Microbial Biotransformation Research Articles

L‐Tryptophan and Bacterial Modulation of Intestinal and Hepatic Gene Expression

Host and microbial metabolites of L‐Tryptophan (Trp) have been shown in vitro to activate aryl hydrocarbon receptor (AhR) and upregulate its prototypical target genes: Cytochrome P450 (CYP) 1A1 and 1A2. Additionally, the Trp microbial metabolite indole‐3‐propionic acid (IPA) produced by Clostridium sporogenes has been shown to be an activator of pregnane X receptor (PXR) in mouse intestine to maintain mucosal integrity. However, very little is known regarding the effect of Trp and its metabolites on host xenobiotic biotransformation in liver and intestine. The objective of this study was to determine the potential crosstalk between microbial Trp metabolism and host xenobiotic biotransformation. To investigate the effect of Trp on the host expression of drug‐processing genes (DPGs), 60‐day‐old conventional (CV) male C57BL/6J mice were orally gavaged 0, 10, 20, or 40 mg/kg L‐Trp once daily for 6 days. Bacterial 16S rRNA qPCR showed that Trp did not alter the abundance of 5 known Trp‐metabolizing bacteria in intestine of CV mice; however, Trp at all 3 doses down‐regulated Bifidobacterium bifidum, which has been shown to be involved in bile acid metabolism. Unexpectedly, Trp did not up‐regulate Cyp1a1 mRNA in any sections of the intestine; in fact, all 3 doses of Trp decreased Cyp1a1 mRNA in jejunum. Conversely, Trp dose‐dependently up‐regulated the PXR‐target gene Cyp3a11, and the constitutive androstane receptor (CAR)‐target gene Cyp2b10 in duodenum at all doses, as well as the PXR‐target gene multidrug resistance transporter 1a (Mdr1a) at the highest dose. In liver, Trp oral treatment did not affect the mRNAs of the prototypical targeted DPGs for AhR, CAR, PXR, or peroxisome proliferator‐activated receptor α (PPARα), although the CAR mRNA was downregulated at the medium and high doses. Furthermore, control germ‐free (GF) mice have lower expression of Cyp3a11. To test that replacing the Trp microbial metabolite IPA in GF mice normalizes the expression of PXR‐targeted DPGs in liver and intestine, 90‐day‐old male GF C57BL/6 mice were administered 0 or 40 mg/kg IPA p.o. or i.p. once daily for 4 days. Neither route of IPA exposure markedly affected Cyp3a11 mRNA expression in intestine or liver. However, Mdr1a was up‐regulated by IPA in jejunum (p.o.) and large intestine (i.p.). The PPARα‐target gene Cyp4a14 was down‐regulated by IPA (i.p.) in duodenum, whereas Cyp1a2 mRNA was up‐regulated by IPA (p.o.) in liver. To recapitulate the effect of Trp oral exposure on human liver, differentiated HepaRG cells were treated with Trp or IPA at a concentration range from 10 to 250 μM for 24h. IPA, but not Trp, dose‐dependently up‐regulated CYP1A1, 2B6, and 3A4 in HepaRG cells, suggesting that the microbial metabolism of Trp is necessary to modulate a hepatic transcriptional response in DPGs. In conclusion, oral exposure to Trp has minimal effect on DPG expression in mouse liver, and preferably up‐regulates the PXR‐ and CAR‐targeted but not the AhR‐targeted DPGs in mouse intestine. The Trp microbial metabolite IPA does not appear to be a major inducer for PXR‐targeted DPGs in liver or intestine of mice, but it may be important for activating AhR, CAR, and PXR pathways in human hepatocytes.Support or Funding InformationNational Institutes of Health grants ES025708, GM111381, ES019487 and the University of Washington Center for Exposures, Diseases, Genomics, and Environment (P30 ES007033)

Microbial Biotransformation: A Process for Chemical Alterations

Microbial Biotransformation: A Process for Chemical Alterations

Regio‐ and Stereospecific O‐Glycosylation of Phenolic Compounds Catalyzed by a Fungal Glycosyltransferase from Mucor hiemalis

