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Abstract Bacterial σI factors of the σ70-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σI paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.0 Å and 3.3 Å of two transcription open complexes formed by two σI factors, SigI1 and SigI6, respectively, from the thermophilic, cellulolytic bacterium, Clostridium thermocellum. These structures reveal a unique, hitherto-unknown recognition mode of bacterial transcriptional promoters, both with respect to domain organization and binding to promoter DNA. The key characteristics that determine the specificities of the σI paralogues were further revealed by comparison of the two structures. Consequently, the σI factors represent a distinct set of the σ70-family σ factors, thus highlighting the diversity of bacterial transcription.

Abstract Background Thraustochytrids have gained attention as a potential source for the production of docosahexaenoic acid (DHA), where DHA is predominantly stored in the form of triacylglycerol (TAG). The TAG biosynthesis pathways, including the acyl-CoA-dependent Kennedy pathway and the acyl-CoA-independent pathway, have been predicted in thraustochytrids, while the specific details regarding their roles are currently uncertain. Results Phospholipid:diacylglycerol acyltransferase (PDAT) plays a key role in the acyl-CoA-independent pathway by transferring acyl-group from phospholipids (PL) to diacylglycerol (DAG) to from TAG. In thraustochytrid Aurantiochytrium sp. SD116, an active AuPDAT was confirmed by heterologous expression in a TAG-deficient yeast strain H1246. Analysis of AuPDAT function in vivo revealed that deletion of AuPDAT led to slow growth and a significant decrease in cell number, but improved PL content in the single cell during the cell growth and lipid accumulation phases. Interestingly, deletion of AuPDAT did not affect total lipid and TAG content, but both were significantly increased within a single cell. Moreover, overexpression of AuPDAT also resulted in a decrease in cell number, while the total lipid and cell diameter of a single cell were markedly increased. Altogether, both up-regulation and down-regulation of AuPDAT expression affected the cell number, which further associated with the total lipid and TAG content in a single cell. Conclusions Our study demonstrates that AuPDAT-mediated pathway play a minor role in TAG synthesis, and that the function of AuPDAT may be involved in regulating PL homeostasis by converting PL to TAG in a controlled manner. These findings expand our understanding of lipid biosynthesis in Aurantiochytrium sp. and open new avenues for developing “customized cell factory” for lipid production.

The hydrolysis and biotransformation of lignocellulose, i.e., biorefinery, can provide human beings with biofuels, bio-based chemicals, and materials, and is an important technology to solve the fossil energy crisis and promote global sustainable development. Biorefinery involves steps such as pretreatment, saccharification, and fermentation, and researchers have developed a variety of biorefinery strategies to optimize the process and reduce process costs in recent years. Lignocellulosic hydrolysates are platforms that connect the saccharification process and downstream fermentation. The hydrolysate composition is closely related to biomass raw materials, the pretreatment process, and the choice of biorefining strategies, and provides not only nutrients but also possible inhibitors for downstream fermentation. In this review, we summarized the effects of each stage of lignocellulosic biorefinery on nutrients and possible inhibitors, analyzed the huge differences in nutrient retention and inhibitor generation among various biorefinery strategies, and emphasized that all steps in lignocellulose biorefinery need to be considered comprehensively to achieve maximum nutrient retention and optimal control of inhibitors at low cost, to provide a reference for the development of biomass energy and chemicals.

Summary: Congo red is hard to remove from dye wastewater due to its structure stability and high chemical oxygen demand. In this study, cationic cellulose nanofibrils (CCNF) prepared from herb residues was physically crosslinked with sodium alginate (SA) in the presence of calcium ions, and the obtained CCNF/SA beads were used to adsorb Congo red. Results showed that CCNF/SA beads with porous internal structure were beneficial to adsorption. The maximum adsorption capacity of Congo red could reach to 518.4 mg/g, which was superior to most cellulose-based adsorption materials. Furthermore, the equilibrium adsorption isotherms and XPS analysis indicated the adsorption for Congo red was a physical process, and hydrogen bond and electrostatic adsorption were proposed as dominant adsorption mechanism. In addition, the Congo red removal efficiency of the beads was still higher than 70% after three cycles. Therefore, this high efficiency and green beads have great potential as adsorbents for anionic dyes removal.

