Search

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.

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.

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.

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.

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.

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 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.

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.

Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular β-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.

To reduce fermentation-associated wastewater discharge and the cost of wastewater treatment, which further reduces the total cost of DHA and ARA production, this study first analyzed the composition of wastewater from Aurantiochytrium (DHA) and Mortierella alpina (ARA) fermentation, after which wastewater recycling technology for these fermentation processes was developed. No negative effects of DHA and ARA production were observed when the two fermentation wastewater methods were cross-recycled. DHA and ARA yields were significantly inhibited when the wastewater from the fermentation process was directly reused. In 5-L fed-batch fermentation experiments, using this cross-recycle technology, the DHA and ARA yields were 30.4 and 5.13 g L−1, respectively, with no significant changes (P > 0.05) compared to the control group, and the water consumption was reduced by half compared to the traditional process. Therefore, this technology has great potential in industrial fermentation for polyunsaturated fatty acid production.

Aurantiochytrium is a commercial docosahexaenoic acid (DHA) producer, and its DHA content can be significantly increased under cold stress. Given this response to low temperature, we examined proteomics changes in Aurantiochytrium under cold stress. We detected approximately 700 protein spots using two-dimensional gels, whereas using iTRAQ technology, we detected 4650 types of proteins and successfully identified > 53%. The results indicated that cold stress inhibits the cellular energy supply from glycolysis and the TCA cycle, to ensure a sufficient supply of NADPH and ribose for anabolism. In contrast, the pentose phosphate pathway was not affected. With respect to lipid synthesis, low temperature led to a significant down-regulation and up-regulation of fatty acid synthase and polyunsaturated fatty acid synthase, respectively, and restricted the protein synthesis of diacylglycerol O-acyltransferase and phospholipid: diacylglycerol acyltransferase. These results show the preferential biosynthesis of polyunsaturated fatty acids and phospholipids by Aurantiochytrium , which collectively serve to increase cell survival rates in cold environments.

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. Electronic supplementary material The online version of this article (10.1186/s13068-019-1374-2) contains supplementary material, which is available to authorized users.

Chromosome microarray analysis (CMA) has proven to be a powerful tool in postnatal patients with intellectual disabilities. However, the diagnostic capability of CMA in patients with congenital oral clefts remain mysterious. Here, we present our clinical experience in implementing whole-genome high-resolution SNP arrays to investigate 33 patients with syndromic and nonsyndromic oral clefts in whom standard karyotyping analyses showed normal karyotypes. We aim to identify the genomic aetiology and candidate genes in patients with congenital oral clefts. CMA revealed copy number variants (CNVs) in every patient, which ranged from 2 to 9 per sample. The size of detected CNVs varied from 100 to 3.2 Mb. In 33 patients, we identified six clinically significant CNVs. The incidence of clinically significant CNVs was 18.2% (6/33). Three of these six CNVs were detected in patients with nonsyndromic clefts, including one who presented with isolated cleft lip with cleft palate (CLP) and two with cleft palate only (CPO). The remaining three CNVs were detected in patients with syndromic clefts. However, no CNV was detected in patients with cleft lip only (CLO). The six clinically significant CNVs were as follows: 8p23.1 microduplication (198 kb); 10q22.2-q22.3 microdeletion (1766 kb); 18q12.3 microduplication (638 kb); 20p12.1 microdeletion (184 kb); 6q26 microdeletion (389 kb); and 22q11.21-q11.23 microdeletion (3163 kb). In addition, two novel candidate genes for oral clefts, KAT6B and MACROD2, were putatively identified. We also found a CNV of unknown clinical significance with a detection rate of 3.0% (1/33). Our results further support the notion that CNVs significantly contributed to the genetic aetiology of oral clefts and emphasize the efficacy of whole-genome high-resolution SNP arrays to detect novel candidate genes in patients with syndromic and nonsyndromic clefts.

Toxin-antitoxin (TA) systems widely exist in bacterial plasmids, phages, and chromosomes and play important roles in growth persistence and host-pathogen interaction. Virulence associated protein BC (VapBC) family TAs are the most abundant TAs in bacteria and many pathogens contain a large number of vapBC loci in the genome which have been extensively studied. Clostridium thermocellum, a cellulolytic anaerobic gram-positive bacterium with promising applications in biofuel production, also contains a VapBC TA in the genome. Despite the structures of several VapBC family TAs have been determined, the toxin and anti-toxin components of C. thermocellum VapBC have very low sequence identity to the proteins in PDB. Therefore, the structure and functional mechanism of this TA is largely unknown. Here we reported the NMR resonance assignments of the VapC toxin from C. thermocellum as a basis for further structural and functional studies.

