Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out ...sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.
The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, ...probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose‐fermenting, lactate‐producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.
An optimized CRISPR/Cas9 system was developed for multiplex genome engineering of polyploid industrial yeast strains. By expressing gRNA on plasmids with increased copy numbers, the authors were able to knock out four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) in a single step with 100% efficiency.
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•111- and 100-oriented grains alternately form as growth proceeds in wedge-shaped cell.•Stripe patterns form between n-layer 100- and n + 1-layer 111-oriented grains.•The stripe ...pattern is formed by continuous change of particle configuration.•The interval of the strip pattern becomes long as the number of layers increase.
The structural evolution of growing thin colloidal crystals in a confined space via the convective assembly technique has been investigated. The thin colloidal crystals were grown in a wedge-shaped cell, where the height of the cell increased with increased crystal growth. Triangle and square patterns, denoted as 111- and 100-oriented grains, respectively, were formed alternately as the height of the cell increased. The structural transformation was associated with an increase in the number of layers when the n-layer 100-oriented grains changed to n + 1-layer 111-oriented grains. Between the different grain structures, a stripe pattern was observed, which was a transitional region, where particle configuration gradually changed. The structural transformation occurred through the continuous change of particle configuration rather than through the abrupt formation of a grain boundary. The interval of the strip pattern lengthened as the number of layers increased, which is understood to be the structure with the highest packing density. The findings of the study give a better insight into convective assembly in a confined space, and also contribute to the greater structural control of colloidal crystals, useful for a number of applications.
► Effect of tungsten on high chromium cast iron has been investigated. ► Microstructure and properties of the high chromium cast iron were analyzed. ► Tungsten element distributed uniformly in the ...matrix and carbides. ► W carbides are composed with WC1−x, W6C2.54, CW3 and W2C. ► Mechanical property and wear resistance are significantly improved by addition of W.
In this study, effect of tungsten on microstructure and properties of high chromium cast iron was investigated. The experimental results indicated that tungsten distributed uniformly in the matrix and carbides. W carbides are composed of WC1−x, W6C2.54 and CW3 and W2C. With the increase of tungsten content, bulk hardness and matrix microhardness both increased gradually and reached the peak at 62.62HRC and 913HV, respectively. All of the tungsten-containing alloys performed better than tungsten-free alloys in impact tests and alloys containing 1.03wt% W showed the highest impact toughness at 8.23Jcm−2. Tungsten considerably improved the performance of high chromium cast iron on wear resistance and alloys containing 1.03wt% W increased 205% compared to tungsten-free alloys. Therefore, tungsten can be used as an alloying element to increase the hardness and wear resistance without scarifying impact toughness in high chromium cast iron. Alloys containing 1.03wt% tungsten showed the optimum properties.
Impurity partitioning at grain boundaries (GBs) during polycrystalline colloidal crystallization has been investigated via direct observation. Polycrystalline grains have a partitioning behavior ...similar to that of single colloidal crystals, which follows the Burton, Prim, and Slichter (BPS) model. We have found that impurities segregate at GBs during colloidal crystal growth, and the impurity concentration at GBs (C GB) for various misorientation angles (θ) between adjacent grains and growth rates (V) has been investigated. C GB was found to increase with either increasing θ or V, and also when the size of the impurity is close to that of the host colloid particle. In situ observations reveal that impurities incorporated into GBs are supplied mostly from the impurities segregated at the solid–liquid interface, and C GB and the growth rate show a BPS-like relationship.
W-Si-C composites with high relative densities and good mechanical and wear properties were successfully prepared by spark plasma sintering. The influence of SiC content on the relative density, ...microstructure, mechanical properties and wear characteristics was investigated. The results indicated that the reaction between SiC and W at their interface produced W2C and W5Si3. SiC also reacted with oxygen impurities at the W grain boundary to form SiO2. The purification of the grain boundaries of W was carried out by SiO2 synthesis. Reactive sintering reduces the free energy of the system and facilitates the densification process of W-Si-C composites. This results in a significant increase in the relative density of W-Si-C composites, which reaches a maximum of 98.12%, higher than the 94.32% of pure tungsten. The hardness significantly increases from 4.33 GPa to 8.40 GPa when the SiC content is 2 wt% compared to pure tungsten due to the generation of the hard ceramic phase and the increase in relative density. The wear resistance of the W-Si-C composites was significantly improved with little SiC addition. The wear rate significantly decreased from 313.27 × 10−3 mm3/N·m of pure tungsten to 5.71 × 10−3 mm3/N·m of W-0.5 wt% SiC. SEM analyses revealed that the dominant wear mechanism of pure tungsten was attributed to fatigue wear, while that of W-Si-C composites was due to abrasive wear.