AbstractGlycosylated small molecules are often bioactive and obtained mainly via microbial biotransformation especially by fungi. However, no responsible glycosylation gene/enzyme has yet been uncovered in a filamentous fungus. We report here the first identification of a phenolic glycosyltransferase MhGT1 from Mucor hiemalis. The substrate promiscuity of the new phenolic O‐glycosyltransferase was explored by using phenols from Traditional Chinese Medicinal herbs as substrates. MhGT1 exhibited robust capabilities for the regio‐ and stereospecific O‐glycosylation of 72 structurally diverse drug‐like scaffolds and sterols with uridine diphosphate (UDP) glucose as a sugar donor. Unprecedentedly, MhGT1 showed higher regiospecificities and activities for prenylated phenols than for their non‐prenylated analogues. Computational modelling of MhGT1 uncovered a truncated N‐terminal domain of the enzyme consisting of hydrophobic and charged amino acid residues which contributed to the broad substrate scope and regiospecificity towards prenylated compounds. Our findings expand the ways to obtain new glycosyltransferases and also effectively apply the enzymatic approach to obtain glycosylated compounds in drug discovery.magnified image

Process for Biotransformation of Androsta-4-ene-3, 17-Dione (4-AD) to Androsta-1,4-Diene-3,17-Dione (ADD).

Androsta-1,4-diene-3,17-dione (androstadienedione, ADD) is key intermediate for the organic synthesis of a variety of female sex hormones such as estrone, estradiol, estriol and other related derivatives. De novo synthesis of this molecule is not yet reported in any form of living system, i.e., microbial, plant, and animal. The structural complexities due to presence of several chiral carbon centers create significant hurdles in chemical synthesis of such molecules. Microbe-mediated biotransformation offer a highly reliable, cost-effective, and relatively non hazardous way for commercial manufacturing of steroidal key intermediates. Currently microbial biotransformations are extensively being exploited for large-scale production of basic intermediates such as androstenedione (AD), ADD, and several types of hydroxylated derivatives of androstane compounds. In this chapter several aspects of microbial biotransformation process of AD to ADD are discussed.

Eawag-Soil in enviPath: a new resource for exploring regulatory pesticide soil biodegradation pathways and half-life data.

Developing models for the prediction of microbial biotransformation pathways and half-lives of trace organic contaminants in different environments requires as training data easily accessible and sufficiently large collections of respective biotransformation data that are annotated with metadata on study conditions. Here, we present the Eawag-Soil package, a public database that has been developed to contain all freely accessible regulatory data on pesticide degradation in laboratory soil simulation studies for pesticides registered in the EU (282 degradation pathways, 1535 reactions, 1619 compounds and 4716 biotransformation half-life values with corresponding metadata on study conditions). We provide a thorough description of this novel data resource, and discuss important features of the pesticide soil degradation data that are relevant for model development. Most notably, the variability of half-life values for individual compounds is large and only about one order of magnitude lower than the entire range of median half-life values spanned by all compounds, demonstrating the need to consider study conditions in the development of more accurate models for biotransformation prediction. We further show how the data can be used to find missing rules relevant for predicting soil biotransformation pathways. From this analysis, eight examples of reaction types were presented that should trigger the formulation of new biotransformation rules, e.g., Ar-OH methylation, or the extension of existing rules, e.g., hydroxylation in aliphatic rings. The data were also used to exemplarily explore the dependence of half-lives of different amide pesticides on chemical class and experimental parameters. This analysis highlighted the value of considering initial transformation reactions for the development of meaningful quantitative-structure biotransformation relationships (QSBR), which is a novel opportunity offered by the simultaneous encoding of transformation reactions and corresponding half-lives in Eawag-Soil. Overall, Eawag-Soil provides an unprecedentedly rich collection of manually extracted and curated biotransformation data, which should be useful in a great variety of applications.

Biotransformation of food spice curcumin by gut bacterium Bacillus megaterium DCMB-002 and its pharmacological implications

ABSTRACTThe food additive curcumin shows many benefits for human health but has low solubility and stability, which leads to its low bioavailability. After oral administration, curcumin undergoes biotransformation by gut microbiota in digestive tracts, and thus it is interesting to characterize the biotransformation products of curcumin and assess their bioactivities to help us understand the pharmacology. Herein a bacterial strain of Bacillus megaterium DCMB-002 isolated from mice feces, showed the capability of transforming curcumin to its various derivatives. The tracing high performance liquid chromatogram coupled with quadrupole time-of-flight tandem mass spectra (HPLC-Q-TOF MSn) led to the identification of seven hydrogenation transformation metabolites through reduction, hydroxylation, demethylation and demethoxylation etc, six of which were reported for the first time. The metabolites exhibited moderate antioxidant activity. The findings provided insights into the microbial biotransformation of curcumin and roles of gut microbiota in its pharmacological effects.

Obtaining of 11α-Hydroxyandrost-4-ene-3,17-dione from Natural Sterols.

