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Cui Qiu,1– 3 Yan Fang1– 3 1First Affiliated Hospital of Anhui University of Science and Technology (Huainan First People’s Hospital) Tianjia ‘an District, Huainan City, People’s Republic of China; 2Anhui University of Science and Technology Medical College Tianjia ‘an District, Huainan City, People’s Republic of China; 3Institute of Ophthalmology, Anhui University of Science and Technology Tianjia ‘an District, Huainan City, People’s Republic of ChinaCorrespondence: Yan Fang, Tel +8613721126292, Fax +8605543320706, Email hnfy@sohu.comPurpose: To investigate the prevalence and influencing factors of symptomatic dry eye disease (DED) in Chinese coal workers.Methods: The prevalence of symptomatic DED in coal workers was investigated by using the questionnaire of Ocular Surface Disease Index (OSDI) and the influencing factors were explored.Results: The prevalence of symptomatic DED was 50.7% in coal workers. Of the influencing factors of symptomatic DED, the level of dust exposure had an odds ratio (OR) of 1.26, the time of dust exposure had an OR of 1.02, and the age had an OR of 1.03.Conclusion: There was a high morbidity of symptomatic DED among coal workers and the level and the time of dust exposure and the age of coal workers had important effects.Keywords: symptomatic DED, prevalence, influencing factors, coal workers

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 Rhodotorulapaludigena 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.

Natural polymer-based sutures have attractive cytocompatibility and degradability in surgical operations. Herein, anionic cellulose nanofibrils (ACNF) and cationic guar gum (CGG) are employed to produce nontoxic CGG/ACNF composite filament with a unique core-shell structure via interfacial polyelectrolyte complexation (IPC) spinning. The comprehensive characterization and application performance of the resultant CGG/ACNF filament as a surgical suture are thoroughly investigated in comparison with silk and PGLA (90% glycolide and 10% l-lactide) sutures in vitro and in vivo, respectively. Results show that the CGG/ACNF filament with the typical core-shell structure and nervation pattern surface exhibits a high orientation index (0.74) and good mechanical properties. The tensile strength and knotting strength of CGG/ACNF suture prepared by twisting CGG/ACNF filaments increase by 69.5%, and CGG/ACNF suture has a similar friction coefficient to silk and PGLA sutures. Moreover, CGG/ACNF suture with antibiosis and cytocompatibility exhibits better growth promotion of cells than silk suture, similar to PGLA suture in vitro. In addition, the stitching experiment of mice with the CGG/ACNF suture further confirms better healing properties and less inflammation in vivo than silk and PGLA sutures do. Hence, the CGG/ACNF suture with a simple preparation method and excellent application properties is promising in surgical operations.

In this work, we successfully explored an unexpected dehydrogenation triggered by Pd/Cu-catalyzed C(sp

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.

High-purity docosahexaenoic acid (DHA) resources are insufficient in the pharmaceutical and food industries. Although many efforts have attempted to obtain the high-purity DHA production, few reports have been successful. Here, a combined metabolic engineering strategy was employed to increase the DHA purity in the oleaginous thraustochytrid Aurantiochytrium. The strategy includes both partial deactivation of the competing pathway of DHA biosynthesis, by disrupting one copy of the fatty acid synthase gene, and strengthening of substrate supply and triacylglycerol synthesis, by the overexpression of acetyl-CoA carboxylase and diacylglycerol acyltransferase. With this strategy, a final mutant was obtained with a DHA purity of 61% in total fatty acids and a content of 331 mg/g dry cell weight. This study provides an advanced strategy for sustainable high-purity DHA production and highlights the strategy for producing designer oils in industrial oleaginous microorganisms.

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.

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

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

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.

