Microstructure and Properties of Hot Pressing Sintered SiC/Y3Al5O12 Composite Ceramics for Dry Gas Seals.

Silicon carbide (SiC) ceramics with high bending strength were prepared by hot pressing sintering (HPS) with yttrium aluminum garnet (Y3Al5O12, YAG) as sintering additive, and the effects of YAG content and sintering temperature on the sintering behavior, microstructure and mechanical properties of SiC ceramics were investigated in detail. The uniform distribution of YAG to form a liquid phase and the driving force provided by hot pressing sintering decrease the sintering temperature, improve the densification of SiC ceramics, and refine the crystal size. By means of suitable sintering conditions with the additional amount of YAG of 5 wt%, the sintering temperature of 1950 °C and a pressure of 30 MPa, the resultant SiC/YAG composite ceramics possesses high sintering and mechanical properties with the relative density of 98.53%, the bending strength of 675 MPa, the Vickers hardness of up to 17.92 GPa, and the elastic modulus of 386 GPa. The as-prepared SiC/YAG composite ceramics are promisingly used as the dry gas seal materials in the centrifugal compressors.

Engineering of 4-hydroxyphenylacetate 3-hydroxylase derived from Pseudomonas aeruginosa for the ortho-hydroxylation of ferulic acid.

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.

Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid.

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (kcat/Km) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 µM-1 s-1, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 µM-1 s-1, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

Enhancing the Catalytic Efficiency of D-lactonohydrolase through the Synergy of Tunnel Engineering, Evolutionary Analysis, and Force-Field Calculations.

Computational design advances enzyme evolution and their use in biocatalysis in a faster and more efficient manner. In this study, a synergistic approach integrating tunnel engineering, evolutionary analysis, and force-field calculations has been employed to enhance the catalytic activity of D-lactonohydrolase (D-Lac), which is a pivotal enzyme involved in the resolution of racemic pantolactone during the production of vitamin B5. The best mutant, N96S/A271E/F274Y/F308G (M3), was obtained and its catalytic efficiency (kcat/KM) was nearly 23-fold higher than that of the wild-type. The M3 whole-cell converted 20 % of DL-pantolactone into D-pantoic acid (D-PA, >99 % e.e.) with a conversion rate of 47 % and space-time yield of 107.1 g L-1 h-1, demonstrating its great potential for industrial-scale D-pantothenic acid production. Molecular dynamics (MD) simulations revealed that the reduction in the steric hindrance within the substrate tunnel and conformational reconstruction of the distal loop resulted in a more favourable"catalytic" conformation, making it easier for the substrate and enzyme to enter their pre-reaction state. This study illustrates the potential of the distal residue on the pivotal loop at the entrance of the D-Lac substrate tunnel as a novel modification hotspot capable of reshaping energy patterns and consequently influencing the enzymatic activity.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Development of highly efficient and specific base editors in Actinobacillus succinogenes for enhancing succinic acid production.

The production of bio-succinic acid (SA) from renewable feedstocks is a promising and sustainable approach to mitigating the high carbon emissions associated with the current energy crisis. Actinobacillus succinogenes was recognized as one of the most promising SA producers; however, lack of genetic background and the scarcity of genetic manipulation tools hinder the improvement in A. succinogenes by metabolic engineering. Here, for the first time, we successfully developed a series of A. succinogenes base editors (BEs) mediated by the fusion of Cas9 nickase and deaminase, including CBE, ABE, Td-GABE, and Td-CBE. Among these, ABE and Td-CBE based on a fusion of Cas9 nickase and TadA-8e variant (Escherichia coli TadA) can efficiently convert A to G and C to T, respectively, with editing efficiencies of up to 100%. We also investigated the multiplex base editing of ABE and Td-CBE, and the results showed that the editing efficiency of ABE reached 100% for six sites and 10% editing efficiency of Td-CBE for two sites. In addition, cytosine base editors were applied to inactivate hypothetical sugar and SA transporters of A. succinogenes. We found that the inactivation of Asuc_0914 encoding sucrose-specific IIBC subunit enhanced SA production, while the inactivation of hypothetical SA transporters Asuc_0715 and Asuc_0716 significantly reduced SA production. Therefore, the tools have great application potential in the metabolic engineering of A. succinogenes.

Cell-type-specific 3D-genome organization and transcription regulation in the brain.