Two-step one-pot microbial transformation enables obtaining of valuable steroids that are difficult to produce chemically. Here we describe a method for obtaining 11α-hydroxyandrost-4-ene-3,17-dione (11α-HAD) from cheap and available natural sterols (phytosterols or cholesterol).11α-HAD is a primary adrenal steroid in mammals and also a key precursor in the syntheses of halogenated corticoids. Conventional routes for its obtaining are based on chemical synthesis, or microbial hydroxylation of androst-4-ene-3,17-dione (AD). AD in turn is produced primarily with microbial biotransformation of natural sterols by some actinobacteria.Consequent bioconversions of sterols using two microbial strains in one bioreactor vessel without separation and purification of AD provides high yield of 11α-HAD. At the first fermentation step, phytosterol is converted to AD with Mycobacterium neoaurum NRRL 3805B, or relative strains, to yield about 70% (mol/mol). At the second step, AD is almost fully (98%) hydroxylated at the position 11α with Aspergillus ochraceus VKM F-830, or other suitable organisms, in the same bioreactor. At the average, 30% (w/w) of the high-purity crystalline 11α-HAD can be obtained.The method can be exploited for production of 11α-HAD for practical use.

Biosynthesis of polyesters and polyamide building blocks using microbial fermentation and biotransformation

Biopolymers can be a green alternative to fossil-based polymers and can contribute to environmental protection because they are produced using renewable raw materials. Biopolymers are composed of various small subunits (building blocks) that are the intermediates or end products of major metabolic pathways. Most building blocks are secreted directly outside of cells, making downstream processes easier and more economic. These molecules can be extracted from fermentation broth and polymerized to produce a variety of biopolymers, e.g., polybutylene terephthalate, polyethylene terephthalate, polytrimethylene terephthalate, nylon-5,4 and nylon-4,6, with applications in medicine, pharmaceuticals, and textiles. Microbes are unable to naturally produce these types of polymers; thus, the production of building blocks and their polymerization is a fascinating approach for the production of these polymers. In comparison to naturally occurring biopolymers, synthesized polymers have improved and controlled structures and higher purity. The production of monomer units provides a new direction for polymer science because new classes of polymers with unique properties that were not previously possible can be prepared. Furthermore, the engineering of microbes for building-block production is an easy process compared to engineering an entire biopolymer synthesis pathway in a single microbe. Polyesters and polyamide polymers have become an important part of human life, and their demand is increasing daily. In this review, recent approaches and technology are discussed for the production of polyester/polyamide building blocks, i.e., 2-hydroxyisobutyric acid, 3-hydroxypropionic acid, mandelic acid, itaconic acid, adipic acid, terephthalic acid, succinic acid, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, 1,3-butanediol, cadaverine, and putrescine.

Investigation of the Chemocatalytic and Biocatalytic Valorization of a Range of Different Lignin Preparations: The Importance of β-O-4 Content

A set of seven different lignin preparations was generated from a range of organosolv (acidic, alkaline, ammonia-treated, and dioxane-based), ionic liquid, autohydrolysis, and Kraft pretreatments of lignocelluloses. Each lignin was characterized by 2D HSQC NMR spectroscopy, showing significant variability in the β-O-4 content of the different lignin samples. Each lignin was then valorised using three biocatalytic methods (microbial biotransformation with Rhodococcus jostii RHA045, treatment with Pseudomonas fluorescens Dyp1B or Sphingobacterium sp. T2 manganese superoxide dismutase) and two chemocatalytic methods (catalytic hydrogenation using Pt/alumina catalyst, DDQ benzylic oxidation/Zn reduction). Highest product yields for DDQ/Zn valorization were observed from poplar ammonia percolation-organosolv lignin, which had the highest β-O-4 content of the investigated lignins and also gave the highest yield of syringaldehyde (243 mg L–1) when using R. jostii RHA045 and the most enzymatic products using P. f...

Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation.

