Biotransformation Experiments Research Articles

Environmental Detoxification of Benzimidazole Fungicide Fuberidazole via Microbial Action in Liquid Cultures

Mitigation of the environment from hazardous pesticides is clamant for all living things. The behavior of the fungicide Fuberidazole was investigated toward biodegradation. Biotransformation experiments were conducted by bacterial strains isolated from soils including, Xanthomonas citri (XC), and Pseudomonas syringae (PS), and fungal strains including, Aspergillus flavus (AF), Aspergillus niger (AN) and Penicillium chrysogenum (PC). Analysis and quantification of Fuberidazole degradation and its metabolites were performed by gas chromatography mass spectrometry and UV-visible spectrophotometry. Pseudomonas syringae and Penicillium chrysogenum displayed great bioremedial potential and degraded 91 and 86% of Fuberidazole after 35 days. Significant metabolites such as Benzimidazole 2-carboxylic acid, Benzimidazole, and 1-(1H-benzimidazole-2-yl)ethanone were produced in the current experiment. The increasing order of half-life for all strains was obtained as, PS (10.3) < PC (10.6) < AN (11) < AF (11.9) = XC (11.9) days. Experimental results demonstrated the capability of bacterial cells to efficiently utilize Fuberidazole as a nutrition source and the significance of favorable environmental conditions for its dissipation.

Sequential Anaerobic-Aerobic Treatment Enhances Sulfamethoxazole Removal: From Batch Cultures to Observations in a Large-Scale Wastewater Treatment Plant.

Sulfamethoxazole (SMX) passes through conventional wastewater treatment plants (WWTPs) mainly unaltered. Under anoxic conditions sulfate-reducing bacteria can transform SMX but the fate of the transformation products (TPs) and their prevalence in WWTPs remain unknown. Here, we report the anaerobic formation and aerobic degradation of SMX TPs. SMX biotransformation was observed in nitrate- and sulfate-reducing enrichment cultures. We identified 10 SMX TPs predominantly showing alterations in the heterocyclic and N4-arylamine moieties. Abiotic oxic incubation of sulfate-reducing culture filtrates led to further degradation of the major anaerobic SMX TPs. Upon reinoculation under oxic conditions, all anaerobically formed TPs, including the secondary TPs, were degraded. In samples collected at different stages of a full-scale municipal WWTP, anaerobically formed SMX TPs were detected at high concentrations in the primary clarifier and digested sludge units, where anoxic conditions were prevalent. Contrarily, their concentrations were lower in oxic zones like the biological treatment and final effluent. Our results suggest that anaerobically formed TPs were eliminated in the aerobic treatment stages, consistent with our observations in batch biotransformation experiments. More generally, our findings highlight the significance of varying redox states determining the fate of SMX and its TPs in engineered environments.

Tracing metabolic route of thymoquinone biosynthesis in diverse Nigella sativa L. cultivars using RP-HPLC technique

Although the therapeutic role of Nigella sativa has been known for a long time, for optimal utilization of plant parts, the developmental profiling of bioactive constituents and their dependence on germplasm diversity need to be assessed.Different cultivars of Nigella sativa(covering 3 samples from northern states of India and 3 reference samples) were taken to explore the relative accumulation of thymol (Thy) and thymoquinone (TQ) in different plant parts, i.e., stem, leaf, bud, flower, fruit, and seeds. Amongst all N. sativa cultivars, the maximum TQ content was found in the seed (45.3885 ± 0.3616 mg/g fresh weight) of the RS cultivar. Seeds of the AN1 cultivar showed the maximum Thy content (2.7853± 0.3535 mg/g fresh weight). The minimum Thy content was found in leaves of Haryana (0.0009 ± 0 mg/g fresh weight) and leaves from RS showed the minimum TQ content (0.0029 ± 0.0001 mg/g fresh weight). There are indications that mature seeds are the best source for both Thy and TQ, followed by fresh seeds collected from the intact fruit of N. sativa. In addition, Thy (precursor) and TQ (product)contentin many plant parts are inversely related to each other, especially in the buds. This indicatedthe interdependence of these two bioactive constituents, but not necessarily in all plant parts. It appears that there might be another functional precursor (in addition to Thy)leading to TQ biosynthesis (speculated to be Carvacrol) in Nigella. Such relative dominance of bioactive compounds and their precursors may be used for designing formulations and setting biotransformation experiments for further value addition.

