Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering.

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.

Effect of Ball Milling Time on the Microstructure and Properties of High-Silicon-Aluminum Composite.

The duration of ball milling greatly influences the characteristics of high-silicon-aluminum composite during the ball milling process. This study examines how the microstructure, thermal conductivity, and hardness of a high-silicon-aluminum composite are affected by different ball milling times. We exposed the powder to various durations of ball milling and employed different pellet ratios. Following this treatment, the powder underwent consolidation via discharge plasma sintering. Our findings show that with a pellet ratio of 10:1 and a milling duration of 8 h, the powder particles were refined, resulting in a more uniform and dense material composition. This refined material boasted a thermal conductivity of 111.6 W/m·K, a Brinell hardness of 136.8 HBW, and a density of 2.304 g/cm3. This method facilitates the creation of a uniform composite powder composition. It encourages the development of a fine-grain structure, which enables the production of particle-reinforced composites with superior properties.

The Synergistic Effect of Trace Ag and Hot Extruding on the Microstructure and Properties of a Biodegradable Mg-Zn-Sr-Ag Alloy.

To further improve the mechanical properties and corrosion resistance of the biodegradable magnesium (Mg) alloy, the Mg-4Zn-0.5Sr-xAg alloy (x = 0.2 wt.%, 0.5 wt.%, 1.0 wt.%, and 2.0 wt.%) was smelted in vacuum under the protection of inert gas. The effect of the Ag content on the microstructure and mechanical properties of Mg-4Zn-0.5Sr was tested. The results show that the comprehensive properties of Mg-4Zn-0.5Sr-0.5Ag are best. The grain size of the Mg-4Zn-0.5Sr-0.5Ag alloy is minimal, that is, 83.28 µm. The average tensile strength (σb), yield strength (σs), elongation (ε), and hardness for the Mg-4Zn-0.5Sr-0.5Ag alloy is 168.00 MPa, 88.00 MPa, 12.20%, and 59.90 HV, respectively. To further improve the properties of cast Mg-4Zn-0.5Sr-0.5Ag alloy, extruding treatment was conducted. After extrusion deformation, the grain size of the alloy was significantly refined to 9 µm; at the same time, fine second phases were formed and evenly distributed in the matrix. And then, the mechanical properties of the alloy are significantly enhanced due to the effect of fine crystal strengthening and dispersion strengthening. The σb, σs, ε, and hardness value for the extruded Mg-4Zn-0.5Sr-0.5Ag alloy are 236.00 MPa, 212.00 MPa, 18.97%, and 65.42 HV, respectively. Under the synergistic action of adding the Ag element and extrusion treatment, the grain size of the alloy was significantly refined and the coarse second phase in the alloy became refined to disperse in the matrix, which benefits the formation of electric couples characterized as small cathode-large anode between the second phase and Mg matrix. During full immersion, corrosion products covered on the large anode surface could reduce the galvanic corrosion tendency.

Effects of Cu/Er on Tensile Properties of Cast Al-Si Alloy at Low Temperature.

The current protocol presents the effects of the addition of Cu, rare earth Er, and Cu-Er composite elements on the microstructure of the Al-10Si-0.3Mg alloy. The variations in their low-temperature tensile properties were also investigated. The addition of rare earth Er elements, Cu elements, and Cu-Er composite elements increased the strength of all three groups of alloys when stretched at low temperatures (-60 °C). Further, the elongation of the alloy increased with the addition of Er, while the elongation of the other two groups decreased. The low-temperature (-60 °C) tensile strength of the alloy with the same composition was higher than that at room temperature (20 °C), but the elongation decreased. Notably, by adding rare earth Er to the Al-10Si-0.3Mg alloy, the three-dimensional morphology was changed from coarse dendritic to fine fibrous, the secondary dendritic arm spacing (SDAS) of the alloy was reduced, and the grains were refined. The Al2Cu phase, Al-Si-Cu-Mg quaternary phase, and Cu-rich phase appeared in the alloy with the addition of Cu elements, but the Si phase morphology and α-Al dendrites were not significantly improved. Interestingly, the Si phase morphology of the alloy was improved by adding Cu-Er composite elements, and SDAS was reduced. Still, the Al2Cu phase, Al-Si-Cu-Mg quaternary phase, and Cu-rich phase were not much improved.

Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development.

The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

Calculation Based on the Formation of Mg2Si and Its Effect on the Microstructure and Properties of Al-Si Alloys.

In order to investigate the effect of Mg2Si formation on the microstructure and properties of an Al-Si alloy, the critical point of a hypereutectic Al-17Si-4Cu-Mg alloy was calculated by Pandat software. The calculation results of the equilibrium phase diagram show that the critical point for Mg2Si phase formation for the alloy was obtained when the Mg content was 2.2%. The contents of 0.5 wt.% Mg and 2.5 wt.% Mg were selected as the research object. The content of Mg increased from 0.5 wt.% to 2.5 wt.%, the eutectic Si in the matrix was reduced, and the Chinese character-like Mg2Si phase appeared in the microstructure. In the peak ageing state, in addition to θ″ and Q' phases that were mainly precipitated, there was also needle-like ß″ precipitation in the 2.5 wt.% Mg content alloy. Larger precipitates were found in 2.5 wt.% content alloys, mainly due to the promotion of the solid solution having the aggregation and segregation of more solute elements in the matrix. The tensile strength, elongation, and hardness of hypereutectic Al-17Si-4Cu-0.5Mg alloy under peak ageing were 331 MPa, 3.11%, and 152.1 HB, respectively. The tensile strength and the elongation decreased while the hardness increased with the 2.5 wt.% Mg content, which is due to the formation of hard and brittle Mg2Si and Al8FeMg3Si, which has a splitting effect on the matrix.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis.