NrdH redoxin is the only hydrogen donor for ribonucleotide reductase in Mycobacterium tuberculosis. Several crystal structures of NrdH redoxins in the oxidized state from different species have been reported, but no structure of the reduced state has yet been reported. Using NMR spectroscopy, we found surprisingly that the reduced NrdH redoxin from M. tuberculosis is largely unfolded at a pH lower than the pKa of its first active site cysteine, and the structural basis of the low stability was analyzed. In addition, a single mutant of the NrdH redoxin suitable to determine the structure in the reduced state was obtained.

Lignocellulosic biomass is the most abundant sustainable carbon source on the planet and has enormous potential to substitute fossil resources on the premise of cost-effective conversion. Efforts have been made to develop various lignocellulosic bioconversion strategies to overcome biomass recalcitrance, promote product conversion efficiency and reduce process cost. Consolidated bio-saccharification (CBS), a consolidated bioprocessing (CBP) derived strategy, is herein proposed for lignocellulose bioconversion by integrating enzyme production and hydrolysis steps but separating fermentation from the integrated process. This strategy employs cellulosome-producing microorganisms as a biocatalyst to enhance lignocellulose solubilization and produces lignocellulose-derived fermentable sugars as a platform product for fermentations aiming at various products. The success of CBS depends on robust biocatalysts with high activity, suitable pretreatments for efficient delignification, and downstream fermentations with process compatibility. The review introduces the updated progress on lignocellulose bioconversion following the CBS route, discusses key factors for optimization of the CBS process, and, more importantly, highlights challenges and promising solutions for the CBS strategy in the industrial application of lignocellulose bioconversion.

Cellulosomes are highly efficient multienzyme complexes for lignocellulose degradation secreted by some lignocellulolytic bacteria. Cellulosomes are assembled through protein modules named cohesin and dockerin, and multiple cohesin modules in the scaffold protein generally determine the complexity of the cellulosomes. Some cellulosomal proteins contain multiple dockerin modules, which may generate more complex cellulosomal architectures. Genome mining revealed that cellulosomal proteins containing double dockerin modules and a protease module exist in many cellulosome-producing bacteria, and these proteins together with cellulosomal protease inhibitors were proposed to have regulatory roles. However, the structures and functions of these multiple-dockerin proteins in cellulosome have not been reported before. In this paper, we present the NMR chemical shift assignments of the double-dockerin of a cellulosomal protease from Clostridium thermocellum DSM1313. The secondary structures predicted from the chemical shifts agree with the structural arrangement of the tandem dockerin modules. The chemical shift assignments here provide the basis for the structural and functional studies of multiple-dockerin proteins in future.

A Gram-stain-negative, motile, aerobic and rod-shaped bacterial strain, designated T17T, was isolated from benthic sediment sampled at Jiaozhou Bay, Bohai Sea, China, and its taxonomic position was investigated. The 16S rRNA gene sequence of strain T17T exhibited the highest similarity values to those of the type strain Marinobacter lacisalsi FP2.5 (96.2 %) and Marinobacter koreensis DD-M3T (96.2 %). Strain T17T grew optimally at 35 °C, pH 7.0–8.0 and in the presence of 6.0–10.0 % (w/v) NaCl. The predominant ubiquinone in strain T17T was identified as Q-9. The major fatty acids of strain T17T were C12 : 0, C16 : 0 and C16 : 0 10-CH3. The major polar lipids of strain T17T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidglycerol, an unidentified aminolipid and an unidentified phospholipid. The DNA G+C content of strain T17T was 63.0 mol%. The draft genome sequence of strain T17T includes 4 755 891 bp in total (N50=2 856 325 bp) with a medium read coverage of 100.0x and 11 scaffolds. In silico DNA–DNA hybridization with the three type strains showed 20.3, 19.7 and 19.9 % relatedness to Marinobacter santoriniensis NKSG1T, Marinobacter segnicrescens SS11B1-4T and Marinobacter daqiaonensis CGMCC 1.9167T, respectively. On the basis of the phenotypic, phylogenetic, genomic and chemotaxonomic properties, strain T17T is considered to represent a novel species within the genus Marinobacter , for which the name Marinobacter bohaiensis sp. nov. is proposed. The type strain is T17T (=KCTC 52710T=MCCC 1K03282T).

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.

