3 were produced to their maximum extent in presence of d-fructose in terms of metabolite per unit quantity of biomass. In the case of nitrogen sources, ammonium acetate, calcium nitrate and barium nitrate produced maximum percentage of M1, M2 and M3 respectively. The results reveal that the carbon and nitrogen source significantly influence the microbial transformation both qualitatively and quantitatively.     

PAH biotransformation in terrestrial invertebrates--a new phase II metabolite in isopods and springtails

Soil-living invertebrates are exposed to high concentrations of contaminants accumulating in dead organic matter, such as polycyclic aromatic hydrocarbons (PAHs). The capacity for PAH biotransformation is not equally developed in all invertebrates. In this paper, we compare three species of invertebrates, Porcellio scaber (Isopoda), Eisenia andrei (Lumbricidae) and Folsomia candida (Collembola), for the metabolites formed upon exposure to pyrene. Metabolic products of pyrene biotransformation in extracts from whole animals or isopod hepatopancreas were compared to those found in fish bile (flounder and plaice). An optimized HPLC method was used with fluorescence detection; excitation/emission spectra were compared to reference samples of 1-hydroxypyrene and enzymatically synthesized conjugates. Enzymatic hydrolysis after fractionation was used to demonstrate that the conjugates originated from 1-hydroxypyrene. All three invertebrates were able to oxidize pyrene to 1-hydroxypyrene, however, isopods and collembolans stood out as more efficient metabolizers compared to earthworms. In contrast to fish, none of the invertebrates produced pyrene-1-glucuronide as a phase II conjugate. Both Collembola and Isopoda produced significant amounts of pyrene-1-glucoside, whereas isopods also produced pyrene-1-sulfate. A third, previously unknown, conjugate was found in both isopods and springtails, and was analysed further using electrospray and atmospheric pressure chemical ionisation mass spectrometry. Based on the obtained mass spectra, a new conjugate is proposed: pyrene-1-O-(6"-O-malonyl)glucoside. The use of glucose-malonate as a conjugant in animal phase II biotransformation has not been described before, but is understandable in the microenvironment of soil-living invertebrates. In the earthworm, three other pyrene metabolites were observed, none of which was shared with the arthropods, although two were conjugates of 1-hydroxypyrene. Our study illustrates the great variety of the still unexplored metabolic diversity of invertebrate xenobiotic metabolism.     

Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

Screening of biotransformation products of carvone enantiomers by headspace-SPME/GC-MS

In the course of our continuing work on transformation of monoterpenes by microorganisms, the biotransformation of (+)- and (-)-carvone was carried out. The metabolites formed during microbial transformations were screened using a simple, rapid and efficient technique: Headspace-solid phase microextraction (SPME)/GC-MS. The results as well as the application of this technique are described.