Abstract Background Lignocellulose is a valuable carbon source for the production of biofuels and biochemicals, thus having the potential to substitute fossil resources. Consolidated bio-saccharification (CBS) is a whole-cell-based catalytic technology previously developed to produce fermentable sugars from lignocellulosic agricultural wastes. The deep-sea yeast strain Rhodotorula paludigena P4R5 can produce extracellular polyol esters of fatty acids (PEFA) and intracellular single-cell oils (SCO) simultaneously. Therefore, the integration of CBS and P4R5 fermentation processes would achieve high-value-added conversion of lignocellulosic biomass. Results The strain P4R5 could co-utilize glucose and xylose, the main monosaccharides from lignocellulose, and also use fructose and arabinose for PEFA and SCO production at high levels. By regulating the sugar metabolism pathways for different monosaccharides, the strain could produce PEFA with a single type of polyol head. The potential use of PEFA as functional micelles was also determined. Most importantly, when sugar-rich CBS hydrolysates derived from corn stover or corncob residues were used to replace grain-derived pure sugars for P4R5 fermentation, similar PEFA and SCO productions were obtained, indicating the robust conversion of non-food corn plant wastes to high-value-added glycolipids and lipids. Since the produced PEFA could be easily collected from the culture via short-time standing, we further developed a semi-continuous process for PEFA production from corncob residue-derived CBS hydrolysate, and the PEFA titer and productivity were enhanced up to 41.1 g/L and 8.22 g/L/day, respectively. Conclusions Here, we integrated the CBS process and the P4R5 fermentation for the robust production of high-value-added PEFA and SCO from non-food corn plant wastes. Therefore, this study suggests a feasible way for lignocellulosic agro-waste utilization and the potential application of P4R5 in industrial PEFA production.

BackgroundHealth-related quality of life (HRQoL) is a critical aspect of overall well-being for patients with lung cancer, particularly those with metastatic spinal cord compression (MSCC). However, there is currently a lack of universal evaluation of HRQoL in this specific patient population. The aim of this study was to develop a nomogram that can accurately predict HRQoL outcomes in patients with lung cancer-related MSCC.MethodsA total of 119 patients diagnosed with MSCC secondary to lung cancer were prospectively collected for analysis in the study. The least absolute shrinkage and selection operator (LASSO) regression analysis, along with 10-fold cross-validation, was employed to select the most significant variables for inclusion in the nomogram. Discriminative and calibration abilities were assessed using the concordance index (C-index), discrimination slope, calibration plots, and goodness-of-fit tests. Net reclassification index (NRI) and integrated discrimination improvement (IDI) analyses were conducted to compare the nomogram’s performance with and without the consideration of comorbidities.ResultsFour variables were selected to construct the final nomogram, including the Eastern Cooperative Oncology Group (ECOG) score, targeted therapy, anxiety scale, and number of comorbidities. The C-index was 0.87, with a discrimination slope of 0.47, indicating a favorable discriminative ability. Calibration plots and goodness-of-fit tests revealed a high level of consistency between the predicted and observed probabilities of poor HRQoL. The NRI (0.404, 95% CI: 0.074–0.734, p = 0.016) and the IDI (0.035, 95% CI: 0.004–0.066, p = 0.027) confirmed the superior performance of the nomogram with the consideration of comorbidities.ConclusionsThis study develops a prediction nomogram that can assist clinicians in evaluating postoperative HRQoL in patients with lung cancer-related MSCC. This nomogram provides a valuable tool for risk stratification and personalized treatment planning in this specific patient population.

Abstract Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production. Graphical Abstract

Abstract Background Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism. Results Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA. Conclusions There are two specific TAG assembly routes in Aurantiochytrium. The “saturated fatty acid (SFA) TAG lane” primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the “polyunsaturated fatty acid (PUFA) TAG lane” primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics.

ABSTRACT Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. In vivo gene inactivation of each transporter and in vitro calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as clo1313_2554, which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of C. thermocellum and will promote rational engineering of the bacterium for industrial application. IMPORTANCE Highly efficient sugar uptake is important to microbial cell factories, and sugar transporters are therefore of great interest in the study of industrially relevant microorganisms. Clostridium thermocellum is a lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery. In this study, we clarify the function and mechanism of substrate specificity of the five reported putative sugar transporters using genetic, biophysical, and structural methods. Intriguingly, the results showed that only one of them, transporter B, is the major cellodextrin transporter, whereas another, transporter A, represents the major glucose transporter. Considering the importance of transporter B, we further identified the missing ATPase gene of transporter B and revealed the correlation between transporter B and cellulosome production. Revealing the mechanism by which C. thermocellum utilizes cellodextrins will help pave the way for engineering the strain for industrial applications.