The effect of the solid–liquid interface morphology on grain boundary (GB) segregation during colloidal polycrystallization was investigated by in situ optical observations.The time evolution of the ...impurity distribution in the liquid in the vicinity of solid–liquid interface was directly observed during crystal growth. Impurities were distributed homogeneously along the direction perpendicular to the growth orientation during the initial stage of the growth and were then gathered in the groove that formed at the solid–liquid interface as crystallization proceeded. GBs were exposed to the liquid at the bottom of the groove, and a high impurity concentration at the GB (C GB) was obtained when the area of the groove was large. The area of the groove changes depending on the crystallographic orientation of neighboring grains across GBs, which results in a difference in the interfacial energies.
Polydimethylsiloxane (PDMS)-based, segmented polyhydroxyurethanes (PHUs) was synthesized from Bisphenol A (BPA) dicarbonate and diamine-terminated PDMS. Two PDMS samples were employed with 875 g/mol ...and 2500 g/mol number-average molecular weight (Mn) values. Due to the inherent hydrophobic nature of PDMS, the PDMS-based PHUs exhibit excellent surface hydrophobicity with a water contact angle >100°. Because of the strong thermodynamic incompatibility of PDMS with BPA-based hard segments, hard-segment content and soft-segment Mn are critical in ensuring macroscopic homogeneity and effective nanophase separation leading to thermoplastic elastomer (TPE) character extending from room temperature to temperatures as low as −100 °C. Nanophase separation was confirmed via small-angle X-ray scattering and dynamic mechanical analysis. With 20–25 wt% hard-segment content, the TPEs derived from 2500 g/mol PDMS exhibit quasi-rubbery plateau regions with tensile storage modulus in the range of 1–10 MPa and the absence of a yield point in tensile tests with strain at break values that can exceed 1000%. With 40–50 wt% hard-segment content, the TPEs derived from 875 g/mol PDMS exhibit quasi-rubbery plateau regions with tensile storage modulus in the range of 100–1000 MPa but yield points in tensile tests at 25–40% strain. This work presents a pathway to achieving hydrophobic, non-isocyanate thermoplastic polyurethane elastomers with highly tunable rubbery moduli that extend to extraordinarily low temperatures.
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•Isocyanate-free, thermoplastic PHU elastomers made with PDMS soft segments.•Effective nanophase separation promoted by thermodynamic incompatibility of PDMS with BPA-based hard segments.•Excellent thermoplastic elastomer character extending from room temperature to temperatures as low as −100 °C.•Excellent surface hydrophobicity with water contact angles >100°.
Despite significant past efforts to exploit green, renewable precursors in polymeric materials and to improve the recyclability and reprocessability of nonisocyanate polyurethane (NIPU) networks, no ...single study has previously investigated biobased polyhydroxyurethane (PHU) network reprocessability. Renewable, dynamic PHU networks were synthesized by reacting bioderived polyfunctional cyclic carbonates, carbonated soybean oil (CSBO), and sorbitol ether carbonate (SEC), with either a synthetic diamine or a biobased diamine. Network reprocessability was studied by dynamic mechanical analysis. With relatively mild reprocessing conditions, CSBO-based PHU networks exhibit complete recovery of crosslink density and associated properties after multiple melt-state recycling steps. In addition to the presence of reversible cyclic carbonate aminolysis and transcarbamoylation exchange reactions, CSBO-based networks were shown via a model reaction to undergo a third dynamic chemistry based on a transesterification exchange reaction. In contrast to the excellent property recovery achieved by CSBO-based PHU networks, as a result of disadvantageous monomer molecular design, SEC-based networks exhibit poor reprocessability even with increased catalyst load and reprocessing temperature and time. This work reveals the effect of monomer structure on the reprocessability of dynamic polymer networks and highlights the effectiveness of CSBO to serve as a precursor of robust, sustainable NIPU networks with excellent reprocessability.
Processing-induced residual stresses in polymer films can cause microscopic defects and macroscopic dimensional changes. Although stress relaxation behavior of polymer films has been studied for ...several decades, little work has addressed random copolymers. We probed the residual stress relaxation in non-equilibrated random copolymer films of styrene (S) and n-butyl acrylate (nBA) using two optical techniques, ellipsometry and fluorescence. Both techniques show that incorporation of very low levels of nBA (2 mol% to 5 mol%) in S/nBA random copolymers can lead to dramatic increases in stress relaxation time of copolymer films relative to neat polystyrene films. For example, relative to polystyrene, the bulk-film stress relaxation time at 130 °C of 95/5 mol% S/nBA copolymer increases by factors of at least six as determined by ellipsometry and three as determined by fluorescence. Nanoconfined, 25-nm-thick films exhibit similar differences. This work provides implications for how low comonomer levels may deleteriously affect stress relaxation in polymer films, and we highlight that these findings are important for applications in which thin polymer films are required.
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•Stress relaxation was characterized in thin and ultrathin spin-coated films.•Ellipsometry and fluorescence provide sensitive measures of stress relaxation.•Studies employed polystyrene and styrene (S)/n-butyl acrylate (nBA) copolymers.•95/5 mol% S/nBA copolymers relaxed by factor of 3–6 more slowly than polystyrene.•Similar effects were observed in bulk and nanoconfined (25 nm thick) films.
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