Bacillaenes are a class of poly-unsaturated enamines produced by Bacillus strains that are notoriously unstable toward light, oxygen, and normal temperature. Herein, in an in-depth study of this highly unstable chemotype, the stability and biological function of bacillaenes were investigated. The structure change of the bacillaene scaffold was tracked by time-course 1H NMR data analysis coupled with the differential analysis of 2D-NMR spectra method, which was demonstrated to be a “domino” effect triggered by 4′,5′-cis (2 and 3) configuration rearranged to trans (2a and 3a). These findings provide the possibility for stabilizing the bacillaene scaffold by chemical modification of its trigger points. In the biofilm assay, compounds 1 and 2 accelerated self-biofilm formation in Bacillus methylotrophicus B-9987 at low concentrations of 1.0 and 0.1 μg/mL. Interestingly, bacillaenes play dual roles as antibiotic and biofilm enhancers in a dose-dependent manner, both of which serve in the self-protection of Bacillus.

Eicosapentaenoic acid (EPA) is an essential nutritional supplement for human health. The most prominent dietary source of EPA is fish oil, which is unsustainable because of the decline in fishery resources and serious environmental pollution. Alternatively, a heterologous polyketide synthase pathway for EPA biosynthesis was assembled in Thraustochytrid Aurantiochytrium. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the EPA biosynthetic gene cluster from Shewanella japonica in Aurantiochytrium. In the shake flask fermentation, the lipid and PUFA yields of the mutant were increased by 26.9 and 36.0%, respectively, and led to about 5-fold increase of the EPA yield. The final EPA titer reached 2.7 g/L in fed-batch fermentation. This study provides a novel metabolic engineering strategy to regulate the EPA ratio in microalgal oil for human nutritional supplementation.

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.

Ammonia-ethanol-water (AEW) pretreatment was adopted to treat wheat straw for ameliorating saccharification with the integrated preparation of submicron lignin spheres (SLS). Results showed that AEW pretreatment could remove 77% of lignin and 90% of extractives, thus increasing specific surface area and porosity of the substrate and finally enhancing the release of fermentable sugars in saccharification. Under the optimal pretreatment conditions (170 °C for 2 h, ethanol concentration 55% (v/v), ammonia concentration 7.5 wt%), the final total sugar yield reached 81.7% after pretreatment and saccharification, which was 2.25 times higher compared to the conventional ethanol organosolv pretreatment. Moreover, washing could be excluded for AEW-pretreated wheat straw before saccharification, and both ethanol and ammonia could be readily recovered and reused, making the AEW pretreatment clean and sustainable. In addition, SLS with hollow structure and average diameter of 161.2 ± 53.6 nm were fabricated using the fractionated lignin, which could offset the overall cost of AEW pretreatment.

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., Promising bioremediation strategies for plastics waste are of great importance and requirements. In our study, we constructed a recombinant Clostridium thermocellum strain expressing a secretory cutinase (LCC) as a thermophilic whole‐cell biocatalyst to degrade PET under high‐temperature condition (60°C). To our knowledge, this biocatalysis system demonstrates the highest PET degradation efficiency compared to reported whole‐cell‐based systems and also enjoys a low‐cost advantage over the free enzyme‐based process.

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

In this study, triazine-degrading strain SB5 was isolated and screened from the activated sludge contaminated with atrazine by enrichment culture technology. Based on its morphology and 16S rRNA gene analysis, strain SB5 was initially identified as本研究采用富集培养技术自莠去津污染的活性污泥中分离筛选到一株具有降解三嗪类除草剂功能的菌株SB5,经形态学和16S rRNA基因分析将其初步鉴定为类节杆菌属细菌。其具有已知莠去津降解相关基因