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure or perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, various artificial small molecules have been successfully used in enzyme engineering. The types of enzymatic reactions and metabolic pathways in cells can be expanded by the incorporation of these artificial small molecules either as cofactors or as building blocks of proteins and nucleic acids, which greatly promotes the development and application of biotechnology. In this review, we summarized research on artificial small molecules including biological metal cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), and non-natural amino acids (nnAAs), focusing on their design, synthesis, and applications as well as the current challenges in synthetic biology.

The alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors.

Summary Polyethylene terephthalate (PET) is a mass‐produced synthetic polyester contributing remarkably to the accumulation of solid plastics waste and plastics pollution in the natural environments. Recently, bioremediation of plastics waste using engineered enzymes has emerged as an eco‐friendly alternative approach for the future plastic circular economy. Here we genetically engineered a thermophilic anaerobic bacterium, Clostridium thermocellum, to enable the secretory expression of a thermophilic cutinase (LCC), which was originally isolated from a plant compost metagenome and can degrade PET at up to 70°C. This engineered whole‐cell biocatalyst allowed a simultaneous high‐level expression of LCC and conspicuous degradation of commercial PET films at 60°C. After 14 days incubation of a batch culture, more than 60% of the initial mass of a PET film (approximately 50 mg) was converted into soluble monomer feedstocks, indicating a markedly higher degradation performance than previously reported whole‐cell‐based PET biodegradation systems using mesophilic bacteria or microalgae. Our findings provide clear evidence that, compared to mesophilic species, thermophilic microbes are a more promising synthetic microbial chassis for developing future biodegradation processes of PET waste.

Abstract Background Phosphopantetheinyl transferase (PPTase) can change the acyl-carrier protein (ACP) from an inactive apo-ACP to an active holo-ACP that plays a key role in fatty acids biosynthesis. Currently, the PPTase has been proved to be involved in the biosynthesis of polyunsaturated fatty acids (PUFAs) via a polyketide synthase (PKS) pathway in Thraustochytrids, while its characteristics are not clarified. Results Here, the heterologous PPTase gene (pfaE) from bacteria was first co-expressed with the PKS system (orfA–orfC) from Thraustochytrid Aurantiochytrium. Then, a new endogenous PPTase (ppt_a) in Aurantiochytrium was identified by homologous alignment and its function was verified in E. coli. Moreover, the endogenous ppt_a was then overexpressed in Aurantiochytrium, and results showed that the production and proportion of PUFAs, especially docosahexaenoic acid (DHA), in the transformant SD116::PPT_A were increased by 35.5% and 17.6%, respectively. Finally, higher DHA and PUFA proportion (53.9% and 64.5% of TFA, respectively) were obtained in SD116::PPT_A using a cerulenin feeding strategy. Conclusions This study has illustrated a PUFAs-synthase-specific PPTase in PKS system and provided a new strategy to improve the PUFA production in Thraustochytrids.

Cellulases from glycoside hydrolase family 48 (GH48) are critical components of natural lignocellulose-degrading systems. GH48 cellulases are broadly distributed in cellulolytic microorganisms. With the development of genomics and metatranscriptomics, diverse GH48 genes have been identified, especially in the highly efficient cellulose-degrading ruminal system. GH48 cellulases utilize an inverting mechanism to hydrolyze cellulose in a processive mode. Although GH48 cellulases are indispensable for cellulolytic bacteria, they exhibit intrinsically low cellulolytic activity. Great efforts have been made to improve their performance. Besides, GH48 cellulases greatly synergize with the complementary endoglucanases in free cellulase systems or cellulosome systems. In this review, we summarized the studies on the diversity of GH48 cellulases, the crystal structures, the catalytic mechanism, the synergy between GH48 cellulases and endocellulases, and the strategies and progress of GH48 engineering. According to the summarized bottlenecks in GH48 research and applications, we suggest that future studies should be focused on mining and characterizing new GH48 enzymes, thoroughly understanding the progressive activity and product inhibition, engineering GH48 enzymes to improve stability, activity, and stress resistance, and designing and developing new biocatalytic system employing the synergies between GH48 and other enzymes.

