INTERACTION OF HYDROXYLATED POLYCYCLIC AROMATIC HYDROCARBONS TO ESTROGEN RECEPTOR

Binding activities and estrogenic/antiestrogenic activities of polycyclic aromatic hydrocarbons (PAHs) and hydroxylated PAHs (OHPAHs) having 2 to 6 rings were evaluated by competition assay and yeast two-hybrid assay expressing human estrogen receptor (hER), respectively. PAHs did not bind to hER and did not show any activity. On the other hand, OHPAHs bound to hER and several OHPAHs showed estrogenic or antiestrogenic activity. Relative estrogenic activity (REP _E ) and antiestrogenic activity (REP _AE ) were calculated from the values of E ₂ and 4-hydroxytamoxyfen as positive controls, respectively. Several OHPAHs having 4 rings showed strongly estrogenic activity. 4-Hydroxybenzo[a]anthracene exhibited the strongest estrogenic activity (REP _E = 7.5 × 10^?3 ) followed by 3-hydroxybenzo[a]anthracene and 2-hydroxychrysene (REP _E = 4.2 × 10^?3 ). Several other 4-ring OHPAHs showed strongly antiestrogenic activity. 3-Hydroxybenzo[c]phenanthrene exhibited the strongest antiestrogenic activity (REP _AE = 190) followed by 2-hydroxybenzo[c]phenanthrene (REP _AE = 69) and 2- hydroxybenz [a]anthracene (REP _AE = 0.42). The results suggested that there is a strong structure – activity relationship.     

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild-type strain MG1655 and an engineered strain for reduced aromatic aldehyde reduction (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24 h. We applied this new strain for the production of para-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared with RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.     

Biodegradation and Metabolic Pathway of 17β-Estradiol by Rhodococcus sp. ED55

Endocrine disrupting compounds (EDCs) in the environment are considered a motif of concern, due to the widespread occurrence and potential adverse ecological and human health effects. The natural estrogen, 17β-estradiol (E2), is frequently detected in receiving water bodies after not being efficiently removed in conventional wastewater treatment plants (WWTPs), promoting a negative impact for both the aquatic ecosystem and human health. In this study, the biodegradation of E2 by Rhodococcus sp. ED55, a bacterial strain isolated from sediments of a discharge point of WWTP in Coloane, Macau, was investigated. Rhodococcus sp. ED55 was able to completely degrade 5 mg/L of E2 in 4 h in a synthetic medium. A similar degradation pattern was observed when the bacterial strain was used in wastewater collected from a WWTP, where a significant improvement in the degradation of the compound occurred. The detection and identification of 17 metabolites was achieved by means of UPLC/ESI/HRMS, which proposed a degradation pathway of E2. The acute test with luminescent marine bacterium Aliivibrio fischeri revealed the elimination of the toxicity of the treated effluent and the standardized yeast estrogenic (S-YES) assay with the recombinant strain of Saccharomyces cerevisiae revealed a decrease in the estrogenic activity of wastewater samples after biodegradation.     

Magnetic polymeric nanoparticles functionalized by mannose‐rhodamine conjugate for detection of E. coli

Mannose‐rhodamine (Rh) conjugate (80% yield) was synthesized in a one‐pot reaction and immobilized onto magnetic polymeric nanoparticles (MPNP; 43% magnetic content) of poly(styrene/divinyl benzene/acrylic acid). The resulting nanoparticles contained MPNP as a substrate, mannose as an E. coli receptor and Rh as a fluorescent signaling unit. TEM imaging clearly demonstrated that multiple mannose‐Rh MPNPs could be captured by E. coli strain ORN178. The fluorescent signal from captured nanoparticles was emitted at 580 nm. These results indicate that mannose‐Rh MPNP offers a simple and rapid strategy for bacterial detection. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2014 , 131, 40012.     

An Engineered Escherichia coli Strain with Synthetic Metabolism for in-Cell Production of Translationally Active Methionine Derivatives

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re-engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans-sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio-orthogonal compounds. Our reprogrammed E. coli strain is capable of the in-cell production of l -azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology-based production.     

Photo-biohydrogen Production by Photosensitization with Biologically Precipitated Cadmium Sulfide in Hydrogen-Forming Recombinant Escherichia coli

An inorganic-biological hybrid system that integrates features of both stable and efficient semiconductors and selective and efficient enzymes is attractive for facilitating the conversion of solar energy to hydrogen. In this study, we aimed to develop a new photocatalytic hydrogen-production system based on Escherichia coli whole-cell genetically engineered as a biocatalysis for highly active hydrogen formation. The photocatalysis part was obtained by bacterial precipitation of cadmium sulfide (CdS), which is a visible-light-responsive semiconductor. The recombinant E. coli cells were sequentially subjected to CdS precipitation and heterologous [FeFe]-hydrogenase synthesis to yield a CdS@E. coli hybrid capable of light energy conversion and hydrogen formation in a single cell. The CdS@E. coli hybrid achieved photocatalytic hydrogen production with a sacrificial electron donor, thus demonstrating the feasibility of our system and expanding the current knowledge of photosensitization using a whole-cell biocatalyst with a bacterially precipitated semiconductor.     

Optimization of the glucose feed rate profile for the production of tryptophan from recombinant E coli

A strain of Escherichia coli was engineered to overproduce L ‐tryptophan. A fed‐batch fermentation process was developed, producing 30.8 ± 1.4 g dm^?3 with a yield on glucose of 0.132 ± 0.010 g g^?1. Specific production rate did not appear to be limited by cloned enzyme activity, but by the carbon flux from central metabolism into the aromatic amino acid pathway. The glucose feed rate profile was modified in an attempt to increase the production rate. Tryptophan production was not affected, but led to glutamic acid excretion at high levels. The high specific glucose consumption rate at the low growth rate led to the high glutamate excretion. A new fermentation process involving modification of the feed profile to limit the formation of by‐products was discovered. The resulting final process increased tryptophan production to 42.3 ± 2.7 g dm^?3 with yield on glucose of 0.176 ± 0.006 g g^?1. The instantaneous yield realized the theoretical maximum for the majority of the fermentation. © 2002 Society of Chemical Industry     

High cell density fermentation via a metabolically engineered Escherichia coli for the enhanced production of succinic acid

BACKGROUND: Succinic acid is a valuable four‐carbon organic chemical with applications in many fields. It was found that cell mass was an important factor in succinic acid production by metabolically engineered Escherichia coli strains. In this work, high cell density fermentation was investigated for succinic acid production by a metabolically engineered strain SD121 with ldhA, pflB, ptsG mutation and heterogenous cyanobacterial ppc overexpression. RESULTS: Under two‐stage cultivation, the controlled DO feeding strategy during the aerobic growth phase facilitated biomass up to a dry cell weight of 19.6 g L^?1, and enhanced succinic acid production in the following anaerobic fermentation phase to a concentration of 116.2 g L^?1. A near theoretical maximum succinic acid yield of 1.73 mol mol^?1 glucose was achieved with an average productivity of 1.55 g L^?1 h^?1. CONCLUSION: The results indicated the potential advantage of high cell density fermentation for improvement of succinic acid production by E. coli. Copyright © 2010 Society of Chemical Industry     

Ultrasound and water-mediated synthesis of bis-thiazoles catalyzed by Fe(SD)3 as Lewis acid-surfactant-combined catalyst

Preparation of bis-aminothiazoles under different conditions including synthesis in EtOH under ultrasound irradiation and also in water in the presence of Fe(SD)₃ as Lewis acid-surfactant-combined catalyst (LASC) under ultrasound irradiation has been studied. The results were compared with the traditional reflux method. Also, the results confirmed the efficiency of the synthesis in water and under ultrasound irradiation technique. Moreover, the antibacterial activity of products was investigated using the well-diffusion method against bacterial strains including Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. All of the products showed good antibacterial activity against M. luteus and E. coli. Most of the products showed antibacterial activity higher than erythromycin against M. luteus, E. coli, and B. subtilis.     

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.     

An Unintentional Discovery of a Fluorogenic DNA Probe for Ribonuclease I

Ribonuclease I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well-known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNase I probe RFA13-1 isolated through in vitro selection with the crude extracellular mixture from Clostridium difficile contaminated with Klebsiella aerogenes as a selection target. Characterization of RFA13-1 reveals that it exhibits high sensitivity to Escherichia coli RNase I with a detection limit of 1.39 pm . Furthermore, RFA13-1 also shows high specificity for RNase I produced only in select bacteria from the Enterobacteriaceae family. As a result, this probe offers a simple tool for RNase I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.     

