Evolving the L-lysine high-producing strain of Escherichia coli using a newly developed high-throughput screening method.

This study provided a new method which applied a selected L-lysine-inducible promoter for evolving lysine industrial strains of E. coli. According to the intracellular levels of the enhanced green fluorescent protein (EGFP) whose expression was controlled by the promoter, 186 strains were preliminarily selected using fluorescence-activated cell sorting from a 10-million-mutant library generated from a L-lysine high-producing E. coli strain. By subsequent multiple parameter evaluation of the 186 selected strains according to the concentration and the yield of lysine, the productivity per unit of cell in 96-deep-well blocks, two mutants MU-1 and MU-2 were obtained. They produced 136.51 ± 1.55 and 133.2 9 ± 1.42 g/L of lysine, respectively, in 5-L jars. Compared with the lysine concentration and the yield of the original strain, those of strain MU-1 improved by 21.00 and 9.05 %, respectively, and those of strain MU-2 improved by 18.14 and 10.41 %, respectively. The mutant selection and evaluation system newly established in our study should be useful for continuous improvement of the current E. coli strains in the lysine industry.

High Performance Mg Alloy with Designed Microstructure and Phases.

A high strength and ductile Mg-Gd-Y-Zn-Zr alloy was designed and fabricated. The local strain evolution of the alloys during plastic deformation was analyzed using high-resolution digital image correlation (DIC). The results showed that the ß particles, nano-sized γ' phases, and LPSO phases were distributed in the as-extruded alloy and a bimodal microstructure was exhibited, including elongated un-dynamic recrystallized grains and fine dynamic recrystallized grains. With increasing extrusion ratio, the grain size remained, with the volume fraction of dynamic recrystallization of the as-extruded alloy increasing from 30% to 75%, and the as-extruded alloy exhibited a high strength-ductility synergy, which is attributed to the grain refinement, extensive ß particles, and elongated block-shaped LPSO phases. The strain evolution analysis showed that a strain-transfer from un-DRXed regions to adjacent DRXed regions and LPSO phases can promote uniform plastic deformation, which tends to improve the ductility of the alloy.

An Expanded Genetic Toolbox to Accelerate the Creation of Acholeplasma laidlawii Driven by Synthetic Genomes.

We have developed genetic tools for the atypical bacterium Acholeplasma laidlawii. A. laidlawii is a member of the class Mollicutes, which lacks cell walls, has small genomes, and has limited metabolic capabilities, requiring many metabolites from their hosts. Several of these traits have facilitated the development of genome transplantation for some Mollicutes, consequently enabling the generation of synthetic cells. Here, we propose the development of genome transplantation for A. laidlawii. We first investigated a donor-recipient relationship between two strains, PG-8A and PG-8195, through whole-genome sequencing. We then created multihost shuttle plasmids and used them to optimize an electroporation protocol. We also evolved a superior strain for DNA uptake via electroporation. We created a PG-8A donor strain with a Tn5 transposon carrying a tetracycline resistance gene. These tools will enhance Acholeplasma research and accelerate the effort toward creating A. laidlawii strains with synthetic genomes.

Multi-Dimensional Revealing the Influence Mechanism of the δ Phase on the Tensile Fracture Behavior of a Nickel-Based Superalloy on the Mesoscopic Scale.

As the key materials of aircraft engines, nickel-based superalloys have excellent comprehensive properties. Mircotensile experiments were carried out based on in situ digital image correlation (DIC) and in situ synchrotron radiation (SR) technique. The effects of the δ phase on the grain orientation, surface roughening, and strain localization were investigated. The results showed that the average kernel average misorientation (KAM) value of the fractured specimens increased significantly compared with that of the heat-treated specimens. The surface roughness decreased with an increasing volume fraction of the δ phase. The strain localization of specimens increased with the increasing ageing time. The size and volume fraction of voids gradually increased with the increase in plastic strain. Some small voids expanded into large voids with a complex morphology during micro-tensile deformation. The needle-like δ phase near the fracture broke into short rods, while the minor spherical δ phase did not break. The rod-like and needle-like δ phases provided channels for the propagation of the microcrack, and the accumulation of the microcrack eventually led to the fracture of specimens.

In Situ Measurements of Strain Evolution in Graphene/Boron Nitride Heterostructures Using a Non-Destructive Raman Spectroscopy Approach.

The mechanical properties of engineered van der Waals (vdW) 2D materials and heterostructures are critically important for their implementation into practical applications. Using a non-destructive Raman spectroscopy approach, this study investigates the strain evolution of single-layer graphene (SLGr) and few-layered boron nitride/graphene (FLBN/SLGr) heterostructures. The prepared 2D materials are synthesized via chemical vapor deposition (CVD) method and then transferred onto flexible polyethylene terephthalate (PET) substrates for subsequent strain measurements. For this study, a custom-built mechanical device-jig is designed and manufactured in-house to be used as an insert for the 3D piezoelectric stage of the Raman system. In situ investigation of the effects of applied strain in graphene detectable via Raman spectral data in characteristic bonds within SLGr and FLBN/SLGr heterostructures is carried out. The in situ strain evolution of the FLBN/SLGr heterostructures is obtained in the range of (0-0.5%) strain. It is found that, under the same strain, SLG exhibits a higher Raman shift in the 2D band as compared with FLBN/SLGr heterostructures. This research leads to a better understanding of strain dissipation in vertical 2D heterostacks, which could help improve the design and engineering of custom interfaces and, subsequently, control lattice structure and electronic properties. Moreover, this study can provide a new systematic approach for precise in situ strain assessment and measurements of other CVD-grown 2D materials and their heterostructures on a large scale for manufacturing a variety of future micro- and nano-scale devices on flexible substrates.