Thraustochytrid is a promising algal oil resource with the potential to meet the demand for docosahexaenoic acid (DHA). However, oils with high DHA content produced by genetic modified thraustochytrids are not accepted by the food and pharmaceutical industries in many countries. Therefore, in order to obtain non-transgenic strains with high DHA content, a two-stage adaptive laboratory evolution (ALE) strategy was applied to the thraustochytrid Aurantiochytrium sp. Heavy-ion irradiation technique was first used before the ALE to increase the genetic diversity of strains, and then two-step ALE: low temperature based ALE and ACCase inhibitor quizalofop-p-ethyl based ALE were employed in enhancing the DHA production. Using this strategy, the end-point strain E-81 with a DHA content 51% higher than that of the parental strain was obtained. The performance of E-81 strain was further analyzed by component analysis and quantitative real-time PCR. The results showed that the enhanced in lipid content was due to the up-regulated expression of key enzymes in lipid accumulation, while the increase in DHA content was due to the increased transcriptional levels of polyunsaturated fatty acid synthase. This study demonstrated a non-genetic approach to enhance lipid and DHA content in non-model industrial oleaginous strains.

Abstract Background The industrial conversion of biomass to high-value biofuels and biochemical is mainly restricted by lignocellulose solubilization. Consolidated bio-saccharification (CBS) is considered a promising process for lignocellulose solubilization depending on whole-cell biocatalysts that simultaneously perform effective cellulase production and hydrolysis. However, it usually takes a long time to reach a high saccharification level using the current CBS biocatalyst and process. Results To promote the saccharification efficiency and reduce the cost, a Clostridium thermocellum recombinant strain ∆pyrF::KBm was constructed as a new CBS biocatalyst in this study. The key CBS factors, including the medium, inoculum size and cultivation, and substrate load, were investigated and optimized. The saccharification process was also stimulated by adding free hemicellulases, suggesting the need to further enhance hemicellulase activity of the whole-cell catalyst. Under the optimal conditions, the CBS process was shortened by 50% with pretreated wheat straw as the substrate. The sugar yield reached 0.795 g/g and the saccharification level was 89.3%. Conclusions This work provided a new biocatalyst and an optimized process of CBS and confirmed that CBS is a feasible strategy for cost-efficient solubilization of lignocellulose, which will greatly promote the industrial utilization of lignocellulosic biomass.

Abstract Background Bacterial insertion sequences (ISs) are ubiquitous mobile genetic elements that play important roles in genome plasticity, cell adaptability, and function evolution. ISs of various families and subgroups contain significantly diverse molecular features and functional mechanisms that are not fully understood. Results IS1447 is a member of the widespread IS3 family and was previously detected to have transposing activity in a typical thermophilic and cellulolytic microorganism Clostridium thermocellum. Phylogenetic analysis showed that IS1447-like elements are widely distributed in Firmicutes and possess unique features in the IS3 family. Therefore, IS1447 may represent a novel subgroup of the IS3 family. Unlike other well-known IS3 subgroups performing programmed − 1 translational frameshifting for the expression of the transposase, IS1447 exhibits transcriptional slippage in both the + 1 and − 1 directions, each with a frequency of ~ 16%, and only + 1 slippage results in full-length and functional transposase. The slippage-prone region of IS1447 contains a run of nine A nucleotides following a stem-loop structure in mRNA, but mutagenesis analysis indicated that seven of them are sufficient for the observed slippage. Western blot analysis indicated that IS1447 produces three types of transposases with alternative initiations. Furthermore, the IS1447-subgroup elements are abundant in the genomes of several cellulolytic bacteria. Conclusion Our result indicated that IS1447 represents a new Firmicutes-enriched subgroup of the IS3 family. The characterization of the novel IS3-family member will enrich our understanding of the transposition behavior of IS elements and may provide insight into developing IS-based mutagenesis tools for thermophiles.

