Overexpression of Mutant Galactose Permease (ScGal2_N376F) Effective for Utilization of Glucose/Xylose or Glucose/ Galactose Mixture by Engineered Kluyveromyces marxianus.

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

Alleviation of catabolite repression in Kluyveromyces marxianus: the thermotolerant SBK1 mutant simultaneously coferments glucose and xylose.

BACKGROUND: Simultaneous cofermentation of glucose and xylose mixtures would be a cost-effective solution for the conversion of cellulosic biomass to high-value products. However, most yeasts ferment glucose and xylose sequentially due to glucose catabolite repression. A well known thermotolerant yeast, Kluyveromyces marxianus, was selected for this work because it possesses cost-effective advantages over Saccharomyces cerevisiae for biofuel production from cellulosic biomass. RESULTS: In the present study, we employed a directed evolutionary approach using 2-deoxyglucose to develop a thermotolerant mutant capable of simultaneous cofermentation of glucose and xylose by alleviating catabolite repression. The selected mutant, K. marxianus SBK1, simultaneously cofermented 40 g/L glucose and 28 g/L xylose to produce 23.82 g/L ethanol at 40 °C. This outcome corresponded to a yield of 0.35 g/g and productivity of 0.33 g/L h, representing an 84% and 129% improvement, respectively, over the parental strain. Interestingly, following mutagenesis the overall transcriptome of the glycolysis pathway was highly downregulated in K. marxianus SBK1, except for glucokinase-1 (GLK1) which was 21-fold upregulated. Amino acid sequence of GLK1 from K. marxianus SBK1 revealed three amino acid mutations which led to more than 22-fold lower enzymatic activity compared to the parental strain. CONCLUSIONS: We herein successfully demonstrated that the cofermentation of a sugar mixture is a promising strategy for the efficient utilization of cellulosic biomass by K. marxianus SBK1. Through introduction of additional biosynthetic pathways, K. marxianus SBK1 could become a chassis-type strain for the production of fuels and chemicals from cellulosic biomass.

Sensitive Surface Enhanced Raman Scattering-Based Detection of a BIGH3 Point Mutation Associated with Avellino Corneal Dystrophy.

Surface enhanced Raman scattering (SERS) is highly useful for sensitive analytical sensing; however, its practical availability for detecting a point mutation associated with disease in clinical sample was rarely proved. Herein, we present a toehold-mediated, DNA displacement-based, SERS sensor for detecting point mutations in the BIGH3 gene associated with the most common corneal dystrophies (CDs) in a clinical setting. To diagnose Avellino corneal dystrophy (ACD), selectivity was ensured by exploring optimal DNA displacement conditions such as length of toehold and hybridization temperature. A SERS-efficient Ag@Au bimetallic nanodendrite was employed to ensure sensitivity. Optimization for a clinical setting showed that discrimination was maximized when toehold length was 6-mer (T6), and hybridization temperature was 36 °C. On the basis of tests that used clinical homozygous and heterozygous CD samples, a single-base mismatched DNA sequence was identifiable within 30 min with a limit of detection (LOD) of 400 fM. From the results, we conclude that our toehold-mediated, DNA displacement-based, SERS sensor allows a rapid and sensitive detection of a BIGH3 gene point mutation associated with Avellino corneal dystrophy, indicating the practical ability of the method to diagnose genetic diseases caused by point mutations.

Roles of phosphoinositide-specific phospholipase Cγ1 in brain development.

Over the past decade, converging evidence suggests that PLCγ1 signaling has key roles in controlling neural development steps. PLCγ1 functions as a signal transducer that converts an extracellular stimulus into intracellular signals by generating second messengers such as DAG and IP3. DAG functions as an activator of either PKC or transient receptor potential cation channels (TRPCs), while IP3 induces the calcium release from intracellular calcium stores. These second messengers regulate the morphological change of neuron, such as neurite outgrowth, migration, axon pathfinding, and synapse formation. These morphological changes depend on finely tuned calcium signaling following receptor tyrosine kinase-mediated PLCγ1 signaling. Thus, deregulation of PLCγ1 signaling causes various abnormalities of neuronal development and it may be associated with diverse neurological disorders. Herein, we discuss the current understanding of the PLCγ1 signaling pathway in neural development and provide recent advances of how PLCγ1 signaling is involved in the formation of neuronal processes for functionally faithful brain development.