3D organization of the genome plays a critical role in regulating gene expression. However, it remains unclear how chromatin organization differs among different cell types in the brain. Here we used genome-scale DNA and RNA imaging to investigate 3D-genome organization in transcriptionally distinct cell types in the primary motor cortex of the mouse brain. We uncovered a wide spectrum of differences in the nuclear architecture and 3D-genome organization among different cell types, ranging from the physical size of the cell nucleus to the active-inactive chromatin compartmentalization and radial positioning of chromatin loci within the nucleus. These cell-type-dependent variations in nuclear architecture and chromatin organization exhibited strong correlation with both total transcriptional activity of the cell and transcriptional regulation of cell-type-specific marker genes. Moreover, we found that the methylated-DNA-binding protein MeCP2 regulates transcription in a divergent manner, depending on the nuclear radial positions of chromatin loci, through modulating active-inactive chromatin compartmentalization.

Biosynthesis of 3-Hydroxyphloretin Using Rational Design of 4-Hydroxyphenylacetate 3-Monooxygenase.

The compound 3-hydroxyphloretin is a typical dihydrochalcone that can be obtained in plants by the 3-hydroxylation of phloretin. Here, the flavin-dependent two-component monooxygenase (HpaBC) derived from Pseudomonas aeruginosa was used to convert phloretin into 3-hydroxyphloretin. Following molecular docking and sequence alignment, modifications to the substrate pocket and loop of PaHpaBC were rationally designed, and mutant residues were selected. The results showed that the mutant Q212G/F292A/Q376N gave the best yield of 3-hydroxyphloretin and showed improved catalytic efficiency. Under optimal reaction condition, 2.03 g/L of 3-hydroxyphloretin was produced in the whole-cell catalysis experiment. Molecular docking and molecular dynamics simulations were used to analyze mutants and elucidate the potential mechanism. It was found that the increase in 3-hydroxyphloretin yield was due to the improvement in the flexibility of the loop and the expansion of its substrate pocket. This strategy based on loop and substrate pocket modification has significance in the engineering of PaHpaB.

Rational Redesign of Chitosanase to Enhance Thermostability and Catalytic Activity to Produce Chitooligosaccharides with a Relatively High Degree of Polymerization.

Chitooligosaccharides (hdpCOS) with a high degree of polymerization (hdp, DP 4-10) generally have greater biological activities than those of low-DP (ldp, DP 2-3) COS. Chitosanase from Bacillus amyloliquefaciens KCP2 (Csn46) can degrade chitosan to more hdpCOS at high temperature (70 °C), but low thermal stability at this temperature makes it unsuitable for industrial application; the wild-type enzyme can only produce COS (DP 2-4) at lower temperatures. Several thermostable mutants were obtained by modifying chitosanase using a comprehensive strategy based on a computer-aided mutant design. A combination of four beneficial single-point mutations (A129L/T175 V/K70T/D34G) to Csn46 was selected to obtain a markedly improved mutant, Mut4, with a half-life at 60 °C extended from 34.31 to 690.80 min, and the specific activity increased from 1671.73 to 3528.77 U/mg. Mut4 produced COS with DPs of 2-4 and 2-7 at 60 and 70 °C, respectively. Therefore, Mut4 has the potential to be applied to the industrial-scale preparation of hdpCOS with high biological activity.

Genome Sequencing and Analysis Reveal Potential High-Valued Metabolites Synthesized by Lasiodiplodia iranensis DWH-2.

Lasiodiplodia sp. is a typical opportunistic plant pathogen, which can also be classified as an endophytic fungus. In this study, the genome of a jasmonic-acid-producing Lasiodiplodia iranensis DWH-2 was sequenced and analyzed to understand its application value. The results showed that the L. iranensis DWH-2 genome was 43.01 Mb in size with a GC content of 54.82%. A total of 11,224 coding genes were predicted, among which 4776 genes were annotated based on Gene Ontology. Furthermore, the core genes involved in the pathogenicity of the genus Lasiodiplodia were determined for the first time based on pathogen-host interactions. Eight Carbohydrate-Active enzymes (CAZymes) genes related to 1,3-ß-glucan synthesis were annotated based on the CAZy database and three relatively complete known biosynthetic gene clusters were identified based on the Antibiotics and Secondary Metabolites Analysis Shell database, which were associated with the synthesis of 1,3,6,8-tetrahydroxynaphthalene, dimethylcoprogen, and (R)-melanin. Moreover, eight genes associated with jasmonic acid synthesis were detected in pathways related to lipid metabolism. These findings fill the gap in the genomic data of high jasmonate-producing strains.

High-Yield Natural Vanillin Production by Amycolatopsis sp. after CRISPR-Cas12a-Mediated Gene Deletion.