Abstract Bacteria can adjust their genetic programs via alternative σ factors to face new environmental pressures. Here, we analyzed a unique set of paralogous alternative σ factors, termed σIs, which fine-tune the regulation of one of the most intricate cellulolytic systems in nature, the bacterial cellulosome, that is involved in degradation of environmental polysaccharides. We combined bioinformatics with experiments to decipher the regulatory networks of five σIs in Clostridium thermocellum, the epitome of cellulolytic microorganisms, and one σI in Pseudobacteroides cellulosolvens which produces the cellulosomal system with the greatest known complexity. Despite high homology between different σIs, our data suggest limited cross-talk among them. Remarkably, the major cross-talk occurs within the main cellulosomal genes which harbor the same σI-dependent promoter elements, suggesting a promoter-based mechanism to guarantee the expression of relevant genes. Our findings provide insights into the mechanisms used by σIs to differentiate among their corresponding regulons, representing a comprehensive overview of the regulation of the cellulosome to date. Finally, we show the advantage of using a heterologous host system for analysis of multiple σIs, since information generated by their analysis in their natural host can be misinterpreted owing to a cascade of interactions among the different σIs.

Abstract Background Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. Results The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L− 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. Conclusions This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Abstract Background Clostridium thermocellum is considered one of the most efficient natural cellulose degraders because of its cellulosomal system. As the major exoglucanase of cellulosome in C. thermocellum, Cel48S plays key roles and influences the activity and features of cellulosome to a great extent. Thus, it is of great importance to reveal the enzymatic features of Cel48S. However, Cel48S has not been well performed due to difficulties in purifying either recombinant or native Cel48S proteins. Results We observed that the soluble fraction of the catalytic domain of Cel48S (Cel48S_CD) obtained by heterologous expression in Escherichia coli and denaturation-refolding treatment contained a large portion of incorrectly folded proteins with low activity. Using a previously developed seamless genome-editing system for C. thermocellum, we achieved direct purification of Cel48S_CD from the culture supernatant of C. thermocellum DSM1313 by inserting a sequence encoding 12 successive histidine residues and a TAA stop codon immediately behind the GH domain of Cel48S. Based on the fully active protein, biochemical and structural analyses were performed to reveal its innate characteristics. The native Cel48S_CD showed high activity of 117.61 ± 2.98 U/mg and apparent substrate preference for crystalline cellulose under the assay conditions. The crystal structure of the native GH48 protein revealed substrate-coupled changes in the residue conformation, indicating induced-fit effects between Cel48S_CD and substrates. Mass spectrum and crystal structural analyses suggested no significant posttranslational modification in the native Cel48S_CD protein. Conclusion Our results confirmed that the high activity and substrate specificity of Cel48S_CD from C. thermocellum were consistent with its importance in the cellulosome. The structure of the native Cel48S_CD protein revealed evidence of conformational changes during substrate binding. In addition, our study provided a reliable method for in situ purification of cellulosomal and other secretive proteins from C. thermocellum.

Abstract Background Arachidonic acid (ARA, C20:4, n-6), which belongs to the omega-6 series of polyunsaturated fatty acids and has a variety of biological activities, is commercially produced in Mortierella alpina. Dissolved oxygen or oxygen utilization efficiency is a critical factor for Mortierella alpina growth and arachidonic acid production in large-scale fermentation. Overexpression of the Vitreoscilla hemoglobin gene is thought to significantly increase the oxygen utilization efficiency of the cells. Results An optimized Vitreoscilla hemoglobin (VHb) gene was introduced into Mortierella alpina via Agrobacterium tumefaciens-mediated transformation. Compared with the parent strain, the VHb-expressing strain, termed VHb-20, grew faster under both limiting and non-limiting oxygen conditions and exhibited dramatic changes in cell morphology. Furthermore, VHb-20 produced 4- and 8-fold higher total lipid and ARA yields than those of the wild-type strain under a microaerobic environment. Furthermore, ARA production of VHb-20 was also 1.6-fold higher than that of the wild type under normal conditions. The results demonstrated that DO utilization was significantly increased by expressing the VHb gene in Mortierella alpina. Conclusions The expression of VHb enhances ARA and lipid production under both lower and normal dissolved oxygen conditions. This study provides a novel strategy and an engineered strain for the cost-efficient production of ARA.