The Promiscuous Flavin-Dependent Monooxygenase PboD from Aspergillus ustus Increases the Structural Diversity of Hydroxylated Pyrroloindoline Diketopiperazines.

The potential of natural products as pharmaceutical and agricultural agents is based on their large structural diversity, resulting in part from modifications of the backbone structure by tailoring enzymes during biosynthesis. Flavin-dependent monooxygenases (FMOs), as one such group of enzymes, play an important role in the biosynthesis of diverse natural products, including cyclodipeptide (CDP) derivatives. The FMO PboD was shown to catalyze C-3 hydroxylation at the indole ring of cyclo-l-Trp-l-Leu in the biosynthesis of protubonines, accompanied by pyrrolidine ring formation. PboD substrate promiscuity was investigated in this study by testing its catalytic activity toward additional tryptophan-containing CDPs in vitro and biotransformation in Aspergillus nidulans transformants bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes. High acceptance of five CDPs was detected for PboD, especially of those with a second aromatic moiety. Isolation and structure elucidation of five pyrrolidine diketopiperazine products, with two new structures, proved the expected stereospecific hydroxylation and pyrrolidine ring formation. Determination of kinetic parameters revealed higher catalytic efficiency of PboD toward three CDPs consisting of aromatic amino acids than of its natural substrate cyclo-l-Trp-l-Leu. In the biotransformation experiments with the A. nidulans transformant, modest formation of hydroxylated and acetylated products was also detected.

Microbial Transformation of Curcumin and Synergistic Investigation of Its Derivatives with Piperine

AbstractCurcumin is associated with a wide range of pharmacological properties for the prevention and treatment of a wide spectrum of diseases. The clinical use of curcumin is limited due to its poor bioavailability. New methods were tried to overcome its limited utility, and one of these is the synthesis of new derivatives. Additionally adjunct compounds are used to enhance the bioavailability of curcumin. In order to improve its bioavailability, different formulation techniques such as co‐administration with piperine and nanoparticles are among the aids. Within the scope of this study, it is aimed to obtain curcumin derivatives by microbial transformation and to reveal their biological activities. Initially 25 different microbial strains were used for biotransformation experiments. Among the evaluated, Alternaria alternata converted three metabolites; namely, hexahydrocurcumin, demethoxyoctahydrocurcumin, octahydrocurcumin, were obtained with yields of 6.9 %, 2.6 % and 5.9 %, respectively. The derivatives were tested against various bacteria using the broth microdilution method, however no remarkable activity was observed (MIC&gt;250 μg/ml). Thus, combination of piperine was evaulated on the bioavailability of curcumin derivatives. As a result, it was noted that the activities of metabolites, individually with piperine, were higher compared to the synergistic effect demonstrated by curcumin‐piperine against bacteria for the first time.

A cytochrome P450 system initiates 4-nitroanisole degradation in Rhodococcus sp. strain JS3073

Nitroanisoles are used widely as synthetic intermediates and explosives. Although bacteria have been reported to degrade 4-nitroanisole (4NA) under aerobic conditions, the key enzymes and the catalytic mechanism have remained elusive. Rhodococcus sp. strain JS3073 was isolated for its ability to grow on 4NA as the sole carbon and energy source. In this study, whole cell biotransformation experiments indicated that 4NA degradation is initiated by O-demethylation to form 4-nitrophenol (PNP), which undergoes subsequent degradation by a previously established pathway involving formation of 1,2,4-benzenetriol and release of nitrite. Based on comparative transcriptomics and heterologous expression, a novel three-component cytochrome P450 system encoded by pnaABC initiates the O-demethylation of 4NA to yield formaldehyde and PNP. The pnaABC genes encode a phthalate dioxygenase type reductase (PnaA), a cytochrome P450 monooxygenase (PnaB), and an EthD family protein (PnaC) with putative function similar to ferredoxins. This unusual P450 system also has a broad substrate specificity for nitroanisole derivatives. Sequence analysis of PnaAB revealed high identity with multiple self-sufficient P450s of the CYP116B subfamily. The findings revealed the molecular basis of the catabolic pathway for 4NA initiated by an unusual O-demethylase PnaABC and extends the understanding of the diversity among P450s and their electron transport chains.

