Search

ABSTRACT A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6–25h assay period with detection and determination limits and EC50 values for 17β-estradiol of 2.8ngL−1, 5.9ngL−1, 33.2ngL−1 (nAES-P) and 3.1ngL−1, 6.7ngL−1 and 39.4ngL−1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors. [ABSTRACT FROM AUTHOR]

Abstract: The EstraMonitor has been developed to provide a device suitable for monitoring estrogens in sewage treatment plants, i.e. provide rapid detection in semi-online operation. This monitor is based on recombinant Arxula adeninivorans yeast cells as the biocomponent. The cells were engineered to co-express the human estrogen receptor α (hERα) gene and the inducible phytase (phyK, derived from Klebsiella sp. ASR1) reporter gene under the control of a promoter with estrogen response elements (EREs). The monitor comprises two measuring chambers containing immobilized transgenic yeast cells – a measuring strain with receptor and reporter gene cassettes and a control strain with only the reporter gene cassette. To measure the estrogenic activity, the measuring and control strains are incubated with the samples. In the presence of estrogenic substances, such as 17β estradiol (E2), the phyK gene is expressed and recombinant phytase is secreted into the sample. Any signal in the control strain is due to background activity and is used to correct the measuring strain signal. Phytase dephosphorylates p-aminophenyl phosphate (pAPP) and the level of phytase was quantified by electrochemically reducing the intermediate product, p-aminophenol (pAP). The electrons are detected as current and are proportional to the level of phytase activity. Since E2 concentration is correlated with phytase activity, the estrogenic activity can be calculated. The EstraMonitor can detect the total estrogenic activity in about 4h with a measurement range between 5 and 100ngL−1, and limit of detection of 5.3ngL−1. [Copyright &y& Elsevier]

The two Arxula adeninivorans -based bioassays (A-YGS and A-YGFS) described here provide sensitive and reliable screening for glucocorticoids in aquatic environments. The biocomponents of A-YGS and A-YGFS were constructed to carry the human glucocorticoid receptor ( hGRα ) and the phytase gene ( phyK , derived from Klebsiella sp. ASR1) or a fluorescence dsRED gene (derived from the Discosoma sp.), as reporter genes. The responses of A-YGS and A-YGFS were measured photometrically and spectrofluorometrically respectively. The half effective concentration (EC 50 ) and limit of detection (LoD) values for dexamethasone obtained with A-YGS and A-YGFS were 0.81 and 0.29 μM, and 9.42 and 0.47 μM, respectively. Furthermore, both bioassays exhibited different binding specificities for several natural and synthetic glucocorticoids: A-YGS – corticosterone > cyproterone acetate > mifepristone > dexamethasone > cortisol > methylprednisolone > prednisolone and A-YGFS – cortisol >corticosterone >dexamethasone >prednisolone >betamethasone >methylprednisolone. As proof of principle, A-YGS was used to assay total glucocorticoids in river water, wastewater influent and effluent taken from a wastewater treatment plant in Germany. Glucocorticoids were not detected in both A-YGS and LC–MS/MS in seven of the eight samples, however a dexamethasone equivalent (DEQ) was detected in an influent wastewater sample using A-YGS (0.055 μM), while LC–MS/MS analysis showed that hydrocortisone and cortisone were present at 0.063 and 0.083 nM, respectively. [ABSTRACT FROM AUTHOR]

An Arxula adeninivorans based microbial biosensor has been developed for the determination of pharmaceuticals and chemicals such as omeprazole, lansoprazole, β-naphtoflavone and methylcholanthrene within 5 h using biochemical detection and 4 h and 10 min using amperometric detection. The biosensor consists of genetically modified A. adeninivorans G1212/YRC102-hAhR-hARNT-phyK ( hAhR – human arylhydrocarbon receptor; hARNT – human arylhydrocarbon receptor nuclear translocator; inducible phyK – derived from Klebsiella sp. ASR1) as the biological component and coupled with either a biochemical or an amperometric detection method. The combination between hAh receptor gene, hARNT and the A. adeninivorans -derived glucoseamylase promoter of the reporter gene ( GAA ) containing specific cyp1A1 -derived core sequence created a construct which enabled specific induction by pharmaceuticals. This offers a new cell-based biosensor for the pharmaceutical determination. The half maximum effective concentration (EC 50 ) and the limit of detection (LoD) were found to be 236.13, 95.01 and 174.72, 83.65 μg/l for omeprazole and lansoprazole, respectively. These two pharmaceuticals are among the most widely used internationally. Additionally A. adeninivorans G1212/YRC102-hAhR-hARNT-phyK cells allow the measurement in raw wastewater, i.e. not concentrated, unpurified and untreated which will allow on-site operation in sewage treatment plants. [ABSTRACT FROM AUTHOR]

The novel A-YAS assay for the detection of androgenic activity in liquid samples such as urine has been developed and assessed. The assay is based on transgenic Arxula adeninivorans yeast cells as the bio-component. The cells were engineered to co-express the human androgen receptor ( hAR ) gene and the inducible phytase reporter gene ( phyK , derived from Klebsiella sp. ASR1 ), under the control of an Arxula derived glucoamylase ( GAA ) promoter, which had been modified by the insertion of hormone-responsive elements (HREs). The Arxula transformation/expression platform Xplor®2 was used to select stable mitotic resistance marker free transformants and the most suitable cells were characterized for performance as a sensor bio-component. The assay is easy-to-use, fast (6–25 h) and is currently the most sensitive yeast-based androgen screen with an EC 50 , limit of detection and of quantification values for 5 α -dihydrotestosterone (DHT) of 277.1 ± 53.0, 56.5 ± 4.1 and 76.5 ± 6.7 ng L − 1 , respectively. Furthermore, the assay allows the determination of androgenic and anti-androgenic activity of various compounds such as naturally occurring androgens and estrogens, pharmaceuticals and biocides. The robustness of the A-YAS assay enables it to be used for analysis of complex samples such as urine. The results of the analysis of a number of cattle urine samples achieved by the A-YAS assay correlate well with GC-MS analysis of the same samples. [ABSTRACT FROM AUTHOR]

Abstract: The ‘EstraMonitor’ was developed to provide an automated system for the detection of estrogenic compounds. It can be used for semi-online and continuous monitoring without additional instrumentation. Previously it was used to detect pure estrogenic molecules. In this investigation, the ‘EstraMonitor’ is used to detect the estrogenic activity in five non-pretreated wastewater samples. The ‘EstraMonitor’ employs genetically modified Arxula adeninivorans G1212/YRC102-hERα-phyK yeast cells as the detection component. Both immobilized and non-immobilized A. adeninivorans G1212/YRC102-hERα-phyK cells remained fully functional in a wide range of samples, including samples that contained up to 5% NaCl. The results demonstrate that it is possible to determine the estrogenic effects in environmental samples without the need for sterilization, extraction or concentration. The 17β-estradiol equivalent quotient (bio-EEQ) values in these samples were measured by both biochemical and amperometric methods and were compared to GC–MS analyses (ana-EEQ). The three methods showed a good correlation over a similar detection range. [Copyright &y& Elsevier]

Abstract: A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor α (hERα) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERα dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERα-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5ngl−1 and a determination limit of 10ngl−1 for 17β-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30h. [Copyright &y& Elsevier]