To improve the economic viability of Chlorella as feedstock for food commodities, a serial of concentrations of low-cost sweet sorghum juice (SSJ), alternative to glucose, were used for the fermentation of Chlorella pyrenoidosa. A high biomass and protein production (8.91 g L−1 biomass and 4.52 g L−1 protein) was revealed with 20% SSJ. To further increase productivity, heavy-ion irradiation-mediated mutagenesis was employed to create mutants where a strain K05, with desired phenotypes (increased biomass and protein production in pilot-scale fermentation), was screened. Compared with the parental strain, the production of biomass, proteins, and chlorophylls of mutant K05 increased by 11.6%, 31.8%, and 7.6%, respectively. Production capacities under industrial scale (two-ton) further pinpoint the stability and scalability of mutant K05. These results suggest that advances in cultivation techniques coupled with artificial strain improvement will further promote microalgae as an attractive platform of functional food.

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. Electronic supplementary material The online version of this article (10.1186/s13068-017-1009-4) contains supplementary material, which is available to authorized users.

Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes.

The difficulty in simultaneously obtaining a high production of neutral lipids and biomass in the oleaginous microalga Nannochloropsis is an attractive challenge that needs to be addressed. To overcome this challenge and unveil the underlying metabolic characteristics, the model oleaginous microalga Nannochloropsis oceanica IMET1, under high light intensity and nitrogen-replete conditions, was prepared via steady-state continuous culture in the present study. The simultaneous accumulation of lipids, particularly neutral lipids, and biomass in N. oceanica IMET1 was triggered at a high light intensity (331.2 mu mol photons m(-2) s(-1)) in the light saturation region accompanied by a sufficient nitrogen supply (2000 mu mol L-1). Moreover, high amounts of valuable fatty acids (arachidonic acid and eicosapentaenoic acid) were also produced. The distribution of fatty acids and intracellular soluble metabolites, the membrane lipid composition and the photosynthetic pathway were associated with the concurrent accumulation of biomass and TAGs in N. oceanica IMET1. The common and different metabolic responses between high-light and nitrogen-depletion strategies for the accumulation of neutral lipids were also discussed. These results provide detailed metabolic clues for the genetic and metabolic engineering of Nannochloropsis for biofuel production. (C) 2015 Elsevier B.V. All rights reserved.

The cellulosome of Clostridium thermocellum is an elegant and efficient multi-enzyme complex for degrading lignocellulose. The cellulosome contains several dozens of carbohydrate hydrolysis enzymes, which are regulated by the presence of environmental substrates through several pairs of sigma and anti-sigma factors. The anti-sigma factors sense the presence of substrates and transduce the signals into the cell. The sigma factors are then released from the corresponding anti-sigma factors, and they recruit RNA polymerase to transcribe specific cellulosomal genes. However, it is not clear how the extracellular signals are transduced into the cell by the anti-sigma factors. The anti-sigma factors of C. thermocellum contain an N-terminal intracellular domain, a trans-membrane helix, a periplasmic domain, a proline-rich region which is probably required for crossing the cell wall, and a C-terminal carbohydrate-binding domain or glycoside hydrolase domain. The periplasmic domain may play a key role in signal transduction; however, its three-dimensional structure is still unknown. Here we report the NMR resonance assignments of the periplasmic domain of anti-sigma factor RsgI2 from C. thermocellum as a basis for further structural determination and functional studies.

Translationally-controlled tumor protein (TCTP) is a eukaryote-conserved protein with crucial roles in cellular growth. It has also been proposed that plant TCTP has functions specific to plant, while no structure of TCTP from photosynthetic organism has been reported. Nannochloropsis is a photosynthetic microalga with high yield of lipid and high-value polyunsaturated fatty acid, which is promising for biodiesel production. Study of growth-related proteins may provide new clue for improving the yield of lipid. TCTP from Nannochloropsis oceanica shares low sequence identity with structure-known TCTPs. Here we reported the NMR resonance assignments of TCTP from N. oceanica for further structural and functional studies.

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.

A Gram-stain-negative, non-motile, aerobic and rod-shaped bacterial strain, designated H-12T, was isolated from a sea squirt (Ciona savignyi) collected from Tsingtao Port, Jiaozhou Bay, China, and its taxonomic position was investigated. Strain H-12T grew optimally at 25–30 °C, at pH 7.0–8.0 and in the presence of 3.0–4.0 % (w/v) NaCl. The 16S rRNA gene sequence of strain H-12T exhibited the highest similarity to that of the type strain of Amylibacter marinus (95.3 %). A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain H-12T clustered with the type strain of A. marinus . The predominant ubiquinone in strain H-12T was identified as Q-10. The major fatty acids of strain H-12T were C18 : 1ω7c and C18 : 1ω7c 11-methyl. The major polar lipids detected in strain H-12T were phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, one unidentified aminolipid, two unidentified phospholipids and five unidentified lipids. The DNA G+C content of strain H-12T was 52.7 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic properties, strain H-12T is considered to represent a novel species within the genus Amylibacter , for which the name Amylibacter cionae sp. nov. is proposed. The type strain is H-12T (=KCTC 52581T=CGMCC 1.15880T).

