Exploration of Zero-Valent Iron Stabilized Calcium-Silicate-Alginate Beads' Catalytic Activity and Stability for Perchlorate Degradation.

Perchlorate contamination in groundwater poses a serious threat to human health, owing to its interference with thyroid function. The high solubility and poor adsorption of perchlorate ions make perchlorate degradation a necessary technology in groundwater contaminant removal. Here, we demonstrate the perchlorate degradation by employing nano zero-valent iron (nZVI) embedded in biocompatible silica alginate hybrid beads fabricated using calcium chloride (1 wt%) as a crosslinker. The concentration of precursors (sodium alginate, sodium silicate) for bead formation was standardized by evaluating the thermal stability of beads prepared at different sodium silicate and alginate concentrations. Thermal degradation of silica alginate hybrid samples showed a stepwise weight loss during the thermal sweep, indicating different types of reactions that occur during the degradation process. The formation of the silica alginate hybrid structure was confirmed by FT-IR spectroscopy. Scanning electron microscopy (SEM) data revealed the surface morphology of silica alginate hybrid changes by varying sodium silicate and alginate concentrations. nZVI-loaded alginate-silicate polymer bead (nZVI-ASB) exhibited excellent perchlorate degradation efficiency by degrading 20 ppm of perchlorate within 4 h. Our study also showed the perchlorate degradation efficiency of nZVI-ASB is maximum at neutral pH conditions.

Gaps-In-Noise Test Performance in Children with Speech Sound Disorder and Cognitive Difficulty.

BACKGROUND AND OBJECTIVES: The Gaps-In-Noise (GIN) test is a clinically effective measure of the integrity of the central auditory nervous system. The GIN procedure can be applied to a pediatric population above 7 years of age. The present study conducted the GIN test to compare the abilities of auditory temporal resolution among typically developing children, children with speech sound disorder (SSD), and children with cognitive difficulty (CD). SUBJECTS AND METHODS: Children aged 8 to 11 years-(total n=30) participated in this study. There were 10 children in each of the following three groups: typically developing children, children with SSD, and children with CD. The Urimal Test of Articulation and Phonology was conducted as a clinical assessment of the children's articulation and phonology. The Korean version of the Wechsler Intelligence Scale for Children-III (K-WISC-III) was administered as a screening test for general cognitive function. According to the procedure of Musiek, the pre-recorded stimuli of the GIN test were presented at 50 dB SL. The results were scored by the approximated threshold and the overall percent correct score (%). RESULTS: All the typically developing children had normal auditory temporal resolution based on the clinical cutoff criteria of the GIN test. The children with SSD or CD had significantly reduced gap detection performance compared to age-matched typically developing children. The children's intelligence score measured by the K-WISC-III test explained 37% of the variance in the percent-correct score. CONCLUSIONS: Children with SSD or CD exhibited poorer ability to resolve rapid temporal acoustic cues over time compared to the age-matched typically developing children. The ability to detect a brief temporal gap embedded in a stimulus may be related to the general cognitive ability or phonological processing.

Simultaneous Determination of Etomidate and Its Major Metabolite, Etomidate Acid, in Urine Using Dilute and Shoot Liquid Chromatography-Tandem Mass Spectrometry.

Etomidate (ET) is a commonly used sedative-hypnotic agent such as propofol to induce anesthesia, and it is rapidly metabolized to etomidate acid (ETA) in liver. Herein, a simple method to determine ET and ETA in urine simultaneously was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A simple sample preparation method reduced the total analysis time. For all analytes, the separation was achieved in 6.5 min using reversed-phase chromatography with gradient elution. The best separation and detection of ETA was achieved using a porous graphitic carbon column. The column temperature was maintained at 30 °C to improve the efficiency and sensitivity. The calibration curves were linear over the concentration ranges of 0.4-120.0 ng/mL (ET) and 1.0-300.0 ng/mL (ETA), obtained with a weighting factor of 1/x2. The coefficients of determination (r2) were greater than 0.9958. The lower limits of quantification were 0.4 ng/mL (ET) and 1.0 ng/mL (ETA), intra-day (n = 6) and inter-day (n = 24) precision values for all compounds were less than 10.2% and 8.4%, respectively, while the intra- and inter-day accuracies were in the -9.9-2.9%, and -7.0-0.6%. The applicability of the method was examined by analyzing the urine samples obtained from ET users.

Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker.

Genome engineering can be used to produce bacterial strains with a wide range of desired phenotypes. However, the incorporation of gene-sized DNA fragments is often challenging due to the intricacy of the procedure, off-target effects, and low insertion efficiency. Here we report a genome engineering method enabling the continuous incorporation of gene-sized double-stranded DNAs (dsDNAs) into the Escherichia coli genome. DNA substrates are inserted without introducing additional marker genes, by synchronously turning an endogenous counter-selectable marker gene ON and OFF. To accomplish this, we utilized λ Red protein-mediated recombination to insert dsDNAs within the promoter region of a counter-selectable marker gene, tolC. By repeatedly switching the marker gene ON and OFF, a number of desired gene-sized dsDNAs can be inserted consecutively. With this method, we successfully inserted approximately 13 kb gene clusters to generate engineered E. coli strains producing 1,4-butanediol (1,4-BDO).

Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria.

Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed.

Tailor-made type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli.

Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct(Cp)) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Ps6-19)). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1( Ps6-19) have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca. 7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue.

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli.

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers.

Polylactic acid (PLA) is a promising biomass-derived polymer, but is currently synthesized by a two-step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl-CoA and incorporation of lactyl-CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB-co-LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome-scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB-co-LA) copolymers containing 55-86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB-co-LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator beta-ketothiolase and acetoacetyl-CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio-based one-step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources.