Ultrasensitive Electrochemiluminescence Biosensor to Detect Ampicillin Resistance Gene (ARGAMP) Based on a Novel Near-Infrared Ruthenium Carbene Complex/TPrA/PEI Ternary ECL System.

The establishment of rapid target identification and analysis methods for antibiotic resistance genes (ARGs) is urgently needed. In this study, we unprecedently designed a target-catalyzed hairpin assembly (CHA) electrochemiluminescent (ECL) biosensor for the ultrasensitive detection of ampicillin resistance genes (ARGAMP) based on a novel, efficient near-infrared ruthenium carbene complex/TPrA/PEI ternary ECL system with low oxidation potential. The ternary NIR-ECL system illustrated in this work displayed double ECL intensity in comparison with their corresponding traditional binary ECL system. The as-prepared ECL biosensor illustrated in this work demonstrates highly selective and sensitive determination of ARGAMP from 1 fM to 1 nM and a low detection limit of 0.23 fM. Importantly, it also exhibits good accuracy and stabilities to identify ARGAMP in plasmid and bacterial genome DNA, which demonstrates its excellent reliability and great potential in detecting ARGAMP in real environmental samples.

Novel Electrochemiluminescent Biosensor to Ultrasensitively Detect U94 Gene in Human Herpesvirus 6 Using Metal-Organic Framework-Based Nanoemitters Comprising Iridium(III) Complexes via One-Pot Coordination Reaction Strategy.

The detection of the U94 gene in human herpesvirus 6 is crucial for early diagnosis of HHV-6 infections, which could induce acute febrile illness in infants. In this work, the first ultrasensitive electrochemiluminescence (ECL) biosensor for detecting U94 gene in Human Herpesvirus 6 was successfully designed by utilizing efficient novel metal-organic framework (MOF)-based ECL nanoemitters comprising iridium(III) complexes (Ir-ZIF-8-NH2) synthesized via one-pot coordination reaction strategy as an ECL indicator and a target-catalyzed hairpin assembly (CHA) signal amplification strategy. The as-prepared ECL indicator Ir-ZIF-8-NH2 exhibited an approximately 2.7-fold ECL intensity compared with its small molecular analogue of emissive iridium(III) complex named IrppymIM formed by in situ coordination reaction between iridium(III) solvent complex and imidazole ligands. In addition, a target-catalyzed hairpin assembly (CHA) strategy was employed to further improve the sensitivity of the proposed ECL biosensor, which demonstrated a wide linear range from 1 fM to 1 µM and the limit of detection as low as 0.113 fM (S/N = 3). Significantly, this biosensor was successfully applied to detect U94 gene in plasmids and real virus samples. The recoveries were in the range of 97.0-109.0% for plasmids and 95.7-107.5% for real virus samples with a relative standard deviation (RSD) of 1.87-2.53%. These satisfactory experimental results from the proposed ECL biosensor in this work would inevitably promote the development of new time/cost-effective and sensitive methods to detect HHV-6 with a major global health threat and substantial burden on healthcare in the future.

Viruses Regulate Microbial Community Assembly Together with Environmental Factors in Acid Mine Drainage.

Viruses are widespread in various ecosystems, and they play important roles in regulating the microbial community via host-virus interactions. Recently, metagenomic studies showed that there are extremely diverse viruses in different environments from the ocean to the human gut, but the influences of viral communities on microbial communities are poorly understood, especially in extreme environments. Here, we used metagenomics to characterize microbial communities and viral communities in acid mine drainage (AMD) and evaluated how viruses shape microbial community constrained by the harsh environments. Our results showed that AMD viral communities are significantly associated with the microbial communities, and viral diversity has positive correlations with microbial diversity. Viral community explained more variations of microbial community composition than environmental factors in AMD of a polymetallic mine. Moreover, we found that viruses harboring adaptive genes regulate a relative abundance of hosts under the modulation of environmental factors, such as pH. We also observed that viral diversity has significant correlations with the global properties of microbial cooccurrence networks, such as modularity. In addition, the results of null modeling analyses revealed that viruses significantly affect microbial community phylogeny and play important roles in regulating ecological processes of community assembly, such as dispersal limitation and homogenous dispersal. Together, these results revealed that AMD viruses are critical forces driving microbial network and community assembly via host-virus interactions. IMPORTANCE Viruses as mobile genetic elements play critical roles in the adaptive evolution of their hosts in extreme environments. However, how viruses further influence microbial community structure and assembly is still unclear. A recent metagenomic study observed diverse viruses unexplored in acid mine drainage, revealing the associations between the viral community and environmental factors. Here, we showed that viruses together with environmental factors can constrain the relative abundance of host and microbial community assembly in AMD of copper mines and polymetallic mines. Our results highlight the importance of viruses in shaping the microbial community from the individual host level to the community level.

Inactivation and process intensification of ß-glucosidase in biomass utilization.

Lignocellulosic biomass has emerged as a promising environmental resource. Enzyme catalysis, as one of the most environmentally friendly and efficient tools among various treatments, is used for the conversion of biomass into chemicals and fuels. Cellulase is a complex enzyme composed of ß-glucosidase (BGL), endo-ß-1,4-glucanase (EG), and exo-ß-1,4-glucanase (CBH), which synergistically hydrolyzes cellulose into monosaccharides. BGL, which further deconstructs cellobiose and short-chain cellooligosaccharides obtained by EG and CBH catalysis into glucose, is the most sensitive component of the synergistic enzyme system constituted by the three enzymes and is highly susceptible to inactivation by external conditions, becoming the rate-limiting component in biomass conversion. This paper firstly introduces the source and catalytic mechanism of BGL used in the process of biomass resource utilization. The focus is on the review of various factors affecting BGL activity during hydrolysis, including competitive adsorption of lignin, gas-liquid interface inactivation, thermal inactivation, and solvent effect. And the methods to improve BGL inactivation are proposed from two aspects-substrate initiation and enzyme initiation. In particular, the screening, modification, and alteration of the enzyme molecules themselves are discussed with emphasis. This review can provide novel ideas for studies of BGL inactivation mechanism, containment of inactivation, and activity enhancement. KEY POINTS: • Factors affecting ß-glucosidase inactivation are described. • Process intensification is presented in terms of substrate and enzyme. • Solvent selection, protein engineering, and immobilization remain topics of interest.

Construction of a xylose metabolic pathway in Trichosporonoides oedocephalis ATCC 16958 for the production of erythritol and xylitol.

PURPOSE: Erythritol is a valuable compound as sweetener and chemical material however cannot be fermented from the abundant substrate xylose. METHODS: The strain Trichosporonoides oedocephalis ATCC 16958 was employed to produce polyols including xylitol and erythritol by metabolic engineering approaches. RESULTS: The introduction of a substrate-specific ribose-5-phosphate isomerase endowed T. oedocephalis with xylose-assimilation activity to produce xylitol, and eliminated glycerol production simultaneously. A more value-added product, erythritol was produced by further introducing a homologous xylulose kinase. The carbon flux was redirected from xylitol to erythritol by adding high osmotic pressure. The production of erythritol was improved to 46.5 g/L in flasks by fermentation adjustment, and the process was scaled up in a 5-L fermentor, with a 40 g/L erythritol production after 120 h, and a time-space yield of 0.56 g/L/h. CONCLUSION: This study demonstrated the potential of T. oedocephalis in the synthesis of multiple useful products from xylose.

Real-time estimation of the remaining surgery duration for cataract surgery using deep convolutional neural networks and long short-term memory.

PURPOSE: Estimating the surgery length has the potential to be utilized as skill assessment, surgical training, or efficient surgical facility utilization especially if it is done in real-time as a remaining surgery duration (RSD). Surgical length reflects a certain level of efficiency and mastery of the surgeon in a well-standardized surgery such as cataract surgery. In this paper, we design and develop a real-time RSD estimation method for cataract surgery that does not require manual labeling and is transferable with minimum fine-tuning. METHODS: A regression method consisting of convolutional neural networks (CNNs) and long short-term memory (LSTM) is designed for RSD estimation. The model is firstly trained and evaluated for the single main surgeon with a large number of surgeries. Then, the fine-tuning strategy is used to transfer the model to the data of the other two surgeons. Mean Absolute Error (MAE in seconds) was used to evaluate the performance of the RSD estimation. The proposed method is compared with the naïve method which is based on the statistic of the historical data. A transferability experiment is also set to demonstrate the generalizability of the method. RESULT: The mean surgical time for the sample videos was 318.7 s (s) (standard deviation 83.4 s) for the main surgeon for the initial training. In our experiments, the lowest MAE of 19.4 s (equal to about 6.4% of the mean surgical time) is achieved by our best-trained model for the independent test data of the main target surgeon. It reduces the MAE by 35.5 s (-10.2%) compared to the naïve method. The fine-tuning strategy transfers the model trained for the main target to the data of other surgeons with only a small number of training data (20% of the pre-training). The MAEs for the other two surgeons are 28.3 s and 30.6 s with the fine-tuning model, which decreased by -8.1 s and -7.5 s than the Per-surgeon model (average declining of -7.8 s and 1.3% of video duration). External validation study with Cataract-101 outperformed 3 reported methods of TimeLSTM, RSDNet, and CataNet. CONCLUSION: An approach to build a pre-trained model for estimating RSD estimation based on a single surgeon and then transfer to other surgeons demonstrated both low prediction error and good transferability with minimum fine-tuning videos.

Development of an enzymatic cascade to systematically utilize lignocellulosic monosaccharide.

BACKGROUND: The fermentation valorization of two main lignocellulosic monosaccharides, glucose and xylose, is extensively developed; however, it is restricted by limited yield and process complexity. An in vitro enzymatic cascade reaction can be an alternative approach. RESULTS: In this study, a three-stage, five-enzyme cascade was developed to convert pretreated biomass to valuable chemicals. First, a ribose-5-phosphate isomerase B mutant isomerized xylose to d-xylulose with high substrate specificity, and a d-arabinose dehydrogenase continued to reduce d-xylulose to d-arabitol. Simultaneously, glucose was utilized for the coenzyme regeneration catalyzed by a glucose dehydrogenase, generating useful gluconic acid and achieving 73% of total conversion rate after 36 h. Then, six kinds of pretreated biomass lignocellulose were hydrolyzed by cellulase and hemicellulase, and corn cob was identified as the initial substrate for providing the highest monosaccharide content. A 65% conversion rate of the lignocellulosic xylose was obtained after 24 h. CONCLUSIONS: This study presents a proof of concept to convert main lignocellulosic monosaccharides systematically by an enzymatic cascade at stoichiometric ratio. © 2022 Society of Chemical Industry.

Efficient production of ε-poly-L-lysine from cassava bagasse hydrolysate used as carbon source by Streptomyces albulus US3-18.

The production of ε-poly-L-lysine (ε-PL) from cassava bagasse hydrolysate (CBH) by Streptomyces albulus US3-18 was investigated in this study. With 30 g/L glucose from CBH, 1.30 g/L ε-PL and 10.68 g/L biomass were obtained in shake flask fermentation. Interestingly, the two values were increased by 14.0% and 21.5%, respectively, compared to the control (1.14 g/L and 8.79 g/L). Simultaneously, the activities of four key enzymes of ε-PL synthesis during CBH fermentation were enhanced to varying degrees. In batch fermentation of 5-L bioreactor, 3.39 g/L ε-PL and 10.17 g/L DCW were harvested with 40 g/L glucose from CBH. The combination of fed-batch fermentation with two-stage pH strategy significantly increased ε-PL titer and biomass to 37.41 g/L and 41.0 g/L, respectively. Moreover, eleven volatile components were detected in CBH by GC-MS, and 6-pentyl-α-pyrone (6PP) was first identified as the most abundant volatile ingredient. The results in CBH fermentation demonstrated that S. albulus US3-18 exhibited high tolerance to these volatile byproducts. Using ICP-MS, the calcium concentration in CBH was determined as 195.0 mg/(kg hydrolyzate), and cobalt, copper, lead, chromium, mercury and arsenic were not detected. By adding 0.05 g/L CaCl2 to M3G medium, ε-PL yield was improved by 28.0%, indicating calcium was one of the factors for the enhanced ε-PL production. The study provides a reference for the efficient production of ε-PL from low-cost agricultural residues.

Efficient production of ε-poly-l-lysine using cassava starch and fish meal by Streptomyces albulus FQC-24.

ε-Poly-l-lysine (ε-PL) is used as a natural food preservative which consists of l-lysine units connected. However, due to the expensive culture medium, the production cost of ε-PL remains high. In this study, cheap raw materials cassava starch (CS) and fish meal (FM) were employed by S. albulus FQC-24 for ε-PL production. In the single factor experiment, the maximum ε-PL production reached 0.97 g/L at 60 g/L CS and 15 g/L FM. The results of screening experiments by Plackett-Burman design showed that three main components affecting ε-PL production were CS, FM, and (NH4)2SO4. And the standardized effects of CS, FM, and (NH4)2SO4 were 0.13, -0.22, and -0.2, respectively. The optimum fermentation medium developed by response surface methodology for ε-PL production contained (g/L) CS, 67.56; FM, 14.70 and (NH4)2SO4, 5.41. Under the optimum conditions, the ε-PL production was achieved 1.30 g/L, with 34.02% higher than that before optimization. Moreover, ε-PL productions of batch and fed-batch fermentation in a 7-L fermentor were improved to 2.13 and 17.17 g/L respectively, which increased by 0.64 and 12.2 times compared with the shake flask culture. The results indicated that FM and CS are promising substrates for the efficient production of ε-PL.

Mobile genetic elements mediate the mixotrophic evolution of novel Alicyclobacillus species for acid mine drainage adaptation.

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions.

Engineering ribose-5-phosphate isomerase B from a central carbon metabolic enzyme to a promising sugar biocatalyst.

Ribose-5-phosphate isomerase B (RpiB) was first identified in the pentose phosphate pathway responsible for the inter-conversion of ribose-5-phosphate and ribulose-5-phosphate. Though there are seldom key enzymes in central carbon metabolic system developed as useful biocatalysts, RpiB with the advantages of wide substrate scope and high stereoselectivity has become a potential biotechnological tool to fulfill the demand of rare sugars currently. In this review, the pivotal roles of RpiB in carbon metabolism are summarized, and their sequence identity and structural similarity are discussed. Substrate binding and catalytic mechanisms are illustrated to provide solid foundations for enzyme engineering. Interesting differences in origin, physiological function, structure, and catalytic mechanism between RpiB and ribose-5-phosphate isomerase A are introduced. Moreover, enzyme engineering efforts for rare sugar production are stressed, and prospects of future development are concluded briefly in the viewpoint of biocatalysis. Aided by the progresses of structural and computational biology, the application of RpiB will be promoted greatly in the preparation of valuable molecules. KEY POINTS: • Detailed illustration of RpiB's vital function in central carbon metabolism. • Potential of RpiB in sequence, substrate scope, and mechanism for application. • Enzyme engineering efforts to promote RpiB in the preparation of rare sugars.

Influence of different linear polarized light on the measurement of water absorption coefficient: a case study in Qujiang Lake, China.

Light absorption by the water column plays an important role in the spatial and temporal distribution of light attenuation, mixed layer heating, primary productivity, and phytoplankton biomass in the water column. The absorption properties of water components are the main influences of the underwater light field and surface spectrum. The study of the absorption properties of the water body contributes to the understanding of its optical properties and the remote sensing inversion of water quality parameters. The effects of light source polarization on the spectral absorption coefficient measurements of colored dissolved organic matter (CDOM) and total particulate matter (TPM) in water were investigated by measuring the polarization of the light source in a UV-Vis spectrophotometer. The results show that the light source of the UV-Vis spectrophotometer is significantly polarized, but the effect of the polarization of the light source on the measurement of the spectral absorption coefficients of CDOM and TPM in water is very small and almost negligible.

Soil heavy metal pollution source analysis based on the land use type in Fengdong District of Xi'an, China.

The soil environment imposes a great influence on human health. Soil heavy metal pollution caused by human activities is an important part of environmental problems in urban areas. Due to an inadequate infrastructure, imperfect management, and intensive human activities, the sources of heavy metals in urban fringe areas are often more complicated than those in other areas, such as mining areas and agricultural irrigation areas. To solve this problem, the first step is to locate the source of pollution. However, the traditional methods of source analysis, such as principal component analysis and positive matrix factorization, always require correlations between elements. This study examined the Hg, Cd, Pb, and Cu contents in the Fengdong District of Xi'an, China, and found that these elements are not correlated in this area. Hence, traditional source analysis methods are not applicable in the study area. In response to this problem, this research proposed a new source analysis method based on Pearson's correlation analysis. The Nemerow index, geoaccumulation index, and ecological risk index were adopted to evaluate soil heavy metal pollution in the study area. Via comparison to the actual situation, it was concluded that the geoaccumulation index is more suitable for source analysis in this area. Through Pearson's correlation analysis, it was found that the geoaccumulation index is significantly correlated with the various land use types. Among them, transportation land exerted a greater impact on Pb pollution, and industrial land exerted a significant impact on the Hg distribution. The Cu distribution was related to construction land, while the Cd distribution was mainly related to urban land and cultivated land. In addition, the demolition of residential areas and abandoned farmlands imposed significant effects on Pb and Cd pollution, respectively.

miR-124 and miR-203 synergistically inactivate EMT pathway via coregulation of ZEB2 in clear cell renal cell carcinoma (ccRCC).

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most aggressive urological malignancies. MicroRNAs (miRNAs) are post-transcriptional gene regulators in tumor pathophysiology. As miRNAs exert cooperative repressive effects on target genes, studying the miRNA synergism is important to elucidate the regulation mechanism of miRNAs. METHODS: We first created a miRNA-mRNA association network based on sequence complementarity and co-expression patterns of miRNA-targets. The synergism between miRNAs was then defined based on their expressional coherence and the concordance between target genes. The miRNA and mRNA expression were detected in RCC cell lines (786-O) using quantitative RT-PCR. Potential miRNA-target interaction was identified by Dual-Luciferase Reporter assay. Cell proliferation and migration were assessed by CCK-8 and transwell assay. RESULTS: A synergistic miRNA-miRNA interaction network of 28 miRNAs (52 miRNA pairs) with high coexpression level were constructed, among which miR-124 and miR-203 were identified as most tightly connected. ZEB2 expression is inversely correlated with miR-124 and miR-203 and verified as direct miRNA target. Cotransfection of miR-124 and miR-203 into 786-O cell lines effectively attenuated ZEB2 level and normalized renal cancer cell proliferation and migration. The inhibitory effects were abolished by ZEB2 knockdown. Furthermore, pathway analysis suggested that miR-124 and miR-203 participated in activation of epithelial-to-mesenchymal transition (EMT) pathway via regulation of ZEB2. CONCLUSIONS: Our findings provided insights into the role of miRNA-miRNA collaboration as well as a novel therapeutic approach in ccRCC.

Complete genome sequencing and comparative genomic analyses of Bacillus sp. S3, a novel hyper Sb(III)-oxidizing bacterium.

BACKGROUND: Antimonite [Sb(III)]-oxidizing bacterium has great potential in the environmental bioremediation of Sb-polluted sites. Bacillus sp. S3 that was previously isolated from antimony-contaminated soil displayed high Sb(III) resistance and Sb(III) oxidation efficiency. However, the genomic information and evolutionary feature of Bacillus sp. S3 are very scarce. RESULTS: Here, we identified a 5,436,472 bp chromosome with 40.30% GC content and a 241,339 bp plasmid with 36.74% GC content in the complete genome of Bacillus sp. S3. Genomic annotation showed that Bacillus sp. S3 contained a key aioB gene potentially encoding As (III)/Sb(III) oxidase, which was not shared with other Bacillus strains. Furthermore, a wide variety of genes associated with Sb(III) and other heavy metal (loid) s were also ascertained in Bacillus sp. S3, reflecting its adaptive advantage for growth in the harsh eco-environment. Based on the analysis of phylogenetic relationship and the average nucleotide identities (ANI), Bacillus sp. S3 was proved to a novel species within the Bacillus genus. The majority of mobile genetic elements (MGEs) mainly distributed on chromosomes within the Bacillus genus. Pan-genome analysis showed that the 45 genomes contained 554 core genes and many unique genes were dissected in analyzed genomes. Whole genomic alignment showed that Bacillus genus underwent frequently large-scale evolutionary events. In addition, the origin and evolution analysis of Sb(III)-resistance genes revealed the evolutionary relationships and horizontal gene transfer (HGT) events among the Bacillus genus. The assessment of functionality of heavy metal (loid) s resistance genes emphasized its indispensable role in the harsh eco-environment of Bacillus genus. Real-time quantitative PCR (RT-qPCR) analysis indicated that Sb(III)-related genes were all induced under the Sb(III) stress, while arsC gene was down-regulated. CONCLUSIONS: The results in this study shed light on the molecular mechanisms of Bacillus sp. S3 coping with Sb(III), extended our understanding on the evolutionary relationships between Bacillus sp. S3 and other closely related species, and further enriched the Sb(III) resistance genetic data sources.

Phylogeny, Divergent Evolution, and Speciation of Sulfur-Oxidizing Acidithiobacillus Populations.

BACKGROUND: Habitats colonized by acidophiles as an ideal physical barrier may induce genetic exchange of microbial members within the common communities, but little is known about how species in extremely acidic environments diverge and evolve. RESULTS: Using the acidophilic sulfur-oxidizer Acidithiobacillus as a case study, taxonomic reclassifications of many isolates provides novel insights into their phylogenetic lineage. Whole-genome-based comparisons were attempted to investigate the intra- and inter-species divergence. Recent studies clarified that functional and structural specificities of bacterial strains might provide opportunities for adaptive evolution responding to local environmental conditions. Acidophilic microorganisms play a key role in the acidification of natural waters and thus the formation of extremely acidic environments, and the feedbacks of the latter might confer the distinct evolutionary patterns of Acidithiobacillus spp. Varied horizontal gene transfer events occurred in different bacterial strains, probably resulting in the expansion of Acidithiobacillus genomes. Gene loss as another evolutionary force might cause the adaptive phenotypic diversity. A conceptual model for potential community-dependent evolutionary adaptation was thus proposed to illustrate the observed genome differentiation. CONCLUSIONS: Collectively, the findings shed light on the phylogeny and divergent evolution of Acidithiobacillus strains, and provided a useful reference for evolutionary studies of other extremophiles.

The antitumor effect of hinesol, extract from Atractylodes lancea (Thunb.) DC. by proliferation, inhibition, and apoptosis induction via MEK/ERK and NF-κB pathway in non-small cell lung cancer cell lines A549 and NCI-H1299.

Lung cancer (especially, non-small cell lung cancer [NSCLC]) is one of the most malignant cancers in the world. Hinesol is the major component of the essential oil of Atractylodes lancea (Thunb.) DC and possesses the most promising anticancer function. However, the effects and molecular mechanism of hinesol on antiproliferation in NSCLC cells has not been well understood. In this study, we found that hinesol effectively inhibited the A549 and NCI-H1299 cells in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. In addition, hinesol induced cell cycle arrest at G0/G1 phase and apoptosis assessed by flow cytometry in A549 cells. Furthermore, Western blot analysis showed that hinesol decreased phosphorylation of mitogen-activated protein kinase, extracellular signal-regulated kinase, IκBα, and p65 inhibited the expressions of Bcl-2, cyclin D1 and upregulated the expression of Bax. Based on these results, hinesol might be a potential drug candidate of anti-NSCLC for therapy.

Comparative Genomic Analysis Reveals the Distribution, Organization, and Evolution of Metal Resistance Genes in the Genus Acidithiobacillus.

Members of the genus Acidithiobacillus, which can adapt to extremely high concentrations of heavy metals, are universally found at acid mine drainage (AMD) sites. Here, we performed a comparative genomic analysis of 37 strains within the genus Acidithiobacillus to answer the untouched questions as to the mechanisms and the evolutionary history of metal resistance genes in Acidithiobacillus spp. The results showed that the evolutionary history of metal resistance genes in Acidithiobacillus spp. involved a combination of gene gains and losses, horizontal gene transfer (HGT), and gene duplication. Phylogenetic analyses revealed that metal resistance genes in Acidithiobacillus spp. were acquired by early HGT events from species that shared habitats with Acidithiobacillus spp., such as Acidihalobacter, Thiobacillus, Acidiferrobacter, and Thiomonas species. Multicopper oxidase genes involved in copper detoxification were lost in iron-oxidizing Acidithiobacillus ferridurans, Acidithiobacillus ferrivorans, and Acidithiobacillus ferrooxidans and were replaced by rusticyanin genes during evolution. In addition, widespread purifying selection and the predicted high expression levels emphasized the indispensable roles of metal resistance genes in the ability of Acidithiobacillus spp. to adapt to harsh environments. Altogether, the results suggested that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. This study sheds light on the distribution, organization, functionality, and complex evolutionary history of metal resistance genes in Acidithiobacillus spp.IMPORTANCE Horizontal gene transfer (HGT), natural selection, and gene duplication are three main engines that drive the adaptive evolution of microbial genomes. Previous studies indicated that HGT was a main adaptive mechanism in acidophiles to cope with heavy-metal-rich environments. However, evidences of HGT in Acidithiobacillus species in response to challenging metal-rich environments and the mechanisms addressing how metal resistance genes originated and evolved in Acidithiobacillus are still lacking. The findings of this study revealed a fascinating phenomenon of putative cross-phylum HGT, suggesting that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. Altogether, the insights gained in this study have improved our understanding of the metal resistance strategies of Acidithiobacillus spp.

Immobilization of cellulase in the non-natural ionic liquid environments to enhance cellulase activity and functional stability.

Ionic liquids (ILs) have been applied as an environmentally friendly solvent in the pretreatment of lignocellulosic biomass for more than a decade. The ILs involved pretreatment processes for cellulases mediated saccharification lead to both the breakdown of cellulose crystallinity and the decrease of lignin content, thereby improving the solubility of cellulose and the accessibility of cellulase. However, most cellulases are partially or completely inactivated in the presence of even low amount of ILs. Immobilized cellulases are found to perform improved stability and higher apparent activity in practical application compared with its free counterparts. Enzyme immobilization therefore has become a promising way to relieve the deactivation of cellulase in ILs. Various immobilization carriers and methods have been developed and achieved satisfactory results in improving the stability, activity, and recycling of cellulases in IL pretreatment systems. This review aims to provide detailed introduction of immobilization methods and carrier materials of cellulase, including natural polysaccharides, synthetic polymers, inorganic materials, magnetic materials, and newly developed composite materials, and illustrate key methodologies in improving the performance of cellulase in the presence of ILs. Especially, novel materials and concepts from the recently representative researches are focused and discussed comprehensively, and future trends in immobilization of cellulases in non-natural ILs environments are speculated in the end.

Enhancement of erythritol production by Trichosporonoides oedocephalis ATCC 16958 through regulating key enzyme activity and the NADPH/NADP ratio with metal ion supplementation.

Erythritol, a well-known natural sweetener, is mainly produced by microbial fermentation. Various metal ions (Al3+, Cu2+, Mn2+, and Ni2+) were added to the culture medium of Trichosporonoides oedocephalis ATCC 16958 at 30 mg/L in shake flask cultures. Compared with controls, Cu2+ increased the erythritol content by 86% and decreased the glycerol by-product by 31%. After 48 hr of shake flask culture, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that expression levels of erythrose reductase (ER) in the presence of 30 mg/L CuSO4 · 5H2O were higher than those obtained after treatment with other examined metal ions. Furthermore, after 108 hr of batch culture in a 5-L bioreactor, supplementation with 30 mg/L of CuSO4 · 5H2O increased the specific erythritol content by 27%. Further studies demonstrated that ER activity under 30 mg/L CuSO4 · 5H2O supplementation in a fermentor was overtly increased compared with the control after 60 hr, while glycerol-3-phosphate dehydrogenase activity was clearly reduced in most of the fermentation process. Furthermore, the NADPH/NADP ratio was slightly lower in T. oedocephalis cells treated with Cu2+ compared with control cells. These results provide further insights into Cu2+ effects on erythritol biosynthesis in T. oedocephalis and should improve the industrial production of erythritol by biological processes.