Occurrence of Per- and Polyfluoroalkyl Substance Contamination of Food Sources and Aquaculture Organisms Used in Aquatic Laboratory Experiments.

When performing basic and translational laboratory studies with aquatic organisms, particularly for bioaccumulation, toxicity, or biotransformation experiments, it is imperative to control the route and dose of exposure. Contamination of feed and the organisms prior to study could alter the results of an experiment. Furthermore, if organisms not exposed in the lab are used for quality assurance/quality control, then blank levels, method detection limits, and limits of quantitation can be affected. In an effort to determine the magnitude of this potential issue for exposure studies involving Pimephales promelas, we analyzed a suite of 24 per- and polyfluoroalkyl substances (PFAS) in four types of feed from three different companies and in organisms from five aquaculture facilities. Contamination with PFAS was found in all types of materials and organisms from all aquaculture farms. The most frequently detected PFAS in fish feed and aquaculture fathead minnows were perfluorocarboxylic acids and perfluorooctane sulfonate (PFOS). Concentrations of total and individual PFAS in feed ranged from nondetect to 76 ng/g and from nondetect to 60 ng/g, respectively. Fathead minnows were contaminated with PFOS and perfluorohexane sulfonate as well as several perflourocarboxylic acids. Concentrations of total and individual PFAS ranged from 1.4 to 351 ng/g and from nondetect to 328 ng/g, respectively. The PFOS measured in food was primarily the linear isomer, consistent with greater bioaccumulation of that isomer in organisms raised as fish food. Future studies are necessary to define the extent of PFAS contamination in aquatic culture facilities and aquaculture production operations. Environ Toxicol Chem 2023;00:1-9. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Aerobic degradation of tetramethyl bisphenol F (TMBPF) with activated sludge: Kinetics and biotransformation products

This study investigated the bio-degradation kinetics of tetramethyl bisphenol F (TMBPF), a non-estrogenic alternative to bisphenol A (BPA). Batch biotransformation experiments were performed whereby samples were inoculated with activated sludge and analysed using liquid chromatography-Orbitrap-tandem mass spectrometry (LC-Orbitrap-MS) utilising two non-targeted workflows (commercial and freely available online) for biotransformation products (BTP) identification. The degradation of TMBPF followed single first-order reaction kinetics and depended on the initial concentration (ci) with faster degradation -kt = 0.16, (half-life = 4.4 days) at lower concentrations ci = 0.1 mg L−1, compared with -kt = 0.02 (half-live = 36.4 days) at ci = 10.0 mg L−1. After 18 days, only 8% of the original TMBPF remained at the lowest tested concentration (0.1 mg L−1). Twelve BTPs were identified, three of which were workflow and one condition-specific. The highest relative quantities of BTPs were observed in nutrient-mineral and mineral media after ten days, while after 14 days, 36 and 31% of TMBPF (ci = 1 mg L−1) remained in the nutrient-mineral and mineral media, respectively. Also, the kinetics of TMBPF and its BTPs were the same with and without an additional carbon source. A newly proposed biodegradation pathway for TMBPF involves cleavage of the methylene bridge, hydroxylation with further oxidation, sulphation, nitrification, nitro reduction with further oxidation, acetylation, and glycine conjugation, providing a deeper insight into the fate of TMBPF during biological wastewater treatment.

Microbial enhanced oil recovery (MEOR) by Pseudomonas sp. under laboratory conditions

The purpose of this work was to propose sustainable solutions for advanced oil recovery by evaluating the ability of the bacterium Pseudomonas sp. in the biotransformation of alkanes, in addition to determining strain growth patterns under extreme conditions. For this, the work was initially carried out under laboratory conditions, in which the crude oil was fractionated to obtain the saturated fraction used in the experiment. The bacterial tolerance to salinity and temperature was also tested to determine the experimental conditions and set up the experiment in regard to these parameters. Additionally, an experiment was performed to produce a biosurfactant through biostimulation. The biotransformation experiment consisted of a triplicate with treatment and a control. For treatments, Erlenmeyers flasks received 100 mL of broth containing the biosurfactant, 10 g (10%) of NaCl, 3% of the strain and 1% of the saturated fraction. Erlenmeyer flasks were incubated at 40 °C and 180 rpm for 18 days with periodic analysis. The results initially showed the bacteria exhibited better tolerance at a temperature of 40 °C, and there was no significant change for the different salinities, which was a nonlimiting parameter. For the final experiment, the bacterial growth analysed by Optical Density (OD). exhibited a low variation, in which the lowest point was in T18 with an absorbance of 0.115 and the highest point was in T6 with an absorbance of 0.149. In the qPCR analysis of the bacterial population, the pattern found was similar to the optical density results, with low variation; the lowest number of copies of the 16S rRNA gene (6.66x 103) was found in T0 and the highest number was found in T12 (7.86x 103). For biotransformation analysis, time 6 was observed to have the highest rate, with 54% oil recovery (C30), followed by 52% (C31) and 51% (C29).