Production of 2,3-butanediol from diverse saccharides via fermentation

The present study aimed to evaluate the potential use of whey to produce 2,3-BD via the fermentation of lactose and its monosaccharides, glucose and galactose, in a synthetic culture medium (medium 9, M9) using a modified strain of Escherichia coli K12 MG1655 (E. coli JFR12) at a 0.1 L/L (10 vol%) inoculum ratio, 37 °C, atmospheric pressure, an initial pH 7.4, and 100 rpm for 72 h varying the saccharide concentration from 12.5, 25, and 50 g/L. The 2,3-BD yield was ∼80 % of the theoretical yield using 25 g/L of glucose and lactose, corresponding to 0.38 g/g saccharides at a fermentation time of 48 h (glucose) and 72 h (lactose). However, the 2,3-BD yield was halved (0.19 g/g galactose), fermenting 25 g/L of galactose at 48 h. Taking into account these results, two important conclusions were determined: i) E. coli JFR12 could transform galactose into 2,3-BD although its yield was half of the yield observed with glucose at 48 h; and ii) E. coli JFR12 was as efficient as other natural 2,3-BD producers such as Klebsiella species fermenting lactose. However, the E. coli strain has the advantage of being an innocuous strain. To the best of our knowledge, there is no other study presenting the production of 2,3-BD from galactose and lactose with a genetically modified E. coli strain.     

Phyto-SERM Constitutes from Flemingia macrophylla

The methanolic extract of Flemingia macrophylla roots exhibited significant estrogenic activity in the transgenic plant assay system which was comparable to the activity of soybean extract. Utilizing estrogenic activity-guided fractionation, one new compound, fleminigin, together with 23 known compounds were isolated from F. macrophylla roots’ methanolic extract. The structure of the new compound was identified based on intensive spectroscopic analysis and the full spectral data for one of the isolated compounds, flemichin E, was introduced for the first time in the current investigation. The estrogenic and anti-estrogenic activities of the isolated compounds were evaluated revealing that the isolated isoflavonoids may act as partial estrogen agonists, as well as antagonists. Additionally, the anti-inflammatory and the cytotoxic activities of the isolated compounds were studied. These results suggested the potential applications of F. macrophylla extract and its isolated compounds as selective estrogen receptor modulators (SERMs).     

Biokatalytische Ganzzellproduktion des Sesquiterpens Presilphiperfolan-8β-ol in stoffwechseloptimierten Escherichia coli

Fermentative whole-cell production in Escherichia coli offers the option of producing complex natural products such as terpenes from simple, inexpensive, and sustainable carbon sources. The aim of this study was to develop such a process for the synthesis of presilphiperfolan-8β-ol (PSP). The biosynthetic pathway of this tricyclic sesquiterpene alcohol was successfully introduced into E. coli. The resulting multi-plasmid strain was able to produce farnesyl pyrophosphate in vivo via the mevalonate pathway, which was then converted to PSP by the sesquiterpene cyclase BcBOT2. The product, which is mainly secreted into the culture medium, was identified via GC-MS and quantified via GC-FID. The constructed strain produced 10 mg L⁻¹ PSP in 48 h at 20 °C directly from carbon feedstock.     

Manipulating Estrogenic/Anti-Estrogenic Activity of Triphenylethylenes towards Development of Novel Anti-Neoplastic SERMs

Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ERα) agonists or antagonists depending on the target issue. Tamoxifen (TAM) (a non-steroidal triphenylethylene derivative) was the first SERM approved as anti-estrogen for the treatment of metastatic breast cancer. On the hunt for novel SERMs with potential growth inhibitory activity on breast cancer cell lines yet no potential to induce endometrial carcinoma, we designed and synthesized 28 novel TAM analogs. The novel analogs bear a triphenylethylene scaffold. Modifications on rings A, B, and C aim to attenuate estrogenic/anti-estrogenic activities of the novel compounds so they can potentially inhibit breast cancer and provide positive, beneficial estrogenic effects on other tissues with no risk of developing endometrial hyperplasia. Compound 12 (E/Z-1-(2-{4-[1-(4-Chloro-phenyl)-2-(4-methoxy-phenyl)-propenyl]-phenoxy}-ethyl)-piperidine) showed an appreciable relative ERα agonistic activity in a yeast estrogen screen (YES) assay. It successfully inhibited the growth of the MCF-7 cell line with GI₅₀ = 0.6 µM, and it was approximately three times more potent than TAM. It showed no potential estrogenicity on Ishikawa endometrial adenocarcinoma cell line via assaying alkaline phosphatase (AlkP) activity. Compound 12 was tested in vivo to assess its estrogenic properties in an uterotrophic assay in an ovariectomized rat model. Compared to TAM, it induced less increase in wet uterine wet weight and showed no uterotrophic effect. Compound 12 is a promising candidate for further development due to its inhibition activity on MCF-7 proliferation with moderate AlkP activity and no potential uterotrophic effects. The in vitro estrogenic activity encourages further investigations toward potential beneficial properties in cardiovascular, bone, and brain tissues.     