Vanillin is an aromatic compound, which is widely used in food flavoring, beverages, perfumes, and pharmaceuticals. Amycolatopsis sp. is considered a good strain for the production of vanillin from ferulic acid by fermentation; however, its high genomic guanine-cytosine (GC) content (>70%) and low transformation and recombination efficiency limit its genetic modification potential to improve vanillin production. Efficient genome editing of Amycolatopsis sp. has been challenging, but this study developed a CRISPR-Cas12a system for efficient, markerless, and scarless genome editing of Amycolatopsis sp. CCTCC NO: M2011265. A mutant, ΔvdhΔphdB, was obtained by the deletion of two genes coding byproduct enzymes from the vanillin biosynthetic pathway. The gene deletion increased vanillin production from 10.60 g/L (wild-type) to 20.44 g/L and reduced byproduct vanillic acid from 2.45 to 0.15 g/L in a 3 L fed-batch fermentation, markedly enhancing vanillin production and reducing byproduct formation; the mutant has great potential for industrial application.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation.

BACKGROUND: Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied. RESULTS: In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0-5.0 and 30-60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below. CONCLUSIONS: In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Thermostability modification of ß-mannanase from Aspergillus niger via flexibility modification engineering.

Introduction: ß-Mannanases can hydrolyze mannans, which are widely available in nature. However, the optimum temperature of most ß-mannanases is too low to be directly utilized in industry. Methods: To further improve the thermostability of Anman (mannanase from Aspergillus niger CBS513.88), B-factor and Gibbs unfolding free energy change were used to modify the flexible of Anman, and then combined with multiple sequence alignment and consensus mutation to generate an excellent mutant. At last, we analyzed the intermolecular forces between Anman and the mutant by molecular dynamics simulation. Results: The thermostability of combined mutant mut5 (E15C/S65P/A84P/A195P/T298P) was increased by 70% than the wild-type Amman at 70°C, and the melting temperature (Tm) and half-life (t1/2) values were increased by 2°C and 7.8-folds, respectively. Molecular dynamics simulation showed reduced flexibility and additional chemical bonds in the region near the mutation site. Discussion: These results indicate that we obtained a Anman mutant that is more suitable for industrial application, and they also confirm that a combination of rational and semi-rational techniques is helpful for screening mutant sites.

Immobilization of Actinobacillus succinogenes on nano- and micro-fiber membranes for efficient and robust production of succinic acid.

This work aimed to study the efficiency of nano- and micro- fiber membranes in immobilizing Actinobacillus succinogenes CCTCC M2012036 for succinic acid production. Among the four kinds of electrospun nanofiber membranes of cellulose acetate, chitosan, poly(vinyl alcohol) (PVA) and chitosan-PVA, the cellulose acetate nanofiber membrane-immobilized cells performed the best with a succinic acid concentration and yield to be 27.3 ± 3.5 g/L and 70.9 ± 5.8%. The cell-immobilized viscose microfiber membrane presented good reuse stability, and 17 batches of fermentation without activity loss were realized with the highest succinic acid yield of 83.20%. A microfiber membrane bioreactor was further constructed with the cell-immobilized viscose microfiber membrane to perform fermentation on a larger scale, and the concentration, yield and productivity of succinic acid were 73.20 g/L, 86.50% and 1.49 g/(L⋅h) using a fed-batch strategy, which were 124.30%, 127.60% and 124.2% of those obtained in the traditional fermenter. This study provided an approach for improving the practicality of biological succinic acid production.

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes.

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.

Multivariate and spatio-temporal groundwater pollution risk assessment: A new long-time serial groundwater environmental impact assessment system.

Groundwater pollution risk assessment is an important part of environmental assessment. Although it has been developed for many years, there has not yet been a multi-dimensional method that takes into account long time series and spatial factors. We proposed a new method combines the advantages of remote sensing cloud computing, long-term groundwater modeling simulation and GIS technology to solve it efficiently. A coastal industrial park in Hainan was used as the study area. The depth of groundwater level, rainfall, topography and geomorphology, soil moisture, pollution source, pollution toxicity and other more than 10 parameters were used as the indexes. A comprehensive model with remote sensing cloud computing, DRASTIC model and Modflow + MT3DMS was established to assess the pollution risk from 2014 to 2021. The multi-year results indicated that the risk assessment of groundwater pollution was usually on the vertical coastal direction, and the risk increased from far away to near coast. With the discharge of pollutants in the industrial park, the pollution risk in the area 5 km away from the centre increased year by year until it became stable in 2019, and the risk in the centre of the park reached 1 level, covered an area of up to 145400 square metres, accounted for 0.012% of the whole study area. The assessment results in 2020 and 2021 fluctuate slightly compared with those in 2019. Therefore, in terms of groundwater resource protection and resource management, it is necessary to focus on the detection of pollution in the coastal zone and the pollution within 5 km of the centre to strictly control pollution discharge. In this study, the comprehensive assessment includes surface indicators, subsurface indicators, and pollutant indicators. Finally, we achieve a multivariate, spatial and long time series groundwater pollution risk assessment system, which is a new groundwater environmental impact assessment (GEIA) system.

