Engineering Strain Research Articles

Rational hyperelastic modelling of elastic poured compound for the failure analysis of embedded rail system

ABSTRACT The continuous homogeneous rail constraint of embedded rail system (ERS) is realized by the encapsulation of rails with the elastic poured compound (EPC) which is a composite material. Previous treatment of EPC as linear elastic material was insufficient in the failure analysis of ERS. In this work, a hyperelastic model is developed to describe the mechanical properties of the EPC with engineering strain up to 150%. Physical tests of uniaxial tension, planar tension and quadruple shear are conducted. A 4-parameter Ogden model is determined by curve fitting and validated with a progressive validation strategy, and then is applied to the failure analysis of ERS. It is found that the material nonlinearity of EPC contributes noticeably to the decrease of the longitudinal stiffness of ERS. The 2nd debonding is more probably caused by the failure of adhesive at the interface between EPC and rail rather than EPC itself.

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

BackgroundBacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability.ResultsTo address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL.ConclusionsWe developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

Enhancement of ergothioneine production by discovering and regulating its metabolic pathway in Cordyceps militaris

BackgroundCordyceps militaris is a traditional medicinal fungus contains a variety of functional ingredients and has been developed as an important mushroom food recently. Ergothioneine, one of the antioxidative compounds in C. militaris, is benefits on aging-related diseases and therefore became a novel functional food nutritive fortifier. Currently, the main diet source of ergothioneine is mushroom food. However, the mushroom farming faces the problems such as rather low ingredient yield and spontaneous degeneration associated fruiting body that restricts large scale production of ergothioneine.ResultsIn this study, we excavated the ergothioneine synthetases in mushroom and modified the genes in C. militaris to construct a new ergothioneine synthesis pathway. By further introducing this pathway into C. militaris genome, we succeeded to increase the ingredients’ production of engineering strain, the highest amount of ergothioneine and cordycepin were up to 2.5 g/kg dry weight and 2 g/L, respectively. Additionally, the expression of ergothioneine synthetase genes in the shape-mutated degenerative C. militaris could recover the ability of degenerative strain to produce high amount of ingredients, suggesting the metabolic regulation of ergothioneine might release the symptom of mushroom degeneration.ConclusionThis study reveals a new pathway to fulfill the market needs of functional mushroom food and food fortifier ergothioneine. It implied the mycelium of C. militaris could be engineered as a novel medicinal mushroom food which could produce higher amount of valuable ingredients.Graphical abstract

Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum.

Acetogenic bacteria are an increasingly popular choice for producing fuels and chemicals from single carbon (C1) substrates. Eubacterium limosum is a promising acetogen with several native advantages, including the ability to catabolize a wide repertoire of C1 feedstocks and the ability to grow well on agar plates. However, despite its promise as a strain for synthetic biology and metabolic engineering, there are insufficient engineering tools and molecular biology knowledge to leverage its native strengths for these applications. To capitalize on the natural advantages of this organism, here we extended its limited engineering toolbox. We evaluated the copy number of three common plasmid origins of replication and devised a method of controlling copy number and heterologous gene expression level by modulating antibiotic concentration. We further quantitatively assessed the strength and regulatory tightness of a panel of promoters, developing a series of well-characterized vectors for gene expression at varying levels. In addition, we developed a black/white colorimetric genetic reporter assay and leveraged the high oxygen tolerance of E. limosum to develop a simple and rapid transformation protocol that enables benchtop transformation. Finally, we developed two new antibiotic selection markers—doubling the number available for this organism. These developments will enable enhanced metabolic engineering and synthetic biology work with E. limosum.

Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius.

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7’-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.

CRISPR-Cas9 Approach Constructed Engineered Saccharomyces cerevisiae with the Deletion of GPD2, FPS1, and ADH2 to Enhance the Production of Ethanol.

Bioethanol plays an important value in renewable liquid fuel. The excessive accumulation of glycerol and organic acids caused the decrease of ethanol content in the process of industrial ethanol production. In this study, the CRISPR-Cas9 approach was used to construct S. cerevisiae engineering strains by the deletion of GPD2, FPS1, and ADH2 for the improvement of ethanol production. RNA sequencing and transcriptome analysis were used to investigate the effect of gene deletion on gene expression. The results indicated that engineered S. cerevisiae SCGFA by the simultaneous deletion of GPD2, FPS1, and ADH2 produced 23.1 g/L ethanol, which increased by 0.18% in comparison with the wild-type strain with 50 g/L of glucose as substrate. SCGFA strain exhibited the ethanol conversion rate of 0.462 g per g of glucose. In addition, the contents of glycerol, lactic acid, acetic acid, and succinic acid in SCGFA decreased by 22.7, 12.7, 8.1, 19.9, and 20.7% compared with the wild-type strain, respectively. The up-regulated gene enrichment showed glycolysis, fatty acid, and carbon metabolism could affect the ethanol production of SCGFA according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Therefore, the engineering strain SCGFA had great potential in the production of bioethanol.

