Analysis of Environmental Endocrine Disrupting Activities in Wastewater Treatment Plant Effluents Using Recombinant Yeast Assays Incorporated with Exogenous Metabolic Activation System

Objective To measure the endocrine disrupting chemicals （EDCs） in wastewater and evaluate the EDCs removal efficiencies in the municipal wastewater treatment plants （WWTP）. Methods A battery of in vitro recombinant yeast bioassays incorporated with exogenous metabolic activation system （rat liver preparation, S9 mix） was conducted to assess the estrogen receptor （ER）, androgen receptor （AR）, progesterone receptor （PR）, and thyroid receptor （TR） ant/agonistic activities of effluents collected from Datansha WWTP. Results The indirect estrogenic, anti‐androgenic, anti‐progesteronic, and anti‐thyroidic activities were observed in the influent. The removal efficiencies of EDCs were above 74%, suggesting that the present wastewater treatment processes were good enough to remove most of these indirect endocrine disrupting chemicals. Conclusion The incorporation of exogenous metabolic capacity into the test system was valid for the study of indirect effects on ER, AR, PR, and TR.

LONG-TERM IMMUNOGENICITY AND EFFICACY OF RECOMBINANT YEAST DERIVED HEPATITIS B VACCINE FOR INTERRUPTION OF MOTHER-INFANT TRANSMISSION OF HEPATITIS B VIRUS

Recombinant DNA Yeast-Derived Hepatitis B Vaccine (RYHB vaccine) is comparable to and can replace Plasma-Derived Hepatitis B Vaccine (PHB vaccine) for the prevention of mother-nfant transmission of hepatitis B virus (HBV), but the duration of immune efficacy of RYHB vaccine is not clear. This study indicates the long-term efficacy for the prevention of mother-infant transmission of HBV. One hundred and six neonates born to HBsAg-arrier mothers with HBeAg positive were randomly divided into two groups, one receiving 20 μg per dose of RYHB vaccine and the another receiving 20 μg per dose of PHB vaccine on the day of birth, at 1 month and at 6 months (three times). Physical examination and blood tests were performed for all infants at 6, 12, 24, 36, 48 and 60 months of age. The results showed that the protective efficacies at 6, 12, 24, 36, 48 and 60 months were 67%, 75%, 63%, 62%, 57% and 56%, respectively for the RYHB vaccine group and 58%, 76%, 51%, 41%, 24% and 18%, respectively for the PHB vaccine group. The protective efficacy was notably significant in the last two years. The study indicates that the duration of protective efficacy is over 5 years with RYHB vaccine, being longer than that of PHB vaccine. These recipients of RYHB vaccine showed no side effects, and the vaccine is regarded as safe and effective.

Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section （influents） and 9 from post-treatment section （effluents）, were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 17β-estradiol （E2）, estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Preparation of Red Component in Secondary Metabolites of Recombinant Hansenula anomala N6076 and Its GC-MS Detection

[ Objective] This study aimed to investigate the preparation of red component in secondary metabolites of recombinant Hansenula anomala strain N6076 and its GC-MS detection. [ Method] Thin-layer chromatography method was applied for large-scale preparation of red component in the secondary metabolites of re- combinant H. anomala strain N6076. The red component was dissolved in anhydrous ethanol for GC-MS detection and chemical structure comparison in the data- base to identify its type. [ Resultl The red component is preliminarily identified as a quinane compound, while no compound with exactly the same structure as the red component has been found in WILEY, Nist and Nbs compound libraries. [ Conclusion] This study laid the foundation for further NMR structure identification of the obtained red component and investigation of the relationship between its structure and biolo#cal effects.

Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture.Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts.We have previously established the TREX system which facilitates the transfer,integration and expression of biosynthetic gene clusters in various bacterial hosts.Here,we describe the yTREX system,a new tool adapted for one-step yeast recombinational cloning of gene clusters.We show that with yTREX,Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition.Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning,transfer and expression of the respective biosynthesis genes from Serratia marcescens.Furthermore,the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum,producing pathway metabolites violacein,deoxyviolacein,prodeoxyviolacein,and deoxychromoviridans.Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype.Finally,the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa.All compounds accumulated to substantial titers in the mg range.We thus corroborate here the suitability of P.putida for the biosynthesis of diverse natural products,and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters,applicable for natural compound discovery and combinatorial biosynthesis.

Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome(YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction(PCR) from the plasmid p BS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination(TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using p GF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

In vitro thyroid disrupting effects of organic extracts from WWTPs in Beijing

It is generally known that there are many endocrine disrupting compounds （EDCs） in the effluents from wastewater treatment plants （WWTPs）. Most research has focused on the occurrence of estrogenic or androgenic activities, while ignoring that there are environmental chemicals disrupting thyroid system, which is essential for growth and development in both humans and animals. In the present work, a two-hybrid yeast assay was conducted to evaluate the removal efficiencies of agonistic or antagonistic thyroid receptor （TR） mediated effects in different treatment processes of three WWTPs located in Beijing. We found no TR agonistic, but TR antagonistic activities in all processes from the WWTPs. The TR antagonistic activities in organic extracts of water samples were then calibrated regarding to a known TR-inhibitor, amiodarone hydrochloride （AH）. The observed concentration of TR disrupting substances ranged from 2.35 × 10 8 to 6.19 × 10 7 mol/L AH in Gaobeidian WWTP, 3.76 × 10 8 to 8.75 × 10 8 mol/L AH in Lugouqiao WWTP, and 4.80 × 10 9 to 2.55 × 10 8 mol/L AH in Beixiaohe WWTP. Of the three WWTPs, the removal rates were 92.7%, 42.2%, and 23.1% respectively. Industrial sewage may contain more TR disrupting substances compared with domestic sewage. The recipient waters were found to contain considerable concentrations of TR disrupting substances that may cause adverse effects on the exposed organisms.

In vitro agonistic and antagonistic endocrine disrupting effects of organic extracts from waste water of different treatment processes

Effluents from wastewater treatment plant （WWTP） have been reported to have a broad spectrum of endocrine disrupting compounds （EDCs）. The majority of studies have focused on the occurrence of estrogenic activity, while ignoring nuclear hormone receptors （NRs） pathways. In the present study, a battery of in vitro yeast bioassays and a cell bioassay, including antagonistic and agonistic effects on estrogen receptor （ER）, androgen receptor （AR）, progesterone receptor （PR）, estrogen- related receptor （ERR） and aryl hydrocarbon receptor （AHR）, were conducted to evaluate the removal efficien- cies of EDCs by different treatment processes of a WWTP located in Beijing. Estrogenic, anti-estrogenic, anti- androgenic, anti-progesteronic, anti-ERR and the activa- tion of AHR activities were detected in samples from all treatment processes and the receiving water. The concen- tration of estrogenic contaminants with estradiol （E2） equivalent concentrations ranged from 0.82 x 10-9 to 3.54 x 10 9g Ee_EQ.L-1. The concentration of anti-estrogenic contaminants with 4-hydroxytamoxifen （4-OHT） equiva- lent concentrations ranged from 1.24 × 10-6 to 2.36 x 10-6 g 4-OHT-EQ.L-1. The concentration of anti-androgenic contaminants ranged from 2.21 x 10-s to 3.52 × 10-6g flutamide-EQ. L-1. The concentration of anti-progesteronic contaminants ranged from 3.15 x 10^-5 to 2.71 x 10^-4g RU486-EQ. L-1. The concentration of anti-ERR contami- nants ranged from 7.09 x 10-5 to 6.50 x 104 g 4-OHT-EQ × L^-10. The concentration of AHR activators ranged from 1.7 × 10-10 to 3.4 × 10^-10g TCDD-EQ-L-1. These processes including secondary clarifier, coagulation, as well as coal and sand filtration could eliminated 67.2% of estrogenic contaminants, 47.0% of anti-estrogenic contaminants, 98.3% of anti-androgenic contaminants, 88.4% of anti- progesteronic contaminants, 65.4% of anti-ERR contami- nants and 46.9% of AHR activators. WWTP effluents contain multiple receptor disruptors may have very complex adverse effects on exposed organisms.