Polysaccharides derived from lignocellulose are promising sustainable carbon sources. Cellulosome is a supramolecular machine integrating multi-function enzymes for effective lignocellulose bio-saccharification. However, how various non-cellulose components of lignocellulose affect the cellulosomal saccharification is hitherto unclear. This study first investigated the stability and oxygen sensitivity of the cellulosome from Clostridium thermocellum during long-term saccharification process. Then, the differential inhibitory effects of non-cellulose components, including lignin, xylan, and arabinoxylan, on the cellulosome-based saccharification were determined. The results showed that lignin played inhibitory roles by non-productively adsorbing extracellular proteins of C. thermocellum. Differently, arabinoxylan preferred to bind with the cellulosomal components. Almost no adsorption of cellulosomal proteins on solid xylan was detected. Instead, xylan in water-dissolved form interacted with the cellulosomal proteins, especially the key exoglucanase Cel48S, leading to the xylan inhibitory effect. Compared to xylan, xylooligosaccharides influenced the cellulosome activity slightly. Hence, this work demonstrates that the timely hydrolysis or removal of dissolved xylan is important for cellulosome-based lignocellulose saccharification.

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.

Chiral compounds are valuable industrial products and intermediates, and the production of chemicals with high enantiopurity is one of the major objects in asymmetric catalysis. Compared with traditional chemical synthesis, enzymatic synthesis can produce chiral molecules under sustainable conditions which are much greener, more economical, and more environmentally friendly. The superfamily of α/β hydrolases includes a lot of diverse enzymes showing excellent chemo-, regio-, and enantio-selectivity in asymmetric synthesis and many of them are biocatalysts in industry. This review outlines the current knowledge of the structures and reaction mechanism of α/β hydrolases and summarizes the screening and protein engineering efforts to develop biocatalysts for chiral chemicals production in recent years. Other strategies such as whole-cell catalysis and protein immobilization to improve the performance of α/β hydrolases are also discussed. The progress in biocatalyst development based on α/β hydrolases will promote the biosynthesis of chiral compounds, thus contributing to the green and sustainable development of the chemical and pharmaceutical industry.

Transition-metal-catalyzed Si-H bond insertion reactions are generally limited to stabilized diazo compounds. An efficient copper-catalyzed Si-H bond insertion reaction of

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.

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.

High-purity docosahexaenoic acid (DHA) resources are insufficient in the pharmaceutical and food industries. Although many efforts have attempted to obtain the high-purity DHA production, few reports have been successful. Here, a combined metabolic engineering strategy was employed to increase the DHA purity in the oleaginous thraustochytrid

Exploration of unstable compounds is a rarely explored area of natural product research. We describe the integration of genomic and metabolomic analyses with bioassay-guided compound mining to effectively explore unstable bacillaenes. New bacillaene structures (2, 4, and 5) were identified from compound mixtures using the DANS-SVI (differential analysis of 2D NMR spectrum-single spectrum with variable intensities) method, which were further verified by the isolation of the pure compounds under strictly controlled conditions. Compound 1 exhibited antibacterial activity against multi-drug-resistant bacterial strains, while glycosylation decreased the activity of the bacillaene scaffold.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a β-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter –35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

The development of a clean and sustainable pretreatment is of great importance for the production of fermentable sugars. In this study, an ammonium sulfite (AS) pretreatment of wheat straw was optimized based on response surface methodology with a three-level, three-factor Box-Behnken design. The investigated factors were AS dosage, pretreatment time, and pretreatment temperature. The effectiveness of the AS pretreatment was evaluated using the standard enzymatic hydrolysis procedure. A second-order polynomial fit was performed to fit the experimental data, and the model analysis showed that the effect of the AS dosage on the final total sugar yields was much more significant than that of the other two factors. Under the optimum pretreatment conditions (27% of the AS dosage (based on the dry wheat straw) at 160 ºC for 63 min), the final total sugar yield achieved was 74.4% after saccharification, which was in agreement with the predicted value (76.5%). Furthermore, it was found that pre-impregnation with acetic acid before AS pretreatment or the post-mechanical refining after AS pretreatment could further increase the fermentable sugar yields to approximately 77%. In addition, the spent liquor containing nitrogen could be used for the production of lignin-based fertilizer, thus making the whole process clean and sustainable.