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. Electronic supplementary material The online version of this article (doi:10.1186/s12896-017-0388-8) contains supplementary material, which is available to authorized users.

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.

Objectives Y chromosome haplogroup Q1a1 is found almost only in Han Chinese populations. However, it has not been found in ancient Han Chinese samples until now. Thus, the origin of haplogroup Q1a1 in Han Chinese is still obscure. This study attempts to provide answer to this question, and to uncover the origin and paternal genetic structure of the ancestors of the Han Chinese. Methods Eighty-nine ancient human remains that were excavated from the presumed geographic source of the Han Chinese and dated to approximately 3,000 years ago were treated by the amelogenin gene polymerase chain reaction test, to determine their sex. Then, Y chromosome single nucleotide polymorphisms were subsequently analyzed from the samples detected as male. Results Samples from 27 individuals were successfully amplified. Their haplotypes could be attributed to haplogroups N, O*, O2a, O3a, and Q1a1. Analyses showed that the assigned haplogroup of each sample is correlated to the suspected social status and observed burial custom associated with the sample. Conclusions The origins of the observed haplotypes and their distribution in present day Han Chinese and in the samples suggest that haplogroup Q1a1 was probably introduced into the Han Chinese population approximately 3,000 years ago. Am. J. Hum. Biol. 26:813–821, 2014. © 2014 Wiley Periodicals, Inc.

Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

Phytohormones are chemical messengers that have a positive effect at low concentrations on the biosynthesis of high-value compounds. Therefore, the effects of phytohormones on lipid and arachidonic acid (ARA) biosynthesis in Mortierella alpina were investigated in this study. At proper concentrations, the stimulatory effects of phytohormones on lipid production were determined to be as follows: 6-benzyl adenine (BA) > indole-3-acetic acid (IAA) > furfuryl adenine (KT) > gibberellin (GA) > indole-3-butyric acid (IBA) > abscisic acid (ABA). The results show that in the presence of 15 mg L−1 BA, the best positive effect was obtained, in which the lipid and ARA yields of M. alpina increased by 20.34% and 29.17%, respectively. Surprisingly, there was no synergy between the addition of two cytokinins (KT and BA), while adding cytokinins (KT or BA) and auxin (IAA) inhibited the growth of M. alpina and the ARA yield decreased by approximately 64%. Additional studies, such as those involving enzyme activity detection and quantitative real time polymerase chain reaction were carried out to check the fatty acid and lipid biosynthesis when the phytohormones were present. The activity of the main NADPH-supplying enzyme, 6-phosphoglucose dehydrogenase (G6PDH), increased by 19.52%. Moreover, the transcription levels of fatty acid synthase (FAS), Δ9-desaturase, and diacylglycerolacyltransferase (DGAT) increased by 9.3, 9.6 and 7.7 times, respectively, when only one type of phytohormone was present, indicating the enhancement of fatty acid and lipid biosynthesis in M. alpina. This study demonstrates the potential application of phytohormones for improving ARA yields of M. alpina.

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.

A new protocol for metabolomic studies was developed by combining liquid chromatography-tandem mass spectrometry and isotope dilution mass spectrometry with universal C-13 labeled internal standards from Escherichia Coli. The multiple reaction monitoring mode of mass spectrometry was used for quantification. Forty-five water-soluble intracellular metabolites, including 20 amino acids, 16 organic acids (primarily from the tricarboxylic acid cycle), and 9 cofactors, were measured and 34 of them were successfully quantified using the C-13-labeled internal standards. The limit of detection, limit of quantification, precision, and linearity of the methods were evaluated. The methods were applied to the quantitative analysis of intracellular metabolites extracted from wild-type and ethanol-adapted strains of Clostridium thermocellum cultivated with and without ethanol stress, and all 34 metabolites including all 9 cofactors were successfully quantified. Further multivariate data analyses of the metabolic differences between wild-type and ethanol-adapted strains were performed on the quantitative data, which can help elucidate the metabolic mechanism behind ethanol adaptation in C. thermocellum. Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.