Abstract Background Cost-efficient saccharification is one of the main bottlenecks for industrial lignocellulose conversion. Clostridium thermocellum naturally degrades lignocellulose efficiently using the cellulosome, a multiprotein supermolecular complex, and thus can be potentially used as a low-cost catalyst for lignocellulose saccharification. The industrial use of C. thermocellum is restrained due largely to the inhibition of the hydrolysate cellobiose to its cellulosome. Although the supplementation of beta-glucosidase may solve the problem, the production of the enzymes greatly complicates the process and may also increase the cost of saccharification. Results To conquer the feedback inhibition and establish an efficient whole-cell catalyst for highly efficient cellulose saccharification, we constructed a recombinant strain of C. thermocellum ∆pyrF::CaBglA which produced a secretory exoglucanase CelS-bearing heterologous BGL using a newly developed seamless genome editing system. Without the extra addition of enzymes, the relative saccharification level of ∆pyrF::CaBglA was stimulated by over twofolds compared to its parent strain ∆pyrF through a two-stage saccharification process with 100 g/L Avicel as the carbon source. The production of reducing sugars and the relative saccharification level were further enhanced to 490 mM and 79.4%, respectively, with increased cell density. Conclusions The high cellulose-degrading ability and sugar productivity suggested that the whole-cell-catalysis strategy for cellulose saccharification is promising, and the C. thermocellum strain ∆pyrF::CaBglA could be potentially used as an efficient whole-cell catalyst for industrial cellulose saccharification.

The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C), sugar unit formation (spcA, B, E, K, J, I), glycosylation (spcN, G), methylation (spcMA, MB), as well as regulation (spcR). Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.

Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.

Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity.Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67-100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate.Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.

The aim of this article was to investigate the correlation between expression of O6-methylguanine-DNA methyltransferase (MGMT) in osteosarcoma and the curative effect of alkylating agent (Cis-diaminodichloroplatinum, CDDP). 42 male patients and 34 female patients with a median age of 17 years (9 to 43 years) were eligible for this study. According to histopathological types, there were 3 cases of telangiectatic osteogenic sarcoma, 22 cases of osteoblastic, 11 cases of chondroblastic and 16 cases of fibroblastic sarcoma. Immunohistochemical method was used to detect the expression of MGMT protein. The correlations between MGMT expression and the curative effect of CDDP on osteosarcoma have been investigated. It was shown by immunohistochemical staining that among 76 osteosarcoma biopsy specimens, 52 (68%) cases were positive, 27 (35%) cases were weak positive, 18 (24%), cases were moderate positive, and 7 (9%) cases were strong positive. There were no significant differences in MGMT expression among different pathological types of tumors (p>0.5). After CDDP chemotherapy, among pathologic specimens in which MGMT expression was positive, necrosis rates were as follows: grade I, 5 cases (38%); grade II, 7 cases (25%); grade III, 15 cases (21%); grade IV, 2 cases (23%). Osteosarcoma necrosis rate was low when the expression of MGMT protein was positive, whereas necrosis rate was high when there was a low level of MGMT expression (p

Objective To explore the value of hybrid surgery in children with cerebral arteriovenous malformation (CAVM). Methods Nineteen cases of pediatric CAVM from December 2015 to December 2019 were included. After the preoperative enhanced MRI and CTA, MRA or DSA examination confirmed the diagnosis, the hybrid surgery (craniotomy combined with endovascular embolization and intraoperative real⁃time DSA) were performed. Operative time, intraoperative blood loss, hospital stays and perioperative complications were recorded. The modified Rankin Scale (mRS) was used to evaluate prognosis 6 months later after surgery. Results In 19 CAVM cases, 2 cases were Spetzler ⁃ Martin grade Ⅰ, 5 cases were grade Ⅱ, 7 cases were grade Ⅲ and 5 cases were grade Ⅳ, and 4 cases dense type, 4 cases intermediate type and 11 cases diffuse type. The average operative time was (307.21 ± 115.64) min and intraoperative blood loss was 150 (100, 260) ml. Intraoperative endovascular embolization was performed in one case, the DSA after CAVM resection revealed 3 cases with residual malformations, and the final DSA cure rate was 100% (19/19). One patient presented with transient nominal aphasia, one with left limb hemiplegia, one with pulmonary infection, and one with epileptic seizure. Average hospital stays was (15.74 ± 5.04) d, followed ⁃ up time was 32 (20, 40) months. Sixteen patients had good prognosis (mRS score < 2), and 3 patients had poor prognosis (mRS score ≥ 2). There were no cases of rebleeding or recurrence. Conclusions Hybrid surgery can safely and effectively improve the cure rate of CAVM in children, especially for children with complex structure and diffuse type or with previous treatment. DOI：10.3969/j.issn.1672⁃6731.2020.04.009