Effect of cyclic straining with various rates on stress softening/hysteresis and structural evolution of filled rubber: A time-resolved SANS study

The effect of cyclic straining on stress softening/hysteresis and structural evolution silica filled deuterated silicone rubber (SFdSR) during cyclic deformation has been investigated. In situ rheo-small-angle neutron scattering (Rheo-SANS) measurements were conducted with engineering strain rates (έ) spanning from 0.001 to 0.1 s−1. Moreover, superimposed time-resolved SANS technique is utilized. The non-linear behaviors all exhibit strong έ and cycle-number (No.C) dependency. Rheo-SANS suggests that the orientation of bound rubber responses sensitively to the strain, έ, and No.C, which presents an accumulative characteristic, associated with cyclic stress relaxation of the engineering stress-nominal strain curves. All the results consistently suggest that some substantial permanent changes occur within bound rubber, filler network, and rubber matrix after deforming the samples of virgin state. Evolutions would drastically develop during the initial couple of cycles, then accumulate and reach to a newly established equilibrium under constant έ, as long as the maximum strain is not exceeded.

Integrated stretchable pneumatic strain gauges for electronics-free soft robots

In soft robotics, actuators, logic and power systems can be entirely fluidic and electronics-free. However, sensors still typically rely on electric or optical principles. This adds complexity to fluidic soft robots because transducers are needed, and electrical materials have to be incorporated. Herein, we show a highly-stretchable pneumatic strain gauge based on a meandering microchannel in a soft elastomer material thus eliminating the need for an electrical signal in soft robots. Using such pneumatic sensors, we demonstrate an all-pneumatic gripper with integrated pneumatic strain gauges that is capable of autonomous closure and object recognition. The gauges can measure at least up to 300% engineering strains. The sensor exhibits a very stable signal over a 12-hour measurement period with no hysteresis. Using pneumatic sensors, all four major components of robots—actuators, logic, power, and sensors—can be pneumatic, enabling all-fluidic soft robots with proprioception and exteroception.

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1 Ps6-19) was commonly utilized in the de novo biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC Cs ) from Chromobacterium sp. USM2 in engineered Escherichia coli for the de novo biosynthesis of PLA from glucose. The results indicated that PhaC Cs had better performance in PLA production than that of class II synthase PhaC1 Ps6-19. In addition, the sulA gene was engineered in PLA-producing strains for morphological engineering. The morphologically engineered strains present increased PLA production. This study also tested fused propionyl-CoA transferase and lactate dehydrogenase A (fused Pct Cp /LdhA) in engineered E. coli and found that fused Pct Cp /LdhA did not apparently improve the PLA production. After systematic engineering, the highest PLA production was achieved by E. coli MS6 (with PhaC Cs and sulA), which could produce up to 955.0 mg/L of PLA in fed-batch fermentation with the cell dry weights of 2.23%, and the average molecular weight of produced PLA could reach 21,000 Da.

Clean synthesis of isoprene with six E. coli engineering strains

Isoprene is one of the most important platform chemicals containing conjugated double bonds and utilized as the precursor material of varieties of chemical raw materials. Currently, isoprene is mostly produced by petroleum smelting, which produces CO2 and some pollution. In that case, the clean production method of isoprene is still the focus of current research. Isoprene synthase (IspS) catalyzes isoprene formation from a metabolic intermediate DMAPP. The expression level and enzyme activity of IspS from different strains affect the amount of isoprene production. In this study, six IspS protein sequences were obtained by searching literatures and NCBI database based on the phylogenetic analysis. We analysed the conservation of their catalytic structures and constructed pBAD expression frameworks for them. The isoprene synthesis systems in E. coli were built and worked in the best induction condition predicted in our study. We used toluene to collect the isoprene produced in the system detected and compared the productivity of each strain. Our results show that the production in cells were 1.55-5.51 μg/L/h.

DIC-based approach to predict post necking behavior for AA6061, AA7075 and their friction stir welded joints

In a tensile test, the precise measurement of the area at the necking section is crucial for predicting the post-necking true stress-strain behavior. In the conventional theoretical method, this area is linked to the engineering strain using the incompressibility criteria, which is not applicable in the post necking domain. The present work proposes a digital image correlation (DIC)-based technique with two cameras for estimating real-time area reduction at a necking section of a rectangular tensile specimen during a uniaxial tensile test. In the current study, the area at a given section is computed by measuring strain synchronously in the width and thickness planes of a tensile specimen and translating the change in lengths in both planes into a global coordinate system. The area computation of the proposed approach is successfully validated by measuring the necking area of a terminated tensile specimen (stopped before fracture) by optical microscope to an error in the range of 0.12–1.10%. Following verification, the post necking behaviors of aluminum alloys such as AA6061-T6 and AA7075-T6 are assessed. The current approach is further implemented to evaluate the area reduction and post necking behavior of similar and dissimilar friction stir welding (FSW) joints of AA6061-T6 and AA7075-T6 that possess process induced microstructural inhomogeneity. The current experimental approach effectively assessed the area reduction of a similar and dissimilar FSW joint influenced by weld inhomogeneity, which is otherwise complicated to contemplate using an analytical approach.