Some widely prescribed drugs are sparsely metabolized and end up in the environment. They can thus be a focal point of ecotoxicity, either themselves or their environmental transformation products. In this context, we present a study concerning furosemide, a diuretic, which is mainly excreted unchanged. We investigated its biotransformation by two environmental fungi, Aspergillus candidus and Cunninghamella echinulata. The assessment of its ecotoxicity and that of its metabolites was performed using the Microtox test (ISO 11348-3) with Vibrio fischeri marine bacteria. Three metabolites were identified by means of HPLC-MS and 1H/13C NMR analysis: saluamine, a known pyridinium derivative and a hydroxy-ketone product, the latter having not been previously described. This hydroxy-ketone metabolite was obtained with C. echinulata and was further slowly transformed into saluamine. The pyridinium derivative was obtained in low amount with both strains. Metabolites, excepting saluamine, exhibited higher toxicity than furosemide, being the pyridinium structure the one with the most elevated toxic levels (EC50=34.40±6.84mgL-1). These results demonstrate that biotic environmental transformation products may present a higher environmental risk than the starting drug, hence highlighting the importance of boosting toxicological risk assessment related to the impact of pharmaceutical waste.

OPTIMAZTION OF GAMMA-DECALACTONE PRODUCTION BY YEAST YARROWIA LIPOLYTICA USING THE TAGUCHI METHOD

The γ-decalactone is one of the lactones with peachy aroma which has been approved as food additive by FDA. The aim of this study was to optimize media composition and conditions for microbial biotransformation of ricinoleic acid and castor oil as substrates to γ-decalactone using the yeast Yarrowia lipolytica. The Y. lipolytica DSM 3286 strain was used as biotransformation agent in different trails designed by Taguchi method. The highest concentration of γ-decalactone was 62.4 and 52.9 mg/L from 1.5% ricinoleic acid and 2.5% castor oil, respectively. Nitrogen sources exhibited significant effect on the biotransformation. The maximum γ-decalactone production occurred at pH 6. The results showed that the composition of biotransformation medium composition is important for γ-decalactone production.

Microbial biotransformation of DON: molecular basis for reduced toxicity

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 μM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

ChemInform Abstract: Research Advances in Deriving Renewable Energy from Biomass in Wastewater Treatment Plants

AbstractReview: 161 refs.

Discovery of tanshinone derivatives with anti-MRSA activity via targeted bio-transformation

Two potent anti-MRSA tanshinone glycosides (1 and 2) were discovered by targeted microbial biotransformation, along with rapid identification via MS/MS networking. Serial reactions including dehydrogenation, demethylations, reduction, glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447. In addition, tanshinosides B (2) showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 μg/mL. This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.

Potassium Aluminium Sulphate (Alum) Inhibits Growth of Human Axillary Malodor-Producing Skin Flora in Vitro

Introduction: Axillary malodor is caused by microbial biotransformation of non-smelling molecules present in apocrine secretions, into volatile odorous molecules. This study aimed to determine the antimicrobial activities of potassium aluminium salts (alum) against four malodorproducing axillary bacterial flora, as an alternative natural product for reducing axillary malodor. Methods: The antimicrobial activity of alum against axillary bacterial flora [Micrococcus luteus (ATCC 49732) (M. luteus), Staphylococcus epidermidis (ATCC 14990) (S. epidermidis), Corynebacterium xerosis (ATCC BAA-1293) C. xerosis and Bacilus subtilis (ATCC 19659) (B. subtilis)], was tested in vitro using broth dilution method for different concentrations (0.937 – 20mg/mL) on Luria-Bertani broth. Subculture was done to determine colony-forming units (CFUs) and the minimum inhibitory concentrations (MICs). Results: Alum showed excellent inhibitory effects on all tested bacteria. The lowest MIC of alum was against C. xerosis, at 1.88 mg/mL. M. luteus, B. subtilis and S.epidermidis showed a higher MIC of 3.75, 5.00 and 7.50 mg/mL, respectively. All of the tested bacteria were completely inhibited at a concentration of 7.50 mg/mL. Conclusions: This study revealed that alum has excellent antimicrobial effects against axillary malodor -producing bacteria and is recommended to be used either directly by topical application or as an active ingredient in deodorants and antiperspirants.