Many studies have shown that (5R)-5-hydroxytriptolide is the optimal modified analogue of triptolide, possessing comparable immunosuppressive activity but much lower cytotoxicity than triptolide. Whether (5R)-5-hydroxytriptolide has preventive effects on neuroinflammation is unclear. This study was designed to pretreat primary astrocytes from the brains of neonatal Sprague-Dawley rats with 20, 100 and 500 nM (5R)-5-hydroxytriptolide for 1 hour before establishing an in vitro neuroinflammation model with 1.0 μg/mL lipopolysaccharide for 24 hours. The generation of nitric oxide was detected by Griess reagents. Astrocyte marker glial fibrillary acidic protein was measured by immunohistochemical staining. The levels of tumor necrosis factor-α and interleukin-1β in the culture supernatant were assayed by enzyme linked immunosorbent assay. Nuclear factor-κB/p65 expression was examined by immunofluorescence staining. The phosphorylation of inhibitor of nuclear factor IκB-α and the location of nuclear factor-κB/P65 were determined using western blot assay. Our data revealed that (5R)-5-hydroxytriptolide inhibited the generation of nitric oxide, tumor necrosis factor-α and interleukin-1β from primary astrocytes activated by lipopolysaccharide, decreased the positive reaction intensity of glial fibrillary acidic protein, reduced the expression of tumor necrosis factor alpha and interleukin-1β in culture supernatant, inhibited the phosphorylation of IκB-α and the translocation of nuclear factor-κB/P65 to the nucleus. These results have confirmed that (5R)-5-hydroxytriptolide inhibits lipopolysaccharide-induced glial inflammatory response and provides cytological experimental data for (5R)-5-hydroxytriptolide in the treatment of neurodegenerative diseases.

This paper presents a two-dimensional infinite dipole array system with a mushroom-like high-impedance surface (HIS) ground plane with wide-angle scanning capability in the E-plane. The unit cell of the proposed antenna array consists of a dipole antenna and a four-by-four HIS ground. The simulation results show that the proposed antenna array can achieve a wide scanning angle of up to 65° in the E-plane with an excellent impedance match and a small S11. Floquet mode analysis is utilized to analyze the active impedance and the reflection coefficient. Good agreement is obtained between the theoretical results and the simulations. Using numerical and theoretical analyses, we reveal the mechanism of such excellent wide scanning properties. For the range of small scanning angles, these excellent properties result mainly from the special reflection phase of the HIS ground, which can cause the mutual coupling between the elements of the real array to be compensated by the mutual coupling effect between the real array and the mirror array. For the range of large scanning angles, since the surface wave (SW) mode could be resonantly excited by a high-order Floquet mode TM−1,0 from the array and since the SW mode could be converted into a leaky wave (LW) mode by the scattering of the array, the radiation field from the LW mode is nearly in phase with the direct radiating field from the array. Therefore, with help from the special reflection phase of the HIS and the designed LW mode of the HIS ground, the antenna array with an HIS ground can achieve a wide-angle scanning performance.

The Han Chinese are the largest ethnic group in the world, and their origins, development, and expansion are complex. Many genetic studies have shown that Han Chinese can be divided into two distinct groups: northern Han Chinese and southern Han Chinese. The genetic history of the southern Han Chinese has been well studied. However, the genetic history of the northern Han Chinese is still obscure. In order to gain insight into the genetic history of the northern Han Chinese, 89 human remains were sampled from the Hengbei site which is located in the Central Plain and dates back to a key transitional period during the rise of the Han Chinese (approximately 3,000 years ago). We used 64 authentic mtDNA data obtained in this study, 27 Y chromosome SNP data profiles from previously studied Hengbei samples, and genetic datasets of the current Chinese populations and two ancient northern Chinese populations to analyze the relationship between the ancient people of Hengbei and present-day northern Han Chinese. We used a wide range of population genetic analyses, including principal component analyses, shared mtDNA haplotype analyses, and geographic mapping of maternal genetic distances. The results show that the ancient people of Hengbei bore a strong genetic resemblance to present-day northern Han Chinese and were genetically distinct from other present-day Chinese populations and two ancient populations. These findings suggest that the genetic structure of northern Han Chinese was already shaped 3,000 years ago in the Central Plain area.