Translationally controlled tumor proteins (TCTPs) are eukaryote-conserved multifunctional proteins, but their primary functions are not well understood yet. Study on TCTP from unicellular species may provide insight into the primary function of the TCTP family. Bioinformatic analysis indicated that the TCTP from Nannochloropsis oceanica (NoTCTP), a model unicellular microalga for biodiesel and polyunsaturated fatty acid production, has low sequence homology to other structure-known TCTPs and two TCTP signature patterns are not detected in NoTCTP. Hence, we determined the solution structure of NoTCTP. The overall structure of NoTCTP, including a long flexible loop, a β-barrel subdomain, and a helical subdomain, is generally similar to other TCTP structures. NoTCTP has a eukaryote-conserved surface for the binding of eukaryotic elongation factor 1B, confirming that TCTP is involved in protein synthesis, which is one of the primary functions of TCTP. Additionally, NoTCTP has distinct features different from other TCTPs. NoTCTP structure lacks a short α-helix which exists in all other known TCTP structures. The helical subdomain and some loops of NoTCTP also have distinct conformations among the TCTP family proteins. Therefore, our study on NoTCTP revealed not only conserved structural features but also the structural diversity of TCTP family proteins.

Corncob residue (CCR) is an economic feedstock with enormous potential for the production of bioethanol. In this work, potassium hydroxide (KOH) pretreatment of CCR was investigated under relatively mild conditions, and the effectiveness of KOH pretreatment was evaluated by enzymatic saccharification at high solid loading. Results showed that after KOH pretreatment (16 wt% KOH dosage at 70 °C for 90 min) and the enzymatic hydrolysis at 20% solids loading and 20 FPU g−1-substrate of cellulase loading, the glucose yield could reach up to about 91%, which was over 90% higher compared to the raw CCR without KOH pretreatment. Correspondingly, about 89% of lignin and 79% of extractives were removed after KOH pretreatment. In addition, the spent liquor of KOH pretreatment containing sylvite could be used as lignin-based fertilizer based on the concept of biorefinery. In this case, the entire process for the production of fermentable sugars was clean and sustainable, which is very vital for the conversion of lignocelluloses to bioenergy or chemicals.

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.

A large and powerful radio frequency (RF) inductively coupled plasma (ICP) source with a Faraday shield (FS) has been investigated by a 3D fluid model, consisting of a plasma module and an electromagnetic field module. Effects of the structure of the FS, the RF current, and the gas pressure on the spatial distributions of plasma parameters including the electron density, electron temperature, and electron potential barrier are investigated systematically. The results show that the FS has a great influence on the spatial distribution and transport mechanism of electrons. The electron density has a maximum value at the center of the ICP source and declines sharply near the FS. Furthermore, it decreases obviously with the decreasing slit width, due to the electron transport mechanism, which is directly related to the electron potential barrier and electron temperature. In addition, the electron density increases with the RF current and gas pressure; it is noteworthy that the stronger induced electric field heating exists at the slit gap under high RF current that makes the electron temperature increase. The data obtained in this paper could lead to a deeper insight into the characteristics of ICP sources, which is extremely essential for optimizing the FS structure and designing large and powerful radio frequency ICPs in the future.

A pH-dependent dual-binding-site switch in a pair of protein modules is discovered, and the molecular mechanism is elucidated., Many important proteins undergo pH-dependent conformational changes resulting in “on-off” switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from Clostridium acetobutylicum, which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4.8 and 7.5 with a 180° rotation of the bound dockerin. Combined analysis by nuclear magnetic resonance spectroscopy, crystal structure determination, mutagenesis, and isothermal titration calorimetry elucidates the chemical and structural mechanism of the pH-dependent switching of the binding sites. The pH-dependent dual-binding-site switch not only represents an elegant example of biological regulation but also provides a new approach for developing pH-dependent protein devices and biomaterials beyond an on-off switch for biotechnological applications.