Indole and p-cresol in feces of healthy subjects: Concentration, kinetics, and correlation with microbiome.

Indole and p-cresol are precursors of the most important uremic toxins, generated from the fermentation of amino acids tryptophan and tyrosine by the proteolytic community of intestinal bacteria. The present study focused on the relationship between the microbiome composition, the fecal levels of indole and p-cresol, and their kinetics of generation/degradation in fecal cultures. The concentration of indole and p-cresol, the volatilome, the dry weight, and the amount of ammonium and carbohydrates were analyzed in the feces of 10 healthy adults. Indole and p-cresol widely differed among samples, laying in the range of 1.0-19.5μg/g and 1.2-173.4μg/g, respectively. Higher fecal levels of indole and p-cresol were associated with lower carbohydrates and higher ammonium levels, that are markers of a more pronounced intestinal proteolytic metabolism. Positive relationship was observed also with the dry/wet weight ratio, indicator of prolonged intestinal retention of feces. p-cresol and indole presented a statistically significant negative correlation with OTUs of uncultured Bacteroidetes and Firmicutes, the former belonging to Bacteroides and the latter to the families Butyricicoccaceae (genus Butyricicoccus), Monoglobaceae (genus Monoglobus), Lachnospiraceae (genera Faecalibacterium, Roseburia, and Eubacterium ventriosum group). The kinetics of formation and/or degradation of indole and p-cresol was investigated in fecal slurries, supplemented with the precursor amino acids tryptophan and tyrosine in strict anaerobiosis. The presence of the precursors bursted indole production but had a lower effect on the rate of p-cresol formation. On the other hand, supplementation with indole reduced the net rate of formation. The taxa that positively correlated with fecal levels of uremic toxins presented a positive correlation also with p-cresol generation rate in biotransformation experiments. Moreover other bacterial groups were positively correlated with generation rate of p-cresol and indole, further expanding the range of taxa associated to production of p-cresol (Bacteroides, Alistipes, Eubacterium xylanophylum, and Barnesiella) and indole (e.g., Bacteroides, Ruminococcus torques, Balutia, Dialister, Butyricicoccus). The information herein presented contributes to disclose the relationships between microbiota composition and the production of uremic toxins, that could provide the basis for probiotic intervention on the gut microbiota, aimed to prevent the onset, hamper the progression, and alleviate the impact of nephropaties.

An enzymatic tandem reaction to produce odor-active fatty aldehydes

Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace–solid phase microextraction–gas chromatography–mass spectrometry–olfactometry (HS–SPME–GC–MS–O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò–Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time.Key points• Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes.• Odors of several Z-unsaturated fatty aldehydes are described for the first time.• Potential for industrial production of aldehyde-based odorants from natural sources.Graphical abstract

Wastewater microorganisms impact the micropollutant biotransformation potential of natural stream biofilms

Biotransformation is the most important process removing manmade chemicals from the environment, yet mechanisms governing this essential ecosystem function are underexplored. To understand these mechanisms, we conducted experiments in flow-through systems, by colonizing stream biofilms under different conditions of mixing river water with treated (and ultrafiltered) wastewater. We performed biotransformation experiments with those biofilms, using a set of 75 micropollutants, and could disentangle potential mechanisms determining the biotransformation potential of stream biofilms. We showed that the increased biotransformation potential downstream of wastewater treatment plants that we observed for specific micropollutants contained in household wastewaters (downstream effect) is caused by microorganisms released with the treated effluent, rather than by the in-stream exposure to those micropollutants. Complementary data from 16S rRNA amplicon-sequencing revealed 146 amplicon sequence variants (ASVs) that followed the observed biotransformation patterns. Our results align with findings for community tolerance, and provide clear experimental evidence that microorganisms released with treated wastewater integrate into downstream biofilms and impact crucial ecosystem functions.