A wide hybrid zone mediated by precipitation contributed to confused geographical structure of Scutiger boulengeri.

Confused geographical structure of a population and mitonuclear discordance are shaped by a combination of rapid changes in population demographics and shifts in ecology. In this study, we generated a time-calibrated phylogeny of Scutiger boulengeri, an endemic Xizang alpine toad occurring in mountain streams on the Qinghai-Xizang (Tibet) Plateau (QTP). Based on three mitochondrial DNA (mtDNA) genes, eight clades were assigned to three deeply divergent lineages. Analysis of nuclear DNA (nuDNA) genes revealed three distinct clusters without geographic structure, indicating significantly high rates of gene flow. Coalescent theory framework analysis (approximate Bayesian computation model DIYABC and Migrate-N) suggested that divergence of the main intraspecific clusters was the result of hybridization after secondary contact in the Holocene around 0.59 million years ago (Ma). The ratio of mtDNA F ST (fixation index) to nuDNA F ST was 2.3, thus failing to show male-biased dispersal. Geographic cline analysis showed that a wide hybrid zone was initially established in southwestern China, without significant reproductive isolation but with strong introgression in S. boulengeri, suggesting high hybrid fitness. Furthermore, mtDNA genes exhibited isolation by distance (IBD) while nuDNA genes exhibited significant isolation by environment (IBE). Results suggested that mitonuclear discordance may have initially been caused by geographic isolation, followed by precipitation-mediated hybridization, producing a wide hybrid zone and geographic structure confusion of nuDNA genes in S. boulengeri. This study indicated that complicated historical processes may have led to specific genetic patterns, with a specific climate factor facilitating gene flow in the system.

Human Arm Motion Prediction for Collision Avoidance in a Shared Workspace.

Industry 4.0 transforms classical industrial systems into more human-centric and digitized systems. Close human-robot collaboration is becoming more frequent, which means security and efficiency issues need to be carefully considered. In this paper, we propose to equip robots with exteroceptive sensors and online motion generation so that the robot is able to perceive and predict human trajectories and react to the motion of the human in order to reduce the occurrence of the collisions. The dataset for training is generated in a real environment in which a human and a robot are sharing their workspace. An Encoder-Decoder based network is proposed to predict the human hand trajectories. A Model Predictive Control (MPC) framework is also proposed, which is able to plan a collision-free trajectory in the shared workspace based on this human motion prediction. The proposed framework is validated in a real environment that ensures collision free collaboration between humans and robots in a shared workspace.

Production of (2S)-sakuranetin from (2S)-naringenin in Escherichia coli by strengthening methylation process and cell resistance.

(2S)-Sakuranetin is a 7-O-methylflavonoid that has anticancer, antiviral, and antimicrobial activities. Methylation process is involved in biosynthesizing (2S)-sakuranetin from (2S)-naringenin, in which S-adenosylmethionine (SAM) serves as the methyl donor. In this study, after methyl donor and substrate inhibition were identified as limiting factors for (2S)-sakuranetin biosynthesis, an efficient (2S)-sakuranetin-producing strain was constructed by enhancing methyl donor supply and cell tolerance to (2S)-naringenin. Firstly, PfOMT3 from Perilla frutescens was selected as the optimal flavonoid 7-O-methyltransferase (F7-OMT) for the conversion of (2S)-naringenin to (2S)-sakuranetin. Then, the methylation process was upregulated by regulating pyridoxal 5'-phosphate (PLP) content, key enzymes in methionine synthesis pathway, and the availability of ATP. Furthermore, genes that can enhance cell resistance to (2S)-naringenin were identified from molecular chaperones and sRNAs. Finally, by optimizing the fermentation process, 681.44 mg/L of (2S)-sakuranetin was obtained in 250-mL shake flasks. The titer of (2S)-sakuranetin reached 2642.38 mg/L in a 5-L bioreactor, which is the highest titer ever reported. This work demonstrates the importance of cofactor PLP in methylation process, and provides insights to biosynthesize other O-methylated flavonoids efficiently in E. coli.