Eicosapentaenoic acid (EPA) is an essential nutritional supplement for human health. The most prominent dietary source of EPA is fish oil, which is unsustainable because of the decline in fishery resources and serious environmental pollution. Alternatively, a heterologous polyketide synthase pathway for EPA biosynthesis was assembled in Thraustochytrid

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy in overcoming multi-drug-resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a β-lactamase homologue BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A and also exhibits substrate promiscuity toward acyl acceptors with different scaffolds. The crystal structure of BmmI covalently linked to the succinyl group and systematic mutagenesis highlighted the role of oxyanion holelike geometry in the recognition of carboxyl-terminated acyl donors. The engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. The structure of BmmI with acyl acceptor MLN A revealed the importance of Arg292 in the recognition of macrolide substrates. Moreover, the mechanism of the BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying the substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.

Marine microalgae are promising sources of novel glycoside hydrolases (GHs), which have great value in biotechnical and industrial applications. Although many GH1 family β-glucosidases have been extensively studied, studies on β-glucosidases from microalgae are rare, and no structure of algal GH1 β-glucosidase has been reported. Here, we report the biochemical and structural study of a GH1 β-glucosidase BGLN1 from Nannochloropsis oceanica, an oleaginous microalga. Phylogenetic analysis of BGLN1, together with the known structures of GH1 β-glucosidases, has indicated that BGLN1 is branched at the root of the eukaryotic part of the phylogenetic tree. BGLN1 showed higher activity against laminaribiose compared to cello-oligosaccharides. Unlike most of the other GH1 β-glucosidases, BGLN1 is partially inhibited by metal ions. The crystal structure of BGLN1 revealed that BGLN1 adopts a typical (α/β)8-barrel fold with variations in loops and N-terminal regions. BGLN1 contains extra residues at the N-terminus, which are essential for maintaining protein stability. BGLN1 has a more acidic substrate-binding pocket than other β-glucosidases, and the variations beyond the conserved -1 site determine the substrate specificity. These results indicate that GH enzymes from microalgae may have unique structural and functional features, which will provide new insight into carbohydrate synthesis and metabolism in marine microalgae.

Persistent key-value (KV) stores are mainly designed based on the Log-Structured Merge-tree (LSM-tree), which suffer from large read and write amplifications, especially when KV stores grow in size. Existing design optimizations for LSM-tree-based KV stores often make certain trade-offs and fail to simultaneously improve both the read and write performance on large KV stores without sacrificing scan performance. We design UniKV, which unifies the key design ideas of hash indexing and the LSM-tree in a single system. Specifically, UniKV leverages data locality to differentiate the indexing management of KV pairs. It also develops multiple techniques to tackle the issues caused by unifying the indexing techniques, so as to simultaneously improve the performance in reads, writes, and scans. Experiments show that UniKV significantly outperforms several state-of-the-art KV stores (e.g., LevelDB, RocksDB, HyperLevelDB, and PebblesDB) in overall throughput under read-write mixed workloads.

The properties of lignocellulosic substrates obtained from different pretreatments have a big impact on downstream saccharification based on both the fungal cellulase system and the cellulosome-based whole-cell biocatalysis system. However the corresponding effect of these two distinct saccharification strategies has not been comparatively analyzed. In this work, three ammonium sulfite (AS)-based pretreatment combinations (i.e., AS + hydrothermal (HT) pretreatment, AS + xylanase (X) pretreatment, and HT + AS pretreatment) were conducted to treat wheat straw. The obtained pretreated substrates with different properties were saccharified using fungal cellulase or an engineered Clostridium thermocellum strain as the whole-cell biocatalyst, and the ability to release sugar was comparatively evaluated. It was found that for the whole-cell saccharification, the total sugar digestibility of AS + HT/X pretreated wheat straw was 10% higher than that of HT + AS pretreated wheat straw. However, for fungal cellulase-based saccharification, the enzymatic hydrolysis efficiency was less susceptible to the sequence of pretreatment combinations. Hence, the whole-cell biocatalysis system was more sensitive to substrate accessibility compared to the free enzymes. In addition, the characterization and analyses showed that AS + HT/X pretreatment could remove more lignin, generating a more accessible surface with a larger external surface and lower surface lignin coverage, compared to the HT + AS pretreatment. Therefore, the AS + HT/X pretreatment was more compatible with the cellulosome-based whole-cell saccharification.