Biosynthetic machineries of anthraquinones and bisanthraquinones in Talaromyces islandicus.

Talaromyces islandicus is a unique fungus that produces more than 20 numbers of anthraquinones (AQs) and their dimeric natural products, bisanthraquinones (BQs). These compounds share a 9,10-anthracenedione core derived from emodin. The biosynthetic pathway of emodin has been firmly established, while that of other AQs and BQs is still unclear. In this study, we identified the biosynthetic gene clusters for chrysophanol and skyrin. The function of key modification enzymes was examined by performing biotransformation experiments and in vitro enzymatic reactions with emodin and its derivatives, allowing us to propose a mechanism for the modification reactions. The present study provides insight into the biosynthesis of AQs and BQs in T. islandicus.

Identification of a diarylpentanoid-producing polyketide synthase revealing an unusual biosynthetic pathway of 2-(2-phenylethyl)chromones in agarwood

2-(2-Phenylethyl)chromones (PECs) are the principal constituents contributing to the distinctive fragrance of agarwood. How PECs are biosynthesized is currently unknown. In this work, we describe a diarylpentanoid-producing polyketide synthase (PECPS) identified from Aquilaria sinensis. Through biotransformation experiments using fluorine-labeled substrate, transient expression of PECPS in Nicotiana benthamiana, and knockdown of PECPS expression in A. sinensis calli, we demonstrate that the C6–C5–C6 scaffold of diarylpentanoid is the common precursor of PECs, and PECPS plays a crucial role in PECs biosynthesis. Crystal structure (1.98 Å) analyses and site-directed mutagenesis reveal that, due to its small active site cavity (247 Å3), PECPS employs a one-pot formation mechanism including a “diketide-CoA intermediate-released” step for the formation of the C6–C5–C6 scaffold. The identification of PECPS, the pivotal enzyme of PECs biosynthesis, provides insight into not only the feasibility of overproduction of pharmaceutically important PECs using metabolic engineering approaches, but also further exploration of how agarwood is formed.

Response surface algorithm for improved biotransformation of 1,4-dioxane using Staphylococcus capitis strain AG

The present investigation reports the biotransformation of an endrocrine disrupting agent; 1,4-dioxane through bacterial metabolism. Initially, potential bacterial isolates capable of surviving with minimum 1,4-dioxane were screened from industrial wastewater. Thereafter, screening was done to isolate a bacteria which can biotransform higher concentration (1000 mg/L) of 1,4-dioxane. Morphological and biochemical features were examined prior establishing their phylogenetic relationships and the bacterium was identified as Staphylococcus capitis strain AG. Biotransformation experiments were tailored using response surface tool and predictions were made to elucidate the opimal conditions. Critical factors influencing bio-transformation efficiency such as tetrahydrofuran, availability of 1,4-dioxane and inoculum size were varied at three different levels as per the central composite design for ameliorating 1,4-dioxane removal. Functional attenuation of 1,4-dioxane by S. capitis strain AG were understood using spectroscopic techniques were significant changes in the peak positions and chemical shifts were visualized. Mass spectral profile revealed that 1.5 (% v/v) S. capitis strain AG could completely (∼99%) remove 1000 mg/L 1,4-dioxane, when incubated with 2 μg/L tetrahydrofuran for 96 h. The toxicity of 1,4-dioxane and biotransformed products by S. capitis strain AG were tested on Artemia salina. The results of toxicity tests revealed that the metabolic products were less toxic as they exerted minimal mortality rate after 48 h exposure. Thus, this research would be the first to report the response prediction and precise tailoring of 1,4-dioxane biotransformation using S. captis strain AG.

Elucidation of the Streptoazine Biosynthetic Pathway in Streptomyces aurantiacus Reveals the Presence of a Promiscuous Prenyltransferase/Cyclase

Heterologous expression of a three-gene cluster from Streptomyces aurantiacus coding for a cyclodipeptide synthase, a prenyltransferase, and a methyltransferase led to the elucidation of the biosynthetic steps of streptoazine C (2). In vivo biotransformation experiments proved the high flexibility of the prenyltransferase SasB toward tryptophan-containing cyclodipeptides for regular C-3-prenylation. Furthermore, their corresponding dehydrogenated derivatives prepared by using cyclodipeptide oxidases were also used for prenylation. This study provides an enzyme with high substrate promiscuity from a less explored group of prenyltransferases for potential use to generate prenylated derivatives.