BACKGROUND: Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. RESULTS: Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. CONCLUSIONS: This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

NaV6O15 microflowers as a stable cathode material for high-performance aqueous zinc-ion batteries.

Reversible aqueous zinc-ion batteries (ZIBs) have great potential for large-scale energy storage owing to their low cost and safety. However, the lack of long-lifetime positive materials severely restricts the development of ZIBs. Herein, we report NaV6O15 microflowers as a cathode material for ZIBs with excellent electrochemical performance, including a high specific capacity of ∼300 mA h g-1 at 100 mA g-1 and 141 mA h g-1 maintained after 2000 cycles at 5 A g-1 with a capacity retention of ∼107%. The high diffusion coefficient and stable tunneled structure of NaV6O15 facilitate Zn2+ intercalation/extraction and long-term cycle stability.

The Magnesium Concentration in Yeast Extracts Is a Major Determinant Affecting Ethanol Fermentation Performance of Zymomonas mobilis.

Zymomonas mobilis is a model ethanologenic bacterium for diverse biochemical production. Rich medium (RM) is a complex medium that is routinely used to cultivate Z. mobilis, which contains carbon sources such as glucose, nitrogen sources such as yeast extract (YE), and KH2PO4. Glucose consumption and cell growth of Z. mobilis is usually coupled during ethanol fermentation. However, sometimes glucose was not consumed during the exponential growth phase, and it took extended time for cells to consume glucose and produce ethanol, which eventually reduced the ethanol productivity. In this study, the effects of different nitrogen sources, as well as the supplementation of an additional nitrogen source into RM and minimal medium (MM), on cell growth and glucose consumption of Z. mobilis were investigated to understand the uncoupled cell growth and glucose consumption. Our results indicated that nitrogen sources such as YE from different companies affected cell growth, glucose utilization, and ethanol production. We also quantified the concentrations of major ion elements in different nitrogen sources using the quantitative analytic approach of Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), and demonstrated that magnesium ion in the media affected cell growth, glucose consumption, and ethanol production. The effect of magnesium on gene expression was further investigated using RNA-Seq transcriptomics. Our results indicated that the lack of Mg2+ triggered stress responses, and the expression of genes involved in energy metabolism was reduced. Our work thus demonstrated that Mg2+concentration in nitrogen sources is essential for vigorous cell growth and ethanol fermentation, and the difference of Mg2+concentration in different YE is one of the major factors affecting the coupled cell growth, glucose consumption and ethanol fermentation in Z. mobilis. We also revealed that genes responsive for Mg2+ deficiency in the medium were majorly related to stress responses and energy conservation. The importance of magnesium on cell growth and ethanol fermentation suggests that metal ions should become one of the parameters for monitoring the quality of commercial nitrogen sources and optimizing microbial culture medium.

Multiple Small RNAs Interact to Co-regulate Ethanol Tolerance in Zymomonas mobilis.

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA-sRNA interactions have been deeply studied in any organism. Zymomonas mobilis is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in Z. mobilis. We conducted multi-omics analysis (combining transcriptomics and sRNA-immunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA-sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA-sRNA direct interactions uncovered so far.

Preparation and electrochemical performance of VO2(A) hollow spheres as a cathode for aqueous zinc ion batteries.

Rechargeable aqueous zinc ion batteries (ZIBs), owing to their low-cost zinc metal, high safety and nontoxic aqueous electrolyte, have the potential to accelerate the development of large-scale energy storage applications. However, the desired development is significantly restricted by cathode materials, which are hampered by the intense charge repulsion of bivalent Zn2+. Herein, the as-prepared VO2(A) hollow spheres via a feasible hydrothermal reaction exhibit superior zinc ion storage performance, large reversible capacity of 357 mA h g-1 at 0.1 A g-1, high rate capability of 165 mA h g-1 at 10 A g-1 and good cycling stability with a capacity retention of 76% over 500 cycles at 5 A g-1. Our study not only provides the possibility of the practical application of ZIBs, but also brings a new prospect of designing high-performance cathode materials.

Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era.

BACKGROUND: Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. RESULTS: In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R 2 values more than 0.7 and 0.9, respectively. CONCLUSIONS: This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.

Identification and Characterization of 5' Untranslated Regions (5'UTRs) in Zymomonas mobilis as Regulatory Biological Parts.

Regulatory RNA regions within a transcript, particularly in the 5' untranslated region (5'UTR), have been shown in a variety of organisms to control the expression levels of these mRNAs in response to various metabolites or environmental conditions. Considering the unique tolerance of Zymomonas mobilis to ethanol and the growing interest in engineering microbial strains with enhanced tolerance to industrial inhibitors, we searched natural cis-regulatory regions in this microorganism using transcriptomic data and bioinformatics analysis. Potential regulatory 5'UTRs were identified and filtered based on length, gene function, relative gene counts, and conservation in other organisms. An in vivo fluorescence-based screening system was developed to confirm the responsiveness of 36 5'UTR candidates to ethanol, acetate, and xylose stresses. UTR_ZMO0347 (5'UTR of gene ZMO0347 encoding the RNA binding protein Hfq) was found to down-regulate downstream gene expression under ethanol stress. Genomic deletion of UTR_ZMO0347 led to a general decrease of hfq expression at the transcript level and increased sensitivity for observed changes in Hfq expression at the protein level. The role of UTR_ZMO0347 and other 5'UTRs gives us insight into the regulatory network of Z. mobilis in response to stress and unlocks new strategies for engineering robust industrial strains as well as for harvesting novel responsive regulatory biological parts for controllable gene expression platforms in this organism.