Donnan Dialysis (DD) is developed in this study for continuous removal of fluoride from Antarctic krill by using seawater or concentrated seawater as the receiving solution. Results show that RO concentrate (1.0 mol L−1 NaCl) got the highest fluoride removal efficiency (93.66% ± 0.81%) with three stages at 0.01 cm s−1 flow velocity. Afterwards, fluoride removal experiment with a real krill solution showed 89.34% ± 0.55% under the optimized conditions. The product has fulfilled the fluoride content of US FDA standard and Canadian Food Inspection Agency in fish and fish products. Results also showed a lower loss of amino acids and proteins in comparison of conventional methods. Finally, a techno-economic evaluation showed that 4.0 USD per ton of Krill solution can be expected by this DD process. This study proves that Donnan Dialysis may be a sustainable defluorination process in removing high fluoride content of seafood by seawater or concentrated seawater.

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. Electronic supplementary material The online version of this article (10.1186/s13068-018-1304-8) contains supplementary material, which is available to authorized users.

Summary Cellulosome is a highly efficient supramolecular machine for lignocellulose degradation, and its substrate‐coupled regulation requires soluble transmembrane signals. However, the inducers for cellulosome synthesis and the inducing effect have not been clarified quantitatively. Values of cellulosome production capacity (CPC) and estimated specific activity (eSA) were calculated based on the primary scaffoldin ScaA to define the stimulating effects on the cellulosome synthesis in terms of quantity and quality respectively. The estimated cellulosome production of Clostridium thermocellum on glucose was at a low housekeeping level. Both Avicel and cellobiose increased CPCs of the cells instead of the eSAs of the cellulosome. The CPC of Avicel‐grown cells was over 20‐fold of that of glucose‐grown cells, while both Avicel‐ and glucose‐derived cellulosomes showed similar eSA. The CPC of cellobiose‐grown cells was also over three times higher than glucose‐grown cells, but the eSA of cellobiose‐derived cellulosome was 16% lower than that of the glucose‐derived cellulosome. Our results indicated that cello‐oligosaccharides played the key roles in inducing the synthesis of the cellulosome, but non‐cellulosic polysaccharides showed no inducing effects.

MoS2 nanodots are emerging as promising semiconductor materials for optoelectronic devices. However, most of the recent attention is focused on the fabrication of MoS2 nanodots, and the survey for exciton dynamics of MoS2 nanodots remains less explored. Herein, we use femtosecond transient absorption spectroscopy to investigate the carrier dynamics of MoS2 nanodots. Our results show that defect-assisted carrier recombination processes are well consistent with the observed dynamics. The photo-excited carriers are captured by defects with at least two different capture rates via Auger scattering. Four processes are deemed to take part in the carrier relaxation. After photoexcitation, carrier cooling occurs instantly within ~0.5 ps. Then most of carriers are fast captured by the defects, and the corresponding time constant increases from ~4.9 ps to ~9.2 ps with increasing pump fluence, which may be interpreted by saturation of the defect states. Next a small quantity of carriers is captured by the other kinds of defects with a relatively slow carrier capture time within ~65 ps. Finally, the remaining small fraction of carriers relaxes via direct interband electron-hole recombination within ~1 ns. Our results may lead to deep insight into the fundamentals of carrier dynamics in MoS2 nanodots, paving the way for their further applications.