Holistic evaluation of biodegradation pathway prediction: assessing multi-step reactions and intermediate products

The prediction of metabolism and biotransformation pathways of xenobiotics is a highly desired tool in environmental sciences, drug discovery, and (eco)toxicology. Several systems predict single transformation steps or complete pathways as series of parallel and subsequent steps. Their performance is commonly evaluated on the level of a single transformation step. Such an approach cannot account for some specific challenges that are caused by specific properties of biotransformation experiments. That is, missing transformation products in the reference data that occur only in low concentrations, e.g. transient intermediates or higher-generation metabolites. Furthermore, some rule-based prediction systems evaluate the performance only based on the defined set of transformation rules. Therefore, the performance of these models cannot be directly compared. In this paper, we introduce a new evaluation framework that extends the evaluation of biotransformation prediction from single transformations to whole pathways, taking into account multiple generations of metabolites. We introduce a procedure to address transient intermediates and propose a weighted scoring system that acknowledges the uncertainty of higher-generation metabolites. We implemented this framework in enviPath and demonstrate its strict performance metrics on predictions of in vitro biotransformation and degradation of xenobiotics in soil. Our approach is model-agnostic and can be transferred to other prediction systems. It is also capable of revealing knowledge gaps in terms of incompletely defined sets of transformation rules.

Organ Specific Differences in Alteration of Aquaporin Expression in Rats Treated with Sennoside A, Senna Anthraquinones and Rhubarb Anthraquinones.

Senna and rhubarb are often used as routine laxatives, but there are differences in mechanism of action and potential side effects. Here, we studied metabolites of senna anthraquinones (SAQ), rhubarb anthraquinones (RAQ) and their chemical marker, sennoside A (SA), in a rat diarrhea model. In in vitro biotransformation experiments, SAQ, RAQ and SA were incubated with rat fecal flora solution and the metabolites produced were analyzed using HPLC. In in vivo studies, the same compounds were investigated for purgation induction, with measurement of histopathology and Aqps gene expression in six organs. The results indicated that SAQ and RAQ had similar principal constituents but could be degraded into different metabolites. A similar profile of Aqps down-regulation for all compounds was seen in the colon, suggesting a similar mechanism of action for purgation. However, in the kidneys and livers of the diarrhea-rats, down-regulation of Aqps was found in the RAQ-rats whereas up-regulation of Aqps was seen in the SAQ-rats. Furthermore, the RAQ-rats showed lower Aqp2 protein expression in the kidneys, whilst the SA-rats and SAQ-rats had higher Aqp2 protein expression in the kidneys. This may have implications for side effects of SAQ or RAQ in patients with chronic kidney or liver diseases.

Application of immersed silicone rubber membrane module for biocatalytic production of 2-phenylethanol and phenylacetic acid

Natural phenylacetic acid can be produced by the biotransformation of 2-phenylethanol by bacterial strains, such as Gluconobacter oxydans, with incomplete oxidative metabolism. 2-Phenylethanol can be produced by the biotransformation from L-phenylalanine using ordinary yeasts such as Saccharomyces cerevisiae. Both compounds are valuable components in flavor industry, where the demand for substances of natural origin has been increasing in recent years and can be satisfied by microbial production. In this work, we proposed two membrane hybrid systems for extractive bioproduction of 2-phenylethanol consisting of a mechanically stirred tank bioreactor, immersed silicone rubber membrane module for product removal, and equipment for the regeneration of water extractant from the membrane module applying two approaches: 1. extract was led to a fixed bed adsorption column for 2-phenylethanol accumulation; 2. extract was led to an airlift bioreactor with the bacterial strain Gluconobacter oxydans, where 2-phenylethanol was oxidized to phenylacetic acid. Biotransformation experiments performed in the proposed hybrid systems proved its functionality and capability to overcome product inhibition and to provide the desired product in high purity. In second approach, phenylacetic acid, as a product of higher value, was produced directly from L-phenylalanine using two strains of microorganisms in a unique double bioreactor membrane hybrid system.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.