Natural rubber components fatigue life estimation through an extreme learning machine

In engineering applications, natural rubber components are subjected to continuous alternating loads, resulting in fatigue failure. Although some theoretical models are used for the fatigue life estimation of rubber materials, they do not comprehensively consider the influence of multiple factors. In the present study, a model based on the extreme learning machine is established to estimate the fatigue life of natural rubber specimens. The mechanical load, peak engineering strain, ambient temperature, and shore hardness of natural rubber specimens are used as the input variables while the measured average fatigue life is used as the output variable of the extreme learning machine model. The performed analyses reveal that the regression results and predicted life of the established extreme learning machine model are encouraging. The backpropagation neural network model and the support vector machine model are implemented to evaluate the performance of the established model. It is concluded that the extreme learning machine model is superior to the backpropagation neural network and support vector machine models in accuracy and efficiency. The extreme learning machine model provides an effective means for accurately predicting the fatigue life of natural rubber components.

Butanol–isopropanol fermentation with oxygen-tolerant Clostridium beijerinckii XH29

Acetone–butanol–ethanol (ABE) fermentation is a traditional way for solvents production through bioconversion by Clostridium species. It is still a challenge to obtain metabolic engineering strains with high ABE yield. Screening strains with remarkable characteristics from nature and improving ABE yield by mutation are viable approaches. Clostridium beijerinckii XH 0906, a newly isolated strain, produces butanol and isopropanol (BI) as the main end-products (9.1 g/L BI) during fermentation with glucose as the sole carbon source. The screening process for this strain was performed under aerobic conditions rather than anaerobic environment. Thus, it is a robust stain capable of oxygen-tolerant BI fermentation. Furthermore, C. beijerinckii XH 0906 fermented xylose and glucose simultaneously to produce BI. A mutant strain obtained by ultraviolet (UV) mutagenesis, C. beijerinckii XH 29, had improved BI production capacity and could produce 17.0 g/L BI and 18.4 g/L BI using glucose or corn stover hydrolysate, respectively as the carbon source. Interestingly, C. beijerinckii XH 29 also produced up to 19.3 g/L isopropanol through fermentation of a glucose–acetone mix. These results indicate that C. beijerinckii XH 29 is an excellent BI producer with great potential for industrial applications.Graphical

Response to Comment on “Cryoforged nanotwinned titanium with ultrahigh strength and ductility”

We address the three main points of Guo et al . They claim that we should have used the engineering stress versus engineering strain curves to infer the mechanical properties of our nanotwinned titanium, question our sample design on the basis of a finite-element analysis, and doubt the immobility of some preexisting grain/twin boundaries in our electron backscatter diffraction micrographs. We find their analysis to be groundless and to contain many inconsistencies.

Effect of solution treatment on microstructure and mechanical properties of as-cast Al-12Si-4Cu-2Ni-0.8Mg-0.2Gd alloy

Effect of solution treatment on microstructure and mechanical properties of Al-12Si-4Cu-2Ni-0.8Mg-0.2Gd alloy was investigated. Results show the Si particles become stable and more intermetallic compounds dissolve in the matrix after solution treatment at 500 °C for 2 h followed by 540 °C for 3 h (T4). The skeleton-like Al3CuNi develops into two parts in the T4 alloy: one is Al3CuNi which has the framework shape; the other is intermetallics including the Al3CuNi (size: 5–10 µm) and AlSiCuNiGd phases (size: ≤5 µm) with complex structure. Adding 0.2% Gd can improve the mechanical properties of the alloys after two-step solution treatment (500 °C/2 h followed by 540 °C/3 h), the hardness of the alloy increases from 130.9 HV to 135.8 HV compared with the alloy with one-step solution treatment (500 °C/2 h), the engineering strength increases from 335.45 MPa to 352.03 MPa and the fracture engineering strain increases from 1.44% to 1.67%.

Recent progress in ergothioneine biosynthesis: a review

Ergothioneine is a multifunctional physiological cytoprotector, with broad application in foods, beverage, medicine, cosmetics and so on. Biosynthesis is an increasingly favored method in the production of ergothioneine. This paper summarizes the new progress in the identification of key pathways, the mining of key enzymes, and the development of natural edible mushroom species and high-yield engineering strains for ergothioneine biosynthesis in recent years. Through this review, we aim to reveal the molecular mechanism of ergothioneine biosynthesis and then employ the methods of fermentation engineering, metabolic engineering, and synthetic biology to greatly increase the yield of ergothioneine.