Fermentative Production of Xylitol: A First Trial on Xylose Bifurcation

Background/Objectives: Xylitol production through chemical processes pathway involves high energy usage and production cost. Alternative method via microbial biotransformation and biocatalyst offer more sustainable and environmental friendly feedstock to be used for xylitol production. Methods: Production of xylitol by Aspergillus niger PY11 using different conditions on 2 carbon source, glucose and xylose, were done for the development of this research. Batch fermentation of A. niger PY11 was conducted for 4 days or 96 hours in temperature set at 30oC and agitation speed of 200 rpm. Samples were taken at 12 hours interval, filtered and analyzed for cell biomass, remaining sugar and D-xylitol concentration. The production of biomass and xylitol was monitored through dry-mass weight of mycelium and by HPLC, respectively. Findings: From the results of the utilization of single carbon source, fermentation of D-xylose produced the highest xylitol yield, which was 0.101 g xylitol/g D-xylose consumed, with the xylitol titre of 1.139 g/l was obtained (equivalent to 0.482 g xylitol/g biomass). However, the highest cell growth was observed when fermentation were conducted using a mixture of D-xylose and D-glucose at the ratio of 3:1, which resulted the biomass yield of 0.239 g biomass/g D-xylose (equivalent to 0.211 g xylitol/g biomass). Total amount of 44.94% of added D-xylose was consumed during the fermentation. Applications/Improvements: This paper shown that the addition of glucose had resulted higher biomass growth of A.niger PY11, thus subsequently increased the bioconversion of xylose to xylitol.

3-Fluorophenmetrazine, a fluorinated analogue of phenmetrazine: Studies on in vivo metabolism in rat and human, in vitro metabolism in human CYP isoenzymes and microbial biotransformation in Pseudomonas Putida and wastewater using GC and LC coupled to (HR)-MS techniques

Wastewater-based epidemiology (WBE) as means to estimate illicit drug and new psychoactive substance (NPS) consumption with spatial and temporal resolution is gaining increasing attention. In order to evaluate a given NPS using WBE, in vivo metabolism and microbial biotransformation of excretion products and unchanged compounds need evaluation. The aims of this study were to identify in vivo phase I and II metabolites of the NPS 3-fluorophenmetrazine (3-FPM) in human and rat urine and study the in vitro contribution of Cytochrome P450 (CYP) isoenzymes in phase I metabolism. Additionally, to study microbial biotransformation products (MBPs) of 3-FPM from incubations in wastewater and in a wastewater isolated Pseudomonas Putida strain. To these aims gas chromatography and liquid chromatography coupled to mass spectrometry were applied. Metabolites and MBPs were isolated from urine and microbial incubations after solid phase extraction and precipitation with or without enzymatic conjungate cleaving. The main transformation pathways were N-oxidation, aryl hydroxylation and subsequent O-methylation, alkyl hydroxylation, oxidation, and degradation of the ethyl-bridge yielding the O/N-bis-dealkylated metabolite, combinations thereof and further glucuronidation or sulfations. The main excretion products in the human urine sample were the unchanged compound and the N-oxide, and the main MBPs were the N-oxide and hydroxylation with subsequent oxidations on the alpha-methyl position. Based on these findings, the proposed strategy for WBE analysis of 3-FPM is quantitative determination of unchanged 3-FPM together with qualitative verification of a number of selected metabolites to verify consumption and rule out discharge.

PWE-061 Dysbiosis in Patients with Bile Acid Diarrhoea (BAD) Demonstrated Using 16S RNA Gene Sequencing

Introduction The gastrointestinal tract harbours a diverse community of approximately 1014 microorganisms comprising 500 to 1000 bacterial species.1 Dysbiosis occurs when pathological imbalances in the gut bacterial community precipitate disease and has been linked to the dysmetabolism of bile acids (BAs) in the gut. 1–2% of primary BAs escape the enterohepatic circulation and undergo microbial biotransformation in the large bowel to form the secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA).2 BA metabolites as a result of microbial transformations act as signalling molecules via the Farnesoid X receptor (FXR). Activation of the ileal FXR stimulates expression of Fibroblast Growth Factor 19 (FGF-19), which binds hepatic FGF-4 and activates JNK1/2 and ERK1/2 to impede the physiological feedback of BA synthesis and maintain a functional BA pool.3 The degree of activation of BA receptors is influenced primarily by the gut microbiota and therefore dysbiosis may result in abnormal BA modification leading to the development of gastrointestinal disease. To enhance our understanding of gut dysbiosis in patients with BAD, 16 S rRNA gene sequencing was undertaken to investigate bacterial communities from faecal samples in patients with BAD and IBS. Methods 35 patients (20 with BAD and 15 with IBS) were recruited from the nuclear medicine department at University Hospitals Coventry and Warwickshire NHS Trust after referral from the gastroenterology clinic for a SeHCAT scan to investigate chronic diarrhoea. The patients with BAD had either Type 1 with a diagnosis of Crohn’s disease, type 2 or type 3 post cholecystectomy. Stool samples were stored at -80oC within two hours of collection and then defrosted for 45 minutes at room temperature prior to analysis. 16 s RNA sequencing was undertaken by isolating DNA from stool samples using the QIAamp Fast DNA Stool Extraction kit. V3-V4 primers and extensor ready mix (Thermo scientific) were used to amplify the 16 s rRNA gene sequences from isolated metagenomic DNA. The eluted DNA was then quantified using the broad-range Qubit kit. DNA was analysed by electrophoresis on 1% agarose gels and purified post-PCR. The DNA was then denatured and sequenced on a MiSeq using the Illumina Miseq V2 2x250 bp paired end protocol. Bioinformatic statistical analysis was undertaken using QUIIME and the UPARSE pipeline. Results The rarefaction plots showed reduced diversity in BAD patients compared with IBS; p = 0.0007216. This was also confirmed using the Shannon’s diversity index. The abundance of certain taxa namely Lachnospiraceae (p = 0.00134), Rumiinococcus (p = 0.0034) and Prevotella (p = 0.0046) were reduced in patients with BAD. Conclusion We have shown that diversity in those with BAD is significantly reduced compared to those with IBS with certain phyla predominating. References 1 Gerard P. Metabolism of Cholesterol and Bile Acids by the Gut Microbiota. Pathogens 2014;3:14–24. 2. Vlahcevic ZR, Heuman DM, Hylemon PB. Physiology and pathophysiology of enterohepatic circulation of bile acids. In D. Zakim and T. Boyer, editors. Hepatology: A Textbook of Liver Disease. 1996, 3rd edition. Vol. 1. Saunders, Philadelphia, PA. 376–417 3 Tsuei J, Chau T, Mills D, et al. Bile acid dysregulation, gut dysbiosis, and gastrointestinal cancer. Exp Biol Med (Maywood). 2014 Nov;239(11):1489–504. Disclosure of Interest None Declared