Recent advances in engineering of microbial cell factories for intelligent pH regulation and tolerance.

pH regulation is a serious concern in the industrial fermentation process as pH adjustment heavily utilizes acid/base and pollutes the environment. Under pH-stress conditions, microbial growth and production of valuable target products may be severely affected. Furthermore, some strains generating acidic or alkaline products require self pH regulation and increased tolerance against pH-stress. For pH control, synthetic biology has provided advanced engineering approaches to construct robust and more intelligent microbial strains, exhibiting tolerance to pH-stress to cope with limitations of pH regulation. This study reviewed the current progress of advanced strain evolution strategies to engineer pH-stress tolerant strains via synthetic biology. In addition, a large number of pH-responsive elements, including promoters, riboswitches, and some proteins have been investigated and applied for construction of pH-responsive genetic circuits and intelligent pH-responsive microbial strains.

Virus Prospecting in Crickets-Discovery and Strain Divergence of a Novel Iflavirus in Wild and Cultivated Acheta domesticus.

Orthopteran insects have high reproductive rates leading to boom-bust population dynamics with high local densities that are ideal for short, episodic disease epidemics. Viruses are particularly well suited for such host population dynamics, due to their supreme ability to adapt to changing transmission criteria. However, very little is known about the viruses of Orthopteran insects. Since Orthopterans are increasingly reared commercially, for animal feed and human consumption, there is a risk that viruses naturally associated with these insects can adapt to commercial rearing conditions, and cause disease. We therefore explored the virome of the house cricket Acheta domesticus, which is both part of the natural Swedish landscape and reared commercially for the pet feed market. Only 1% of the faecal RNA and DNA from wild-caught A. domesticus consisted of viruses. These included both known and novel viruses associated with crickets/insects, their bacterial-fungal microbiome, or their plant food. Relatively abundant among these viral Operational Taxonomic Units (OTUs) was a novel Iflavirus, tentatively named Acheta domesticus Iflavirus (AdIV). Quantitative analyses showed that AdIV was also abundant in frass and insect samples from commercially reared crickets. Interestingly, the wild and commercial AdIV strains had short, extremely divergent variation hotspots throughout the genome, which may indicate specific adaptation to their hosts' distinct rearing environments.

Tensile Deformation and Fracture Behaviors of a Nickel-Based Superalloy via In Situ Digital Image Correlation and Synchrotron Radiation X-ray Tomography.

Nickel-based superalloys have become key materials for turbine disks and other aerospace components due to their excellent mechanical properties at high temperatures. Mechanical properties of nickel-based superalloys are closely related to their microstructures. Various heat treatment processes were conducted to obtain the desired microstructures of a nickel-based superalloy in this study. The effect of the initial microstructures on the tensile deformation and fracture behaviors was investigated via in situ digital image correlation (DIC) and synchrotron radiation X-ray tomography (SRXT). The results showed that the size and volume fraction of γ″ and γ' phases increased with the aging time. The yield strength and the ultimate tensile strength increased due to the precipitation strengthening at the expense of ductility. The surface strain analysis showed severely inhomogeneous deformation. The local strains at the edge of specimens were corresponded to higher void densities. The fracture of carbides occurred owing to the stress concentration, which was caused by the dislocation accumulation. The fracture mode was dimple coalescence ductile fracture.

Transcription Factor-Based Biosensors in High-Throughput Screening: Advances and Applications.

The molecular mechanisms that cells use to sense changes in the intra- and extracellular environment are increasingly utilized in synthetic biology to build genetic reporter constructs for various applications. Although in nature sensing can be RNA-mediated, most existing genetically-encoded biosensors are based on transcription factors (TF) and cognate DNA sequences. Here, the recent advances in the integration of TF-based biosensors in metabolic and protein engineering screens whereas distinction is made between production-driven and competitive screening systems for enzyme evolution under physiological conditions are discussed. Furthermore, the advantages and disadvantages of existing TF-based biosensors are examined with respects to dynamic range, sensitivity, and robustness, and compared to other screening approaches. The application examples discussed in this review demonstrate the promising potential TF-based biosensors hold as screening tools in laboratory evolution of proteins and metabolic pathways, alike.

Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic colonization with in vivo strain evolution over time.

Pandoraea apista in the family Burkholderiaceae is an emerging opportunistic pathogen in cystic fibrosis patients. Here, we describe a case from which 3 separate isolates of P. apista were recovered over a 1-year period. Using a combination of first-, second-, and third-generation sequencing technologies, we sequenced and de novo assembled the complete genomes of these 3 P. apista isolates. The genome of P. apista TF81F4 sequenced in this study was 5.58 Mb with a GC% of 62.3%, differed in sequence from other Pandoraea species by >20%, and included a number of previously undescribed loci. Three P. apista isolates cultured over a 12-month period were >99.999% identical by nucleotide, consistent with a model of chronic colonization by a single strain. Over time, the isolates accumulated point mutations, deletions, and insertions in a stepwise fashion, indicating in vivo strain evolution within the cystic fibrosis lung niche.