Increased NADPH Supply Enhances Glycolysis Metabolic Flux and L-methionine Production in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important strain for the industrial production of amino acids, but the fermentation of L-methionine has not been realized. The purpose of this study is to clarify the effect of reducing power NADPH on L-methionine synthesis. Site-directed mutagenesis of zwf and gnd genes in pentose phosphate pathway relieved feedback inhibition, increased NADPH supply by 151.8%, and increased L-methionine production by 28.3%; Heterologous expression of gapC gene to introduce NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase increased NADPH supply by 75.0% and L-methionine production by 48.7%; Heterologous expression of pntAB gene to introduce membrane-integral nicotinamide nucleotide transhydrogenase increased NADPH by 89.2% and L-methionine production by 35.9%. Finally, the engineering strain YM6 with a high NADPH supply was constructed, which increased the NADPH supply by 348.2% and the L-methionine production by 64.1%. The analysis of metabolic flux showed that YM6 significantly increased the glycolytic flux, including the metabolic flux of metabolites such as glycosyldehyde-3-phosphate, dihydroxyacetate phosphate, 3-phosphoglycate and pyruvate, and the significant increase of L-methionine flux also confirmed the increase of its synthesis. This study provides a research basis for the systematic metabolic engineering construction of L-methionine high-yield engineering strains.

Engineering piezoelectricity and strain sensitivity in CdS to promote piezocatalytic hydrogen evolution

Piezocatalytic hydrogen evolution has emerged as a promising direction for the collection and utilization of mechanical energy and the efficient generation of sustainable energy throughout the day. Hexagonal CdS, as an established semiconductor photocatalyst, has been widely investigated for splitting water into H 2 , while its piezocatalytic performance has attracted less attention, and the relationship between the structure and piezocatalytic activity remains unclear. Herein, two types of CdS nanostructures, namely CdS nanorods and CdS nanospheres, were prepared to probe the above-mentioned issues. Under ultrasonic vibration, the CdS nanorods afforded a superior piezocatalytic H 2 evolution rate of 157 μmol g −1 h −1 in the absence of any co-catalyst, which is nearly 2.8 times that of the CdS nanospheres. The higher piezocatalytic activity of the CdS nanorods is derived from their larger piezoelectric coefficient and stronger mechanical energy harvesting capability, affording a greater piezoelectric potential and more efficient separation and transfer of intrinsic charge carriers, as elucidated through piezoelectric response force microscopy, finite element method, and piezoelectrochemical tests. This study provides a new concept for the design of efficient piezocatalytic materials for converting mechanical energy into sustainable energy via microstructure regulation. CdS spheres and CdS nanorods with different lengths were constructed by hydrothermal method and solvothermal process varying reaction time, respectively. The medium-length CdS nanorods subjected to ultrasonic stimulation exhibits excellent piezocatalytic H 2 evolution performance due to the strong induced piezoelectric potential and benign mechanical strain collecting ability.

Accurate characterization of triaxial deformation and strength properties of salt rock based on logarithmic strain

Energy storage is an important strategic issue. Salt caverns have good physical properties and are suitable for energy storage, so it is necessary to study the long-term mechanical behavior of salt rock. In this paper, the conventional triaxial compression test on the salt rock was carried out. By analyzing the deformation of salt rock under triaxial stress, the deformation characteristics of rock salt under different confining pressures were summarized. The logarithmic strain is introduced into the analysis of experimental data and compared with the engineering strain. And find that using logarithmic strain to describe specimen deformation has many advantages and is more suitable for describing the deformation of rock specimens. Finally, the accuracy of Hou's model is verified based on experimental results.

Fatigue life prediction of natural rubber components using an artificial neural network

AbstractUniaxial tension fatigue tests conducted on dumbbell‐shaped specimens made of vulcanized natural rubber are carried out. Using the fatigue test data, a back‐propagation neural network (BPNN) model for estimating the fatigue life of natural rubber specimens is established. An improved sine‐cosine algorithm (ISCA) is proposed to optimize the parameters of the BPNN model. The peak engineering strain, ambient temperature, and Shore hardness of natural rubber specimens are used as the input variables while the rubber fatigue life as the output variable of the BPNN model. The regression results and predicted life distribution of the established BPNN model are encouraging. For comparison, a genetic algorithm, a particle swarm algorithm, and a standard sine‐cosine algorithm are also used to obtain the BPNN model parameters, respectively. The results showed that the prediction accuracy and efficiency of ISCA are better than other three algorithms. In addition, the sensitivity analysis is introduced to quantify the relative influence of the model inputs on the output.