Incorporation of exogenous docosahexaenoic acid into various bacterial phospholipids

Incorporation of exogenous docosahexaenoic acid (DHA) into bacterial phospholipids was examined as a method for DHA-linked phospholipid production. The cultivation of 23 bacterial strains in medium with DHA showed that an eicosapentaenoic acid-producing bacteriumShewanella sp. strain SCRC-2738 (strain SCRC-2738),Bacillus subtilis W23,B. cereus, an Antarctic marine bacterium strain S-7 (strain S-7), photosynthesis bacterium (PSB)Rhodopseudomonas capsulatus utilized for the production of larval marine fish,Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens andEscherichia coli K12 all incorporated DHA into their polar lipids. The polar lipids of the strain SCRC-2738, strain S-7, PSB andE. coli K12 were identified to be phospholipids. DHA was localized at thesn-2 position in the phospholipids of the four strains. Incorporation of exogenous DHA into their phospholipids produced an increase in saturated fatty acids and a decrease in monounsaturated fatty acids exceptE. coli K12. The strain SCRC-2738 incorporated the largest amount of DHA into their phospholipids among the tested bacterial strains in this study: DHA was 16% of the total fatty acids in the phosphatidylethanolamine (PE) and 29% in the phosphatidylglycerol (PG). In the PSB, incorporated DHA was 12% of the total fatty acids in the PE, 10% in the PG and phosophatidylcholine so that the PSB was nutritionally fortified.     

Multiple Site‐Selective Insertions of Noncanonical Amino Acids into Sequence‐Repetitive Polypeptides

A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin‐mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl‐tRNA synthetase/tRNA^CUA pair and the noncanonical amino acid. Elastin‐mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full‐length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein‐based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.     

Synthesis and Evaluation of N‐Phenylpyrrolamides as DNA Gyrase B Inhibitors

ATP‐competitive inhibitors of DNA gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N‐phenylpyrrolamides and evaluated them against DNA gyrase and topoisomerase IV from E. coli and Staphylococcus aureus. Antibacterial activities were studied against Gram‐positive and Gram‐negative bacterial strains. The most potent compound displayed an IC₅₀ of 47 nm against E. coli DNA gyrase, and a minimum inhibitory concentration (MIC) of 12.5 μm against the Gram‐positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux‐pump‐deficient E. coli strain (MIC=6.25 μm ) and against wild‐type E. coli in the presence of efflux pump inhibitor PAβN (MIC=3.13 μm ). Here we describe new findings regarding the structure–activity relationships of N‐phenylpyrrolamide DNA gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.     

Impedimetric microbial biosensor based on single wall carbon nanotube modified microelectrodes for trichloroethylene detection

Contamination of soils and groundwaters with persistent organic pollutants is a matter of increasing concern. The most common organic pollutants are chlorinated hydrocarbons such as perchloroethylene and trichloroethylene (TCE). In this study, we developed a bacterial impedimetric biosensor for TCE detection, based on the immobilization of Pseudomonas putida F1 strain on gold microelectrodes functionalized with single wall carbon nanotubes covalently linked to anti-Pseudomonas antibodies. The different steps of microelectrodes functionalization were characterized by electrochemical impedance and atomic force spectroscopies, and analytical performances of the developed microbial biosensor were determined. The impedimetric biosensor response was linear with TCE concentration up to 150 μg L⁻¹ and a low limit of detection (20 μg L⁻¹) was achieved. No significant loss of signal was observed after 4 weeks of storage at 4 °C in phosphate buffer saline pH 7 (three to four measurements a week). After 5 weeks, 90% of the initial value still remained. cis-1,2-Dichloroethylene and vinylchloride, the main TCE degradation products, did not significantly interfere with TCE. The microbial sensor was finally applied to the determination of TCE in natural water samples spiked at the 30, 50 and 75 μg L⁻¹ levels. Recoveries were very good, ranging from 100 to 103%.     

An Engineered E. coli Strain for Direct in Vivo Fluorination

Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25–30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C−F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-l -methionine (SAM) and fluoride in live E. coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E. coli host that has the potential for future engineering of more elaborate fluorometabolites.