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au-Ag bimetallic nanodendrite substrate.

A simple and sensitive surface enhanced Raman scattering (SERS)-based DNA sensor that utilizes the toehold-mediated DNA displacement reaction as a target-capturing scheme has been demonstrated. For a SERS substrate, Au-Ag bimetallic nanodendrites were electrochemically synthesized and used as a sensor platform. The incorporation of both Ag and Au was employed to simultaneously secure high sensitivity and stability of the substrate. An optimal composition of Ag and Au that satisfied these needs was determined. A double-strand composed of 'a probe DNA (pDNA)' complementary to 'a target DNA (tDNA)' and 'an indicator DNA tagged with a Raman reporter (iDNA)' was conjugated on the substrate. The conjugation made the reporter molecule close to the surface and induced generation of the Raman signal. The tDNA released the pre-hybridized iDNA from the pDNA via toehold-mediated displacement, and the displacement of the iDNA resulted in the decrease of Raman intensity. The variation of percent intensity change was sensitive and linear in the concentration range from 200fM to 20nM, and the achieved limit of detection (LOD) was 96.3fM, superior to those reported in previous studies that adopted different signal taggings based on such as fluorescence and electrochemistry.

Measurement of polyethylene pellets near the glass transition temperature to enhance Raman spectral selectivity among samples and improve accuracy for density determination.

A simple and effective strategy for improving the accuracy of the multivariate determination of polyethylene (PE) density using Raman spectroscopy has been demonstrated. This strategy is based on the possibility that varied polymeric structures of the PE samples, especially at a sub-zero temperature range, would enhance their spectral selectivity, thereby potentially improving the multivariate correlation with their pre-determined physical properties such as density. For the evaluation, Raman spectra were collected at regular intervals during continuous increase of the PE temperature from cryogenic to near room temperature. Then, using partial least squares (PLS) regression, calibration models were developed to correlate the Raman spectral features collected at each time period with the reference PE density values. Interestingly, the accuracy was improved when the temperature of the PE pellets was -35 °C, near the glass transition temperature (Tg). To explain the improved accuracy, a two-dimensional (2D) correlation analysis was employed to detail the spectral variation induced by temperature change. Diverse segmental chain motions (so called micro-Brownian motion) predominantly occurring in the amorphous section of the PEs around Tg greatly enhanced the spectral selectivity among PE samples. In addition, minor ß-relaxation occurring around this temperature was an additional source of the enhanced spectral selectivity. In parallel, differential scanning calorimetry (DSC) curves of the samples were also examined to check the existence of the phase transitions.

Feasibility Study for Detection of Turnip yellow mosaic virus (TYMV) Infection of Chinese Cabbage Plants Using Raman Spectroscopy.

Raman spectroscopy provides many advantages compared to other common analytical techniques due to its ability of rapid and accurate identification of unknown specimens as well as simple sample preparation. Here, we described potential of Raman spectroscopic technique as an efficient and high throughput method to detect plants infected by economically important viruses. To enhance the detection sensitivity of Raman measurement, surface enhanced Raman scattering (SERS) was employed. Spectra of extracts from healthy and Turnip yellow mosaic virus (TYMV) infected Chinese cabbage leaves were collected by mixing with gold (Au) nanoparticles. Our result showed that TYMV infected plants could be discriminated from non-infected healthy plants, suggesting the current method described here would be an alternative potential tool to screen virus-infection of plants in fields although it needs more studies to generalize the technique.

A three-dimensional gold nanodendrite network porous structure and its application for an electrochemical sensing.

A three dimensional (3D) gold (Au) nanodendrite network porous structure constructed by a simple electrochemical synthetic method has been presented, and its utility for sensitive electrochemical measurement was demonstrated in this study. The 3D nanodendrite network porous structure was constructed on a platinum surface through electrodeposition of Au under the presence of hydrogen bubbles generated from the same surface. Iodide, used as a co-reagent, played an important role in the construction of the nanodendrite network by preventing continual growth of Au into larger agglomerates as well as inhibiting coalescence of neighboring nanodendrites. An electrochemical sensor incorporating the structure was built and used to detect As(III) in ultra low concentration range. For the purpose of comparison, bare gold and gold nanoparticle-incorporated electrodes were also prepared. With the use of 3D nanodendrite network porous structure, a much more sensitive detection of As(III) was possible due to its large surface area.