Single step biotransformation of corn oil phytosterols to boldenone by a newly isolated Pseudomonas aeruginosa

A new potent Pseudomonas aeruginosa isolate capable for biotransformation of corn oil phytosterol (PS) to 4-androstene-3, 17-dione (AD), testosterone (T) and boldenone (BOL) was identified by phenotypic analysis and 16S rRNA gene sequencing. Sequential statistical strategy was used to optimize the biotransformation process mainly concerning BOL using Factorial design and response surface methodology (RSM). The production of BOL in single step microbial biotransformation from corn oil phytosterols by P. aeruginosa was not previously reported. Results showed that the pH concentration of the medium, (NH4)2SO4 and KH2PO4 were the most significant factors affecting BOL production. By analyzing the statistical model of three-dimensional surface plot, BOL production increased from 36.8% to 42.4% after the first step of optimization, and the overall biotransformation increased to 51.9%. After applying the second step of the sequential statistical strategy BOL production increased to 53.6%, and the overall biotransformation increased to 91.9% using the following optimized medium composition (g/l distilled water) (NH4)2SO4, 2; KH2PO4, 4; Na2HPO4. 1; MgSO4·7H2O, 0.3; NaCl, 0.1; CaCl2·2H2O, 0.1; FeSO4·7H2O, 0.001; ammonium acetate 0.001; Tween 80, 0.05%; corn oil 0.5%; 8-hydroxyquinoline 0.016; pH 8; 200rpm agitation speed and incubation time 36h at 30°C. Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values.

Microbial conversion of ginsenoside Rd from Rb1 by the fungus mutant Aspergillus niger strain TH-10a

ABSTRACTGinsenoside Rd, one of the ginsenosides with significant pharmaceutical activities, is getting more and more attractions on its biotransformation. In this study, a novel fungus mutant, the Aspergillus niger strain TH-10a, which can efficiently convert ginsenoside Rd from Rb1, was obtained through screening survival library of LiCl and ultraviolet (UV) irradiation. The transformation product ginsenoside Rd, generated by removing the outer glucose residue from the position C20 of ginsenoside Rb1, was identified through high-performance liquid chromatography (HPLC) analysis. Factors for the microbial culture and biotransformation were investigated in terms of the carbon sources, the nitrogen sources, pH values, and temperatures. This showed that maximum mycelia growth could be obtained at 28°C and pH 6.0 with cellobiose and tryptone as the carbon source and the nitrogen source, respectively. The highest transformation rate (∼86%) has been achieved at 32°C and pH 5.0 with the feeding time of substrate 48 hr. Also, Aspergillus niger strain TH-10a could tolerate even 40 mg/mL ginseng root extract as substrate with 60% bioconversion rate after 72 hr of treatment at the optimal condition. Our results highlight a novel ginsenoside Rd transformation fungus and illuminate its potentially practical application in the pharmaceutical industries.