Aurantiochytrium is an important docosahexaenoic acid (DHA) producer containing two kinds of fatty acid synthesis pathways, that is, the fatty acid synthase pathway (FAS) for saturated fatty acid synthesis and the polyketide synthase pathway (PKS) for polyunsaturated fatty acid synthesis. To understand the regulation mechanism between the two pathways, the impacts of six short-chain fatty acids on the fatty acid synthesis of Aurantiochytrium sp. SD116 were studied. All short-chain fatty acids showed little effect on the cell growth, but some of them significantly affected lipid accumulation and fatty acid composition. Pentanoic acid and isovaleric acid greatly inhibited the synthesis of saturated fatty acids, whereas the polyunsaturated fatty acid synthesis was not affected. Analysis of malic enzyme activity, which supplied NADPH for saturated fatty acids biosynthesis, indicated that the two fatty acid synthesis pathways can utilize different substrates and possess independent sources of NADPH.

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.

Extensive heterogeneity is a defining hallmark of glioblastoma multiforme (GBM) at the cellular and molecular levels. EGFRvIII, the most common EGFR mutant, is expressed in 24-67% of cases and strongly indicates a poor survival prognosis. By co-expressing EGFRvIII and EGFRwt, we established an EGFRvIII/wt heterogenic model. Using this approach, we confirmed that a mixture of EGFRvIII and EGFRwt at a certain ratio could clearly enhance tumor growth in vitro and in vivo compared with EGFRwt cells, thereby indicating that EGFRvIII cells promote tumor growth. Furthermore, we demonstrated that the EGFRvIII cells could support the growth of EGFRwt cells by secreting growth factors, thus acting as the principal source for maintaining tumor survival. F25P preproinsulin effectively reduced the concentrations of EGF, VEGF, and MMP-9 in the blood of tumor-bearing mice by competitively inhibiting the endoplasmic reticulum signal peptidase and increased the overall survival in orthotopic models. Taken together, our results provided an effective therapy of F25P preproinsulin in the EGFRvIII/wt heterogenic model.

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form D: (-)-tartaric acid or L: (+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[D: ] and CESH[L: ], producing D: (-)- and L: (+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver-Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[L: ] showed much higher catalytic efficiency than CESH[D: ], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[L: ] and CESH[D: ] have different structural folds and potential active site residues. CESH[L: ] adopted a typical α/β-hydrolase fold with a cap domain and a core domain, whereas CESH[D: ] possessed a unique TIM barrel fold composed of 8 α-helices and 8 β-strands, and 2 extra short α-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[D: ], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

A yeast-like fungus, termed strain SD301, with the ability to produce a high concentration of squalene, was isolated from Shuidong Bay, China. The nucleotide sequence analysis of the internal transcribed spacer (ITS) region of SD301 indicated the strain belonged to Pseudozyma species. The highest biomass and squalene production of SD301 were obtained when glucose and yeast extracts were used as the carbon and nitrogen sources, respectively, with a C/N ratio of 3. The optimal pH and temperature were 6 and 25 °C, with 15 g L(-1) of supplemented sea salt. The maximum squalene productivity reached 0.039 g L(-1) h(-1) in batch fermentation, while the maximum squalene yield of 2.445 g L(-1) was obtained in fed-batch fermentation. According to our knowledge, this is the highest squalene yield produced thus far using fermentation technology, and the newly isolated strain Pseudozyma sp. SD301 is a promising candidate for commercial squalene production.

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes., Selective RNA processing and stabilization (SRPS) can regulate bacterial operons, but the process is not well understood. Here, the authors show that the stoichiometry of cellulosome, a 12-subunit protein complex expressed from an operon in Gram-positive Clostridium cellullolyticum, is regulated by SRPS.

Ancient DNA was extracted from 13 skeletal remains from the burial groups of Khitan nobles, which were excavated in northeast China. The hypervariable segment I sequences (HVS I) of the mitochondrial DNA control region, in the 13 individuals, were used as genetic markers to determine the genetic relationships between the individuals and the genetic affinity to other interrelated populations by using the known database of mtDNA. Based on the phylogenetic analysis of these ancient DNA sequences, the genetic structures of two Khitan noble kindreds were obtained, including the Yel Yuzhi's kindred and the Xiao He's kindred. Furthermore, the relationships between the Khitan nobles and some modern interrelated populations were analyzed. On the basis of the result of the analysis, the gene flows of the ancient Khitans and their demographic expansion in history was deduced.

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a β1β2α1α2α3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the 1H-15N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought. Proteins 2006. © 2005 Wiley-Liss, Inc.